1-: ’e t“): ,9 v3. .. .. a . . . :3}. . safflififi: ....... .Haq...4....w.u,....~ ., a ‘ \Cyl.J- 4 1m? : ‘ 4 :5 :53... 5, mm r7: 4?“??? c 2 144$. t... .u JV? 5.1:: J: 4 r (5r. “.1 }ofv_ . .H , {e .523: 55.3 w? 4x". y » g 4.43% 5A. 4.3. 4.0. "7' it??? ”:3 3 5003 5447 44/3? LIBRARY ' Michigan State ' University This is to certify that the dissertation entitled AN ANALYSIS OF THE EXPRESSION, FUNCTION, AND EVOLUTION OF THE FTSZ PLASTID DIVISION GENES presented by KEVIN D. STOKES has been accepted towards fulfillment of the requirements for Ph.D. degreein Plant Biology ‘i’é‘pu/m/ 0461/ Major professor a Date 28 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 9 -f v'v ~- —.—~—. PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 cJCIRC/DateDue.p65—p. 1 5 AN .\ AN ANALYSIS OF THE EXPRESSION, FUNCTION, AND EVOLUTION OF THE FTSZ PLASTID DIVISION GENES By Kevin David Stokes A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Plant Biology 2003 AN -“ mpfilClfi.’ [CID (ill Chli‘il‘fl“ ek‘ill and Name diI‘NHn F7 [0.1\\Cllli" that entirt FI~Z ring « functii‘wnai . In t nudeur cm hiiimolngut 03:7]?qu \I I" an!) mt: :ihrit: ah 4 .I\{‘ TL ABSTRACT AN ANALYSIS OF THE EXPRESSION, FUNCTION, AND EVOLUTION OF THE FTSZ PLASTID DIVISION GENES By Kevin David Stokes The photosynthetic capabilities of plants cells are dependent on the presence and maintenance ofchloroplasts. The chloroplast complement of higher plant mesophyll cells, often very numerous, is maintained by division as cells differentiate and expand. Chloroplasts are evolutionarily derived from cyanobacteria through an endosymbiotic event and continue to bear several ancestral characteristics. The prokaryotic origins became even more evident when a chloroplast-targeted homologue of the bacterial cell division protein FtsZ was identified in plants. In bacteria, FtsZ is the first known protein to assemble at the division site. FtsZ has GTPase activity and polymerizes into a ring that encircles the cell at the cyoplasmic membrane surface. Following assembly of the FtsZ ring at the division site, several other proteins are recruited for assembly of a functional cell division apparatus. In contrast to most bacteria that encode a single F (32 gene, plants have multiple nuclear encoded FtsZ proteins that are localized to the chloroplast. The plant homologues have been grouped into two families, Ftle and FtsZZ, based on sequence comparisons. Arabidopsis plants express three different FtsZ homologues: one FtsZ] family member, AtFtle-l, and two FtsZZ family members, AtFtsZZ-l and AtFtsZZ-Z. Antisense repression experiments indicate members of both families are required for chitin»; Cilit‘lup and ti . 5 protein. OWN; Llhf‘dpn. ,. iU\iHn g. u Arabldnp CDNA d1. COOMWJIL' leiix 6‘. F: nipuidnnn In ; “E militim Ctlfncncd I menu‘s C :1ng \ 3K? {‘0an \ Slim-mm: a chloroplast division, suggesting that chloroplasts have evolved a more complex division apparatus than is present in bacteria. In an effort to better understand the role of the Ftle and FtsZZ proteins in chloroplast division. experiments were designed to investigate their function, expression, and evolution. To investigate some of the FtsZ functions in plastid division, the FtsZ proteins were overexpressed in Arabidopsis and the effects on chloroplast division were observed. The results indicate a stoichiometric balance is required for division and that disruption of that balance inhibits chloroplast division. Experiments with promoter-GUS fusion constructs were used to determine where and when FtsZ is expressed in Arabidopsis. Some of the FtsZ expression patterns were confirmed by measuring the cDNA distribution patterns. The results indicate the three F (32 homologues are coordinately expressed in several plant tissues including roots, meristems, and young leaves. FtsZ expression occurs in tissue regions with rapidly dividing chloroplast populations and is consistent with the role of FtsZ in chloroplast division. In an effort to understand when and why chloroplasts evolved two FtsZ families, we performed phylogenetic analyses, compared genetic structures, and compared conserved protein sequences. Phylogenetic analyses indicate the F tle and F1322 sequences diverged before the split between the chlorophycean and charophycean green algal lineages and possibly earlier. Genetic structure comparisons reveal intron positions are conserved within the F tle and F IsZ2 family members but differ between them, also supporting an early divergence. Comparison of conserved protein sequences indicated several regions in which the Ftle and FtsZZ family members differ. These conserved differences may define functional differences between the Ftle and FtsZZ proteins. OPPOIILL hull C (I " [hruttgt the gut: -. Anti lms' ‘UPI‘UFI .1 ifl'ClltL ill lllcse \Cdf ACKNOWLEDGEMENTS It is with deepest gratitude that I thank Dr. Katherine W. Osteryoung for the opportunity to work under her expert direction. without which this dissertation could not have come to fruition. For her time, patience. experience. and advice that guided me through my education and this body of work. opening to my view the world of plants and the gateway to a lifetime of exploration. 1 also want to thank her forjust being a friend and trusting rne beyond the laboratory. I am also grateful to Drs. Dean DellaPenna, Barbara Sears. and Tao Sang for their support and guidance as part of my committee. Thanks to the numerous co-workers and friends that supported, encouraged, had patience. and never gave up on me throughout these years of work. Finally, I am most grateful and indepted to my family for their love and support throughout my life. For their encouragement to follow my heart and dreams, always wanting to be a part of this incredible journey. They have been great examples to me and I am proud to be a part of their lives. TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... xii LIST OF FIGURES ....................................................................................................... xiii KEY TO ABBREVIATIONS ......................................................................................... xv CHAPTER 1 ...................................................................................................................... l FtsZ Ring Formation in Bacteria ................................................................................. 2 Bacterial FtsZ Binds and Hydrolyzes GTP .................................................................. 3 Bacterial FtsZ Proteins Form Polymers ....................................................................... 4 FtsZ and Tubulin Proteins are Structurally Similar ..................................................... 5 Dynamics of Bacterial FtsZ Polymer Formation ......................................................... 8 Other Bacterial Cell Division Proteins ....................................................................... 12 MinC, D, and E ................................................................................................... 12 ZapA ................................................................................................................... l4 FtsA ..................................................................................................................... l7 ZipA .................................................................................................................... l7 FtsK ..................................................................................................................... 18 FtsQ ..................................................................................................................... l9 FtsL ..................................................................................................................... 19 FtsB (ngQ) ....................................................................................................... 2O FtsW .................................................................................................................... 21 FtsI ...................................................................................................................... 22 FtsN ..................................................................................................................... 22 Stiochiometric Ratios are Important Among Bacterial Cell Division Components .. 23 [h Ft» Or E\; E\; Sh: Fl \ f" (IIIPTI AIM' lnh~ Rest .firn .4 Dhtu. Ft C} ha PL C05 L Developmental Patterns of Chloroplasts .................................................................... 24 The Arc Mutants and Chloroplast Division ............................................................... 28 FtsZ Function in Chloroplast Division ....................................................................... 31 FtsZ Rings in Chloroplasts ......................................................................................... 33 Other Potential FtsZ Functions .................................................................................. 34 Expression of F tsZ in Cyanobacteria ......................................................................... 35 Expression of F ml in Plants ...................................................................................... 36 Mitochondrial Division and FtsZ ............................................................................... 38 FtsZ Studies Reported Hereafter ................................................................................ 39 CHAPTER 2 .................................................................................................................... 43 Abstract ...................................................................................................................... 44 Introduction ................................................................................................................ 45 Results ........................................................................................................................ 50 FtsZ Proteins are Encoded by a Small Gene Family in Arabidopsis .................. 50 AtFtsZZ-l Is Not Imported into Isolated Chloroplasts ....................................... 52 Expression of AIF ml] -1 or AtFtsZZ-l Antisense Constructs Disrupts Chloroplast Division in Transgenic Arabidopsis ................................................ 54 AtFtle-l and AtFtsZ2-1 Have Distinct Functions in Chloroplast Division ..... 60 Two Nuclear F tsZ Gene Families in Plants ........................................................ 62 Discussion .................................................................................................................. 67 Functional Divergence of F tsZ Genes in Plants ................................................. 67 Concordance between Functional and Structural Studies ................................... 69 Implications of Transgenic Plant Phenotypes for Developmental Patterns of Plastid Division ................................................................................................... 71 Different Division Mechanisms in Chloroplasts and Mitochondria? ................. 72 Conclusion ................................................................................................................. 73 vi M. Methods ...................................................................................................................... 74 Plant Material ...................................................................................................... 74 cDNA Library Screening .................................................................................... 74 Hybridization Analysis ....................................................................................... 75 Chloroplast Import Assays .................................................................................. 75 Construction of Antisense Genes and Plant Transformation .............................. 75 Selection of Transgenic Plants ............................................................................ 76 Analysis of Transgenic Phenotypes by Microscopy ........................................... 76 RNase Protection Assays .................................................................................... 77 Identification of Other Plant FtsZ Genes and DNA Sequence Analysis. ........... 79 Acknowledgments ...................................................................................................... 79 Note Added In Proof .................................................................................................. 80 CHAPTER 3 .................................................................................................................... 81 Abstract ...................................................................................................................... 82 Introduction ................................................................................................................ 83 Results ........................................................................................................................ 85 Production of Antibodies Specific for Recognition of AtFtle-l or AtFtsZZ-l 85 Overproduction of AtFtle-l Inhibits Chloroplast Division in Transgenic Arabidopsis ......................................................................................................... 87 The Severity of Chloroplast Division Inhibition Is Proportional to AtFtle -1 Protein Level ....................................................................................................... 91 AtFtle-l Overexpression Produces a Novel Chloroplast Morphology in Some Transgenic Plants ................................................................................................ 95 Slight Overexpression of AtFtsZZ-l Does Not Disrupt Chloroplast Division 96 AtFtle-l and AtFtsZZ-l Accumulation Are Regulated Independently of One Another ............................................................................................................... 98 vii Db Nht Askh Char Afifix ("APT 1. like Discussion .................................................................................................................. 99 Correlation between AtFtle -1 Accumulation and Plastid Number .................. 99 Expression of the AtFtsZ2~l Sense Transgene Does Not Produce a Plastid Division Phenotype ........................................................................................... 101 Aberrant Chloroplast Morphology Associated with High Levels of AtFtle-l Protein ............................................................................................................... 102 Materials and Methods ............................................................................................. 103 Construction of Sense Transgenes and Plant Transformation .......................... 103 Selection and Propagation of Transgenic Plants ............................................... 103 Microscopic Analysis ....................................................................................... 104 Generation of Antipeptide Antibodies .............................................................. 104 Immunoblotting Procedures .............................................................................. 105 Competition Binding Assays ............................................................................ 107 AtFtle-l Quantification .................................................................................. 107 Acknowledgements .................................................................................................. 108 Chapter 3 - Supplemental Data ................................................................................ 109 AtFtsZZ-Z Cosuppression Inhibits Chloroplast Division ......................................... 109 Background ....................................................................................................... 109 Material and Methods ....................................................................................... l 10 Preparation of AtFtsZZ-Z Overexpressing Construct ................................. l 10 Plant Transformation and Screening for Kanamycin Resistant Plants ...... 1 10 Results ............................................................................................................... l 1 1 Discussion ......................................................................................................... l 13 CHAPTER 4 - - -- - -- -- -- - - - - - .............. - 114 Abstract .................................................................................................................... l 15 Introduction .............................................................................................................. l 16 viii .\1. Re‘li Materials and Methods ............................................................................................. 1 19 Determination of the FtsZ’s 5’-UTR Using 5’-RACE ..................................... 1 19 Construction of the F tsZ Promoter-GUS Constructs ........................................ l 19 Plant Transformation and Growth .................................................................... 121 GUS Staining .................................................................................................... 121 Detecting GUS Transcript by RT-PCR ............................................................. 122 Immunoblot Analysis of the FtsZ Proteins in Roots ......................................... 123 Plant Material for Real-Time RT-PCR Analysis .............................................. 124 Nucleic Acid Isolation for Real~Time RT-PCR Analysis ................................ 124 Real-Time RT-PCR Analysis ........................................................................... 124 Results ...................................................................................................................... 126 The Structures of the 5’—UTR Differ Between the F tsZ Genes in Arabidopsis 126 GUS Reporter Gene Expression Patterns. ......................................................... 128 The GUS Reporter Gene Is Not Transcribed Under Control of the AtFtsZI-l Promoter ............................................................................................................ 132 Arabidopsis Roots Express FtsZ Proteins ......................................................... 135 Relative Expression Levels of the Arabidopsis F {52 Genes Remain Constant Throughout Leaf Development ......................................................................... 137 Discussion ................................................................................................................ 141 CHAPTER 5 .................................................................................................................. 144 Abstract .................................................................................................................... 145 Introduction .............................................................................................................. 146 Materials and Methods ............................................................................................. 150 Sequences and Their Alignment ....................................................................... 150 Phylogenetic Analyses ...................................................................................... 150 Testing Constraint Trees ................................................................................... 152 ix Dl\\’ APPE \l Relative Rate Test ............................................................................................. 153 Genetic Structure Comparisons ........................................................................ 153 Protein Comparisons ......................................................................................... 154 Results ...................................................................................................................... 154 Phylogenetic Analysis of FtsZ Proteins from Photosynthetic Eukaryotes ....... 154 Phylogenetic Analysis of FtsZ cDNA Sequences from Photosynthetic Eukaryotes ........................................................................................................ 158 Testing for Differences in The Ftle and FtsZ2 Evolutionary Rates ............... 162 Genetic Structure Is Conserved Within the Plant FtsZ Clades ......................... 162 Ftle and FtsZ2 Protein Comparisons ............................................................. 166 Discussion ................................................................................................................ 169 Early Divergence of the Ftle and FtsZZ Families .......................................... 169 Relationship of the FtsZ] Clade to the Other Three Clades ............................. 169 Evolutionary Origins of the Chloroplastic Ftle and FtsZZ Sequences .......... I71 Acknowledgements .................................................................................................. 174 CHAPTER 6 - ................................ .............................. 175 Summary .................................................................................................................. 176 Chapter Summaries and Future Directions .............................................................. 177 Function of Ftle and FtsZZ Proteins in Chloroplast Division ........................ 177 Altered Ftle or FtsZZ Levels Disrupt Chloroplast Division .......................... 178 F tsZI and F tsZZ Genes are Coordinately Expressed ........................................ 180 Ftle and FtsZZ Sequence Comparisons Identify Potential Functional Differences ........................................................................................................ 184 APPENDIX A .......................................................................................................... 188 Al} LITER FtsZ Protein Alignment .................................................................................... 188 APPENDIX B .......................................................................................................... 197 FtsZ cDNA Alignment ...................................................................................... 197 APPENDIX C .......................................................................................................... 220 Conserved Residues of the FtsZ Proteins ......................................................... 220 LITERATURE CITED ................................................................................................. 230 xi Table 1 Table 1, Table 1 Table 2_ LIST OF TABLES CHAPTER 1 CHAPTER 2 Table 1. Percentage of Identitya between the Arabidopsis FtsZ Proteins and Those of Several Prokaryotes. ........................................................................................... 51 CHAPTER 3 Table 1. Phenotypic distribution of transgenic Tl plants ................................................. 90 CHAPTER 4 CHAPTER 5 Table l. Accession numbers for FtsZ protein and cDNA sequences ............................. 151 Table 2. Comparison of F tsZ intron lengths .................................................................. 165 CHAPTER 6 xii Figure 1. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 1. Figure 2. Figure 3. LIST OF FIGURES CHAPTER 1 The sequential assembly pathway of the cell division components at mid cell of E. coli .......................................................................................................... 16 CHAPTER 2 Alignment Showing Homology of AtFtsZ2-l to AtFtle -l and Several Prokaryotic FtsZ Proteins ................................................................................ 49 Hybridization Analysis ofAtFtsZI-I and AIFtsZZ-I in Arabidopsis .............. 53 In Vitro Assay for Post-Translational Import of AtFtsZZ-l to the Chloroplast..55 Phenotypes of Transgenic Plants Expressing Antisense Constructs ofAtFtle- I or AtFtsZZ-I ................................................................................................. 57 Relationship between Mesophyll Cell Plan Area and Total Chloroplast Plan Area in Transgenic and Wild-Type Plants ...................................................... 59 RNase Protection Assays of AtFtsZI-I and AtFtsZZ-l Expression Levels in Independent Transgenic Antisense Lines ........................................................ 61 Plant F tsZ Genes Can Be Grouped into Two Families on the Basis of Their Deduced Amino Acid Sequences .................................................................... 64 Phylogenetic Relationships among Plant FtsZ Proteins .................................. 66 A Tentative Model for FtsZ Localization and Function in Division of Higher Plant Chloroplasts ........................................................................................... 70 CHAPTER 3 Specificity of AtFtsZ antipeptide antibodies ................................................... 86 Phenotypes of transgenic plants overexpressing AtFtsZ] -l or AtFtsZZ—l ...... 88 Immunoblot analysis of plant extracts overexpressing AtFtle-l .................. 92 xiii figuri- Figu re Figure Figure Figure Figure 3 FigUre 4, Fiilllre 5, “gun ]. Figllre l Figllre 3. “flat nflwi Figure 6. Figure 4. Figure 5. Figure 6. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Relative levels of the 40-kD AtFtle-l polypeptide in plants expressing the AtFtsZI-I transgene ........................................................................................ 94 Immunoblot analysis of transgenic plant extracts expressing the AtFtsZZ-I transgene .......................................................................................................... 97 Immunoblot analysis of transgenic plant extracts expressing the AtFtsZZ-Z transgene ........................................................................................................ 1 12 CHAPTER 4 Genetic structure of the 5’-UTR region of the three F tsZ genes ................... 127 Histochemical localization of FtsZ-promoter activity during development of transgenic Arabidopsis .................................................................................. 130 Detection of GUS expression in transgenic Arabidopsis seedlings with the AtFtle-I promoter-GUS fusions ................................................................. 133 Immunoblot detection of FtsZ protein in root, stem, shoot apex, and immature leaf extracts ................................................................................................... 136 Quantification of FtsZ mRNA in various Arabidopsis tissues ...................... 139 CHAPTER 5 Phylogenetic analysis of FtsZ proteins ......................................................... 156 Phylogenetic analysis of F tsZ cDNA sequences ........................................... 159 Testing the maximum likelihood tree against two constraint trees ............... 161 Comparison of F tsZI and F tsZ2 genetic structures ...................................... 163 Comparison of FtsZ proteins from photosynthetic organisms ...................... 167 Two scenarios for the evolutionary origins of the plant FtsZ gene families. 172 CHAPTER 6 xiv Ah ac ATP .AIPasc BAC BLAST Cterm i :1 cDNA mm tarp dGTP 0x. D17 EDTA EST EMS Eu, its Ala arc ATP ATPase BAC BLAST bp C-terminus cDNA cpm CTP d dATP dGTP DNA DTT EDTA EST EMS FAM fts KEY TO ABBREVIATIONS Alanine Accumulation and replication ofchloroplasts Adenosine triphosphate Adenosine triphosphotase Bacterial artificial chromosome Basic local alignment search tool Base pair Carboxy terminus Completmentary DNA Counts per minute Cytidine tripohosphate Day Deoxyadenosine triphosphate Deoxyguanosine triphosphate Deoxyribonucleic acid Dithiothreitol Ethylenediaminetetraacetic acid Expressed sequence tag Ethyl methanesulfonate 6-carboxyfluorescein Filamentation temperature-sensitive XV (IQ GDP GFP GTP GTP...- GI’S HPLC hr IPTG Km" kl) or 1,1,. .\1 g GDP GFP GTP GTPase GUS HPLC hr IPTG Kanr kD or kDa L M mg min mM mRNA MS N-terminus ng nm PAGE PCR Gram Guanosine diphosphate Green fluorescence protein Guanosine triphosphate Guanosine triphosphatase B-glucuronidase High-performance liquid chromatography Hour Isopropylthio-B-galactoside Kanamycin resistant Kilodalton Liter Molar Milligram Minute Millimolar Messenger RNA Murashige and Skoog Amino terminus Nanogram Nanometer Polyacrylamide gel electrophoresis Polymerase chain reaction xvi PD Phe RAC E RT RNA SEDS SDS Sue T~D.\‘A TBR TA.\1R,\ L'TP [TR X‘glUc PD Phe RACE RT RNA SEDS SDS Suc T-DN A TBR TAMRA UTP UTR X-gluc Plastid dividing Phenylalanine Rapid amplification of cDNA ends Reverse transcription Ribonucleic acid Second Shape, elongation, division, and sporulation Sodium dodecyl sulfate Sucrose Transfer DNA Tree bisection-reconnection 6-carboxy-N,N.N’,N’-tetramethylrhodamine Uridine triphosphate Untranslated region 5—bromo-4-chloro-3-indolyl-B-D-glucuronid xvii CHAPTER 1 Introduction Cell division is a critical process for the proliferation of any organism. In 1968 Hirota et al. (1968) reported an experiment designed to decipher the molecular basis of bacterial cell division by isolation of bacterial mutants that were unable to divide at elevated temperatures. Due to their filamentous morphology. the mutants were calledfts forfilamentation temperature-sensitive. One of the proteins identified from the screen, FtsZ, has been shown to be a key component of the bacterial cell division machinery (Lutkenhaus et al., 1980). FtsZ proteins are structurally homologous to tubulins, have GTPase activity, and polymerize (Lutkenhaus and Addinall, 1997). FtsZ forms a ring at the cell division site and is an early step in the assembly of the cell division apparatus, which includes at least ten other components: ZapA, FtsA. ZipA, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, and FtsN (Errington et al., 2003; Margolin, 2003). To date, only homologues of FtsZ have been identified in higher plants, where they have been shown to function in the division of chloroplasts. FtsZ Ring Formation in Bacteria Although the filamentous phenotype observed in bacterial mutant screens indicated FtsZ had a role in cell division, it was a report by Bi and Lutkenhaus (1991) that suggested FtsZ formed a ring at the division site. Immunoelectron microscopy experiments localized FtsZ protein to a region at the midcell of Escherichia coli, a position that correlated to the division site. Cross-sectional micrographs of bacterial cells indicated the protein formed a ring at the inner surface of the outer membrane, which remained at the leading edge of the septum throughout constriction. FtsZ rings seemed to form just prior to septa] constriction, since localization was only observed in longer cells Obserx 8.1441} 4 gch\. liltllt‘dl ;, dDISNY Batten, Other G f prOFWISC. inlhé F1- e“'l‘i‘rim. GTp tdc ”mills" m. eipre\\jr The” fit. in C611 41] ATP QSC‘Q and not in shorter cells that had recently divided (Bi and Lutkenhaus, 1991). Quantification by immunoblotting indicated there are about 5,000—20,000 FtsZ molecules in each E. coli cell, which is enough protein to encircle the bacterium about 20 times at the division site (Pla et al., 1991; Dai and Lutkenhaus, 1992: Lu et al., 1998). Similar observations of the FtsZ protein localizing to the division septum have also been made in Bacillus subtilis (Wang and Lutkenhaus. 1993). Mutations in other downstream division genes, likefIsQ andftsl, produced cells with slightly constricted phenotypes and indicated FtsZ ring formation is one of the first steps that regulates the assembly of the division apparatus (Begg and Donachie. 1985). Bacterial FtsZ Binds and Hydrolyzes GTP Sequence comparisons identified motifs within the FtsZ protein that are similar to other GTP binding proteins. In eukaryotic tubulins the tubulin signature motif is proposed to be involved in GTP binding (Bourne et al., 1991) and this motif is conserved in the FtsZ protein (Erickson, 1995). Several groups used nucleotide-binding experiments to determine whether, like the tubulins, FtsZ also bound and hydrolyzed GTP (de Boer et al., 1992; RayChaudhuri and Park, 1992; Mukherjee et al., 1993). A single mutation in the tubulin signature motif significantly reduced GTP binding and cells expressing this mutant FtsZ protein failed to initiate division (de Boer et al., 1992). These findings indicated that GTP binding and hydrolysis are important for FtsZ function in cell division. Interestingly, this single mutation changed FtsZ from a GTPase to an ATPase, at least in vitro (RayChaudhuri and Park, 1994). 11.15 L mutar‘. that It (Mal/7. trump. filament “ihi‘ffi‘ds . Ml men. 181;:th FE\Z PERL WWW 311'): , Bacterial FtsZ Proteins Form Polymers In addition to displaying GTPase activity, tubulins polymerize into multimeric structures (Weisenberg, 1972). Since FtsZ has protein sequences and GTPase activity that are similar to tubulins, the polymerization capabilities of bacterial FtsZ were investigated. After incubating purified FtsZ protein with Mg2+ and GTP, protein filaments were observed by electron microscopy (Mukherjee and Lutkenhaus, 1994). This filamentation was not observed when GTP was absent or when mutant protein that was unable to bind GTP was used in the assay. However, filaments were observed when mutant protein was used that could bind but not hydrolyze GTP. These results indicate that FtsZ polymerization is dependent on nucleotide binding but not hydrolysis (Mukherjee and Lutkenhaus, 1994). Similar conclusions were reported by Bramhill and Thompson (1994) who observed GTP—dependent polymerization of FtsZ protein filaments by electron microscopy. Large, polymerized FtsZ protein structures will sediment by centrifugation whereas free protein will not. Using the amount of protein that sediments as a measure of polymerization, various nucleotide cofactors such as GTP, GDP, GTPyS, dGTP, and ATP were tested for their ability to promote polymerization of FtsZ into large structures (Bramhill and Thompson, 1994). Only when both Mg2+ and GTP were incubated with FtsZ protein did significant sedimentation occur. These results indicated hydrolysis is important for polymerization into large structures (Bramhill and Thompson, 1994), but polymerization was later determined not to require hydrolysis (Scheffers and Driessen, 2002). It was reported that polymerization into these larger structures was enhanced by a drop (FTP unknt 1996. 1 It‘sttlul enhan. 70.”) large ,1 tilamcn more ('11 ‘E‘EFalI Thernia 5h€€ls or “med 1‘: [Ubulin tubulin ,5 Th he re. flat, 19. Fszand 7:2,.“ .ggfined I. drop in the pH during the polymerization reaction (Erickson et al., 1996). Addition of GTP in the Bramhill and Thompson (1994) experiments actually lowered the pH and unknowingly enhanced formation of these large FtsZ polymer structures (Erickson et al., 1996). Detailed analysis of these large FtsZ polymer structures was done with high- resolution electron microscopy (Erickson et al.. 1996). Although formation was enhanced at lower pH, large complexes were observed to form in the pH range of 5.5 to 7.0, in the presence of either GTP or GDP. In these polymerization experiments several large structures were reported. One of the simplest structures was a long. straight filament. More complex structures consisted of two—dimensional sheets made of two or more of these filaments. Even more complex tubular polymers that are composed of several filaments were observed. Filaments were also observed that formed minirings. The miniring curvature was also manifested as filaments spiraling away from polymer sheets or tubular structures. All these structures, including the long straight filaments, curved filaments and minirings, and filament sheets, are similar to structures formed by tubulin. In addition, optical diffraction and computer image reconstruction of FtsZ and tubulin sheets indicated the lattice spacing is very similar between the two proteins. These results supported the similarity between the tubulin and FtsZ structures (Erickson et al., 1996). FtsZ and Tubulin Proteins are Structurally Similar Additional support for tubulin and FtsZ functional and structural similarity was reported by de Pereda et al. (1996). The structures of (1. B. and y tubulin as well as FtsZ orgal regio Clillltt protet C‘kprm lsZ p Flsz Ic‘ Chan The Fls. Malian. each pr and An;- were predicted using alignments of several sequences of each protein from various organisms. Although tubulin and FtsZ protein sequences overall are only slightly similar, regions of the predicted secondary structures were very similar, including the region containing the tubulin signature motif. To support these structural predictions, limited proteolysis experiments confirmed that regions of the tubulin proteins predicted to be exposed could be cleaved. Although proteolysis experiments were not done with the FtsZ protein, the predictions supported similar protein structures for both tubulin and FtsZ (de Pereda et al., 1996). The similarity between FtsZ and tubulin was graphically demonstrated by the determination of their respective crystal structures. Nogales et al. (1998) determined the crystal structure of the OLD tubulin heterodimers that had been polymerized into sheets. The FtsZ crystal structure was determined by Lowe and Amos (1998) using protein from Methanococcus jamzaschii. Both the tubulin and FtsZ structures are very similar, with each protein consisting of two domains that are connected by a linker (Lowe, 1998; Lowe and Amos, 1998; Nogales et al., 1998; Nogales et al.. 1998). The N-terrninal portion of FtsZ is called the GTPase domain (Lowe, 1998; Lowe and Amos, 1998) and consists of residues 38-227 of the M. jannaschii protein that form a six-stranded B-sheet with two and three helices on either side. The arrangement of the B-sheet and helices is consistent with the Rossman-fold topology (Rossmann et al., 1974). The GTP nucleotide is bound to one side of the B-sheet and makes contact with six loops. Because the structure of this domain in FtsZ and tubulin were distinct from that in other classical GTPases, it was proposed that FtsZ and tubulin form a new family of GTP hydrolyzin g enzymes (Lowe, 1998; Nogales et al.. 1998). The extreme N-terminal residues of tubulin protrude from the molecule and make important crystal contacts (Lowe and Amos, 1998). However, because this extension is highly variable among FtsZ proteins, being very short in E. coli FtsZ, it is unlikely to have a significant role in FtsZ polymerization. The C-terminal domain of the FtsZ protein consists of residues 228-356 and is connected to the N—terminal domain by a long helix. This region has a parallel four— stranded central B-sheet with two helices supporting it on one side (Lowe, 1998; Lowe and Amos, 1998). This domain has sequence similarity to calmodulins that bind calcium, and make binding of calcium feasible in the FtsZ protein (Lewe and Amos. 1998). The residues at the extreme C-terminus protrude from the molecule and are disordered. Comparisons among the FtsZ proteins indicate the C-termini are very divergent among different organisms. In tubulin, the corresponding region forms two helices that sit on the surface of the molecule (Nogales et al., 1998) and are located outside of the microtubule (Wolf et al., 1996). It was proposed that this C-terminal region of tubulin contacted microtubule-associated proteins or motor proteins (Nogales et al., 1998). By analogy, the C-terminus of FtsZ is likely to be important for interactions with other proteins. The divergence of the C—terminal region in both tubulin and FtsZ may reflect differences in the contacts that are required in different organisms. A region within the N-terminal domain of (it-tubulin. called the T3 loop, makes important contacts with GTP. Specifically, the loop contacts the y—phosphate of the GTP molecule (Nogales et al., 1998). Analysis of a molecular model of the microtubule, based on the crystal structure of the (LB-tubulin dimer (Nogales et al., 1998), indicates that a conformational change in the T3 loop might cause hydrolysis of the y—phosphate (Nogales et al., 1999). A similar conformational change was predicted from molecular mllthl mutat; had Lil mutate 0r GI) lL'l'OlL llidl Cur models of the FtsZ T3 loop (Diaz et al.. 2001). To test this prediction Diaz et al. (2001) mutated a threonine residue in the T3 loop to a typtophan. making an FtsZ protein that had different spectral characteristics when GTP or GDP were bound. Analysis of this mutated FtsZ protein indicate there is a change in the position of the T3 loop when GTP or GDP is bound (Diaz et al.. 2001 ). Analysis of the crystal structure of 0t,B—tubulin (Nogales et al., 1998) and microtubule modeling (Nogales et al., 1999) revealed a region that includes the T7 loop that contacted the GTP nucleotide bound to an adjacent monomer. The T7 loop region of FtsZ is also determined to be near the GTP nucleotide of an adjacent monomer by three- dimensional reconstruction of FtsZ polymerized into sheets (Lowe and Amos, 1999). Furthermore, mutation of residues within the T7 loop region severely reduces both polymerization and GTP hydrolysis (Lu et al., 2001; Scheffers and Driessen, 2001; Scheffers et al., 2002). These results indicate that GTP hydrolysis occurs in an active site that is composed of two FtsZ monomers. Dynamics of Bacterial F tsZ Polymer Formation Evidence that FtsZ dimers are present in vivo was reported by Di Lallo et al. (1999). They constructed a chimeric gene that consisted of FtsZ and a portion of the A repressor that is active only as a dimer. The repressor fragments alone were unable to dimerize, but functional repressor was present when the FtsZ-repressor chimera was expressed in E. coli. This indicated FtsZ formed dimers that allowed the repressor to become active. Using this as an assay, F tsZ mutants were isolated that could no longer dimerize. Mapping of the mutations provided insights into regions that were involved in “uh; on the lmpttfl tLueL GTPht Whine: C(M‘ipcrd Strandckj p0llmer Pill. mcr. millllttnk. ‘ the lonc. “mama 31ml... L, meS‘UIC j 1r. aMEmbh UX‘U 1A1)”: ‘ FtsZ dimerization (Di Lallo et al., 1999). In another study, point mutations within FtsZ genes were isolated that either affected GTP binding or polymerization of the FtsZ protein (Lu et al., 2001). The polymerization mutants had normal, or near normal, GTPase activity and filament assembly in WHO, but were unable to complement an F ml with a different temperature sensitive mutation. These results indicated mutations located on the lateral surface of FtsZ are not required for filament polymerization but are important for filament-to-filament interactions that make up the hi gher-order structures (Lu et al., 2001). Several reports have investigated the dynamics of FtsZ polymer formation and GTP hydrolysis. One question has been whether FtsZ monomers are added to a growing polymer in a cooperative or isodesmic manner (Romberg et al., 2001). Assembly of cooperative polymers requires a nucleation event followed by rapid growth of a multi- stranded complex; the monomer concentration is critical in this type of assembly. The polymers also tend to be very long whereas isodesmic polymers are short. Isodesmic polymers are single—stranded, require no nucleation event, and require no critical monomer concentration. Using electron microscopy and light scattering measurements (the longer the polymer the more light is scattered) to analyze the kinetics of polymer formation, Romberg et al. (2001) suggested that FtsZ polymer formation is isodesmic. Similar conclusions were reached by Rivas et al. (2000) using sedimentation analysis to measure the kinectics of polymer formation. In a recent report however, Caplan and Erickson (2003) suggest that FtsZ polymer assembly is cooperative while disassembly is isodesmic. Caplan and Erickson (2003) used isothermal titration calorimetry to measure the heat of FtsZ self-association to intest TClCds CODCCI IHCJ\U sednnt change all of ti FtsZ u. Theren “hen l- WQUth‘; mennnfi Fl-‘Z P1) investigate the thermodynamics of FtsZ polymerization. When proteins interact, heat is released. By measuring the amount of heat generated in reactions with different FtsZ concentrations the total number of protein interfaces could be inferred and these measurements were not biased toward longer molecules. as were the light scattering and sedimentation experiments. When FtsZ and GDP are injected into the calorimeter, the change in heat followed an isodesmic model. Under the conditions that were used almost all of the GDP-bound FtsZ protein was in a polymerized form. When the polymerized FtsZ was injected into the calorimeter, the change in heat was due to disassembly. Therefore, the authors suggest disassembly of FtsZ polymers is isodesmic. However. when FtsZ and GTP were injected into the calorimeter, a critical FtsZ concentration was required for polymerization and an isodesmic process could not model the thermodynamics of polymer formation. Therefore, the authors concluded assembly of FtsZ polymers is apparently cooperative (Caplan and Erickson, 2003). Understanding the exact role that GTP hydrolysis plays in FtsZ polymer formation has been difficult to determine, although progress has been made. Mingorance et al. (2001) incubated radiolabeled GTP with FtsZ protein, filtered the reaction to remove free nucleotide and protein, and collected polymerized protein. By analyzing the amount of bound radiolabel, they determined that most of the nucleotide in the FtsZ polymers was GTP. When labeled GTP was added to preformed polymers, they measured a rapid exchange of bound nucleotide. From these results it was suggested that hydrolysis of GTP destabilizes the polymers, but by quickly exchanging GDP with fresh GTP the polymers were restabilized (Mingorance et al., 2001). However, their method was flawed as it assumed the phosphate released by hydrolysis was rapidly removed from 10 13015111 Thh R" Stabilix. the proteins (Scheffers and Driessen, 2002). Scheffers et a1. (2000) performed similar nucleotide binding assays as Mingorance et al. (2001), but they extracted the bound nucleotide from the protein and analyzed the nucleotides by thin layer chromatography. They found that almost all of the bound nucleotide is hydrolyzed to GDP. In addition. they also found the phosphate released by the hydrolysis remains present in the FtsZ proteins. This indicated hydrolysis of GTP is very rapid and, by itself, does not destabilize polymers. In addition, Scheffers et al. (2000) found that GDP-containing polymers are stabilized by non-hydrolyzable GTP analogues in polymerization reactions. This result indicated that the FtsZ polymers, like tubulin, might have a GTP cap that stabilizes the polymerized structure. Although several properties of FtsZ have been described, many of the functions and structures that are physiologically relevant are still unclear. FtsZ proteins bind GTP and hydrolysis occurs very rapidly upon dimerization or polymerization (L6we and Amos, 1998; Scheffers and Driessen, 2002). Polymerization of FtsZ into filaments is likely an important step in the formation of the division ring. However, it is unclear whether the filaments form de novo at the site or whether short oligomers form in the cytosol and then aggregate at the division site (Stricker et al., 2002). The structure of the FtsZ ring is also unclear, but probably consists of linear filaments bundled together, since tubular structures have never been observed in bacteria by electron microscopy (Lu et al., 2000). Although the structure of the FtsZ ring is still unresolved, it seems to be very dynamic with subunits being exchanged throughout its existence (Stricker et al., 2002). Using green fluorescent protein (GFP)-labeled FtsZ and fluorescence recovery after 11 phttlt' slfUt‘i slmcl‘. \kllll L~ photobleaching, Stricker et al. (2002) observed that bleached signal in FtsZ ring structures recovers rather rapidly. even in constricting cells. This indicates that the ring structure, even during constriction. is dynamic with its components rapidly exchanging with cytoplasmic pools of FtsZ protein. The force-generating component of the ring structure is unknown. However, the FtsZ ring itself has been implicated based on observations that GTP hydrolysis causes FtsZ filaments to favor a curved conformation (Lu et al., 2000). Whether FtsZ is the force-generating component or not. one important function of the FtsZ ring seems to be the recruitment or localization of other protein components to the cell division apparatus (Errington et al.. 2003). Other Bacterial Cell Division Proteins MinC, D, and E The positioning of the FtsZ ring at the division site involves the functions of three proteins that make up the min system, MinC, MinD, and MinE. In E coli, these three Min proteins are encoded by and expressed from a single operon (de Boer et al., 1989). When MinC or MinD is mutated, FtsZ ring formation occurs not only at midcell but also at the poles, but no ring forms when MinE is mutated (de Boer et al., 1989, 1992). These and other results (Rothfield et al., 1999) indicate that MinC and MinD act in a complex as inhibitors of FtsZ ring formation, while MinE acts as a topological inhibitor of MinCD. A specific mutation in MinC reduces the ability of the protein to inhibit division, reduces its affinity for FtsZ, and reduces the inhibition of FtsZ polymerization (Hu et al., 1999). These observations indicate MinC interacts directly with FtsZ to prevent polymerization. l2 In 1.4. FtsZ 301K! Iv. o-li 21.1) 1 Very (,1 111m] l since 1: 111616111 CCll pnj MinC j, k“CID th. and de ] Sldliund In fact, fusion experiments with domains of MinC indicated the N-terminus interacts with FtsZ while the C-terminus was involved in dimerization with MinD (Hu and Lutkenhaus, 2000). Interactions between MinC and MinD have also been observed with the yeast two-hybrid system (Huang et al., 1996) and by proteolysis experiments (Szeto et al., 2001). The process by which the Min system regulates placement of the FtsZ ring is a very dynamic. Localization studies of GFP-labeled MinC indicate the protein oscillates from pole to pole (Hu and Lutkenhaus, 1999). The oscillation is dependent on MinD since its removal results in MinC becoming diffuse throughout the cytoplasm. MinD, therefore, seems to be the oscillating component that binds MinC to the membrane at the cell poles (Raskin and de Boer, 1999; Raskin and de Boer, 1999). The oscillation of MinC from pole to pole likely inhibits FtsZ ring formation. MinE, which functions to keep the MinCD protein complex from the mid-cell, is also localized to a ring (Raskin and de Boer, 1997). Careful analysis of GFP-tagged MinE indicates the ring is not stationary but oscillates from one side of mid-cell to the other. The oscillation seems to define a boundary beyond which MinCD is excluded. These results indicate there is a complex oscillating system that is important to properly place the FtsZ ring at mid—cell for cell division and keep FtsZ rings from forming at cell poles. Following the placement and assembly of the FtsZ ring, at least ten other components of the cell division apparatus localize to the division site (Chen and Beckwith, 2001; Errington et al., 2003). Nine of the components assemble in a specific order that starts with ZipA and FtsA and is followed by FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, and FtsN. The tenth component, the recently discovered ZapA. is likely one of the 13 first c" uncle. lt‘thN fun-ctr the lit 1: ZapA antagt =7 31.10: I. apparct: dll Mitt ll 15 ink Fl\Z_ 3. domain Fitting,“ fUnL'llriir; anKKCI F32 Illa-H l 16:44.,” 0, Alt UUUOI C“ mar. H\ . . 14,, T Jan; “410'“; first components to assemble following FtsZ ring formation, although the exact timing is unclear. The order of assembly of the cell division components and a schematic representation and their predicted membrane topology are diagrammed in Figure 1. The functions of each component of this intricately orchestrated assembly will be discussed in the following sections. ZapA ZapA was recently identified in a screen for genes whose overexpression could antagonize a MinD overexpression block on cell division (Gueiros-Filho and Losick, 2002). ZapA is not essential for division since knocking out the gene resulted in no apparent defect in septum formation. However, when FtsZ levels are reduced. cell division requires ZapA. Fluorescence microscopy of a GFP-tagged ZapA protein showed it is localized to the division site and affinity chromatography indicated it interacts with FtsZ. Structural predictions suggest ZapA is a cytoplasmic protein with a coiled-coil domain, which indicates it may dimerize. Polymerization studies showed that ZapA promotes assembly of FtsZ into bundled filaments in vitro, which indicated ZapA might function in binding filaments together in the FtsZ division ring. One mechanism proposed for inhibition of cell division by MinD is that MinD inhibits the association of FtsZ filaments. ZapA, however. induces association of FtsZ filaments, which may be the reason overexpression of ZapA antagonizes the effects of MinD overexpression. Although ZapA was originally identified in Bacillus subtilis, orthologues are present in many bacterial species, including one in E. coli that also localizes to the division site (Gueiros—Filho and Losick, 2002). 14 Fts Figure 1. The sequential assembly pathway of the cell division components at mid cell of E. coli. A, The dependency pathway for protein localization to the division site. Arrows point from proteins that are required before the protein is localized to the division site. B, Schematic representation of the 1 1 known E. coli cell division proteins and their predicted topology in the cell membrane. Components that have been shown to interact with FtsZ are diagramed in contact with the FtsZ ring. OM, outer memberane; PG, peptidoglycan; CM, cell memberane. Fts Fiall re A B OM '0 CD FtsZ / \‘ZapA FtsA ZipA V FtsK FtsQ /\ FtsL<+FtsB V FtsW Ftsl Ftsl FtsN Figure 1 l6 FtsA 1905 only 1 not 11.: (Pla ct FDA 1t 151a at; been d: fAlilltJU dun no. LO“ Q ; Zipx FtsA FtsA was identified in the initial screen for filamenting mutants (Hirota et al., 1968). Immunofluorescence microscopy showed that FtsA localizes to the division site only if FtsZ is located at the division site (Addinall and Lutkenhaus, 1996). FtsA does not have a membrane-spanning domain. but is associated with the cytoplasmic membrane (Pla et al., 1990). Mutations in the C-terminus of the FtsZ protein prevent localization of FtsA to the division site and indicate there is a direct interaction between the two proteins (Ma and Margolin, 1999). The crystal structure of FtsA from Thermotoga muritima has been determined and its structure resembles that of actin (van den Ent and Lowe, 2000). Although the function of FtsA is unknown, it is required for the recruitment of other downstream components of the division apparatus. Analysis of the crystal structure led Lowe and van den Ent (2001) to suggest a peptide located at the C-terminus of FtsA may interact with and be involved in the recruitment of the other cell division components. ZipA ZipA was identified in an affinity-blotting screen for proteins that bound FtsZ (Hale and de Boer, 1997). GFP-tagged ZipA localizes to the ring structure at the division site and is required for septum formation. Additional localization studies indicated FtsZ polymerization does not require ZipA, but without ZipA the other components of the division apparatus are not recruited to the septum (Hale and de Boer, 1999; Liu et al., 1999; Pichoff and Lutkenhaus, 2002). The localization of ZipA and FtsA to the FtsZ ring is independent, but recruitment of the other division components requires the presence of both. FtsZ polymerization studies indicated that ZipA stabilizes FtsZ filaments and even 17 pron: OIZU prulci dams 10 lllt' dnma 11 mm. Emmi mthe. Anna! mqut FtsA h. promotes bundling (RayChaudhuri, 1999). Furthermore, 143 residues at the C-terminus of ZipA are sufficient to induce bundling of FtsZ filaments (Hale et al., 2000). The ZipA protein has a flexible tether between a membrane-spanning domain and an FtsZ binding domain (Hale and de Boer, 1997; Ohashi et al., 2002). suggesting that ZipA anchors FtsZ to the cell membrane. Sequence comparisons indicate ZipA has microtubule binding domains (RayChaudhuri, 1999) and X—ray crystallography shows that ZipA interacts with a small fragment of FtsZ (Mosyak et al.. 2000). Specifically, ZipA interacts with a 17 amino acid peptide that is located at the C-terminus of FtsZ. This C-terminal peptide is in the same region where FtsA binds to FtsZ. Although ZipA has a membrane-spanning domain but FtsA does not, a single amino acid change in FtsA bypasses the requirement for ZipA in cell division (Geissler et al.. 2003). These results indicate both ZipA and FtsA have functional overlap although there are significant structural differences between them. FtsK By mapping a temperature-sensitive cell division mutant Begg et al. (1995) identified the gene ftsK . FtsK is localized to the division septum (Wang and Lutkenhaus, 1998; Yu et al., 1998) and its localization requires the prior localization of FtsZ, ZipA. and FtsA (Hale and de Boer, 2002; Pichoff and Lutkenhaus, 2002). FtsK has a domain that is predicted to span the membrane several times and a cytosolic domain that has homology to other nucleotide-binding domains (Begg et al., 1995). Several studies have indicated FtsK interacts with chromosomes and that this interaction is important for cell division (Liu et al., 1998; Yu et al.. 1998; Steiner et al., 1999; Barre et al., 2000; Boyle et 18 al.: that l can 1: FtsQ Fl—‘Q lt 3001; l mlCIm, Ell\ N”, d“ 1510; (ll—hen a P‘lxtu‘ld, PDQ pr 10 1he d FtsL “I‘m“ A." r/ , (It) .21 (It: al., 2000). A recent report showed by analytical gel filtration and electron microscopy that FtsK proteins form multimers (Aussel et al.. 2002). In addition. the FtsK multimers can translocate along DNA duplexes in vitm (Aussel et al.. 2002) and indicate FtsK may be a motor protein that moves replicated chromosomes through the division septum. Another function of the FtsK—chromosome interaction may be to keep the division apparatus between the replicated chromosomes. F tsQ FtsQ was described by Begg et al. (1985) in a screen for cell division mutants. FtsQ localizes at the division septum in an FtsK-dependent manner (Chen and Beckwith. 2001; Hale and de Boer, 2002; Geissler et al.. 2003) and immunofluorescence microscopy and GFP-tagging experiments confirmed FtsK is localized to a ring at the cell division site (Buddelmeijer et al., 1998; Ghigo et al., 1999). Although essential for division, the specific function of FtsQ is unclear (Chen et al., 1999). Various observations of the B. subtilis FtsQ homologue, DivIB, led Errington et a1. (2003) to postulate DivIB recruites FtsL to the division site. In support of this hypothesis, when FtsQ proteins with site-specific mutations were expressed in cells, FtsL failed to localize to the division septum (Chen et al., 2002). FtsL FtsL was identified in a screen for envelope proteins that are required for cell division (Guzman et al., 1992). The protein has been localized to the septum by GFP- tagging experiments in an FtsQ-, but not FtsI-, dependent manner (Ghigo et al., 1999). 19 The 1 PC”? iGtt/t pILlICll l \ldl‘lt‘ FtsL \ culled three 1 tranxm Bcck‘w FLSB (h “01km, Beck“; CI (ii. 2” B Vila. Itl Semm 1: mm b i‘ The FtsL protein has a domain that is located in the cytosol. a domain located in the periplasm, and a membrane-spanning domain that separates the other two domains (Guzman et al., 1992). The periplasmic domain has a coiled—coil motif that indicates the protein may multimerize. Mutagenesis of the coiled-coil motif makes FtsL dimers less stable in SDS treatment (Ghigo and Beckwith, 2000). Experiments where the domains of FtsL were swapped with similar domains from other proteins (for example, swapping the coiled—coil domain of FtsL with a coiled-coil domain from another protein) indicated all three of the FtsL protein domains are required for function, even though the cytosolic and transmembrane domains are not required for localization or dimerization (Ghigo and Beckwith, 2000). However, the role of FtsL in septum formation is still unclear. FtsB (ngQ) ngQ was identified in two different laboratories at about the same time, one working in Vibrio cholerae and the other in E. coli (Buddelmeijer et al., 2002). The Beckwith laboratory predicted FtsL would not dimerize in vivo very well and did computer-based searches for a structurally similar protein as a possible dimerization partner. ngQ was the most promising protein identified from the search (Buddelmeijer et al., 2002). The protein was renamed FtsB in a recent review (Buddelmeijer and Beckwith, 2002). GFP-tagging of FtsB showed the protein was localized to the division septum in an FtsQ-dependent manner, whereas mutations in downstream components, such as FtsW, did not disrupt FtsB localization (Buddelmeijer et al., 2002). When cells were depleted of FtsB, FtsL becomes unstable. which indicates that FtsB stabilizes FtsL and suggests that both assemble at the division site at about the same time. 20 Fa\t locah requxr “(mg F tsW FtsW was identified in a mutagenesis screen for cell division mutants and is localized to the mra region of the E. coli chromosome (Ishino et al., 1989). FtsW is required for cell division and is localized to the division septum (Boyle et al., 1997; Wang et al., 1998). Although Khattar et al. (_ 1997) observed the presence of FtsZ rings when one mutant FtsW protein was expressed. the FtsZ ring was not always observed with all the different FtsW mutants (Boyle et al., 1997; Khattar et al., 1997; Wang et al., 1998). Therefore, the exact point at which FtsW localized to the septum was unclear, but the results suggested that it might be quite early in the assembly of the division apparatus. Khattar et a1. (1997) also identified two different polypeptide products of the ftsW gene. It was suggested that one of these products might be involved in the early steps of assembling the division apparatus and that it stabilizes the FtsZ ring. However, Mercer and Wiess (2002) disputed this finding because they found that FtsW mutantions did not destabilize the FtsZ ring, at least not significantly. Furthermore, GFP-tagging indicated that FtsW is recruited to the septum after FtsL and FtsB, but before Ftsl (Buddelmeijer and Beckwith, 2002; Mercer and Weiss, 2002). Structural analysis of the Streptococcus pneumoniae FtsW protein indicates it has 10 membrane-spanning segments and a large extracytoplasmic domain (Gerard et al., 2002). FtsW, along with RodA and SpoVE, are founding members of the SEDS (shape, elongation, division, and sporulation) protein family (Henriques et al., 1998) and each SEDS protein seems to work in conjunction with a transpeptidase (Matsuhashi et al., 1990). Since FtsW and FtsI, a transpeptidase, are 21 CAPT; . ,r, AULg. ks ftsl anttht. be Ida lmmc lfrmint 1989;. 10¢;th €1.11” 1. RH pol. Unlnmt prllklll; 19981. \cpidll() FtsN expressed from the same operon (Ishino et al., 1989; Boyle et al., 1997) it has been suggested that FtsW may function in conjunction with Ftsl (Mercer and Weiss, 2002). Ftsl Ftsl was purified based on its ability to bind Cephalexin, a penicillin-type antibiotic (Tamura et al., 1980). Because it bound penicillin and was the third protein to be identified with this property, Ftsl was originally called PBP—3. Proteolyic digestion of inverted membranes suggested Ftsl is anchored to the cell membrane at the amino terminus with the bulk of the protein located in the periplasmic space (Bowler and Spratt, 1989). Wang et al. (1998) showed that Ftsl localizes to the division septum and that localization is dependent on the previous localization of FtsL to the division site (Weiss et al., 1999). In addition to septum localization, FtsI was also observed to localize at the cell poles by immunofluorescence microscopy. although the significance of this was unknown (Weiss et al., 1997). The PBP proteins, to which Ftsl belongs, are peptidoglycan transpeptidases and synthesize murein (Spratt, 1977; Goffin and Ghuysen, 1998). Specifically, synthesis of septal peptidoglycan by Ftsl is required during cell septation but not during cell elongation (Botta and Park, 1981). F tsN FtsN was identified in a screen for suppressors of an ftsA temperature-sensitive mutation (Dai et al., 1993). A late recruit to the division site, FtsN requires FtsK, FtsQ, FtsL, and Ftsl for localization to the septum (Chen and Beckwith, 2001). The protein has a noncleavable hydrophobic sequence that binds it to the membrane (Dai et al., 1993). 22 SCPllll Sliot'l maint.‘ of cell offunt An 1m letek ‘1“st Cells .5 ”WM 1. Cell d1. lt‘w], ‘. CC’Uld 199‘ .1, The function of FtsN is unclear. but it does have sequence similarity to cell wall amidases. Because of this similarity, Errington et al. (2003) suggested that FtsN allows septum constriction by hydrolyzing certain bonds in the cell wall. Stiochiometric Ratios are Important Among Bacterial Cell Division Components One important factor in the functionality of the cell division machinery is the maintanance of stoichiometric ratios among the different protein components. Inhibition of cell division in the FtsZ mutant (Lutkenhaus et al.. 1980) is due to decreased amounts of functional protein, which causes an imbalance in the ratio of the division components. An imbalance can also occur when protein expression is increased. When FtsZ protein levels were slightly increased, the number of initiated division events increased (Ward Jr and Lutkenhaus, 1985). This increase was manifested by constriction and separation of cells at both the cell center and at abnormal positions near the cell poles that resulted in non-viable minicells. However, dramatically increasing FtsZ protein levels inhibited all cell division and resulted in a filamentous phenotype similar to that observed when FtsZ levels were reduced. The inhibition of cell division observed with high FtsZ expression could be rescued by simultaneous overexpression of FtsA protein (Dai and Lutkenhaus, 1992). Overexpression of ZipA also inhibits cell division, but division can be rescued by concomitant overexpression of FtsZ (Hale and de Boer, 1997). These results suggest that the components of the bacterial cell division machinery are in a strict stoichiometric balance and that disruption of that balance is detrimental to cell division. Developmental Patterns of Chloroplasts The chloroplast complement of a mature mesophyll cell results from both proplastid (undifferentiated plastids) and chloroplast division. Proplastids are located in the root and shoot meristems. Proplastids that have constricted centers have been observed in shoots of spinach as well as in the roots of spinach, pea, barley, tomato, maize, lettuce, pine, watermelon, and wheat (Chaly and Possingham, 1981). The constrictions are likely an indication that the proplastids they are undergoing division. Further support for proplastid division comes from microscopic observations of Arabidopsis arc6 mutant plants (Robertson et al.. 1995). The chloroplasts in the arc6 mutants are very large, and only one or two are present in each mesophyll cell. Electron micrographs of the shoot apex from the arc6 mutant plants reveal very few and large proplastids in each cell, whereas proplastids in the shoot apex of wild-type Arabidopsis plants are smaller and more numerous. This indicates proplastid division is severely reduced in the arc6 mutant plants. During leaf development, mesophyll cells differentiate and expand. While the cells expand, the chloroplasts (differentiated proplastids) divide. Possingham and Lawrence ( 1983) summarized several studies that reported the number of chloroplasts in cells at different developmental stages. In spinach, beet, pea, bean, and wheat as mesophyll cells expand there is an increase in the number of chloroplasts in each cell (Possingham and Smith, 1972; Boffey et al., 1979; Lamppa et al., 1980; Scott and Possingham, 1980; Whatley, 1980; Tymms et al., 1983). In addition, measurements of cell size and chloroplast number in Arabidopsis and tobacco mesophyll cells indicate that the bigger the cell, the more chloroplasts it contains (Boasson et al., 1972; Kameya, 1972; 24 P}AL 01’ [1' bd Smtt Chlttf lias~ “hit. €\Pat Pyke and Leech. 1992; Pyke et al., 1994; Robertson et al., 1996). When cultured spinach or tobacco leaf disks are treated with cytokinin the size of the mesophyll cells increases. as does the number of chloroplasts in each cell (Boasson et al.. 1972; Possingham and Smith, 1972; Possingham et al., 1988). This relationship between cell size and chloroplast numbers has also been observed in Polytriclumz moss cells (Paolillo and Kass, 1977). This increase in chloroplast numbers indicates the chloroplasts are dividing while the mesophyll cells expand and suggest there is a close relationship between cell expansion and chloroplast division. In the monocot wheat, chloroplast division occurs in a relatively small region of the leaves where cell expansion is greatest (Pyke, 1997). Microscopic examination of the leaves of Arabidopsis, a dicot, indicates chloroplast division occurs over a longer period of cell development and expansion than in wheat (Pyke and Leech, 1992; Pyke, 1997). Therefore, the region where most chloroplast division occurs is less pronounced in Arabidopsis leaves. and other dicot leaves, than it is in wheat leaves. However. a chloroplast division gradient is still observed in dicots from leaf base to tip, with most of the division occurring in the basal portion of the Arabidopsis leaf (Possingham and Smith, 1972; Possingham, 1973; Leech et al., 1981; Pyke et al., 1991). Besides young leaves, cells in the rapidly expanding cotyledons and the basal stock of developing petals also have numerous chloroplasts undergoing division (Pyke, 1997; Pyke and Page, 1998). Dividing chloroplasts with several different shapes have been observed in electron micrographs (Leech et al., 1981; Leech, 1986; Leech and Pyke, 1988). From these observations, predictions have been made regarding the morphological changes that occur to the chloroplasts during division. The first proposed morphological change is a 25 cons ends 1981 bactc ester: nudig data. tPEhi lllash “'l‘tlt‘h Unglct andjn rmS ltji Chlorky Ewe“; mil. s " l (153% If) dlxlshw- the \1an 'Ahtdx l t I constriction at the center of the plastid. followed by twisting around this constriction that ends with separation of the two daughter chloroplasts (Chaly et al., 1980; Leech et al.. 1981). The constriction suggests chloroplasts divide by binary fission. similar to bacteria. The significance of the chloroplast twisting is unknown, but may be a result of external forces encountered by the chloroplast near the end of division. Ultrastructural studies in ferns identified the presence of electron-dense rings at the constriction of dividing chloroplasts (Duckett and Ligrone, 1993). These rings, called plastid dividing (PD) rings, have also been observed in red algae, green algae, and angiosperms (Hashimoto, 1986; Kuroiwa, 1989; Oross and Possingham, 1989; Ogawa et al., 1994), which indicates they may be ubiquitous in all plants. Although most of the observed PD rings in the various plants appear to be singlets, a double-ring structure was observed in Avena sativa (barley) (Hashimoto, 1986) and in the red alga Cyanidioschyzon merolae (Miyagishima et al., 1998), with one PD ring located on the cytosolic surface and a second on the stromal surface of the chloroplast membrane. Careful studies of the unicellular red alga Cyanidium caldarium revealed not only the double PD ring structure, but the presence of a third PD ring (the middle PD ring) located in the interrnembrane space (Miyagishima et al., 1998). Although the PD rings have been identified in many plants, following their presence throughout chloroplast division has been difficult. Kuroiwa et a1. (1998) were able to observe PD ring formation, contraction, and dispersion throughout the entire division process using synchronized cultures of the red alga C. caldarium. Formation of the stromal PD ring occurs first, followed by formation of the middle and outer PD rings (Miyagishima et al., 1998, 1998; Miyagishima et al., 1999; Miyagishima et al., 2001; 26 Miyagishima et al., 2001). Disassembly of the three rings is just the reverse of their formation. Throughout chloroplast division, the PD rings remain associated with the leading edge of the constricting plastid until dispersal upon chloroplast separation (Kuroiwa et al., 1998; Kuroiwa et al., 2002). It should be noted that the presence of a middle PD ring has been questioned, due to difficulty in resolving the inner and outer leaflets of the membrane (Hashimoto, 2003). Even so, the presence of an inner and outer PD ring suggests at least two complexes are involved in chloroplast division. It had been hypothesized that the stromal PD ring could be at least partly composed of FtsZ protein, because both the PD and FtsZ rings form at the stromal surface of the inner chloroplast envelope membrane (Miyagishima et al., 1998; McAndrew et al., 2001). However, careful examination of the FtsZ and PD rings in C. merolae by Miyagishima et al. (2001) determined they are separate and distinct structures. Furthermore, analysis indicates the FtsZ ring forms first and is followed by the formation of the stromal and cytoplasmic PD rings. In the late stages of chloroplast division, just prior to separation, the FtsZ ring disappears before the two PD rings (Miyagishima et al., 2001). Electron microscopic observations in Pelargonium zonale (geranium) also indicate the FtsZ ring forms before the stromal or cytoplasmic PD rings and disappear in the late stages of chloroplast division in higher plants (Kuroiwa et al., 2002). Measurements of the diameter of the rings indicate the thickness of the outer PD ring increases while the FtsZ and inner PD ring diameters remain constant throughout division (Kuroiwa et al., 2002). From these observations the authors suggested that the FtsZ and inner PD rings decompose (lose components) throughout division while the 27 0111: ind: Intar Imn: outer 1311} micrt 51mm struct pllflm The A 3 xet u mutarp and t. outer PD ring does not. They also suggest that the thickening of the outer PD ring might indicate it generates the force for chloroplast constriction (Kuroiwa et al., 2002). Although the composition of the cytoplasmic PD ring is unknown, isolation of intact outer PD rings from C. molerac has allowed for some protein characterization. Immunoblot analysis failed to detect FtsZ protein in samples enriched in the isolated outer PD rings, providing evidence that the outer PD ring is not composed of FtsZ protein (Miyagishima et al., 2001; Kuroiwa et al., 2002). Furthermore, immuno—electron microscopy with FtsZ antibodies determined the FtsZ ring is located in the chloroplast stroma and not the cytoplasm. Negative staining of isolated outer rings revealed the structure is a bundle of 5-nm filaments and SDS—PAGE analysis indicated a 56-kDa protein is a major component (Miyagishima et al.. 2001). The Arc Mutants and Chloroplast Division The first evidence for a genetic control of chloroplast division was the isolation of a set of arc (accumulation and replication of chloroplasts) mutants in Arabidopsis. These mutants were isolated through a screen of T-DNA— and EMS-mutagenized plants (Pyke and Leech, 1992; Pyke et al., 1994; Robertson et al., 1995; Robertson et al., 1996; Marrison et al., 1999) and show a range of defects in chloroplast division. Plants with the arc] mutation have an increase in the number of chloroplasts, while the size of those chloroplasts are reduced (Pyke and Leech, 1992). In contrast, the acm, arc3, and arc6 mutant plants have increased chloroplast size but decreased chloroplast number (Pyke and Leech, 1992; Pyke et al., 1994). The most severe chloroplast division mutant is the arc6 mutant, which has one or two greatly enlarged chloroplasts in each mesophyll cell. 28 :Antt ClllOl (311101 of lllt 1992 chlor. CDlUTI me \0' lelSir are In: a£lstr EEHe , all ha, arE6Ix e311m ,. d'ftuh 1,. Another interesting mutant is arc5, which also has reduced numbers of enlarged chloroplasts that are constricted in the center and seem to be arrested in the final stage of chloroplast division (Robertson et al.. 1996). Several reports describe a strong correlation in the arc mutants between the size of the mesophyll cell and the number of the chloroplasts they contain (Pyke and Leech, 1992; Pyke et al., 1994; Robertson et al., 1996; Marrison et al., 1999). As the number of chloroplasts in a cell decreases, the chloroplast size increases. This keeps the total chloroplast area within the cells constant. Compared to the 80 chloroplasts in a mature mesophyll cell of wild—type Arabidopsis plants, for instance, plants with the arc6 mutation have one greatly enlarged chloroplast, while plants with the arc] mutation have increased numbers of smaller chloroplasts. These results suggest the arc gene products are not involved in maintaining the chloroplast volume during cell expansion (Pyke and Leech, 1992; Pyke et al., 1994; Robertson et al., 1996; Marrison et al., 1999). Studies with the arc mutants have provided insights into the process of plastid division. Double mutants of arc] with arc3, arc5, arc6, or arc] l have phenotypes that are intermediate between the two parental mutant plants, suggesting that the ARC I gene acts on a pathway independent of the other genes (Marrison et al., 1999). The ARC6 gene seems to function upstream of ARC3, ARCS, and ARC I 1 since the double mutants all have one or two enlarged chloroplasts in the mesophyll cells, similar to the parental arc6 phenotype. The ARC3 protein is predicted to have a role in chloroplast division, but not proplastid division, since arc3 mutant plants have about 16 chloroplasts, which is the estimated number of proplastids that differentiate into chloroplasts. Most of the arc5 double mutant crosses resulted in chloroplasts that have a constricted phenotype and 29 pIOl Cll\lf Sequ bran Chlt’j‘tr deser the t") atthe based Chltfiir. that t 3003 ARTE. 3111039 Sinai, llnmh. memlr: in'fier; ”amt, Chlglhr DTP '74 . I "its”: provided further support that the ARC5 gene affects the final stages of chloroplast division. The gene encoding ARC5 was identified and reported by Gao et al. (2003). Sequence analysis indicated the gene codes for a dynamin-like protein of eukaryotic origin, since no prokaryotic homologues were identified. These results indicate the chloroplast divison apparatus is composed of proteins of both prokaryotic and eukaryotic desent. Chloroplast import assays indicate ARCS is located on the cytoplasmic surface of the outer chloroplast envelope, and studies with GFP-tagged ARC5 localized it to a ring at the division site (Gao et al., 2003). Although the exact function of ARCS is unclear. based on studies with other dynamin proteins it may function in constriction of the chloroplast, possibly as a force generating protein (Sweitzer and Hinshaw, 1998), or it may be a molecular switch (Sever et al., 2000; Gao et al., 2003; Miyagishima et al., 2003). Another recently identified protein that is required for chloroplast division was ARTEMIS (Fulgosi et al., 2002). In contrast to ARCS, ARTEMIS was of prokaryotic ancestry. When the cyanobacterial homologue is mutated, bacterial division is inhibited. Similarly, in Arabidopsis ARTEMIS mutants, chloroplast division is inhibited. Immunoelectron analysis, immunoblot analysis of isolated envelopes and thylakoid membranes, and envelope extraction experiments indicated ARTEMIS is an integral inner envelope membrane protein. ARTEMIS has some sequence similarity to translocases, which led the authors to suggest ARTEMIS may not have a direct role in chloroplast division but may influence the translocation of other chloroplast division proteins (Fulgosi et al., 2002). 30 rin idc onl prote ll‘l‘tt‘; tech, Sldbl ll “82 I Chili,“ . Plant i- Elite. P113143” Gimp; Willem The gene encoding ARC6 has also been recently identified (Vitha et al., in press). ARC6 is located in the inner envelope membrane and has a large domain that faces the chloroplast stroma. In addition, an ARC6-GFP fusion protein localizes to the division ring. Although ARC6 homologues are present in cyanobacteria, they have not been identified in other bacteria (Vitha et al., in press). This indicates ARC6 and its orthologues may be unique to photosynthetic organisms. Chloroplast division is inhibited in arc6 mutants and in plants overexpressing an ARC6 transgene, resulting in plants with one or two chloroplasts per mesophyll cell. Immunofluorescent microscopy of chloroplasts from the arc6 plants shows that Ftle and FtsZ2 proteins form short filaments, while plants overexpressing wild-type ARC6 have long Ftle and FtsZ2 protein filaments. Also, sequence comparisons identified a J-domain in ARC6 that is typical of DnaJ co-chaperones. The microscopy results along with sequence similarity to co-chaperones indicates ARC6 might have a role, either directly or indirectly, in stabilizing FtsZ rings in chloroplasts (Vitha et al., in press). FtsZ Function in Chloroplast Division Identification of an FtsZ homologue in plants indicated the proteins involved in chloroplast division have a prokaryotic origin (Osteryoung and Vierling. 1995). The first plant FtsZ homologue was identified in Arabidopsis through a homology search of the Expressed Sequence Tag database dbEST (Newman et al., 1994) using the E. coli FtsZ protein as a query sequence. Of the known prokaryotic FtsZ proteins, sequence comparisons indicated the plant homologue is most closely related to cyanobacterial FtsZ proteins, which is consistent with chloroplasts being ancestors of a cyanobacterial 31 en \\1 ch th chl and QUIZ undt Cl’lloi Is; sin 1995 Ytduc Bach Arabi dulcr dl\l\] endosymbiont (McFadden, 2001). The F [52 gene is located in the eukaryotic nucleus, which suggests it was transferred from the cyanobacterial endosymbiont during chloroplast evolution (Osteryoung and Vierling. 1995). Chloroplast import assays followed by protease digestion demonstrated that the plant FtsZ protein is localized to the chloroplast stroma. where it is processed by removal of the transit peptide (Osteryoung and Vierling, 1995). Proof that plant FtsZ proteins function in chloroplast division came from studies in moss and Arabidopsis. Strepp et al. (1998) used homologous recombination to knock out an F tsZ gene in the moss Plzyscomitrella patens, which resulted in cells with large, undivided chloroplasts. The large chloroplasts have diameters similar to wild—type chloroplasts, but their lengths were much longer. This lengthened chloroplast phenotype is similar to the filamentous phenotype observed in bacterial F {52 mutants (Strepp et al., 1998). In Arabidopsis, antisense repression of either of two nuclear-encoded F tsZ genes reduces chloroplast numbers from 100 to as few as one greatly enlarged chloroplast in each cell (see Chapter 2; Osteryoung et al., 1998). Sequence comparisons of the two Arabidopsis FtsZ proteins with other plant FtsZ homologues indicate they belong to two different families, called Ftle and FtsZ2 These results established that chloroplast division requires members of two FtsZ families. To further investigate the functional properties of the FtsZ proteins, transgenic plants were constructed with transgenes that overexpress either AtFtsZI-l or AtFtsZZ-l (see Chapter 3; Stokes et al., 2000). Immunoblot analyses and microscopic examination of the chloroplasts indicate increased AtFtsZ] —1 levels inhibits chloroplast division, and that AtFtle-l levels are directly correlated with the severity of the division defect. 32 et al. all th Yesh deter addit. high 1 there, 1 Prttt FtsZ; Sleci Ftsl] Slight increases in AtFtsZZ—l levels do not inhibit chloroplast division (Stokes et al.. 2000). However, analysis of the genomic AtFtsZZ-l sequence, and comparisons with other plant FtsZ sequences that had become available, indicated the AtFtsZZ-l cDNA used in the overexpression study was truncated. missing the amino terminus containing the chloroplast import signal. Therefore. I isolated a full-length cDNA clone for AtFtsZZ-l as well as for a second Arabidopsis F tsZ2 homologue. AIFIsZZ-Z (McAndrew et al., 2001). Import assays using the full-length Arabidopsis FtsZ proteins indicate that all three are localized to the chloroplast stroma (McAndrew et al., 2001). Fujiwara and Yoshida (2001) independently isolated cDNA clones for AtFtsZZ-l and AtFtsZZ-Z and determined that both proteins, tagged with GFP, are targeted to tobacco chloroplasts. In addition to localization studies, McAndrew et al. (2001) observed that overexpressing high levels of full-length AtFtsZ2-l protein inhibits chloroplast division, whereas slight increases do not inhibit division. These results indicate that both AtFtle-l and AtFtsZ2- 1 proteins have similar properties when their expression is increased. Overexpression of Ftle or FtsZ2 proteins probably inhibits chloroplast division by disrupting a stoichiometric balance that is required among the division proteins. F tsZ Rings in Chloroplasts Using an Ftle protein from Pisum sativum (pea), Gaikwad et al. (2000) showed that the plant FtsZ protein can polymerize, in Vitro, to form multimers. The pea FtsZ protein can also complement a mutant E. coli FtsZ protein (Gaikwad et al., 2000). Mori et al. (Mori et al., 2001) used fluorescence microcopy and immunogold localization to show that Ftle protein assembles into a ring structure in Lilium longiflorum 33 Chlt) ring \\ he O\Cl' the f THEN but ti 1“ 0 t Ollie A1Ft.) C3,.” . ”9'1? 1:. chloroplasts. In Arabidopsis plants, Vitha et al. (2001) observed both Ftle and FtsZ2 rings that colocalize at the chloroplast midpoint. Colocalization is also observed in plants where chloroplast division is inhibited due to overexpression ofAIFtle—l, overexpression ofAIFtsZZ-l, or mutations in arc6. In their studies on the PD rings in geranium, Kuroiwa et a1. (2002) not only determined that the FtsZ ring is distinct from the inner and outer PD rings but that antibodies against Ftle and FtsZ2 proteins react to the FtsZ ring, providing further evidence that the ring was composed of both proteins. These studies indicate that plant FtsZ proteins can polymerize, like the bacterial protein, but that chloroplast division involves a complex ring structure that is composed of at least two different FtsZ proteins. Other Potential FtsZ Functions Besides rings and filaments, FtsZ protein has also been observed in other complex formations in plant cells. One structure is a cytoskeleton-like network of FtsZ filaments in chloroplasts of P. patens overexpressing an FtsZZ protein (Kiessling et al., 2000). Other researchers (Vitha et al., 2001) have also observed these networks of FtsZ filaments in Arabidopsis. However, the network is likely an artifact of FtsZ overexpression since it is only observed in plants overexpressing FtsZ protein (Kiesslin g et al., 2000; Fujiwara and Yoshida, 2001; Vitha et al., 2001). During analysis of FtsZ-GFP fusion experiments, Vitha et al. (2001) noticed that AtFtle-l could be detected in “stromules”, which are thin connections between two chloroplasts (Kohler et al., 1997). Plant FtsZ proteins, therefore, could have a structural role in the formation of these thin chloroplast—to-chloroplast connections. However. At} pnj 111.1 C011 Tan ~41; kLt.‘ [1‘ St) proti It‘q L; {1’sz . AtFtle-l was only detected in the stromules of plants overexpressing the AtFtle-l protein and not in wild-type plants. Therefore, the localization of FtsZ to the stromules may also be an artifact of high AtFtle -1 levels. Another piece of evidence that FtsZ may function outside of plastid division comes from the isolation of a L. longiflormn cDNA from generative cells (Mori and Tanaka, 2000). Generative cells have no plastids and isolation of FtsZ cDNA from these cells indicated to the authors that the F tsZ genes might have functions that are not associated with plastid division. Since the experiments only detected FtsZ cDNA and not protein, the transcript may be produced but not translated. Further experiments will be required to determine if FtsZ protein is present in the generative cells that lack plastids. Ftle and FtsZ2 rings have also been observed in chloroplasts that do not seem to be undergoing division in mature leaf mesophyll cells (Vitha et al., 2001). If the chloroplasts are not dividing, do the FtsZ proteins have other, as yet unidentified function? In addition, the amount of Ftle transcript increased in abundance during petal development and was higher in yellow and orange varieties of Tegetes erecta L. (marigold) than in white varieties (Moehs et al., 2000). Although the presence of chloroplasts in the petals, especially at the base, cannot be ruled out, the increase in FtsZ transcript as petals develop and in the yellow and orange marigold varieties suggests FtsZ proteins may have a function in chromoplasts. EXpression of F tsZ in Cyanobacteria F tsZ expression in Prochlorococcus. a cyanobacterium, has been shown to be coordinated with cell division (Holtzendorff et al., 2001; Holtzendorff et al., 2002). Cell 35 Lll\ the s} n and ("if \ exp expt slUtl tHnl mR.‘ in th. dlt'] ‘ been dlfle Else thz . Oh)€f division of Prochlorococcus sp. strain PCC 951 1 cultures becomes synchronized when they are grown in a turbidostat with 12 hour day and night cycles. When the cultures are synchronized under these light conditions DNA replication occurs in the late afternoon and is followed by cell division at night (Holtzendorff et al., 2001). Immunoblot analysis of samples removed from the turbidostat over a three-day period indicates F tsZ expression is at a minimum during the day and maximum at night. Therefore, FtsZ expression seems to be correlated with the cell cycle. These results are supported by studies of naturally occurring synchronous Proclzlorococcus populations in the Red Sea (Holtzendorff et al., 2002). In these natural populations, the amount of expressed F tsZ mRNA was measured by real-time reverse transcriptase-polymerase chain reaction. As in the previous experiment, F tsZ expression is maximal at night when the cells are dividing, and at a minimum during the day. Differential expression of FtsZ has also been observed in Anabaena, where vegetative cells express FtsZ, but terminally differentiated heterocysts do not (Kuhn et al.. 2000). Expression of F tsZ in Plants A few studies have investigated the regulation of F tsZ gene expression in plants. El-Shami et a1. (2002) used a synchronized culture of Nicotiana tabacum cells to study FtsZ expression during the cell cycle. They observed oscillations of F tsZ expression that coincide with the cell cycle. In another set of experiments, Gaikwad et al. (2000) observed that the highest levels of a P. sativum F tsZ gene is in young leaves while old leaves have significantly less FtsZ expression. These expression results, along with the 36 cyanobacterial studies, suggest FtsZ expression in plants correlates with the cell cycle, but there may also be other factors that affect expression. Light and the growth hormone cytokinin are two other factors that may have a role in regulating FtsZ expression. When cultured spinach leaf disks were incubated with cytokinin not only was cell expansion induced. but chloroplast division was also induced (Possingham et al., 1988). Measurements of transcript amount indicate F IsZ expression is increased in excised cytokinin treated cucumber cotyledons, but not auxin treated cotyledons (Ullanat and Jayabaskaran, 2002). Also, Ullanat and Jayabaskaran (2002) observed increased F tsZ expression in light-treated cucumber cotyledons. Similarly, Gaikwad et al. (2000) noted that F tsZ expression increases when pea seedlings are exposed to light. Although informative, the plant studies only investigated the expression of a subset of the F tsZ genes encoded in these plants. In fact, only one F tsZI gene was analyzed in the P. sativum study (Gaikwad et al., 2000) even though FtsZ2 protein has been detected in peas (McAndrew et al., 2001). The N. tabacum study only investigated one of at least four F tle and one of at least two F IsZZ genes (El-Shami et al., 2002). And the expression of only one of at least three F tsZZ genes was analyzed in the cucumber study (Ullanat and Jayabaskaran, 2002). Analysis of the expression of all FtsZ genes in a plant is required before it can be concluded that all F tsZ genes are regulated in the same manner. The expression pattern of the three Arabidopsis F tsZ genes is the subject of chapter 4. 37 Ali! entlt et 31 All“, J Lilli.) Eithf ClO'v, \Ihic al.2 Ill Illt ll3eet "htcx Sam I .ird}: ECHO? fUnCt Olfar Mitochondrial Division and FtsZ Mitochondria are thought to have originated from an a-proteobacterial endosymbiont, rather than a cyanobacterium as has been proposed for chloroplasts (Gray et al., 2001). Two FtsZ proteins have been described in the golden—brown alga Mallomonas splcmlcns and the red alga Cyanidioschyzon merolae: one localizing to the chloroplast and the other localize to the mitochondria (Beech et al., 2000; Takahara et al., 2000). Sequence comparisons indicate that the mitrochondrial FtsZ proteins are more closely related to oz-proteobacterial FtsZ proteins than to the cyanobacterial proteins, which is in accordance with the presumed mitochondrial endosymbiotic origin (Gray et al., 2001). Although FtsZ proteins that localized to the mitochondria have been identified in these algae and protists, they are the exception rather than the rule among eukaryotes (Beech and Gilson, 2000; Beech et al., 2000; Gilson and Beech, 2001). In fact, few mitochondria actually use FtsZ for organellar division. For example, the yeast Saccharomyccs cercvisiac, the nematode C acnorhabditis elegans, and plants like Arabidopsis thaliana do not encode an FtsZ protein in their nuclear or mitochondrial genomes (Goffeau et al., 1996; Osteryoung, 2000). These results indicate that FtsZ did function in mitochondrial division anciently. but was lost during the evolution of the organelle. Instead of an FtsZ-based apparatus. mitochondrial division in plants, animals. and yeast involves systems where dynamins perform critical roles. In yeast, Dnml is a dynamin-like protein that is required for mitochondrial division (Bleazard et al., 1999; Sesaki and Jensen, 1999). In mutant dnml cells, mitochondria aggregate into networks or highly branched structures. Dnml is localized to the cytoplasmic surface of the 38 mitochondria, either at the site of constriction in dividing mitochondria or at the ends. if the mitochondria have recently divided. These results indicate Dnml functions in mitochondrial division. In addition to Dnm 1. several other proteins required for mitochondrial division have now been identified. including onl, Mdvlp, and Fislp (Bleazard et al., 1999; Sesaki and Jensen. 1999; Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000; Cerveny et al., 2001; Tieu et al., 2002). Besides yeast, dynamin-like proteins involved in mitochonrial division have been identified in the protozoan Dictyostelium discoideum (Wienke et al.. 1999), Caenorlzabditis elegans (Labrousse et al., 1999), humans (Smirnova et al., 1998; Pitts et al., 1999), and Arabidopsis (Arimura and Tsutsumi, 2002). These results indicate that in fungi, animals, and plants dynamin-like proteins. and not FtsZ. are involved in mitochondrial division. FtsZ Studies Reported Hereafter The primary aim of the work described in this thesis is to better understand the function, expression, and phylogenetic relationships of the plant FtsZ proteins. Work by Osteryoung and Vierling (1995) reported an FtsZ homologue in Arabidopsis is imported into the chloroplast. From this result it was hypothesized that the Arabidopsis FtsZ protein is involved in chloroplast division. Chapter 2 describes the isolation of a second Arabidopsis FtsZ homologue. Sequence comparisons indicate that the two Arabidopsis FtsZ proteins are grouped into different families, Ftle and FtsZ2 To investigate whether the two F tsZ genes are required for chloroplast division, plants expressing antisense constructs of each gene were analyzed. Microscopic examination of the mesophyll cells from these transgenic plants revealed that chloroplast numbers were greatly reduced and enlarged. The results from this chapter indicate that members of both the Ftle and FtsZ2 family are required for chloroplast division. Further investigation into the functions and relationships of the Ftle and FtsZ2 proteins are described in chapter 3. In bacteria. increasing FtsZ protein levels inhibited cell division (Bi and Lutkenhaus. 1990). We postulated that increased Ftle or FtsZZ protein levels in Arabidopsis plants would inhibit chloroplast division. To test this hypothesis, transgenic plants were prepared with constructs that overexpress the Arabidopsis FtsZ genes. Microscopic examinations in combination with immunoblot analysis indicated greatly increased AtFtle-l levels inhibit chloroplast division. In plants with the AtFtsZZ-l overexpressing transgene, only slight increases of AtFtsZ2-1 expression were observed and chloroplast division did not seem to be affected in these plants. Inhibition of chloroplast division was observed in some AtFtsZZ-l transgenic plants, but immunoblot analysis indicated expression of the AtFtsZZ-I gene was reduced, not increased. Transgenic plants with constructs designed to overexpress the AtFtsZ2-2 gene have now been prepared and are described in the final section of chapter 3. However, the transgenic plants did not overexpress AtFtsZZ-Z. Most transgenic plants had a wild-type- like chloroplast phenotype and immunoblot analysis indicated FtsZ protein levels were similar to those in wild-type plants. A few transgenic plants did have severely reduced chloroplast division, but instead of overexpressing the protein, the level of both AtFtsZZ- 1 and AtFtsZZ-2 protein were reduced. This AIFtsZZ-Z overexpression data were not available at the time the AtFtle-l and AtFtsZZ-l overexpression studies were published, but are now included at the end of chapter three. 40 The work in chapter 4 investigates the expression of the three Arabidopsis FtsZ genes. Because Ftle and FtsZ2 proteins are both required for chloroplast division, we postulated that the three Arabidopsis FtsZ genes are expressed in tissues where chloroplasts are dividing. To investigate expression of the FtsZ genes in Arabidopsis, transgenic plants with the GUS reporter gene fused to the promoters for the Arabidopsis FtsZ genes were prepared. Histochemical staining of these plants indicated that both FtsZ2 genes are coordinately expressed in many tissues, especially in tissues where chloroplast division is common. Very little staining was observed in the plants with the AtFtle-l promoter-GUS fusion construct and evidence is discussed that there are sequences after the start codon that are required for AtFtle-l expression. However, measurements of RNA indicated all three Arabidopsis FtsZ genes are coordinately expressed. Measurements of transcript amounts also indicate the Arabidopsis F tsZ genes are expressed at a constant stoichiometric ratio throughout leaf development, and this ratio may be important for chloroplast division. Most bacteria encode a single FtsZ gene, while plants encode multiple homologues that belong to two families. In an effort to better define the differences between the Ftle and FtsZZ family members. phylogenetic and sequence comparisons were performed and the results are reported in chapter 5. Phylogenetic analysis of FtsZ protein or cDNA sequences supports the grouping of the plant proteins into the Ftle and FtsZ2 families and indicates the two families diverged before the chlorophycean and charophycean green algae lineages split. Genetic structure comparison indicated that the intron positions are conserved within, but not between, the Ftle and FtsZ2 family members. These results further support an early divergence of the two FtsZ families. 41 Finally, we determined the amino acid residues that are conserved among the members of the Ftle and FtsZZ families. The residues that are conserved in each family, but that differ between them, may define their functional differences and will guide future work to determine why plants have two FtsZ families. 42 CHAPTER 2 Osteryoung KW, Stokes KD, Rutherford SM, Percival AL, Lee WY (1998) Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. Plant Cell 10: 1991-2004 43 " “‘ “’71 Abstract The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplastlocalized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis F tsZ gene (AtFtsZI-l or AtFtsZZ-l) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent F tsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process. Introduction A number of metabolic pathways crucial for plant growth and development are housed in plastids. Among the various types of plastids present in plants, chloroplasts have been studied most extensively because of their role in photosynthesis. However, plastids also synthesize various amino acids, lipids, and plant growth regulators and so are assumed to be essential for viability of all plant cells (Mullet, 1988). For plastid F continuity to be maintained during cell division and for photosynthetic tissues to accumulate the high numbers of chloroplasts required for maximum photosynthetic productivity, plastids must divide. Most of the available information concerning the process of plastid division is based on morphological and ultrastructural observations of dividing chloroplasts. During division, chloroplasts exhibit a dumbbell—shaped appearance in which the division furrow becomes progressively narrower. It is therefore generally agreed that chloroplast division occurs by a binary fission mechanism involving constriction of the envelope membranes (Leech, 1976; Possingham et al., 1988; Whatley. 1988). In plastids from a variety of higher plant and algal species, an electron-dense “plastid dividing ring" of unknown composition has been described in association with the zone of constriction. The electron—dense material often can be resolved into two concentric rings, one on the stromal face of the inner envelope and one on the cytosolic face of the outer envelope (Hashimoto, 1986; Oross and Possingham, 1989; Duckett and Ligrone, 1993; Kuroiwa et al., 1998). Little else is known regarding the structure of the division apparatus. Valuable information on the plasticity and genetic complexity of plastid division has been obtained from study of the arc mutations (for accumulation and 45 1’1 .a All"d My site .4' 1994 (1th“.- I 1 Riki h . . . 4}“ at [eplication of ghloroplasts) in the model plant system Arabidopsis (Pyke and Leech, 1992, 1994; Robertson et al., 1996; Pyke, 1997). These mutations define at least seven nuclear genes important in the control of plastid number in higher plants. However, none of the arc loci has as yet been isolated. and until recently, the molecular events underlying chloroplast division in plants have remained entirely undefined. A significant development in understanding the mechanistic basis for chloroplast division was the discovery that the nuclear genome of Arabidopsis encodes a eukaryotic homolog of the bacterial cell division protein FtsZ (Osteryoung and Vierling, 1995). The ftsZ gene was originally identified in a temperature-sensitive mutant of Escherichia coli that formed bacterial filaments at the restrictive temperature due to incomplete septum formation (Lutkenhaus et al., 1980), hence the designation fts (for filamenting temperature-sensitive). FtsZ is a rate-limiting cytoskeletal component of the cell division apparatus in prokaryotes (Ward Jr and Lutkenhaus, 1985; Baumann and Jackson, 1996; Margolin et al., 1996; Wang and Lutkenhaus, 1996), assembling at the nascent division site into a contractile ring just inside the cytoplasmic membrane (Bi and Lutkenhaus, 1991; Lutkenhaus and Addinall, 1997). Recent studies have revealed that FtsZ is a structural homolog and possibly the evolutionary progenitor of the eukaryotic tubulins (Erickson, 1995; de Pereda et al., 1996; Erickson et al., 1996; Erickson, 1998; L6we and Amos, 1998), and it can undergo dynamic GTP-dependent assembly into long polymers in vitro (de Boer et al., 1992; RayChaudhuri and Park, 1992; Mukherjee et al., 1993; Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994; Bramhill, 1997; Yu and Margolin, 1997; Mukherjee and Lutkenhaus, 1998). In a previous study (Osteryoung and Vierling, 1995), we 46 ltlt ltlt 1‘“ identified an F tsZ gene (AtFtle-l ) from Arabidopsis whose putative product exhibited between 40 and 50% amino acid identity with most of its prokaryotic counterparts. We further demonstrated that the gene product is synthesized as a precursor in the cytosol and post-translationally targeted to the chloroplast by virtue of an N—terminal chloroplast transit peptide. These findings provided direct evidence that the chloroplast division machinery in photosynthetic eukaryotes evolved from the endosymbiotic ancestor of chloroplasts and led us to hypothesize that this chloroplast-localized FtsZ has a function analogous to that of FtsZ in prokaryotes. that is, that it is a key structural component of the chloroplast division apparatus in plants (Osteryoung and Vierling, 1995). A role for a plant FtsZ in the division of chloroplasts was recently confirmed by Strepp et a1. (1998), who reported that a targeted knockout of PthsZ , an F tsZ homolog from the nonvascular plant Physcomitrella patens , resulted in severe disruption of chloroplast division in that organism. The localization of the PthsZ gene product was not investigated; however, the finding that it differs from the previously published Arabidopsis FtsZ in that it lacks an N—terminal extension that could function as a chloroplast transit peptide is an interesting observation. In this report, we describe the isolation of an additional cDNA from Arabidopsis encoding an FtsZ protein that also lacks a potential chloroplast targeting sequence. In addition, we provide experimental evidence that this protein is not localized in the chloroplast and demonstrate that both the chloroplast-targeted and nontargeted forms of Arabidopsis FtsZ are critical for the division of higher plant chloroplasts. Finally, we propose that plant F tsZ genes can be grouped into two families whose products are localized in different subcelluar 47 Figure 1. Alignment Showing Homology of AtFtsZ2-1 to AtFtle—l and Several Prokaryotic FtsZ Proteins. Identical amino acids are boxed. Asterisks indicate residues conserved among tubulins and FtsZ proteins (Erickson, 1998); double underlining indicates the tubulin signature motif(de Boer et al., 1992); dots indicate gaps in the alignment. The alignment was performed using CLUSTAL W 1.7 (Thompson et al., 1994). GenBank accession numbers for the proteins in the alignment are as follows: Bacillus subtilis. M22630; Staphylococcus aureus, [106462; Anabaena sp, U 14408; AtFtle-l, U39877; AtFtsZ2-l, AF089738; E. coli, AEOOOl l9; and Rhizobium meliloti, L25440. 48 Figure l AtFtsZZ-l ........................... MLRGEGTSTIVNPRKETSSGPVVEDFEEPS... Anabaena .................................... MENNR....IGEIVPGR ....... AtFtle-l MAIIPLAQLNELTISSSSSSFLTKSISSHSLHSSCICASSRISQFRGGFSKRRSDSTRSK B. subtilis ............................................. MLEFETNID ...... S. aureus E. coli R. meliloti . meliloti AtFtsZZ-l Anabaena AtFtle-l B. subtilis s. aureus E. coli R. meliloti AtFtsZZ-l Anabaena AtFtle-l B. aubtilis s. auraus E. coli B. meliloti AtFtsZZ-l ..EGEGRTVQMVQADAASVGATRR ............ Anabaena .QAAP...QQNAANARVVSAPPKRTPTQ ....... AtFtle-l .QKTLLTDPRAAKLLDKMGSSGQQ ........... B. subtilis .EKDVTKPQRPSLNQSIKTHNQSVPKRD...AKRE S. aureus .TSHGRKSGSTGFGTSVNTSSNATSKDESFTSNSS E. coli .RPEITLVTNKQVQQPVMDRXQQHGMAPLTQEQKP R. maliloti AIFDRSLDGTFRVS. ATGIDSNRSAQPTAPEAMNGQTAAKVPSRTLQ ............ AtFtsZZ-l ......... PSSSFRESGSVEIPEFLKKKGSSRYPRV... 397 Anabaena .TPLTNSPAPTPEPKEKSGLDIPDFLQRR...RPPKN... 379 AtFtle-l ......... ENKGMSLPHQKQSPSTISTK..SSSPRRLFF 433 B. subtilis EPQQQNTVSRHTSQPADDTLDIPTFLRNR.NKRG ...... 382 S. aureus NAQATDSVSERTHTTKED..DIPSFIRNR.EERRSRRTRR 390 E. coli VAKVVN.DNAPQTAKEPDYLDIPAFLRKQAD ......... 383 49 30 144 119 178 116 116 115 118 204 179 238 176 176 175 178 264 239 298 236 236 235 238 323 298 357 295 295 295 298 369 346 404 349 353 353 345 Ct“ Rt‘ its 861.. ch, tor l\ 10 L, tillm l} compartments and suggest a model for the functional role of plant FtsZ proteins in the division of chloroplasts that takes all of these findings into account. Results FtsZ Proteins are Encoded by a Small Gene Family in Arabidopsis We identified the first eukaryotic F tsZ gene from Arabiodpsis in the expressed sequence tag (EST) database (Newman et al.. 1994) on the basis of its high degree of conservation with bacterial fIsZ. Because the gene product was imported into chloroplasts, we originally referred to it as chtsZ. However, in keeping with guidelines for a standard nomenclature for plant genes (Lonsdale and Price, 1997), we have now renamed this gene ARATH;Ftle-I , which we henceforth designate as AtFtle-l. The existence of at least one additional nuclear F tsZ gene in Arabidopsis was subsequently indicated by the appearance in the database of a second EST with partial homology to both AtFtsZI-I and prokaryotic F tsZ genes. Because the sequence of this EST suggested that the cDNA from which it was derived was rearranged, a polymerase chain reaction fragment containing the region of homology with F 152 was amplified from Arabidopsis genomic DNA and used to screen an Arabidopsis cDNA library in an effort to isolate a nonrearranged clone. Three identical full-length cDNAs encoding a second homolog of Arabidopsis FtsZ were isolated. Comparisons between the encoded polypeptide, designated AtFtsZZ- 1, and several other FtsZ proteins are shown in Figure l and Table l. The amino acid sequence exhibits ~50% identity to both AtFtle-l and to bacterial FtsZ proteins and contains all of the features conserved among FtsZ proteins. These include the tubulin signature motif involved in GTP binding (de Boer et al., 1992; 50 Table 1. Percentage of Identity11 between the Arabidopsis FtsZ Proteins and Those of Several Prokaryotes Species AtFtsZ] -1b AtFtsZ2-lb Anabaena sp 64.5 62.3 Bacillus subtilis 54.1 60.5 Staphylococcus aurcus 59.9 58.4 E. coli 51.0 51.0 Rhizohium meliloti 49.5 45.9 aCalculated in pairwise comparisons by using the SIM local alignment algorithm (Huang and Miller, 1991) with the default parameters specified on the ExPASy Molecular Biology Server, Swiss Institute of Bioinformatics (http://expasy.hcuge.ch/sprot/sim-prot.html). Accession numbers are provided in the legend to Figure 1. bAtFtle-l and AtFtsZ2-l share 59.4% identity.127 51 RayChaudhuri and Park, 1992; Mukherjee et al., 1993), other residues conserved among tubulins and FtsZ proteins, and a stretch near the N terminus that is highly conserved among all FtsZ proteins (Figure 1). Among the prokaryotic FtsZ sequences, AtFtsZ2-1 is most closely related to that of the cyanobacterial species Anabaena (Table 1). This suggests that like AtFtle-l, AtFtsZZ-l probably had an endosymbiotic origin. However, a notable difference between the two Arabidopsis FtsZ proteins is that AtFtle-l contains a long extension at its N terminus relative to most prokaryotic FtsZ proteins that was shown previously to function as a chloroplast transit peptide (Osteryoung and Vierling, 1995). AtFtsZ2-l lacks an N-terminal extension. To investigate whether additional F tsZ genes exist in Arabidopsis, we probed DNA and RNA gel blots at moderate stringency with either the AtFtsZI-I or AtFtsZZ-l cDNA. On both DNA and RNA gel blots, AIFIsZI-I hybridized with a single band, as shown in Figures 2A and 2B (lanes 1), indicating that AtFtle-l in Arabidopsis is likely encoded by a single gene. In contrast, AtFtsZZ-l hybridized with two bands, which were distinct from those recognized by AtFtsZ] -1 (lanes 2). These results indicate the existence of at least three genes encoding FtsZ proteins in Arabidopsis: one encoding AtFtle - l , one encoding AtFtsZZ—l. and one encoding a protein that is closely related to AtFtsZZ-l. Recent sequence data from the Arabidopsis genome project have confirmed this conclusion (described below). AtFtsZZ-l Is Not Imported into Isolated Chloroplasts To test whether AtFtsZZ-l could be targeted to the chloroplast, we performed an in vitro chloroplast import experiment identical to that conducted previously for AtFtle- 52 A DNA B RNA ‘1-1' 2-1 1-1 2-1 Figure 2. Hybridization Analysis of AtFtle-l and AtFtsZZ-l in Arabidopsis. Hybridizations with either the AtFtle-l (l-l) cDNA (lanes 1) or the AtFtsZZ-I (2-1) cDNA (lanes 2) were conducted at moderate stringency. Length markers in kilobases are indicated at right. (A) DNA gel blot. Genomic DNA (1.5 ug) was digested with BamHI, separated on a 0.7% agarose gel, and transferred to a nylon membrane for hybridization. The signal shown for the faster migrating fragment in lane 2 disappeared when the blot was washed at high stringency (data not shown). (B) RNA gel blot. Poly(A)+ RNA was isolated from leaf tissue, and 1.5 pg was separated on a 1.5% agarose gel containing formaldehyde and transferred to a nylon membrane for hybridization. 53 l (Osteryoung and Vierling, 1995). The results are shown in Figure 3. A full-length radiolabeled AtFtsZ2-1 translation product was synthesized in vitro (Figure 3A, lane 1). The addition of isolated pea chloroplasts failed to effect import or proteolytic processing of the translation product (Figure 3A, lane 2). In a control reaction run at the same time, the AtFtle-l translation product was imported and processed (Figure 3B, lanes 1 and 2), and the import product could be protected from degradation during a postimport treatment with protease (Figure 3B, lane 3), as has been shown previously (Osteryoung and Vierling, 1995). Thus, the chloroplasts used for the experiment were import competent and intact. These results indicate that AtFtsZ2-1 cannot be posttranslationally targeted to the chloroplast in vitro as is AtFtsZ] -1, which is consistent with the apparent absence of a transit peptide at its N terminus. Neither could AtFtsZZ-l be imported into isolated yeast mitochondria (R. Jensen, personal communication). Analysis of the amino acid sequence by PSORT, a computer algorithm designed to identify potential intracellular sorting signals (Nakai and Kanehisa, 1992), did not predict other targeting sequences. We conclude from these findings that AtFtsZZ-l is probably not localized in chloroplasts or mitochondria in vivo but is likely a cytosolic protein. Expression of AtF tsZI -1 or AtFtsZZ-I Antisense Constructs Disrupts Chloroplast Division in Transgenic Arabidopsis We previously hypothesized a role for AtFtle-l in chloroplast division based on its high degree of conservation with the bacterial FtsZs and the demonstration that it is localized in the chloroplast (Osteryoun g and Vierling, 1995). To test whether AtFtle-l 54 A AtFtsZ2-1 B AtFtsZ1-1 lm Tr lm Tr Im +P """ . -68 o '43 -29 -. -18.4 1 2 3 Figure 3. In Vitro Assay for Post-Translational Import of AtFtsZZ-l to the Chloroplast. In vitro transcription and translation reactions were performed to obtain full—length, radiolabeled translation products (lanes 1; full-length products indicated by diamonds). Chloroplasts isolated from pea seedlings were incubated with the translation products for 30 min and then reisolated by centrifugation through Percoll to remove unimported radioactivity (lanes 2). Reisolated chloroplasts were dissolved in SDS sample buffer, and import products were analyzed by SDS—PAGE and fluorography. Equal amounts of chloroplast extract, as determined by chlorophyll measurements, were loaded on the gel (Chen et al., 1994). Molecular mass standards are indicated at right in kilodaltons. Tr indicates translation products; Im indicates import products. (A) Import assay for AtFtsZ2— 1. No radioactivity was recovered with the chloroplasts after reisolation, indicating that none of the radiolabeled translation product was imported. (B) Control import assay for AtFtle—l. As shown previously (Osteryoung and Vierling, 1995), the radiolabeled precursor (lane 1) was processed upon import (lane 2), and the import product (asterisk) was protected from proteolysis by thermolysin (lane 3). Im+P indicates import product protected from proteolysis. 55 and AtFtsZZ-l function as chloroplast division proteins, we conducted experiments to determine whether expression of antisense versions of either gene in transgenic Arabidopsis plants would inhibit chloroplast division. yielding plants with reduced numbers of chloroplasts. Antisense genes were constructed in the binary vector pART27 (Gleave, 1992), which incorporates the constitutive cauliflower mosaic virus 35S promoter to drive transgene expression as well as a selectable marker conferring plant resistance to kanamycin. Seeds were collected from vacuum-infiltrated (Bechtold et al., 1993; Bent et al., 1994) individuals, and transformants were selected by germination on kanamycin-containing media. Kanamycin-resistant (Kan') seedlings were transferred to soil for subsequent growth and analysis. To analyze chloroplast number and size, tissue samples from the first fully expanded leaves from multiple transgenic lines were prepared to allow visualization of individual leaf mesophyll cells under the microscope (Pyke and Leech, 1991). In both the AtFtsZI-l and AtFtsZZ-I antisense plants, a significant proportion of the Kanr Tl individuals exhibited drastically reduced numbers of chloroplasts. The phenotypes of these plants fell into two distinct classes, shown in Figure 4. The most commonly observed phenotypic class consisted of plants in which the mesophyll cells contained between one and three greatly enlarged chloroplasts, indicating that chloroplast division was severely disrupted. Indeed, the cells in most of these plants appeared to contain only a single enormous chloroplast (Figures 4A and 4C), compared with ~ 100 smaller chloroplasts in mature mesophyll cells from wild-type plants (Figures 4E and 4F). The other phenotypic class consisted of plants having between 10 and 30 chloroplasts of intermediate size in mature mesophyll cells (Figures 4B and 4D). Within an individual 56 Figure 4. Phenotypes of Transgenic Plants Expressing Antisense Constructs of AtFtsZ]- I or AtFtsZZ- 1. Tissue from the first leaves of 23-day-old plants was prepared for visualization of individual mesophyll cells by using Nomarski interference contrast optics, as described by (Pyke and Leech, 1991). (A) and (B) Cells from transgenic plants expressing the AtFtsZI-l antisense gene. (C) and (D) Cells from transgenic plants expressing the AtFtsZZ-l antisense gene. (E) and (F) Cells from wild-type Arabidopsis. Bars in (A) to (F) = 25 um. Images in this dissertation are presented in color. 57 transformant, most of the mesophyll cells appeared to be affected to the same extent. Interestingly, a continuous series in numbers of chloroplasts was not observed among the antisense plants. as might be expected from variations in transgene expression (Hooykaas and Schilperoort, 1992). Of 184 T1 plants showing reduced chloroplast numbers in the two antisense experiments, 165 had one to three chloroplasts and 19 had 10 to 30 chloroplasts. In all plants exhibiting reduced numbers of chloroplasts, visual inspection suggested that the reduction in chloroplast number was closely compensated for by a corresponding increase in chloroplast size (Figure 4). This observation was confirmed by measurements of chloroplast plan area (Pyke and Leech, 1992) as a function of mesophyll cell size in several transgenic lines having the most severe phenotypes. The data shown in Figure 5 demonstrate that the relationship between total chloroplast plan area and cell size over a wide range of cell sizes was almost the same in the transgenic and wild—type plants. These data indicate that despite the presence of only a single chloroplast in most of the mesophyll cells, the total chloroplast compartment volume was conserved. Similar results have been reported for several of the arc mutants (Pyke and Leech, 1992). DNA gel blot analysis on a subset of plants from both antisense experiments yielded distinct hybridization patterns, confirming that the reduced chloroplast numbers in different transgenic lines were the result of independent T-DNA insertion events (results not shown). Most of the transgenes segregated as single loci based on analysis of segregation for Kanr in the T3 and T3 generations. The phenotypes have remained heritable through the T4 generation, although in some lines a proportion of the Kanr 58 00 e ‘9 M1 00 A 08:; of o E 4000-1 0. - m 0 CD 0 9 I < D 0 f5 3000- I o. 9 2 .C 9 2000.. Slope R2 g Wt .89 .89 1*- 1-1 .05 .71 1000. 2-1 .34 .72 0 V I I U I 0 1000 2000 3000 4000 5000 6000 Mesophyll Cell Plan Area (umz) Figure 5. Relationship between Mesophyll Cell Plan Area and Total Chloroplast Plan Area in Transgenic and Wild-Type Plants. Chloroplast plan areas were measured over a wide range of cell sizes in several AtFtle-l (l-l, filled boxes) and AtF tsZ2-1 (2-1, filled triangles) antisense and wild-type (WT, open circles) plants. The slopes and R2 values calculated from linear regressions are shown. 59 progeny in each generation have reverted to wild type with regard to chloroplast number, suggesting the possibility of transgene silencing in those individuals (Matzke and Matzke, 1998). Despite the greatly reduced numbers and enlarged sizes of the chloroplasts in the antisense plants, their outward appearance under the conditions used in this study did not differ noticeably from the wild type. Flowering, seed production, and seed viability appeared normal for all transgenic plants, although subsequent analysis may reveal small variations, as has been observed for arc6, which grows somewhat more slowly than the wild type (Pyke et al., 1994). AtFtle-l and AtFtsZ2-1 Have Distinct Functions in Chloroplast Division A significant result of the transgenic plant experiments was that chloroplast numbers were reduced in plants expressing antisense copies of either AtFtle-l or AtFtsZZ-l. An important question arising from these findings was whether in each experiment this phenotype was due to downregulation of only the gene targeted for antisense suppression or whether expression of both genes was inhibited. To address this issue, we performed ribonuclease protection assays to investigate levels of AtFtsZ] -l and AtFtsZZ-I RNA in several transgenic lines showing the most severe phenotypes. In the AtFtle-l antisense plants, the levels of AtFtsZI-l RNA were reduced significantly below those present in the wild type, as shown in Figure 6A (lanes 1 to 3). However, AtFtsZZ-I RNA levels in these same plants were unaffected (Figure 6B, lanes 1 to 3). Similarly, in the AtFtsZZ-I antisense lines, only AtFtsZZ-l RNA levels were reduced (Figure 6B, lanes 4 and 5); AtFtle-I RNA remained at wild-type levels (Figure 6A, 60 WT AtFtle -1 AtFtSZZ-l A AtFtsZ1-1 -> B Figure 6. RNase Protection Assays of AtFtle-l and AtFtsZZ-l Expression Levels in Independent Transgenic Antisense Lines. Total RNA was isolated from 18-day-old plants exhibiting one to three large chloroplasts in mesophyll cells and hybridized with a radiolabeled RNA probe specific for either AtFtsZ/J (A) or AtFtsZZ-l (B) RNA. After treatment with RNase, the protected fragments were separated on a sequencing gel and detected by autoradiography. Lanes 1, wild type (WT); lanes 2 and 3, two independent AtFtle-I antisense lines; and lanes 4 and 5, two independent AIF tsZ2-1 antisense lines. Arrows indicate positions of fully protected fragments. (A) RN ase protection of AtFtle-l RNA. (B) RN ase protection of AtFtsZZ-l RNA. 61 lanes 4 and 5). These results confirm that chloroplast division could be inhibited in the transgenic plants by downregulation of either AtFtle-l or AtFtsZZ-l. Taken together. the results from the antisense experiments establish that AtFtsZ/- l and AtFtsZZ-l are not functionally redundant with regard to their roles in chloroplast division. Expression of both genes is necessary to achieve wild-type numbers of mesophyll cell chloroplasts in Arabidopsis. Two Nuclear F tsZ Gene Families in Plants Recently, a candidate for the second AtFtsZZ gene whose existence was predicted from hybridization studies (Figure 2) was revealed as a consequence of the Arabidopsis genome sequencing program. In a BLAST sequence similarity search (Altschul et al., 1990), we identified two independent genomic clones derived from different bacterial artificial chromosome (BAC) libraries that had overlapping end sequences with significant homology to AtFtsZZ-l. Further sequence analysis of these BAC clones yielded a partial amino acid sequence sharing 83% identity with AtFtsZZ-l in the region of overlap, confirming the existence of a second gene closely related to AtFtsZZ-l in Arabidopsis. We designate this newly identified gene as AtFIsZZ-Z. Six nuclear FtsZ genes from four plant species representing dicots, monocots. and lower plants, including the three Arabidopsis sequences described above, are currently documented in the database. Their deduced amino acid sequences are aligned in Figure 7A, and the percentages of amino acid and DNA identity in pairwise comparisons among them are presented in Figure 7B. Both AtFtsZ2—1 and AtFtsZZ-Z share >75% amino acid identity with PthsZ from the moss P. patcns. In contrast, these three proteins share 62 Figure 7. Plant FtsZ Genes Can Be Grouped into Two Families on the Basis of Their Deduced Amino Acid Sequences. GenBank accession numbers for the proteins used in these analyses are as follows: AtFtsZ2-1, AF089738; AtFtsZ2-2. B25544+B96663 (overlapping BAC clones); PthsZ, AJ001586; AtFtle-l. U39877; PsFtsZ. Y15383; and rice EST, C27863. (A) Alignment of the deduced amino acid sequences of six plant FtsZ genes currently represented in the databases, performed using CLUSTAL W 1.7 (Thompson et al., 1994 ) - Identical residues within the Ftle family are boxed in gray. Identical residues within the FtsZ2 family are boxed in black. AtFtsZ2-2 and the rice EST are partial sequences- Asterisks indicate identity among all plant FtsZ proteins shown at that position in the alignment. Dots represent gaps in the alignment. (B) Amino acid and DNA sequence identities in pairwise comparisons among six plant F tsZ genes. Percentages of identity were calculated as described in Table 1. Darker shading indicates amino acid sequence comparisons. Lighter shading indicates DNA sequence comparisons. Boxed groupings at left and right, respectively. show identities within the FtsZ2 and Ftle families. Asterisks indicate partial sequences. Dashes indicate partial sequences that could not be compared because they do not overlap. l 63 Figure 7 AtFtle-l AtFtsz2-1 Pthsz AtFtle-l AtFtaZZ-Z AtFtsZZ—l PthaZ fi .9. I I... Qtfififii i i I I Ii! .. AtFtszl-l PsFtsz AtFtsZZ‘Z AtFtIZZ—l ppm-z t on tut-«a toot a 0' not at a a It 0 antenna a... AtFtle -1 ‘7' tats Tvetapaaass. smears: 222222“ straws? amasmm I. lit! ti ’U Rice 25-: GISDIITIPGLVN’VDFAD K~v co scrum—1 OGISDIITIPGLVNVDFAD - 297 Purtsz EPTW mg asrsmrnpcnvworm xiv ‘ zaa xtrcazz-l ISDIITIPGLVNVDFAD an I 4 263 ypnaz 246 O. D... it... OQQOIIO IQOIOliIDII ‘ t I Q i O 'o) v hi 9 - K “iii?! . ~. - R I t... B i Q... AtFtle—l TIS'I'KSS 92;“ 433 Plrtsz KVESRPP 423 AtFtsZZ-l :fifliggmv 397 PthsZ ..... 377 Rice AtFtsZZ-1 AtFtsZZ-Z " PthsZ AtFtsZ1-1 PsFtsZ EST " AtFBZZ-1 AtFtsZZ-Z " Rice EST * 361% identity with AtFtle- 1. However, the proteins encoded by PsFtsZ. a full—length F tsZ cDNA from pea (Pisum sativmn), and by a partial rice cDNA identified in the EST database (rice EST) share >85% identity with AtFtle—l (Figure 78). Furthermore, like AtFtle -l , PsFtsZ has an obvious N-terminal extension (Figure 7A), with all of the hallmarks of a chloroplast targeting sequence (Von Heijne et al., 1991), but neither PsFtsZ nor the partial rice protein exhibit >6l% identity with either AtFtsZ2-1 or AtFtsZ2-2. Comparisons of DNA sequences among the six plant FtsZ sequences show similar trends (Figure 7B). The comparative data shown in Figures 7A and 78 provide evidence that the existing plant FtsZ sequences can be classified into two distinct families on the basis of their overall amino acid sequence similarities. This proposal is strongly supported by the results of parsimony analysis for phylogenetic relatedness. shown in Figure 8. One FtsZ family appears to comprise precursor proteins with N-terminal extensions (Figure 7A) that are synthesized in the cytosol and post-translationally delivered to the chloroplasts where the transit peptide is processed. Based on sequence comparisons (Figures 7A and 7B) and parsimony analysis (Figure 8), AtFtle -l and PsFtsZ are both members of this family, as is the partial protein defined by the rice EST. We designate this group the Ftle family. The other family, which we designate FtsZ2, includes AtFtsZ2-1. AtFtsZ2-2, and PthsZ. None of the FtsZ2 family members contains an obvious extension at the N terminus (Figure 7A) that would suggest localization in the chloroplasts, which is consistent with the inability of AtFtsZ2-l to be imported into isolated chloroplasts in vitro (Figure 3). These data suggest that the FtsZ2 proteins so far identified are localized in the cytosol. 65 Rice EST .. I AtFtsZ1-1 FtSZ‘I a 78 PsFtsZ AtFtsZZ~1 . 99 I 100 AtFtsZZ-Z F1522 L— PthsZ Anabaena FtsZ _9°_l Synechocystis FtsZ Figure 8. Phylogenetic Relationships among Plant FtsZ Proteins. Weighted maximum parsimony analysis was performed with PAUP 3.1.1 (Swofford, 1998), using the exhaustive search option. Gaps were treated as missing. A single most parsimonious tree was produced. Horizontal branch lengths are proportional to amino acid step differences. Numbers above the horizontal branches indicate bootstrap confidence limits (>50%) supporting each Clade of 1000 branch-and-bound replicates. The Anabaena sp (GenBank accession number Z31371) and Synechocystis sp (GenBank accession number D90906) sequences were defined as the outgroup. 66 Despite the apparent localization of the Ftle and FtsZZ family members in different subcellular compartments, our antisense experiments with transgenic plants clearly establish that members of both families. AtFtle -1 and AtFtsZ2- 1, play critical roles in the division of chloroplasts in Arabidopsis. Based on these findings, in conjunction with previous ultrastructural observations of plastid dividing rings both inside and outside the chloroplast (Kuroiwa et al., 1998), we propose that chloroplast division in higher plants is mediated by at least two functionally distinct forms of FtsZ: one represented by AtFtle-l, which is localized inside the chloroplast and functions on the stromal surface of the inner envelope, and one represented by AtFtsZZ-l, which is localized in the cytosol and functions on the cytosolic surface of the outer envelope. Discussion Functional Divergence of F tsZ Genes in Plants Our discovery that multiple F tsZ genes are present in the Arabidopsis nuclear genome provided the first clue that the FtsZ family in plants is functionally more complex than in prokaryotes. In prokaryotes, FtsZ is almost always encoded by a single gene (Lutkenhaus and Addinall, 1997). The amino acid sequence data derived from the four full-length cDNAs available from Arabidopsis (AtFtle-l and AtFtsZZ-l ), pea (PsFtsZ), and moss (PthsZ) and the two partial sequences available from Arabidopsis (AtFtsZ2-2) and rice (rice EST) provide evidence that plant FtsZ proteins from evolutionarily divergent species can be grouped into two families—FtsZ] and FtsZ2. Within each family, the proteins analyzed thus far share amino acid sequence identities ranging from 76 to 91%, whereas between families, the amino acid identities drop to 67 361%. The antisense experiments demonstrating similar phenotypes in plants that underexpress members of either family reveal that both FtsZ families contribute to and are essential for chlorOplast division in higher plants but indicate that they have unique functions in that process. Further analysis suggests that the functional distinction between the two FtsZ families in plants is at least partially a consequence of their differential subcellular localizations. At least two members of the Ftle family, AtFtle—l and PsFtsZ, possess extensions at their N termini when compared with most prokaryotic FtsZ proteins, whereas at least three members of the FtsZZ family, AtFtsZ2- l , AtFtsZ2-2, and PthsZ, have no such extensions. This correlation is evident even though the parsimony analysis producing the two Clades was based only on regions of overlap and therefore excluded the poorly conserved N termini. Consistent with the presence of N—terminal extensions in Ftle proteins, our in vitro chloroplast import results indicated that AtFtle-l can be imported into isolated chloroplasts (Osteryoung and Vierling, 1995). In addition, the database entry for PsFtsZ (GenBank accession number Y15383) indicates that this protein also is localized in the chloroplast. In contrast, AtFtsZZ-l lacks an N-terminal extension and cannot be imported into isolated chloroplasts, despite its demonstrated role in chloroplast division. PthsZ, which we have assigned to the FtsZZ family, also lacks an N-terminal extension but has been shown to function in chloroplast division (Strepp et al., 1998). We have been unable to find convincing evidence in the literature of nuclear- encoded chloroplast proteins that lack N-terminal targeting sequences. Therefore, although definitive localization of the plant FtsZ proteins awaits further experimentation, it seems probable that the FtsZ2 family members, which clearly function in chloroplast 68 division, are localized in the cytosol, whereas the Ftle family members are localized in the chloroplast. Concordance between Functional and Structural Studies Many ultrastructural studies of chloroplast division have reported the appearance of an electron-dense ring, termed the plastid dividing ring, in the zone of constriction before separation of the daughter plastids (reviewed in Kuroiwa et al., 1998). A similar electron-dense ring in dividing bacteria has been shown to contain FtsZ (Bi and Lutkenhaus, 1991 ). In algae and land plants, the plastid dividing ring can be resolved into two concentric rings that appear to reside on opposite sides of the chloroplast envelope (Hashimoto, 1986; Oross and Possingham, 1989; Duckett and Ligrone, 1993; Kuroiwa et al., 1998). A simple model incorporating these cytological observations with our findings on the role of FtsZ proteins in the division of chloroplasts is depicted in Figure 9. The model predicts that chloroplast-localized FtsZ] is a component of the inner plastid dividing ring, which is localized in a position analogous to that of the FtsZ ring in dividing bacteria. Although our in vitro import data indicated that newly imported AtFtle-l protein was in the soluble chloroplast fraction (Osteryoung and Vierling, 1995), we expect to find that during plastid division in vivo at least a portion of the protein is localized to the inner envelope surface. This is consistent with the finding that the FtsZ ring in bacteria is able to undergo assembly and disassembly in vivo (Addinall et al., 1997; Pogliano et al., 1997). With regard to the composition of the outer plastid dividing ring, several reports have described the appearance of fine filaments around the isthmus of dividing chloroplasts (Chida and Ueda, 1991; Ogawa et al., 1994; Kuroiwa et 69 Cflosol Outer Envelope FtsZZ—> 2 Figure 9. A Tentative Model for FtsZ Localization and Function in Division of Higher Plant Chloroplasts. The model predicts that (1) Ftle family members are post—translationally targeted to the stroma, where they assemble at the surface of the inner chloroplast envelope to become a component of the inner plastid dividing ring; (2) FtsZZ family members remain in the cytosol and assemble at the surface of the outer envelope to become a component of the outer plastid dividing ring; and (3) the two FtsZ containing rings function together on opposite sides of the chloroplast envelope to effect constriction of the organelle. Other molecules, such as actin, may also participate. 7O al., 1998), supporting the idea that cytoskeletal elements participate in chloroplast division at the cytosolic surface. There has been some speculation that actin may be a component of the outer ring, although the evidence is inconclusive (Kuroiwa et al., 1998). Our model predicts that the outer ring is composed at least partially of FtsZ2, although actin could also be a component or may interact with it. The fact that both chloroplast and putative cytosolic forms of FtsZ are required for chloroplast division suggests that the two FtsZ-containing plastid dividing rings proposed in the model function together in some way to accomplish constriction of the chloroplast. Whether FtsZ proteins in plants or bacteria are themselves force generating or whether other molecules, such as motor proteins, provide the motive force necessary for division remains unknown. Experiments to test and further refine this model are under way. Implications of Transgenic Plant Phenotypes for DeveIOpmental Patterns of Plastid Division An interesting observation from our antisense experiments is that reduced expression of the F tsZ genes does not yield a continuum in the number of mesophyll cell chloroplasts present in different transgenic lines, as might be expected from variations in transgene expression (Hooykaas and Schilperoort, 1992). Rather, the phenotypes fall into two discrete classes in which the cells contain either one to three large chloroplasts or 10 to 30 intermediate-sized chloroplasts. Analysis of plastid division patterns in several species indicates that division of proplastids in the shoot apical meristem is sufficeint to maintain the proplastid population at 10 to 20 per cell, whereas increased division of 71 developing or fully differentiated chloroplasts results in the proliferation of mesophyll cell chloroplasts that normally accompanies leaf maturation (Pyke, 1999). Consequently, we infer from the phenotypes of the transgenic plants that both proplastid and chloroplast division are inhibited in plants with the most severe phenotypes, whereas plants with intermediate phenotypes are apparently inhibited primarily in division of differentiated chloroplasts. Because similar phenotypes are observed among AtFtsZ] -1 and AtFtsZZ-l antisense lines, these findings further imply that both of these genes function in proplastid as well as in chloroplast division. In addition, the lack of a continuum among the observed phenotypic classes suggests that threshold levels of FtsZ expression may be necessary for plastid division, possibly for complete formation of the plastid dividing rings, and that constriction of a few small plastids in the meristem and young leaf cells may require lower levels of FtsZ than does constriction of larger chloroplasts later in leaf development. These conjectures can be investigated by further analysis of the transgenic plants for FtsZ expression levels and plastid numbers in different cell types. Different Division Mechanisms in Chloroplasts and Mitochondria? It seems reasonable to suppose that chloroplasts and mitochondria, both of which presumably evolved from eubacterialike endosymbionts (Gray, 1989, 1993), would divide by similar mechanisms. Therefore, it is surprising that BLAST searches to date have failed to identify FtsZ homologs in the recently completed Saccharomyces cerevisiae nuclear or mitchondrial genomes or in the genomes of any other nonphotosynthetic eukaryote (K.W. Osteryoun g, unpublished results). These findings suggest that the mechanism of mitochondrial division differs fundamentally from that of chloroplasts and bacteria. Because F tsZ is an ancient gene family, predating the split between the eubacteria and archaebacteria (Margolin et al., 1996; Wang and Lutkenhaus, 1996), this observation further implies that an FtsZ-based division apparatus may have been present early in the evolution of mitochondria but was supplanted over evolutionary time by a different system. Perhaps studies of primitive eukaryotes will eventually reveal remnants of a prokaryotically derived mitochondrial division apparatus. Conclusion The data presented here confirm that the mechanism of chloroplast division in higher plants has its evolutionary origins in the mechanism of prokaryotic cell division, as was first suggested by the discovery of a chloroplast-localized form of FtsZ. Evidence already exists that some additional components of the chloroplast division machinery are also of prokaryotic origin. For example, homologs of MinD, a gene involved in the positioning of the FtsZ ring in bacteria (de Boer et al., 1991; de Boer et al., 1992), are present in the plastid genome of the unicellular green alga Chlorella vulgaris (Wakasugi et al., 1997) and the nuclear genome of Arabidopsis (K.W. Osteryoung and K.A. Pyke, unpublished data). However, the demonstration that multiple forms of FtsZ are involved in chloroplast division suggests that the prokaryotic division apparatus has been elaborated during the evolution of photosynthetic eukaryotes, resulting in a more complex machinery for chloroplast division in which constituents of both the chloroplast and cytosol cooperate to achieve constriction of the organelle. Further studies to firmly establish the subcellular localization of different FtsZ proteins in plants and define other components of the plastid division apparatus will no doubt reveal additional similarities 73 and differences between the mechanisms of chloroplast and prokaryotic cell division. Methods Plant Material Arabidopsis thaliana ecotype Columbia was used for all experiments. Seeds were sown on Supersoil (Rod McLellan Co., South San Francisco, CA) potting mix and vermiculite (4:1 mix) and stratified at 4°C for 48 hr in the dark before germination. Plants were grown in controlled environment chambers at a relative humidity of 40% and provided daily with 16 hr of light (125 umol m~2 sec_') at 20°C and 8 hr of dark at 18°C. The age of the plants was taken from the date the seeds were placed in the light. cDNA Library Screening Primers flanking the region of homology to FtsZ present in an expressed sequence tag (EST) (accession number 248464) were used to amplify the corresponding region of Arabidopsis genomic DNA. The primers had the following sequences: foward primer, 5’—CCAGGCTATGAGAATGTCT- 3’; and reverse primer, 5’- CTGTGACAAAGACCATATCTGAGC- 3‘. The amplified fragment was gel purified, radiolabeled with 32P-dATP by the random primer method (Feinberg and Vogelstein, 1983), and used as a probe to screen the 7LPRL2 cDNA library (Newman et al., 1994) at high stringency (65°C, 0.2 X SSC [1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate]). The library, obtained from the Arabidopsis Biological Resource Center (Columbus, OH; stock number CD4-7), was constructed in the hZipLox vector (Gibco BRL). Three strongly hybridizing clones were obtained. The pZLl plasmids containing the 74 hybridizing cDNAs were excised from the phage DNA according to the manufacturer’s instructions. End sequencing of the inserts indicated that all three clones were identical. One clone was sequenced completely on both strands and designated AIFtsZZ-l. Hybridization Analysis The methods used for DNA extraction, RNA extraction, poly(A)+ RNA isolation, and RNA and DNA gel blot analyses were performed as described previously (Osteryoung et al., 1993). For DNA gel blot analysis, 1.5 ug of genomic DNA was digested with BamHI and electrophoresed through a 0.7% agarose gel. Blots were washed in 0.2 x SSC at 46°C before being exposed to film at —80°C with an intensifying screen. For RNA gel blot analysis, 1.5 ug of poly(A)+ RNA isolated from rosette leaves of 3- to 4-week-old plants was separated on a 1.5% agarose gel. Blots were washed in 0.1 X SSC at 60°C and exposed to film for 1 week. Chloroplast Import Assays In vitro chloroplast import assays were performed for AtFtle —1 and AtFtsZ2-l as described by (Osteryoung and Vierling, 1995) and (Chen et al., 1994). Construction of Antisense Genes and Plant Transformation The plasmid SN506 (Norris et al., 1998). a derivative of the binary plant transformation vector pART27 (Gleave, 1992). was digested with XhoI and Xbal to remove the insert. The gel-purified vector fragment was ligated directionally to a 743-bp XbaI-AVaI fragment spanning nucleotides 132 to 875 of the AtFtle-l cDNA or to a 75 l 164-bp SpeI—Aval fragment spanning nucleotides 126 to 1290 of the AtFtsZZ-I cDNA. The ligation products were amplified in Escherichia coli, and the resulting plasmids were purified and transferred to Agrobacterium tumcfacicns GV3101 (Koncz and Schell, 1986). Transformation of Arabidopsis was done using the vacuum infiltration method (Bechtold et al., 1993; Bent et al., 1994). Selection of Transgenic Plants T. seeds collected from vacuum-infiltrated plants were sown on Rockwool (GrodanHP; Agro Dynamics, East Brunswick, NJ) (Gibeaut et al., 1997), saturated with nutrient solution containing 100 mg/L kanamycin (Fisher Scientific, Hampton, NH), covered with a clear plastic lid, and stratified and grown as described above. Kanamycin- resistant (Kan') seedlings were transplanted to soil at 14 days by cutting the surrounding Rockwool and placing both Rockwool and seedlings in soil. T3 seed were collected from individual T, plants at maturity. For analysis of transgene segregation ratios, T3 or T3 seed was surface sterilized, sown on plates containing 100 mg/L kanamycin in nutrient medium (4.3 g/L Murashige and Skoog salts [Gibco BRL], 1% sucrose, B5 vitamins [Gibco BRL], and 0.8% Phytagar [Gibco BRL]), incubated at 4°C for 2 days. moved to the light for germination, and grown as described above; resistance or sensitivity to kanamycin was scored 10 days later. Lines segregating as homozygous were used for subsequent studies. Analysis of Transgenic Phenotypes by Microscopy Analyses of the transgenic phenotypes were performed initially with 14—day-old 76 Kanr Tl seedlings and later with established homozygous lines at 14 and 23 days. The first leaf was removed with a razor blade and prepared for microscopy as described by Pyke and Leech (1991). A longitudinal strip was cut along the center of the leaf to ensure a representative sample of cell sizes. Fixed tissue samples were macerated on a microscope slide by using the blunt end of a scalpel, then suspended in a drop of 0.1 M Nag—EDTA, pH 9, and covered with a coverslip. Samples were analyzed with Nomarski (Olympus Optical, Tokyo, Japan) differential interference contrast optics, using an Olympus (Olympus America, Melville. NY) BH-2 microscope. Images for analysis and publication were captured by computer using an Optronics (Goleta, CA) DEI-750 digital CCD camera and Adobe Premiere (Adobe Systems, San Jose, CA) imaging software. Chloroplast numbers were counted by eye under the microscope. Mesophyll cell and chloroplast plan areas were analyzed from captured CCD images on a Macintosh- type (Power Computing Corporation, Round Rock, TX) computer using the NIH-Image public domain software (http://rsb.info.nih.gov/nih-image/). Total chloroplast plan area was taken as the product of chloroplast number and mean chloroplast size per cell (Pyke and Leech, 1994). RNase Protection Assays Total RNA was isolated from 18-day—old plants, as described previously (Logemann et al., 1987), using 1 g of leaf tissue from independent transgenic lines (T3) or from the wild type. Only transgenic individuals exhibiting severely reduced numbers of chloroplasts were used for RNA isolation. After precipitation. the RNA pellet was resuspended in buffer (50 mM Mes, pH 7.0, and 2.5 mM magnesium acetate) and treated 77 for 15 min with 4 units of RNase-free DNase (Promega), then extracted with phenol— chloroform, and precipitated with sodium acetate and isopropanol. The final RNA pellet was washed with 70% ethanol and resuspended in 50 ILL of sterile H30. Plasmids used for probe synthesis were constructed as follows. For the AtFtle-l probe, the pZLl plasmid (Gibco BRL) containing the AtFtle-l cDNA (GenBank accession number U39877), which was provided by the Arabidopsis Biological Resource Center (stock number 105K17T7), was digested with ClaI and XbaI to remove from the insert all but the 88 nucleotides at the 5’ end of the cDNA. The overhangs were filled in with the Klenow fragment of DNA polymerase I (Promega) and ligated back together. For the AtFtsZZ-l probe, the pZLl plasmid containing the AtFtsZZ-l cDNA, obtained from the library screen described above, was digested with SpeI and XbaI to remove from the insert all but 129 nucleotides at the 5‘ end of the cDNA, and the ends were ligated back together. For probe synthesis, the plasmids were linearized with SmaI upstream of the inserts. Radiolabeled antisense RNA was generated by in vitro transcription. The reactions contained 2 pg of linearized plasmid, 500 2 11M each of ATP, GTP, and CTP, 1 mM DTT, 20 units of RNasin (Promega), 2 11L of 5 X transcription buffer (Promega), 2.5 uL of 32Port) (800 Ci/mmol; ICN. Costa Mesa. CA), and 18 units of SP6 RNA polymerase (Promega). After a 1~hr incubation at 37°C. the reactions were treated with 4 units of DNase (Promega) for 15 min. The probes were purified by electrophoresis through a 6% polyacrylamide gel. The full-length probes were excised from the gel and eluted by incubation in 50 1.1L of elution buffer (Ambion Inc.. Austin, TX) overnight at 37°C. 78 RNase protection assays were performed using the RPA II ribonuclease protection assay kit (Ambion, Inc.), as described in the Streamlined Procedure in the manual supplied by the manufacturer. Hybridizations were conducted overnight at 42°C by using 30 pg of total RNA and ~5 x 10'1 cpm of probe. The final RNA pellet was resuspended in 3 uL of loading buffer and heated at 95°C for 2 min before being electrophoresed through a 6% polyacrylamide sequencing gel. The gel was transferred to filter paper and dried. 1“”fl The protected RNA fragments were detected by autoradiography on XAR-5 (Eastman Kodak Co., Rochester, NY) or Bio—Max (Eastman Kodak Co.) film for 2 days at —80°C by using an intensifying screen. Identification of Other Plant F tsZ Genes and DNA Sequence Analysis The additional plant FtsZ sequences used in Figure 7 were initially identified by using the BLAST sequence similarity search (Altschul et al., 1990). The partial sequence shown for AtFtsZ2-2 represents a conti g formed by two bacterial artificial chromosome (BAC) end sequences (GenBank accession numbers B25544 and B96663) that share an overlap of ~85 nucleotides on opposite strands. Pairwise comparisons, multiple sequence alignments, and parsimony analysis (Swofford, 1998) were performed as indicated in the legends for Figures 1, 7. and 8. Acknowledgments We gratefully acknowledge Drs. Kevin Pyke, Stanislav Vitha, and Dean DellaPenna for critical reading of the manuscript, Dr. Rob Jensen for performing the mitochondrial import assays, Joe Gera for lending time and technical expertise with the 79 RNase protection assays, Dr. Sergey Morzunov for advice on parsimony analysis, Kelly Gallaher for DNA sequencing, Elizabeth Tattersall for generous help in proofreading. and Caroline Idema, Jennifer Holder, and Nikole Steele for exceptional assistance in the care, processing, and analysis of the transgenic plants. This work was supported by grants to K.W.O. from the National Science Foundation (No. MCB-9604412) and the Nevada Agricultural Experiment Station (No. 1 106-152- 032R). Note Added In Proof A recent update of the F tsZ sequence from Physcomitrella patens (Strepp et al., 1998; GenBank accession number AJ001586) indicates that the encoded protein contains an N-terminal extension. This additional sequence information does not influence the assignment of PthsZ to the F tsZ2 gene family. However, the conclusion that N-terminal extensions are attributes of Ftle, but not FtsZ2, family members may be premature, or may apply only to higher plant FtsZ proteins. 80 CHAPTER 3 Stokes KD, McAndrew RS, Figueroa R, Vitha S, Osteryoung KW (2000) Chloroplast division and morphology are differentially affected by overexpression of Ftle and FtsZ2 genes in Arabidopsis. Plant Physiol 124: 1668-1677 81 ##i M Abstract In higher plants. two nuclear gene families, FtsZ] and FIsZZ, encode homologues of the bacterial protein FtsZ, a key component of the prokaryotic cell division machinery. We previously demonstrated that members of both gene families are essential for plastid division, but are functionally distinct. To further explore differences between Ftle and FtsZZ proteins we investigated the phenotypes of transgenic plants overexpressing AtFtsZI-I or AtFtsZZ-l, Arabidopsis members of the FtsZ] and FtsZ2 families, respectively. Increasing the level of AtFtle —1 protein as little as three-fold inhibited chloroplast division. Plants with the most severe plastid division defects had 13- to 26- fold increases in AtFtle -1 levels over wild type, and some of these also exhibited a novel chloroplast morphology. Quantitative immunoblotting revealed a correlation between the degree of plastid division inhibition and the extent to which the AtFtle-l protein level was elevated. In contrast. expression of an AtFtsZZ-I sense transgene had no obvious effect on plastid division or morphology, though AtFtsZZ-l protein levels were elevated only slightly over wild-type levels. This may indicate that AtFtsZZ-l accumulation is more tightly regulated than that of AtFtle-l. Plants expressing the AtFtsZZ-l transgene did accumulate a form of the protein smaller than those detected in wild-type plants. AtFtsZZ-l levels were unaffected by increased or decreased accumulation of AtFtle-l and vice versa, suggesting that the levels of these two plastid division proteins are regulated independently. Taken together, our results provide additional evidence for the functional divergence of the F tle and F tsZ2 plant gene families. Introduction The first identified proteins of the chloroplast division machinery were homologues of the essential bacterial cell division protein FtsZ (Osteryoung and Vierling, 1995; Osteryoung et al., 1998; Strepp et al., 1998). In contrast with most bacterial genomes that contain only a single gene encoding FtsZ, the nuclear genome of Arabidopsis contains at least three F tsZ genes encoding members of two distinct protein families, Ftle and FtsZ2. AtFtsZ] -l and AtFtsZZ—l, members of the F tle and F tsZ2 families, respectively, have been shown to be essential for chloroplast division. When the level of either gene is diminished, chloroplast division is inhibited, yielding cells with as few as one very large chloroplast. One important difference between AtFtle-l and AtFtsZZ-l is their predicted localization. AtFtle-l is targeted to the chloroplast, as is a closely related FtsZ protein from pea (Gaikwad et al., 2000), and is thought to be a component of a division ring that forms on the stromal side of the inner envelope membrane. AtFtsZ2-1, in contrast, lacks a chloroplast transit peptide, is not targeted to the chloroplast in vitro, and is hypothesized to be a constituent of a division ring that assembles on the cytoplasmic surface of the outer envelope membrane. The two rings together are postulated to coordinate chloroplast division (Osteryoung et al., 1998). Bacterial FtsZ is a self-associating cytoskeletal GTPase evolutionarily and structurally related to the eukaryotic tubulins (de Boer et al., 1992; RayChaudhuri and Park, 1992; Mukherjee and Lutkenhaus, 1994; Erickson, 1997; Yu and Margolin, 1997; Lowe and Amos, 1998; Nogales et al., 1998; Nogales et al., 1998). Early in the bacterial division cycle, prior to the onset of cytokinesis, FtsZ assembles into a ring at the division plane that encircles the cell on the inner surface of the cytoplasmic membrane. At least 83 eight other essential proteins are then recruited to the division site and function to complete cytokinesis. Throughout this process. the FtsZ ring remains localized at the leading edge of the division septum (Bi and Lutkenhaus, 1991; Bramhill, 1997; Lutkenhaus and Addinall, 1997; Pogliano et al., 1997; Rothfield and Justice, 1997; Nanninga, 1998; Rothfield et al., 1999). Overexpression of FtsZ in bacteria has yielded important insights into the properties of this protein. First, FtsZ appears to be rate limiting in the division process since slight overproduction of FtsZ increases cell division (Ward Jr and Lutkenhaus, 1985). This increased division results in the formation of small, inviable “minicells” that lack chromosomes due to the occurrence of divisions not only at midcell, but also near the cell poles. Second, high overexpression of FtsZ inhibits cell division and results in the formation of bacterial filaments (Ward Jr and Lutkenhaus, 1985). This appears to be due in part to a stoichiometric imbalance between FtsZ and other division proteins because the filamentation phenotype can be relieved by simultaneous overexpression of FtsA or ZipA, two other bacterial cell division proteins that interact with FtsZ directly and colocalize with FtsZ to the midcell (Ward Jr and Lutkenhaus, 1985; Wang and Gayda, 1990; Dai and Lutkenhaus. 1992; Hale and de Boer, 1997; Wang et al.. 1997; Hale and de Boer, 1999; Mosyak et al., 2000). Although chloroplast division involves some proteins homologous to components of the bacterial division machinery, division of higher plant chloroplasts differs from bacterial cell division since it requires the coordinated activities of at least two FtsZ proteins and does not involve cell wall ingrowth at the division site (Osteryoung and Pyke, 1998; Osteryoun g et al., 1998). In the studies described here we sought to further 84 explore the functional differences between Ftle and FtsZ2 proteins by analyzing the phenotypes of Arabidopsis plants overexpressing AtFtle-l or AtFtsZZ—l. The results reveal additional parallels between chloroplast and bacterial cell division with regard to the behavior of FtsZ, but support a difference in the roles played by Ftle and FtsZ2 in chloroplast division. The data also suggest that the levels of AtFtle -l and AtFtsZZ-l are regulated independently in Arabidopsis, and that Ftle may have an additional function inside the organelle in regulating chloroplast morphology. Results Production of Antibodies Specific for Recognition of AtFtle-l or AtFtsZZ-l In preparation for investigating AtFtle -1 and AtFtsZZ-l protein levels in this and other studies we produced antipeptide antibodies specific for detection of these two polypeptides on immunoblots. The specificities and reactivities of the affinity-purified antibodies, designated 1- 1A and 2-1A, respectively, were analyzed in a series of immunoblotting and competition binding assays (Figure 1). Each antibody was highly selective for its target protein. In Arabidopsis leaf extracts the l-lA antibodies reacted with a single polypeptide of 40 kD (Figure 1, A, lanes 1 and 3 and B, lanes 1 and 5), whereas the 2-1A antibodies reacted primarily with a polypeptide of 46 kD, though one of 45 kD was also detected (Figure 1, A, lanes 4 and 5 and B, lanes 2 and 4). In competition binding assays, immunoreactivity of the l-lA antibody with the 40-kD protein was prevented when the antibodies were preincubated with recombinant AtFtle - 1 protein (Figure 1A, lane 2), but not with recombinant AtFtsZ2-l protein (Figure 1A, lane 3). Likewise, immunoreactivity of the 2-1A antibodies with the 46- and 45- kD A Antibody Probe 1-1A 2-1A Prelncubation a, U 8 a) '31 El Treatment ——) g E E g E E Z < < Z < < kD ‘ 46 > .4- ---v 40 > "'" ""‘ B 5‘ to" 5‘ 9° 41> 0° (313' 0“ Plant «x. ‘5‘) «x. 0““: Genotype 4“ v‘ v“ ‘1'" v“ Antibody 57 S. 5. S. 5. S. AtFtsZ2-1 > I? - 4 46 AtFtsZt-1> 2" - < 40 Figure 1. Specificity of AtFtsZ antipeptide antibodies. A, Immunoblots of proteins isolated from wild-type Arabidopsis leaf extracts were probed with l-lA (lanes 1-3) or 2-1A (lanes 4-6) antibodies raised against peptide from AtFtle -l or AtFtsZZ- 1, respectively. Antibodies were preincubated with purified, recombinant AtFtle-l protein (lanes 2 and 5) or AtFtsZZ-l protein (lanes 3 and 6). B. Immunoblot of proteins isolated from leaf extracts of wild-type (lanes 1 and 2), AtFtsZ] - I antisense (lanes 3 and 4), or AtFtsZZ-l antisense (lanes 5 and 6) plants. Blots were probed with either 1-1A (lanes 1, 3, and 5) or 2-1A (lanes 2, 4, and 6) antibodies. The 46- and 40-kD polypeptides are indicated by markers. Equivalent volumes of plant extracts were loaded in each lane. 86 polypeptides was blocked when the antibodies were preincubated with recombinant AtFtsZZ-l protein (Figure 1A, lane 6). but not with AtFtle-l protein (Figure 1A, lane 5). Furthermore, the l- l A antibodies detected the 40-kD polypeptide in extracts from wild-type plants and transgenic plants expressing an AtFtsZZ-l antisense construct (Figure 18, lanes 1 and 5), but not in plants expressing an antisense AtFtle-I construct (Figure 18, lane 3). Conversely, the 2-lA antibodies detected the 46- and 45-kD polypeptides in extracts from wild-type plants and transgenic plants expressing an null-Fm AIF tle -1 antisense construct (Figure 1B, lanes 2 and 4), but not in plants expressing an antisense AtFtsZZ—l construct (Figure 13, lane 6). Neither antibody cross—reacted with prokaryotic FtsZ proteins in bacterial extracts (not shown). From these results, we conclude that the l-IA antibodies specifically recognize AtFtle-l, which migrates at 40 kD, whereas the 2-1 A antibodies specifically recognize either two distinct forms of AtFtsZZ-l, which migrate at 46 and 45 kD, or AtFtsZZ-l and a closely related polypeptide. The absence of both bands in the AtFtsZZ—l antisense lines (Figure 1B, lane 6) is most consistent with the former possibility. Overproduction of AtFtle-l Inhibits Chloroplast Division in Transgenic Arabidopsis AtF ml] -1 was overexpressed in Arabidopsis under the control of the cauliflower mosaic virus 358 (358) promoter in the vector pART27 (Gleave, 1992). The kanamycin- resistant (kan') transgenic plant lines used in this report were confirmed to be independent transformants by Southern-blot analysis (data not shown). With the exception of a slight twist in some of the leaves. all kanr plants exhibited normal growth 87 Figure 2. Phenotypes of transgenic plants overexpressing AtFtsZI-I or AtFtsZZ-I . Mesophyll cells are shown from the first leaves of 23-d—old plants transformed with the empty pART27 vector (A), the AtFtsZI-l sense transgene (B-E), or the AtFtsZZ-I sense transgene (F). Tissue was prepared for imaging with differential interference contrast optics using methods described previously (Pyke and Leech, 1991). Bar=25 pm in all figures. A three—dimensional rotating reconstruction of cells with phenotypes similar to those shown in C and D can be found in a video supplement at www.plantphysiol.org. 88 and development. However, microscopic examination of mesophyll cells revealed distinct phenotypes in plants overexpressing AIFtsZI-I when compared with wild-type plants of the Columbia ecotype, which typically contain 80— 100 chloroplasts in fully expanded mesophyll cells (Osteryoun g et al., 1998). or to control plants transformed with the empty pART27 vector (Figure 2A). The phenotypes of the transgenic plants consistently fell into three categories defined by chloroplast number and size. Transgenic plants were classified as “wild-type-like“ if the size and number of chloroplasts in mesophyll cells were similar to those in cells from wild-type or vector controls. Plants with 15 to 30 enlarged chloroplasts per cell were classified as “intermediate” (Figure 2B), and those with five or fewer very large chloroplasts per cell were termed “severe” (Figure 2C). Most plants with severe phenotypes contained only one or two chloroplasts per cell and these organelles usually appeared flattened, filling the thin layer of cytoplasm surrounding the vacuole. Despite the enlarged size, chloroplast ultrastructure consistently appeared normal (data not shown). Similar “severe” phenotypes have been described for the Arabidopsis plastid division mutant arc6 (Pyke and Leech, 1994), for plants transformed with antisense AtFtsZ] -1 or AtFtsZ2- l transgenes (Osteryoung et al., 1998), and for F tsZ knockout mutants in the moss Pl'zi-‘sconzitrella patens (Strepp et al., 1998). The proportion of Tl individuals exhibiting wild-type-like, intermediate, and severe phenotypes were 19%, 39%, and 42%, respectively (Table 1). Therefore, over 80% of the kan’ plants expressing the AtFtsZ/J sense transgene displayed defects in chloroplast division. 89 ‘beem . l Table 1. Phenotypic distribution of transgenic T1 plants. Percentage of Transgenic Plantsa Phenotype AtFtsZ] -l AtFtsZZ-l Wild-type-like 19 72 Intermediate 39 14 Severe 42 14 aTotal number of plants analyzed: AtFtsZ/J, 129; AtFtsZZ—I, 90 The Severity of Chloroplast Division Inhibition Is Proportional to AtFtle-l Protein Level AtFtsZ] -1 protein levels in T3 kanr plants representing wild-type-like, intermediate, and severe phenotypes were investigated by immunoblot analysis. Proteins in leaf homogenates were separated by SDS—PAGE, transferred to nitrocellulose, and probed with the affinity-purified AtFtsZ] —l antipeptide antibodies. An immunoreactive 40-kD polypeptide was detected that varied in amount among different transgenic lines (Figure 3A, lanes 5-10), but comigrated with authentic AtFtle-l from wild-type and empty-vector control extracts (Figure 3A, lanes l-4). These results indicate that most of the AtFtsZ] -1 protein in the overexpression lines was properly targeted to the chloroplast and processed. However, a slower-mi grating immunoreactive polypeptide of 72 kD was also detected in plants with increased levels of the 40-kD polypeptide (Figure 3A, lanes 7-10). Competition binding assays have shown that this polypeptide does contain AtFtsZ] -1 (not shown), and may represent a non—dissociated form of the protein, possibly a dimer. A similarly migrating polypeptide is occasionally observed in wild-type plants as well (data not shown). The increased levels of the 72-kD polypeptide in overexpression lines with high AtFtle-l levels may be related to the ability of FtsZ proteins to form dimers and multimers in a concentration-dependent fashion (Di Lallo et al., 1999; Sossong et al., 1999; Rivas et al., 2000; White et al., 2000). An Ftle homologue from pea has also been shown to form multimers in vitro (Gaikwad et al., 2000). Visual inspection of immunoblots from the AtFtle-l overexpression lines suggested a correlation between the level of AtFtsZ] -l accumulation and the severity of 91 A12345678910 k[) '111 *~-~-73 - 47.5 -33.9 -28.8 E C CCWWI S SSPhenotype 312345678910 '---~-"‘5"45 Figure 3. Immunoblot analysis of plant extracts overexpressing AtFtsZ] -1 . Proteins in extracts from 23-d-old transgenic plants were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antipeptide antibodies raised against AtFtle-l (A) or AtFtsZ2-l (B). Lane 1, Empty vector control (B); lanes 2 through 4, wild type (C); lanes 5 through 10, transgenic plants with wild-type-like (W, lanes 5 and 6), intermediate (1, lane 7), or severe (S, lanes 8-10) phenotypes. Equal loading of all samples was continued by staining the membranes with ponceau S (data not shown). chloroplast division defect. In extracts from plants with wild-type-like (Figure 3A, lane 5 and 6) and intermediate (Figure 3A, lane 7) phenotypes. the levels of the 40-kD AtFtle- l polypeptide were similar to. or slightly higher than, those seen in extracts from the vector controls (Figure 3A, lane 1) and non-transformed wild-type plants (Figure 3A, lanes 2-4), whereas in plants with severe phenotypes (Figure 3A, lanes 8-10), AtFtle-l protein levels were noticeably elevated. To further analyze this relationship, the level of AtFtle-l in the transgenic plants relative to that in control plants was quantified by immunoblotting (Figure 4). For this purpose, a calibration curve was constructed from densitometric analysis of the 40-kD polypeptide in four lanes loaded with increasing volumes of whole leaf extract from control plants transformed with the empty vector (Figure 4, lanes l—4). For transgenic extracts, the volumes analyzed were adjusted to maintain protein levels within the linear range of the standard curve (Figure 4, lanes 5- 13). The relative level of the 40-kD AtFtle-l polypeptide in each sample was then calculated from the standard curve based on the densitometry readings and sample volume. The calculated protein levels in extracts from three separate non-transformed wild-type plants (Figure 4, lanes 5-7) were comparable with those from empty vector control plants, indicating that the increased protein levels observed in the transgenic plants were due to expression of the AtFtle-l transgene and not to the vector. In transgenic plants classified as wild-type-like, the relative level of AtFtle-l protein was similar to the control level or elevated no more than two-fold (Figure 4, lanes 8 and 9). Protein levels between three-fold (Figure 4, lane 10) and 6-fold (not shown) above control levels were measured in plants with an intermediate chloroplast phenotype. Although data from only a single intermediate line are shown in Figures 3 and 4, two 93 Empty Vector Control Transgenic AtFtsZt -1 Control WT er INT SEVERE Lane12 3 4 5 6 7 8 910111213 Vol.Loaded(pL) 4 12 22 32 15 15 15 15 15 5 0.8 0.8 0.8 AtFtsZ1-1 (40 kD) “- b h - ._, an and - . an .fl Relative AtFtsZ1 -1 Level 1 1 1 1 2 1 3 26 13 21 Figure 4. Relative levels of the 40-kD AtFtsZ] -l polypeptide in plants expressing the AtFtsZ1-1 transgene. Densitometry readings from an immunoblot loaded with increasing amounts of extract from an empty vector control plant (lanes 1-4) were used to construct a standard concentration curve for AtFtle-l. AtFtle-l levels in the other plant extracts (lanes 5- 13), all loaded so that the densitometry reading produced by the 40-kD AtFtsZ1-1 polypeptide on immunoblots fell within the linear range of the standard curve, were then calculated, taking into account the volume loaded. The volume loaded, signal produced on immunoblots, and calculated level of AtFtsZ1-l relative to that in the empty-vector controls are shown for three Columbia wild type plants (lanes 5-7), and transgenic plants with wild-type-like (WTL, lanes 8 and 9), intermediate (INT, lane 10), or severe (SEVERE, lanes 1 1-13) phenotypes. 94 additional lines with intermediate phenotypes also had approximately 3-fold more AtFtle-l than controls, whereas one line had 6-fold more (not shown). Plants exhibiting the most severe phenotypes had AtFtle~l levels ranging from 13— to 26-fold over control levels (Figures 3A, lanes 8-10, and 4, lanes 1 1-13). Our data indicate a correlation between the level of AtFtsZ] -1 and the severity of chloroplast division inhibition. However, we cannot rule out the possibility that the observed division defects resulted from accumulation of the 72-kD form of AtFtsZ] -l rather than from overproduction of the protein per se. This 72-kD band was not quantified, but its levels in the transgenic lines appeared to correlate with those of the 40-kD polypeptide (Figure 3, lanes7—10, and data not shown). AtFtsZ1-1 Overexpression Produces a Novel Chloroplast Morphology in Some Transgenic Plants In addition to the phenotypes described above, an interesting and unusual phenotype was encountered in three independent AtFtsZ] -l overexpression lines with severe phenotypes. A small number of cells in these plants contained chloroplasts that appeared long and narrow. giving the impression of worms or noodles (Figure 2, D and E). Two of these plant lines had only a few noodle-like chloroplasts per cell (Figure 2D), whereas one line had about 15 (Figure 2E). The diameter of these chloroplasts varied somewhat, but was comparable to that of wild-type chloroplasts. The length, however. was many times longer than in wild type, and the chloroplasts meandered in unique patterns around the cytoplasm of the cell. Only a small proportion of the cells in these plants displayed the noodle-like phenotype; the vast majority of cells exhibited a typically 95 severe chloroplast morphology. The noodle-shaped chloroplasts were only observed in transgenic plants with high levels of AtFtle-l protein, including those represented in lanes 8 through 10 of Figure 3. but have not been found in all such lines. Slight Overexpression of AtFtsZZ-l Does Not Disrupt Chloroplast Division The effect of AtFtsZZ-I overexpression in transgenic plants was also investigated. The T1 generation of kanr plants transformed with the AtFtsZZ-I transgene contained cells with wild-type-like, intermediate, and severe chloroplast phenotypes, similar to those described for the AtFtsZ1-l transgenic plants, and had no obvious abnormalities in growth or development. However. in contrast to the AtFtsZ1-l overexpression lines, 72% of the AtFtsZZ-I kanr T1 individuals exhibited a wild-type-like phenotype (Figure 2F), whereas plants with intermediate and severe phenotypes each constituted only 14% of the total (Table 1). Furthermore, in subsequent generations the lines with intermediate and severe phenotypes reverted to the wild-type-like phenotype. The extent of this reversion was such that only one transgenic line retained reduced chloroplast numbers by the T3 generation. Because of this trend, T1 and T3 plants were studied further. AtFtsZZ-l protein levels were analyzed in whole leaf extracts from T I and T3 plants by immunoblotting. In extracts from transgenic plants exhibiting wild-type-like phenotypes (Figure 5A, lanes 2, 3. 6-9, 1 1, 13, and 16-18), the same 46- and 45-kD polypeptides present in wild-type and empty-vector control plants (Figure 5A, lanes 1 and 2) were detected. The levels of the 46-kD polypeptide in these lines were comparable with control levels, although some extracts exhibited a slight increase (Figure 5A, lanes 4 and 8). We have observed that the intensity of the 45-kD band varies 96 A12 3 45 6 7 89101112131415161718 kD ‘111 '73 :' "~ '1 m? 3 Z: . mfg \44 E CWWSWWWWI WSW I S WWWPhenotype i T1 IL T3 —-I Generation B123 4 5.6 7 89101112131415161718 “-~--~‘~-1~w M~--‘-~'4O Figure 5. Immunoblot analysis of transgenic plant extracts expressing the AtFtsZZ-l transgene. Proteins in extracts from 23-d—old transgenic plants were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antipeptide antibodies raised against AtFtsZ2-l (A) or AtFtle-l (B). Lane 1, Empty vector control (E); lane 2, wild type (C): lanes 3 through 18, T. or T3 transgenic plants with wild-type-like (W, lanes, 3, 4, 6-9, I l, 13, 16-18), intermediate (I, lanes 10 and 14), or severe (S, lanes 5 and 15) phenotypes. Equal loading of all samples was confirmed by staining the membranes with ponceau S (data not shown). 97 considerably among individuals in wild type (not shown), and the levels of the 45-kD protein in the transgenic plants did not appear to vary outside this range. The more obvious result of AtFtsZZ-l overexpression was the accumulation of a 44-kD polypeptide. detected in all ltanr plants with a wild-type-like phenotype, but not detected in any of the controls. Accumulation of this polypeptide was not correlated with any noticeable plastid division defect. In contrast. all transgenic lines with intermediate or severe phenotypes (Figure 5A, lanes 5, 10. 12. and 14), including the line retaining a severe phenotype into the T3 generation (lane 15), had reduced levels of the 46- and 45— kD species present in controls, and did not accumulate the 44—kD polypeptide present in lines with the wild-type-like phenotypes. These data suggest that inhibition of chloroplast division in the AtFtsZZ-I transgenic plants resulted from cosuppression rather than overexpression of AtFtsZZ-l . AtFtsZ1-1 and AtF tsZ2-1 Accumulation Are Regulated Independently of One Another To learn whether overproduction of AtFtle-l was accompanied by a change in AtFtsZ2-l levels or vice versa, the levels of both proteins in extracts from each set of transgenic plants were analyzed on duplicate immunoblots. In all AtFtsZ1-1 overexpression lines analyzed, AtFtsZ2-l protein remained at wild-type levels (Figure 3B). In a converse manner, the level of AtFtle-l protein in the AtFtsZZ-I transgenic lines was unaffected by the level of AtFtsZ2-l protein (Figure SB). Further, antisense repression of AtFtsZ1—1, though reducing AtFtle-l protein to nearly undetectable levels (Figure 1B, lane 3) and severely inhibiting chloroplast division (Osteryoung et al., 1998), 98 had no affect on accumulation of AtFtsZ2—l (Figure 1B, lane 4), or vice versa (Figure 18, lanes 5 and 6). Therefore, we conclude that the phenotypes associated with manipulation of AtFtsZ1-1 or AtFtsZZ-l expression levels. whether from an increase or decrease, result from altered accumulation of only one and not both proteins. In addition, although AtFtsZ1-I and AtFtsZZ-l are co-expressed in wild-type plants (Figure 1B, lanes 1 and 2; Osteryoung et al., 1998), the collective results of overexpression and antisense experiments suggest that Ftle and FtsZ2 protein levels are regulated independently in Arabidopsis. Discussion Correlation between AtFtsZ1-1 Accumulation and Plastid Number Plants producing AtFtle-l at levels ranging from 13- to as high as 26-fold over wild-type levels exhibited drastically reduced numbers of enlarged chloroplasts, indicating a severe inhibition of chloroplast division. This phenotype is comparable with the filamentation phenotype observed in E. coli cells expressing F tsZ at high levels. However, when bacterial FtsZ levels were only slightly elevated, extra divisions were induced near the cell poles, resulting in the formation of minicells. Furthermore, when levels of overexpression were below those resulting in filamentation, the minicell phenotype was proportional to the degree of FtsZ overexpression (Ward Jr and Lutkenhaus, 1985). Based on these data we anticipated that slightly elevated AtFtsZ1 -1 protein levels might increase the frequency of chloroplast division, yielding plants with cells containing smaller, more numerous chloroplasts. Instead, AtFtsZ] -1 levels as little as three-fold over wild—type levels inhibited, rather than increased, chloroplast division. 99 However, we have observed a few transgenic lines with more than 150 tiny chloroplasts in mesophyll cells (data not shown). but these plants were chlorotic and died as seedlings, preventing further analysis of their phenotypes or AtFtsZI-I expression levels. Nevertheless, these observations suggest that elevated AtFtsZ1—l levels could, under a limited set ofcircumstances, increase the frequency of chloroplast division. Based on the observation that plants with two-fold more AtFtsZ1-l than wild type had wild-type numbers of chloroplasts. whereas plants with 3—fold more protein had intermediate numbers, indicating partial inhibition of division, we would predict that AtFtsZ] -l accumulation only within this narrow range would lead to increased numbers of chloroplasts, and that levels above this are inhibitory for plastid division. This idea is consistent with the behavior of FtsZ in bacteria and with the possibility that the plastid division defect resulting from AtFtle -1 overproduction reflects a stoichiometric imbalance in plastid division components. It is possible that the use of a weak promoter instead of the strong 358 promoter might allow identification of more plants with only slightly elevated levels of AtFtle-l, and perhaps increased chloroplast numbers, for further study. It is also possible that simultaneous overexpression of additional chloroplast division proteins (including AtFtsZZ-l) might be required to permit an increase in the plastid division frequency. This is suggested by the finding that the increased frequency of cell divisions observed when F tsZ is overexpressed in E. coli only occurs when FtsA is also overexpressed at similar levels (Begg et al., 1998). Plant survival alternatively may be compromised by increased chloroplast numbers, which could account for the small number of plants identified with this phenotype. This conjecture is supported in part by the phenotype of the Arabidopsis arcl chloroplast 100 division mutant, which is characterized by slightly increased numbers of small chloroplasts (Pyke and Leech, I992; Marrison et al.. 1999). arcl grows more slowly and is pale early in its development compared with either wild type or other arc mutants that have reduced numbers of enlarged chloroplasts. Expression of the AtFtsZZ-I Sense Transgene Does Not Produce a Plastid Division Phenotype In contrast to the dramatic phenotypes associated with AtFtsZ1-l overexpression, more than 70% of the plants transformed with the AtFtsZZ-l sense transgene displayed a wild—type-like phenotype. In fact. immunoblotting results (Figure 5A, lanes 5, 10. 12. I4. and 15) indicated that all plastid division defects observed among these transgenic lines were due to cosuppression of endogenous AtFtsZZ-l expression rather than to overexpression, similar to the plastid division defects observed in plants expressing an AIFIsZZ-l antisense transgene (Figure 18. lane 6; Osteryoung et al., 1998). However, authentic AtFtsZZ-l protein did not accumulate substantially over wild-type levels in the overexpression experiments (Figure 5A), which may indicate that higher levels are lethal or that AtFtsZZ-l accumulation is more tightly regulated than that of AtFtle-l. The only phenotype associated with AtFtsZZ-I transgene expression (when it did not result in cosuppression) was the presence of a 44-kD form of AtFtsZ2-l not detected in wild-type plants. Because this polypeptide was smaller than the one produced by in vitro translation of the predicted AtFtsZZ-l open reading frame (Osteryoung et al.. 1998; RS. McAndrew, S. Vitha and K.W. Osteryoung, unpublished data). it may represent a degradation product or an aberrantly processed form of AtFtsZ2-l. Accumulation of this 101 polypeptide at the observed levels had no effect on plastid division. however. Overall, the differences in the phenotypes of plants expressing AtFtsZ1-l and AIFISZZ-I transgenes further support a difference in the functions of Ftle and FtsZ2 proteins. Aberrant Chloroplast Morphology Associated with High Levels of AtFtle-l Protein The long. narrow chloroplasts observed in some of the AtFtsZ1-1 overexpression lines occurred only in transgenic lines exhibiting severe plastid division defects and high AtFtsZ1-l protein levels. Although at present we cannot be certain of the biological relevance of this unique phenotype, it could suggest an additional role for chloroplast- localized Ftle proteins in the control of chloroplast shape. Immunofluorescence data indicating the presence of longitudinally oriented AtFtsZ1-l—containing filaments in the noodle-shaped chloroplasts (S. Vitha and R. McAndrew, unpublished observations) are consistent with this idea. The assembly of many such filaments in plastids with high AtFtsZ] —1 levels could restrict their ability to expand laterally, allowing plastid expansion to occur only longitudinally to produce narrow. elongated chloroplasts. The noodle phenotype alternatively could reflect an abnormality in the formation of stromules, narrow tubular connections between plastids through which protein molecules can pass (Kohler et al., 1997). At present there is no direct evidence that plant FtsZ proteins participate in chloroplast shape determination or stromule biogenesis, but the notion that FtsZs function in multiple processes, like their tubulin structural homologues, is not incompatible with their cytoskeletal properties. 102 Materials and Methods Construction of Sense Transgenes and Plant Transformation Full—length cDNAs for AtFtsZ1-l (accession no. U39877) and AtFtsZZ-I (accession no. AF089738) in the plasmid vector pZLl (Gibco BRL) were obtained as described previously (Osteryoung et al.. 1998). A gel-purified SmaI-Clal fragment containing the complete AtFtsZ1-l cDNA sequence was ligated directionally behind the 358 promoter in pART7 (Gleave, 1992) digested with the same enzymes. The resulting plasmid was digested with N011 and the fragment containing the AtFtsZ1—1 insert was ligated into Natl-digested pART27 (Gleave, 1992) to create the plasmid pAP202 containing the AtFtsZ1-l transgene. The AtFtsZZ-I transgene was constructed by digesting the pART27 derivative pSN506 (Norris et al., 1998) with EcoRI and HindIII, and replacing the insert with an EmRI-HindIII fragment containing the complete AtFIs‘ZZ-l cDNA to create pRFSOl. pAP202 and pRFSOl were purified from Escherichia coli and transferred to Agrobacterium tumefaciens GV3101 (Koncz and Schell, 1986). Restriction analysis confirmed that no rearrangements occurred in the transfer to Agrobacterizmz. Arabidopsis ecotype Columbia was transformed by vacuum infiltration (Bechtold et al., 1993; Bent et al., 1994) with pAP202, and by floral dip (Clough and Bent, 1998) with pRFSOl or the pART27 empty vector. Selection and Propagation of Transgenic Plants T1 seed from the inoculated plants were collected, sown on plates containing nutrient medium (4.3 g/L Murashige and Skoog salts, 1% [w/v] Suc, B5 vitamins, and 0.8% [w/v] Phytagar [Gibco—BRL]) and 100 mg/L kanamycin (Fisher Scientific, 103 Hampton, NH). incubated at 4°C for 2 d. and moved to controlled environment chambers for germination. Chambers were set at a relative humidity of 40%, with 16 h of light daily (l25 umol m2 s") at 20°C. and 8 h of darkness at 18°C. The age of the plants was taken from the date of seed transfer to the growth chamber following cold treatment. After 10 d in the growth chamber, kanr plants were transferred to a mixture of Supersoil potting mix (Rod McLellan Co., San Francisco) and vermiculite (4:1). T3 and T3 seeds were sown on Rockwool (GrodanHP; Agro Dynamics, East Brunswick, NJ) saturated with Hoagland nutrient solution containing 100 mg/L kanamycin (Gibeaut et al., 1997). Seeds were covered with plastic and incubated at 4°C for 2 d, then transferred to growth chambers for germination. After 14 d, kanr plants were transferred to soil and grown as described above. Microscopic Analysis When plants were 18 d post-germination. the first leaf was removed with a razor blade and prepared for microscopic analysis as described (Pyke and Leech, 199] ). Samples were then viewed with differential interference contrast optics using a BH-2 microscope (Olympus America. Melville, NY). Images were captured by computer using a DEI-750 digital charged-coupled device camera (Optronics, Goleta, CA) and Adobe Photoshop (Adobe Systems, San Jose, CA) software. Generation of Antipeptide Antibodies Peptides corresponding to regions of AtFtle-l and AtFtsZ2-l predicted to constitute highly accessible and immunogenic epitopes were designed using the crystal 104 structure of M€I}l(lll()(.'()(‘Cll.S' janasc'hii (Lowe and Amos, 1998), epitope mapping data for monoclonal antibodies against E. coli FtsZ (Voskuil et al.. 1994), and molecular modeling programs. Peptides corresponding to residues 201 through 215 in AtFtle-l (EGRKRSLZALEAIEK) and residues 168 through 184 in AtFtsZZ-l (RRRTVQAQEGLASLRD) were synthesized. purified by HPLC, and coupled to keyhole limpet hemocyanin (Pierce, Rockford. IL). These peptides, designated l-lA and 2—1A, respectively, were injected into rabbits (nos. 4164 and 4166, respectively) for the production of polyclonal antibodies (Alpha Diagnostics, San Antonio, TX). The antisera obtained were partially purified by ammonium sulfate precipitation (50% final saturation) followed by dialysis against phosphate-buffered saline (140 mM NaCl, 3 mM KCI, 10 mM N83HPO4, 2 mM KHgPO4, pH 7.4). as described (Harlow and Lane, 1988). Antibodies were further purified on affinity columns prepared by immobilizing the peptide antigens to SulfoLink Coupling Gel (Pierce) according to the manufacturer’s standard protocol, yielding final protein concentrations of 0.7 and 0.9 mg ml'I for antibodies l-lA and 2-1A. respectively. determined using the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). These preparations were diluted for immunoblotting as described below. Immunoblotting Procedures Tissue for immunoblot analysis was collected from leaves of 21-d-old plants with microscopically verified mesophyll cell phenotypes. Tissue was homogenized in a microcentrifuge tube with 10 11L of grinding buffer {60 mM Tris[tris(hydroxymethy1)- aminomethane]—HC1. pH 8.0, 100 mM dithiothreitol. 2% [w/v] SDS, 15% [w/v] Suc, 5 105 mM e—amino-N-caproic acid, 1 mM benzamidine HCl. and 0.01% [w/v] bromophenol blue} per milligram of leaf tissue plus a few grains of sterilized sand. Homogenized tissue was heated for 15 min at 700C and stored at -20°C until use. Prior to electrophoresis. samples were reheated to 700C for 5 min and centrifuged (3 min, 140003,!) to remove particulates. Proteins were separated by standard SDS-PAGE on 1 1% (w/v) polyacrylamide gels (Bio—Rad. Richmond. CA) and transferred to nitrocellulose membranes (0.45 pm. Micron Separations, Westborough, MA). Except where indicated, sample volumes loaded on gels were equivalent. Membranes were blocked for 30 min in TBST (50 mM Tris—HCl, pH 7.4, 200 mM NaCl, and 0.2% [v/v] Tween 20) containing 2% (w/v) Camation non—fat dry milk (Nestle Food Company, Glendale, CA), then incubated in TBST plus 2% (w/v) nonfat dry milk (TBST-milk) containing affinity-purified AtFtsZ1—1 or AtFtsZ2—1 antibodies at dilutions of 1:1,500 and 1:3.000, respectively. Incubations with primary antibody were carried out in Seal-a—Meal bags (Dazey Corp, Century, KS) shaken vigorously overnight at room temperature. After four 10-min washes in TBST, membranes were incubated with horseradish peroxidase-conjugated goat-anti-rabbit secondary antibodies (Fisher, Pittsburgh) for 15 min at 124.000 dilution in TBST-milk. Following four 10-min washes in TBST, membranes were developed using Renaissance Western Blot Chemiluminescence Reagent (NEN Life Science Products. Boston) and the signal was recorded on X-OMAT ls film (Kodak. Rochester, NY). 106 Competition Binding Assays To determine antibody specificity, immunoblotting experiments were performed as described above. except that prior to probing membranes, each diluted antibody was preincubated for 2 to 4 h in TBST-milk with an equimolar amount of purified, recombinant AtFtsZ1-1 (residues 41-269) or AtFtsZZ-l (residues 92-282) on a rocking platform. These truncated versions of AtFtle-l and AtFtsZ2-1 were obtained by expressing the corresponding cDNA fragments in the expression vector pJC40 (C105 and Brandau, 1994) as lO—histidine-tagged FtsZ fusion proteins in E. coli BL21(7LDE3)/plysS cells. following induction with isopropylthio-flgalactoside (1 mM) at 37°C. The recombinant proteins were purified from inclusion bodies in cell lysates using metal chelation chromatography (His-Bind Resin. Novagen. Madison, WI) according to the manufacturer‘s protocol for denaturing conditions. Protein concentrations of truncated AtFtsZ] -l or AtFtsZ2-1 following chromatography. determined using the Bio-Rad Protein Assay reagent (Bio-Rad), were 1.75 and 1.24 mg ml'l, respectively. AtFtsZ1-1 Quantification Quantification of FtsZ protein levels was performed by scanning the film used for chemiluminescent detection of signals on immunoblots into the computer using a Mirage Ilse imager (UMAX Technologies, Fremont, CA) and Binuscan software (Binuscan, New York). Densitometry measurements were used to quantify the levels of AtFtle-l from a standard curve prepared by evaluating the intensities of the 40-kD polypeptide in four lanes loaded with increasing amounts of an AtFtsZ1—l-containing plant extract prepared from a control plant transformed with the empty pART27 vector. A best-fit curve 107 . ’3 . . calculated from the data had an R“ value of 0.98. This curve was used to calculate the relative amount of protein in the other samples, which were loaded so that the signal generated on the immunoblot was in the linear range of the standard curve. The level of AtFtsZ1-l protein in each extract was calculated relative to the control sample. Acknowledgements We gratefully acknowledge Travis Gallagher for excellent care and feeding of plants. 108 Chapter 3 - Supplemental Data AtFtsZZ-Z Cosuppression Inhibits Chloroplast Division Background Wild—type Arabidopsis plants express three different FtsZ proteins, AtFtle - l , AtFtsZZ-l and AtFtsZ2-2, which are localized to the chloroplast stroma (McAndrew et al., 2001). Previous research indicated overexpression ofAtFtle-l inhibited chloroplast division (Stokes et al., 2000). Attempts to overexpress AtFtsZZ-l did not result in any inhibition of chloroplast division. However, immunoblots indicate that only slight increases in protein expression were obtained with the transgenic plants. In the AtFtsZZ- I transgenic plants in which chloroplast division was inhibited, the amount of AtFtsZ2-1 protein was reduced due to cosuppression. At the time the overexpression experiments were published by Stokes et al. (2000) the AIFtsZZ-Z gene had not been isolated and characterized. Since that report the AtFtsZZ-Z transcript has been isolated and reported (McAndrew et al., 2001). In an effort to determine the effect of increased AtFtsZZ—Z protein on chloroplast division, plants were prepared with an AtFtsZZ-Z overexpressing transgene. Most of the transgenic plants had a normal chloroplast phenotype, but FtsZ protein levels were similar to those in wild-type plants. Severe inhibition of chloroplast division was only observed in transgenic plants where AtFtsZZ-l and AtFtsZ2—2 levels were both reduced. 109 Material and Methods Preparation of AtFtsZ2-2 Overexpressing Construct Total RNA was isolated from 19—day-old soil grown plants as described by McAndrew et al. (2001 ). Reverse transcription (RT) reactions were performed using the Superscript II (Gibco-BRL) enzyme as instructed by the manufacturer, with 10 pg of total RNA in a 30 uL reaction volume and the reverse primer 5’- AGTGGGTCTAGAGGCGAGGA. PCR amplification of the AtFtsZ2-2 gene was performed on 600 ng of RNA from the above RT reaction using the forward primer 5’- CAGAATGGCAGCTTATGTTTCTCC and the reverse primer used in the RT reactions. The amplified AtFtsZZ-Z gene was cloned into pBuescript digested with SmaI. The resulting plasmid. called pKS 135. was digested with XbaI and the fragment containing the AtFtsZZ-Z gene was purified and cloned into the shuttle vector pART7 (Gleave, 1992) that had been digested with the same enzyme. This plasmid. called pKS 168, was digested with Natl and the fragment with the AtFtsZ2-2 insert cloned into pART27 (Gleave, 1992) that had been digested with the same enzyme. The final plasmid was called sz 170. Plant Transformation and Screening for Kanamycin Resistant Plants The pKSl70 plasmid was purified from Escherichia coli, transferred to A grobacterium runwfaciens GV3101 (Koncz and Schell, 1986), and then transformed into Arabidopsis thaliana ecotype Columbia (Col-0) plants by floral dip (Clough and Bent, 1998). T1 and T3 seed were sown on MS plates as described by Stokes et al. (2000), except 50 mg/L kanamycin was used for selection of transgenic plants. After the 110 transfer oftransgenic plants to soil, the growth conditions. microscopic analysis, and immunoblotting procedure were as described by Stokes et al. (2000). Results Transgenic plants were generated with a construct designed to overexpress AtFtsZZ-Z in an effort to determine the effect of increased AtFtsZ2-2 on chloroplast division. Numerous T3 plants from three independently transformed lines (Figure 6; transgenic lines A, B, C) were analyzed for both their chloroplast phenotypes in mesophyll cells and FtsZ protein levels. When the transgenic plants are compared to wild-type plants (Fig 6; Wt), the transgenic plants grouped into three different FtsZ protein profiles and chloroplast phenotype combinations. One group of transgenic plants had a normal chloroplast phenotype and normal levels of AtFtsZ1-l, AtFtsZZ-l. and AtFts22-2 protein (Figure 6, lanes 2—5). Although the four plants in this group are from the same transgenic line. this FtsZ protein profile and chloroplast phenotype was the most commonly observed among all the transformed lines that were tested (data not shown). A second group of plants had decreased levels of AtFtsZZ-Z, but near-normal levels of AtFtle-l and AtFtsZZ-l (Figure 6, lanes 8- 10). These plants, from two independent lines, also had normal chloroplast phenotypes. The third group had a severe chloroplast phenotype, which was characterized by one very enlarged chloroplast per cell. FtsZ protein profiles for these plants had reduced AtFtsZZ—2 and AtFtsZ2-l protein levels, but near normal AtFtle-l levels (Figure 6, lanes 6 and 7). Plants with this severe Chloroplast phenotype were only observed in one transgenic line (data not shown). 11] Lane12345678910 Transgenic line Wt A1 A2 A3 A4 B1 82 B3 C1 C2 (X-AtFtSZl-1 *----- «an» up * a... a—AtFtsZ2-1 '- ‘I' - u- - II- “ our Antibody a—AtFtSZZ-Z - - ‘- - - ”" Phenotype C WWWWS 8 WWW Figure 6. Immunoblot analysis of transgenic plant extracts expressing the AtFtsZZ-Z transgene. Protein extracts from T3 transgenic plants were separated by SDS-PAGE, transferred to PVDF, and probed with antipeptide antibodies raised against AtFtle-l, AtFtsZ2-l, or AtFtsZ2-2. Samples are from three independent transformed lines (A, lanes 2-5; B, lanes 6 and 7; C, lanes 8-10): lane 1, Wild-type control (C); lanes 2-10, transgenic plants with wild-type-like (W, lanes 2-5 and 8-10) or severe (S, lanes 6 and 7) phenotypes. Equal amounts of extract were loaded on four gels and confirmed by Coomassie staining one of those gels (data not shown). 112 I. Discussion Instead of increased amounts of AtFtsZZ-Z protein, transgenic plants with the overexpressing AtFtsZZ—Z construct had near normal or reduced FtsZ protein levels. The only lines in which chloroplast division was severely inhibited had undetectable levels of AtFtsZ2-l and AtFtsZ2-2, indicating the chloroplast phenotype is a result of cosuppression. A few plants had reduced AtFtsZ2-2 levels and near—normal levels of AtFtle -l and AtFtsZZ- 1. but chloroplast division did not seem to be affected. This result indicates that a reduction in AtFtsZZ-Z protein levels by itselfdoes not inhibit chloroplast division. A recently isolated AtFtsZZ—Z knockout plant that has no detectable AtFtsZ2-2 protein but normal levels of AtFtsZZ-l protein supports this result (unpublished results). The chloroplast phenotype in the mesophyll cells of this AtFtsZ2- 2 knockout plant is intermediate. with about 15 enlarged chloroplasts. The intermediate phenotype indicates there is some chloroplast division. but the frequency of chloroplast division has been reduced. The most common phenotype among the transgenic lines was normal FtsZ protein levels and normal chloroplast size and number. Although the exact reason for this result is unclear. it may be that expressing AtFtsZ2~2 with a strong promoter is detrimental to plant viability. Cosuppression that resulted in inhibition of chloroplast division was also observed when AtFtsZZ-I was overexpressed with a similar construct (Stokes et al., 2000). However. overexpression ofArFtsZZ-l was achieved using the native promoter (McAndrew et al., 2001). Therefore. overexpression of AtFtsZZ-Z may also require a construct that uses the native AIFIsZZ-Z promoter. CHAPTER 4 Coordinate expression of the F tsZ I and F rsZ2 genes in Arabidopsis thaliana plants 114 P" Abstract The Arabidopsis genome encodes three chloroplast-localized FtsZ proteins that belong to two different families, Ftle and FtsZ2. Members of both families perform roles in chloroplast division but little is known about their developmental patterns of expression. We used ,B-glucurmiidase (GUS) reporter gene assays, real—time reverse transcriptase—polymerase chain reaction, and immunoblot assays to investigate RNA and protein expression patterns for all three Arabidopsis FtsZ family members. Analysis of GUS reporter staining patterns as well as the FtsZ transcript distribution indicates that the F tle and F tsZZ genes are coordinately expressed throughout the plant. A high level of FtsZ gene expression occurs in the young expanding leaves but expression is reduced in the older, fully expanded leaves. These expression patterns correspond to reported patterns of plastid division (Pyke and Leech, 1992; Pyke, 1997), and are consistent with the role of the FtsZ’s in plastid division. Measurements of the F tsZ transcript amounts indicate AtFtsZZ-l is the most abundant, followed by AtFtsZ1-1, and AtFtsZZ-Z. In addition, the ratio of F ml] to F tsZZ transcript is constant throughout leaf develOpment. 115 Introduction Plastid division is important for the maintenance of plant photosynthetic competence. The chloroplast complement of a cell results from division of undifferentiated proplastids and green chloroplasts (Chaly and Possingham, 1981; Whatley, 1993; Robertson et al., 1995; Pyke. 1997). Proplastid division in root and shoot meristems is likely important for maintaining proplastid numbers in these rapidly dividing cell populations (Possingham and Lawrence, 1983), while chloroplast division in monocot and dicot leaves is correlated with cell expansion (Boasson et al., 1972; Possingham and Smith. 1972; Possingham and Lawrence, 1983; Pyke et al., 1994; Robertson et al.. 1996). However. the region of the leaf where chloroplast division occurs differs between monocots and dicots. In the monocot wheat, chloroplast division occurs in a relatively small region of the leaf where cell expansion is greatest (Leech and Pyke, 1988; Pyke, 1997). In contrast. chloroplast division in Arabidopsis. a dicot, occurs over a longer period of cell development and expansion (Pyke and Leech, 1992: Pyke. 1997). This makes the region where chloroplast division occurs less pronounced in Arabidopsis than in wheat leaves. However. most chloroplast division still occurs in cells in the basal portion of both monocot and dicot leaves (Possingham and Smith, 1972; Possingham, 1973; Leech et al., 1981; Pyke et al., 1991). In Arabidopsis dividing chloroplasts have also been observed at the base of developing petals and in the rapidly expanding cotyledons (Pyke, 1997; Pyke and Page, 1998). It is in the tissues where plastid division is actively occurring that we anticipate chloroplast division genes, like F tsZ, to be expressed. 116 In bacteria, FtsZ is a cytoskeletal GTPase protein with structural homology to the eukaryotic tubulins that localizes to a ring at the cell division site (Bi and Lutkenhaus, 1991; Bramhill. 1997: Lutkenhaus and Addinall, 1997; Lewe and Amos, 1998). Most bacteria encode a single FtsZ protein, whereas plants contain multiple nuclear encoded copies of the FtsZ proteins. The plant FtsZ proteins have been grouped into two families, Ftle and FtsZZ, based on conserved molecular features (Osteryoung et al., 1998,). Members of both families are localized to rings at the chloroplast division site inside the stroma (Osteryoung et al.. 1998; Gaikwad et al.. 2000; Fujiwara and Yoshida, 2001; McAndrew et al., 2001 ), and are required for chloroplast division (Osteryoung et al., 1998; Strepp et al., 1998). In Arabidopsis. there are three FtsZ genes, one FtsZ] gene, AtFtsZ1-l, and two FtsZ2 genes, AtFtsZZ-l and AtFts‘ZZ-Z. Expression ofAtFIsZI-l and AIFIsZZ—I has been reported in Arabidopsis leaves by detecting the proteins by immunofluorescence microscopy as well as immunoblot analysis (Stokes et al., 2000; McAndrew et al., 2001; Vitha et al., 2001). No information regarding the expression of the third Arabidopsis FtsZ gene AtFtsZZ-Z has been reported except that the gene product is imported into the chloroplast (McAndrew et al., 2001). Very little has been reported about patterns of FtsZ expression in Arabidopsis. Although roles for both F ml] and F tsZ2 genes in chloroplast division have been established (Osteryoung and Vierling, 1995; Osteryoung et al., 1998; Strepp et al., 1998), the relationship of their expression patterns to the patterns of plastid division during development has not been determined. In marigold (Tugetes erecta L.) an F Ile transcript was isolated from petals and was expressed at a higher level in the petals than in leaves (Moehs et al., 2000). There is some evidence indicating that FtsZ expression 117 responds to light in cucumbers and peas (Gaikwad et al., 2000; Ullanat and Jayabaskaran, 2002), cytokinin treatment in cucumbers (Ullanat and Jayabaskaran, 2002) and to the cell cycle in tobacco (El-Shami et al., 2002). However. the studies with cucumber investigated the expression of only one, of several, FtsZ2 genes (Ullanat and Jayabaskaran, 2002), while the study in peas involved only an FtsZ] gene (Gaikwad et al., 2000). The tobacco study investigated one, of several, FtsZ] genes and one, of two, FtsZ2 genes (El-Shami et al., 2002). Furthermore, the significance of cell cycle- dependent FtsZ expression is unclear since the study used a non-photosynthetic tobacco cell culture (El-Shami et al., 2002,). None of these studies investigated the expression patterns of both F tsZ] and F tsZZ or their relationship to FtsZ protein levels and patterns of plastid division in the whole plants. In an effort to better understand the expression of the F tsZ genes in Arabidopsis, experiments were performed that included promoter fusions, transcript quantification, and immunoblot analysis. To understand the spatial and temporal expression of the three Arabidopsis F tsZ genes. we used the promoters from each to drive expression of the ,6 glucuronidase (GUS) reporter gene in transgenic Arabidopsis plants. We report here that the GUS staining pattern, in conjunction with transcript quantification, indicates the three Arabidopsis F tsZ genes are coordinately expressed in roots, stems, shoot apex, and expanding leaves. Measurements of the relative levels of the three F IsZ gene transcripts indicate AtF tsZ2-1 is the most abundant, followed by AtFtsZI-l, and AtFtsZZ-Z. Furthermore, the ratio of F 1‘le to F tsZZ transcripts remains constant throughout leaf development. These results indicate that expression of the F tsZ genes is coordinated and that their relative stoichiometric ratio may be important for chloroplast division. 118 Materials and Methods Determination of the FtsZ’s 5’-UTR Using 5’-RACE Amplification of the 5’-UTR for the FtsZ genes was performed using the FirstChoice RLM-RACE kit (Ambion. Austin. TX) as described by the manufacturer using 10 ug of total RNA that had been isolated from l9-day-old Arabidopsis thaliana ecotype Columbia (Col-0) plants. Two serial PCR amplification reactions utilized nested forward primers provided in the kit with sequence—specific nested reverse primers. The reverse primers for the AtFtsZ1-l gene were, in order of use. 5‘- CGCATAGAAATCAACACTCTG and 5‘-AACGGCATTGTTACCACCAC. The 5’— UTR of the AtFtsZZ-l gene was amplified with the reverse primers 5’- ACCACCTCCCACACCAATAACC and 5‘-AGTCCCTTCACCTCTAAGCAT. The reverse primers for the AtFtsZZ-Z gene were 5‘-TGAACCACCACCTCCAACGCC and 5’-AGTAGATAACTCATCCAAATCCTC. The amplified products were gel-purified and ligated into pBluescript 11 KS (Stratagene) for sequencing. Construction of the F tsZ Promoter-GUS Constructs All promoter constructs were made using pK31207. a gift from Dr. Dean Dellapenna (Michigan State University). pKSl207 is a derivative of pART27 (Gleave, 1992) in which the pBIlOl GUS gene has been insterted into the Natl restriction site. The GUS gene was amplified from pBIlOl with the forward primer 5’- AGTCGGCCGAAGCTTGCATGCCTGCAGGTC and the reverse primer 5’- TGCCGGCCGGAATTCCCGATCTAGTAACAT, each primer has an EagI restriction 119 site engineered at the end. The PCR-amplified GUS gene was digested with the enzyme EagI and ligated into pART27 that had been digested with the Natl enzyme. The final pKS 1207 plasmid was sequenced for accuracy. Plants transformed with pKS1207 served as a promoterless control. The promoter for AtFtsZ1-I was amplified from genomic Arabidopsis DNA using the primers 5’- GAAGGATCCCAGACACTTTCTC (forward) and 5’- GTTTACTTCCTCTGCTTTCAGAGAAG (reverse). The amplified product was ligated into a SmaI-digested pBluescript 11 KS (Stratagene, La Jolla, CA) vector. The promoter was isolated from the resulting plasmid by digestion with EcoRI, the overhangs were then filled in with Klenow, and then digested with Xbal. The isolated AtFtsZ1-1 promoter fragment was then directionally ligated into pKS 1207 that had been digested with HindIII, the overhangs filled in, and then digested with XbaI. The resulting plasmid, pKS174, contains 1679 bp of AtFtsZ1-l promoter sequence, but sequencing indicated one base pair immediately upstream of the AtFtsZ1-l start codon (position —1 of the promoter) was incorrect. The reporter gene for AtF tsZ2-1 was constructed similarly, except that the primers 5’-TTCTCTGCTCTCTTGATGATCA (forward) and 5’- AATGAGACCAATCACTGCAGG (reverse) were used for promoter amplification. The final construct, pKSlS4, contains 1775 bp of the AtF tsZ2-1 promoter sequence. For the AtFtsZZ-Z reporter gene, the primers 5’-TCTCGAAACGTTTATGCCAT (forward) and 5’~TCTGAGACTACAGAAGCAACCAAA (reverse) were used to amplify the promoter region, which was cloned into pBluescript as described above. The fragment with the AtFtsZZ—Z promoter was excised from the resulting plasmid by digestion with HindIII and XbaI and then directionally cloned into pKS 1207 that had been digested with the same 120 enzymes. The final plasmid, pKSlS6. contains 131 1 bp of the AtFtsZtsZZ-Z promoter sequence. The promoters in pKS154 (AtFtsZZ-l ) and pKS 156 (AtFtsZZ—Z) were sequenced to confirm sequence integrity. Plant Transformation and Growth The plasmids pKS1207. pKS 174, pKSlS4, and pKS156 were purified from Escherichia coli, transferred to Agra/mcterium tumefliciens GV3101 (Koncz and Schell, 1986), and then transformed into Arubidapsis thaliuna ecotype Columbia (Col-0) plants by floral dip (Clough and Bent, 1998). T1 and T3 seed were sown on MS plates as described by Stokes et al. (2000), except 50 mg/L kanamycin was used for selection of transgenic plants. After transferring the transgenic plants to soil. growth conditions were as described by Stokes et al. (2000). For etiolated seedlings, seeds were grown on plates that were treated identically to the light-grown seedlings, except the plates were covered with aluminum foil. The age of the plants was taken from the date of plate transfer to the growth chamber, following 2 days of cold treatment. After 8 days in the growth chamber, etiolated and 1i ght—grown seedlings were harvested for GUS staining and kanr plants were transferred to soil. Whole plants and inflorescence tips were harvested for histochemical staining after growing for 19 or 32 days. respectively. GUS Staining The GUS staining proceedure is adapted from protocols by Jefferson et al. (1987) and Rodrigues-Pousada et al. (1993). Harvested tissue was immediately fixed in 90% acetone for 15 minutes, then rinsed in wash solution (50 mM NaPOi pH 7.2, 0.5 mM K3Fe(CN)6, and 0.5 mM K4FC(CN)6) five times. 15 minutes each wash. The wash solution was then replaced with stain solution (wash solution, 0.1% Triton X-100, and 1 mM 5-bromo-4-chloro-3—indolyl-B—D-glucuronide [X-Gluc]). Before addition to the stain solution the X-gluc (ANGUS Buffers and Biochemicals, Niagara Falls, NY) was dissolved in N-N-dimethylformamide to a concentration of 100 mM. After the stain solution was added to the tissue. the samples were briefly placed under vacuum then incubated overnight at 37°C. Tissue was then cleared with several changes of 70% ethanol and stored in 50 mM NaPOi pH 7.2 at room temperature. GUS expression analysis was performed on three independently transformed lines for each construct. Only one representative line for each construct is reported, since the staining pattern for each of the three tested lines was the same. Detecting GUS Transcript by RT-PCR Seeds were grown on MS plates with 50 mg/L kanamycin, or without kanamycin for wild-type seed, as described above. RNA was isolated from the shoots of 10-day-old plate-grown seedlings using the RNeasy plant mini kit (Qiagen, Valencia, CA) as directed, except about 400 mg of tissue was used and volumes of the kit solutions RLT, RPE, and ethanol were doubled from that described in the first five steps of the manual. Contaminating genomic DNA was removed by treating the RNA samples with DNase, then re-isolating the RNA with the kit described above. Reverse transcription (RT) reactions were performed using the Superscript II (Gibco-BRL) enzyme as instructed by the manufacturer, with 15 ug of total RNA in a 60 1.1L reaction volume and the reverse primers 5’- TGATCCCAI 1 1TGTCAAGGAGTT (AtFtsZ1-l) and 5’- 122 TTCGTTGGCAATACTCCACA (GUS). PCR amplification of a 382 bp AtFtsZ1-I gene fragment used the AtFtsZ1-1 reverse primer indicated above with the forward primer 5’- TTGCAGATGTGAAGGCAGTC. PCR amplification of a 467 bp GUS gene fragment used the GUS reverse primer indicated above with forward primer 5’- TGCAACTGGACAAGGCACTA. Since the GUS gene used in our constructs does not have any introns, the amplified fragments from RNA or contaminating genomic DNA would yield the same size product. Therefore, to test for contamination by genomic DNA, we also amplified a fragment of the AtFtsZ1-I gene. which has introns that are removed from the sequence. Another purpose of the AtFtsZ1-l control was to confirm whether endogenous AtFtsZ1 -l transcript levels could be detected in the samples. Isolation of genomic DNA for use in the PCR reactions was done as described by Neff et al. (1998). Immunoblot Analysis of the FtsZ Proteins in Roots Protein extracts were prepared from 19—day-old Arabidopsis ecotype Columbia (Col-0) plants as described previsously (Stokes et al., 2000). Total protein in each sample was measured using the RC DC Protein Assay kit (Bio-Rad, Hercules, CA) as directed by the manufacturer. Four polyacrylamide gels were loaded with equal amounts of total protein and the extracts separated by SDS-PAGE. Comparison of the loaded protein samples was visually determined by staining one of the gels with Coomassie Brilliant Blue R250. Immunoblot analysis was performed according to procedures described by Vitha et a1. (2001). 123 Plant Material for Real-Time RT-PCR Analysis Wild-type Arabidopsis ecotype Columbia (Col-0) seed was sown in soil, vemalized 2 days at 4°C, and then grown in growth chambers under the same conditions as above. After 19 days, the plants were harvested and tissue was separated into three fractions consisting of the first and second pair of leaves. the third leaf pair, and the remaining tissue (which included the fourth leaf pair, shoot apex, stems, and some root fragments). The separated tissue was immediately frozen in liquid nitrogen. Nucleic Acid Isolation for Real-Time RT-PCR Analysis The separated and frozen tissue, described above, was ground in a mortar and pestle that had been pre-chilled with liquid nitrogen. Total RNA was isolated from the ground tissue as described by McAndrew et a1. (2001). Isolated RNA was treated with RNase-free DNase (Promega, Madison, WI) and then extracted with phenol/chloroform to remove proteins. Isolated RNA was again treated with DNase and then purified on RNeasy plant mini kit (Qiagen, Valencia, CA) as directed. These RNA samples were used as the template for the real-time RT-PCR analysis ofAtFtsZI—I, AtFtsZZ-l and AtFtsZZ-Z transcript levels. Real-Time RT-PCR Analysis Reverse transcription (RT) of 5 pg total RNA was performed using the Taqman® RT-PCR Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s directions. Amplification and detection of AtFtsZ1-I transcript during the Taqman® 124 assay involved the primers 5’-CCACAGGCTTCTCTCAGTCATTC (forward) and 5’- TGATCCCATTTTGTCAAGGAGTT (reverse) along with the Taqman® probe 5’— AGAAGACACTTCTGACTGATCCAAGAGCAGCT, at concentrations of 300 nM. 300nM, and 150 nM respectively. The AtFtsZZ-l transcripts were detected with the primers 5‘-GCTACGGGTTTCAAACGACAA (forward) and 5'- AGCTCCAACTGACGCAGCAT (reverse) with the Taqman® probe 5’— AGAAGGACGAACAGTTCAGATGGTACAAGCA. at concentrations of 900 nM, 300 nM, and 150 nM respectively. For detection ofAtFtsZZ-Z transcripts, primers 5’- GAAGGAGAAGGGAGGCCACT (forward) and 5’- GACGTCTTGTGGCTCCCATT (reverse) were used with the Taqman® probe 5’- CAGGCGACACAAGCGGATGCAT at concentrations of 900 nM. 300 nM. and 150 nM respectively. The three Taqman® probes were synthesized and labeled with the 5’ fluorescent reporter dye FAM (6- carboxyfluorescein) and the 3’ quencher dye TAMRA (6-carboxy-N,N,N’,N’- tetramethylrhodamine) by the manufacturer (Applied Biosystems). Primers, probe, and optimized conditions for EFIOL detection were as described by Tian and DellaPenna (2001). Real-time RT-PCR reactions used 200 ng of total RNA from the RT reactions, gene-specific primers and probe, and Taqman® Universal PCR Master Mix (Applied Biosystems) as described in the kit‘s manual. Optimization of primer and probe concentrations was done as directed by the manufacturer. Quantification of transcripts for each gene in the samples was based on a comparison to a calibration curve consisting of a dilution series of a plasmid containing the cDNA for the gene of interest. Measurements were repeated on plant samples taken from four biological replications. 125 Transcript amounts were either compared based on total RNA or on the EFlatranscript amount for each sample in each biological replication. Results The Structures of the 5’-UTR Differ Between the F tsZ Genes in Arabidopsis Before the promoter expression experiments were initiated, there was some question as to the sequence of the FtsZ genes in Arabidopsis. Specifically, the start codon for the AtFtsZ2-l gene was in question. To identify the correct start codon of each FtsZ gene we determined the sequence of each 5"-UTR (untranslated region) by 5’- RACE (rapid amplification of cDNA ends). The 5’-RACE experiments confirmed the start codon for each Arabidopsis F tsZ gene and determined the AtFtsZZ-I cDNA used in the overexpression construct described in chapter 3 (Stokes et al., 2000) was truncated. Subsequently, a full length AtFtsZZ-I cDNA was isolated and characterized (McAndrew et al., 2001). Although the 5’-RACE experiments were important in determining the full- length transcripts (McAndrew et al., 2001), the sequence of the 5’-UTR’s were not reported. Figure l diagrams the structure the AtFtsZ1-l, AtFtsZZ-l, and AtFtsZZ-Z primary transcripts upstream of the start codon. The 5’-UTR of AtFtsZ1-1 extends 89 bp upstream from the translation start site, and does not contain any introns. The AIFIsZZ-l gene has a 452 bp intron that is removed, to produce a final 5’-UTR of 164 bp. The 5’- UTR of the AtFtsZZ-l gene has been determined by Fujiwara and Yoshida (2001) who reported a similar length and the removal of same 5’-UTR intron. Introns were also detected in the 5’-UTR region of the AtFtsZZ-Z gene, but in this case three differentially 126 AtFtSZ‘l -1 _ATG ..... 75 84 At FtsZZ—‘l _—fi—ATG ..... 58 ' 7 AtFtsZZ-Z A — ,4, 1ATG ..... 58 ' ' 48 B — 408 _ATG ..... Figure 1. Genetic structure of the 5'-UTR region of the three FtsZ genes. The exon and intron structure of the 5’-UTR region for each F tsZ gene up to the translation start site (ATG) are diagramed. Exons are represented as bars and are drawn to scale with the size (bp) above, while introns are shown as lines with their size (bp) below, but are not drawn to scale. Three AtFtsZ2-2 transcripts that have differentially spliced 5’-UTR regions (AtFtsZ2-2 A. B, and C) were isolated. Dashed lines indicate junctions common among the AtFtsZZ—Z species. 127 spliced species were isolated (Figure l; AtFtsZZ—ZA, B, and C). One of these (A) has a 449 bp intron that is removed, while another species (B) has a 408 bp intron that is removed. In contrast to the first two. species (C) has two removed introns. The intron junctions nearest the 5‘ end of the UTR‘s are identical in all three AtFtsZZ-Z species, as well as the intron junction nearest the start codon of species B and C (Figure 1, dashed lines). The results indicate that both AtFIsZZ-l and AtFtsZ2-2 transcripts have introns that are removed from the 5’-UTR region whereas the AtFtsZ1-I does not. GUS Reporter Gene Expression Patterns To study the expression patterns of the three Arabidopsis FtsZ genes, we generated transgenic plants expressing a GUS reporter gene under control of the AtFtsZ/- I (pKSl74),AtFtsZ2-1 (pKS154). or AtFtsZZ-Z (pKSlS6) promoter. The promoter regions were chosen based on the results of the 5’-UTR analysis, which confirmed the position of the start codon for each FtsZ gene. To analyze the developmental and tissue- specific expression patterns of the different FtsZ genes, transgenic plants at various stages of development were histochemically stained for GUS expression (Figure 2). Both light-grown and etiolated whole seedlings were stained at 8 days. whole plants at 19 days. and floral shoots at 32 days. No GUS staining was detected in transgenic plants with a promoterless GUS construct (pKS1207). which served as a negative staining control (Figure 2, A0-H0). Transgenic plants expressing the AtFtsZZ-l and AtFtsZZ-Z reporter construct exhibited identical patterns of GUS staining (Figure 2, A2-12 and A3-I3 respectively). Expression was observed in the roots. shoot apex, and cotyledons of etiolated seedlings 128 Figure 2. Histochemical localization of FtsZ-promoter activity during development of transgenic Arabidopsis. Histochemical GUS staining in Arabidopsis plants transformed with constructs containing no promoter (A0-H0), the AtFtsZ1-l promoter (Al-H1), the AtFtsZZ-I promoter (A2-H2), or the AtFtsZ2-2 promoter (A3-H3) fused to the GUS reporter gene. Histochemically stained tissue from (A0-A3) 8-day etiolated seedlings; (BO-B3) 8-day light-grown seedlings; (C0-C3) l9-day lSt leaf pair; (D0-D3) 19-day 2nd leaf pair; (E0- E3) l9-day 3rd leaf pair; (F0-F3) l9-day 4lh leaf pair; (GO-G3) 19-day root, stem, meristem, and immature leaves; and (HO-H3) 32-day floral shoots with inserts showing an opened floral bud. Note that pollen grains are stained in H1 but not H2 or H3. Bars 2 5 mm. Images in this dissertation are presented in color. 129 pKS1207 pKS174 (Vector Control (AtFtsZ1-1 No Promoter Promoter a-Day Etiolated Seedling May Light-grown Seedling 19-Doy 1st Loaf set 19-Day 2nd Loaf set 19m 3rd Leaf set 19-Day 4th Leaf set 19—Day Root, Stem. Morletom, Immature Loews 32-Day Floral Shoot Figure 2 130 gar (Fig 2; A2, BZ, A3. B3). suggesting light stimulation is not required for the FtsZ promoters to drive expression. Intense staining of cotyledons from etiolated seedlings compared to the staining at the margins of light—grown seedling cotyledons indicates the F tsZ2 promoters drive reporter gene expression prior to or during cotyledon expansion, when there is significant chloroplast division (Pyke. 1997) but expression is reduced when the cotyledons have reached full expansion. The roots, shoot apex, and first leaf pair of light-grown seedlings also have an intense GUS staining pattern, consistent with FtsZ expression in tissues with actively dividing proplastids and chloroplasts (Pyke, 1997). In the l9-day—old plants with the FtsZ2 promoter constructs, the GUS staining intensity was strongest in the roots, stems, shoot apex. and fourth (youngest) leaf pair (Figure 2; F2, G2, F3, G3). Portions of the third leaf pair were also stained, but the staining was generally restricted to the outside edges of the leaf base (Figure 2, E2 and E3). In the older, fully expanded leaves, very little GUS staining was observed (Figure 2; C2, D2, C3, D3). Therefore, the reporter gene assays suggest that FtsZ2 genes are expressed at higher levels in young, expanding leaves than in mature leaf tissue. Much of the floral shoot tissue in plants with the F tsZZ promoter constructs (Fig 2, H2 and H3) showed intense staining, including the floral buds. Staining of the petal base indicates AtFtsZZ-I and AtFtsZZ-Z are expressed in this tissue, which has a significant population of cells with constricted chloroplasts (Pyke and Leech, 1992; Pyke, 1997; Pyke and Page, 1998). No staining was observed in the pollen sac or pollen grains of plants with either FtsZ2 promoter-GUS construct. 131 In contrast, transgenic plants with the AtFtsZ1-l reporter gene had staining in only the pollen grains (Figure 2, H1 and inset). GUS staining was not observed in the other floral tissues. in the l9-day-old plants, or in either of the seedlings (Figure 2, Al- H1). These results are inconsistent with those of Vitha et al. (2001), who used the same promoter fragment to expression a GFP-tagged AtFtsZ1-l protein in leaves of transgenic Arabidopsis plants. Furthermore. AtFtsZ1-I RNA was also detected in vegetative tissue by real-time reverse transcriptase-polymerase chain reaction (RT-PCR, described below) and AtFtle—l protein has been detected in leaf extracts (Stokes et al., 2000; McAndrew et al., 2001; Vitha et al., 2001). These data indicate that the staining pattern observed with the GUS reporter construct does not accurately represent expression of the endogenous AtFtsZ1-l gene. The GUS Reporter Gene Is Not Transcribed Under Control of the AtFtsZ1-I Promoter One possible explaination for the lack of GUS staining in the transgenic plants with the AtFtsZ1-l promoter-GUS construct could be that expression is below the level required for histochemical detection. Therefore, we performed RT-PCR to determine whether GUS transcript could be detected in transgenic seedlings with the AtFtsZ1-l promoter-GUS construct, which would indicate whether the GUS reporter gene is being transcribed. Total RNA was isolated for this experiment from lO-day-old plate-grown wild-type Arabidopsis plants, transgenic plants with the AtFtsZZ-I promoter-GUS construct, and two independently transformed lines with the AtFtsZ1-l promoter-GUS construct. Figure 3 shows the RT—PCR-amplified AtFtsZ1-I and GUS products that were 132 Primer Set AtFtsZ1-1 GUS RNA E RNA § Sample a Sample 0 - < CD < h - <2: :13 < 8 “J ‘T ‘T ‘T g 8 “.1 ‘7 ‘7 *7 "‘ 9‘. 1: 1: .‘2 m *" 9‘. S S 1: E < < < < 2 E < < < < GUS- , 9P - -600bp GenomicAt1-1- ' a i . -500 bp At1-1RNA- ’ ‘ ' ‘400 '09 Lane1 2 3 4 5 6 7 8 9 1O 11 Figure 3. Detection of GUS expression in transgenic Arabidopsis seedlings with the AtFtsZ1-l promoter-GUS fusions. An agarose gel with the RT-PCR amplified AtFtsZ1-l (Atl-l, Lanes 1-5) or GUS (GUS, lanes 7-1 1) gene fragments from RNA isolated from lO-day-old seedling shoots of wild- type Arabidopsis (Wt Col, lanes 1 and 7), a transgenic line with the AtFtsZZ-Z promoter- GUS construct (At2-2. lanes 2 and 8), and two independently transformed lines with the AtFtsZ1-I promoter-GUS construct (Atl-lA, lanes 3 and 9: Atl-lB, lanes 4 and 10). PCR amplification of the AtFtsZ1-I and GUS fragments from genomic DNA from one of the AtFtle-l promoter lines (Atl-lA, lanes 5 and 1 1) served as a control. 133 separated on an agarose gel from these plant samples. When primers for AtFtsZ1-1 are used, PCR amplification ofAtFtle-l from the genomic DNA results in a product that is 96 bp longer, due to the presence of an intron, than the corresponding fragment amplified from RNA by RT-PCR. Comparison of the fragment amplified from genomic DNA (Figure 3, lane 5) to that from the RNA samples (Figure 3, lanes 1—4) indicates there is no detectable genomic DNA contamination in the RNA preparations. Since the endogenous AtFtsZI—l transcript is detected in all the RNA samples, even those from wild-type Arabidopsis plants, the GUS transcript should be detectable if it is present at similar levels. Samples from wild-type plants were used as a negative control for GUS expression, since no GUS gene is present in these plants. Expression of GUS transcript is not detected in this negative control sample (Figure 3, lane 7). As a positive control, a transgenic plant line with the AtFtsZZ-l promoter-GUS construct was used because expression of GUS in this plant line was detected histochemically. As expected, GUS transcript was detected in this positive control sample (Figure 3, lane 8). When two independently transformed AtFtsZ1-1 promoter-GUS lines were tested, no GUS transcript was detected (Figure 3, lanes 9 and 10), although the GUS gene fragment was amplified from genomic DNA (Figure 3, lane 1 1). These results indicate that the GUS gene, although present in the plants, is either not transcribed under the control of the AtFtsZ1-I promoter that was used in the GUS reporter experiments or is very unstable. Because the construct used by Vitha et a1. (2001) utilized the identical promoter to express the GFP- tagged AtFtle-l protein but included the entire genomic sequence of AtFtsZ1-I, these 134 results suggest that expression ofAtFtle-l requires regulatory elements located be downstream of the 5’-UTR. Arabidopsis Roots Express FtsZ Proteins The staining patterns in the promoter-GUS experiments indicated the FtsZ2 genes are expressed in the roots, stems, shoot apex and young leaves of 19-day-old plants. In pea plants, Gaikwad et a1. (2000) reported that expression of an FtsZ] gene in pea was highest in the leaves but very low in roots and stems. In order to determine if all three Arabidopsis FtsZ genes are expressed in roots we performed immunoblot analysis on extracts from roots, shoot apex and stems, and young leaves of wild-type Arabidopsis plants (Figure 4A). For these immunoblot analyses, tissue similar to that shown in Figure 2G but from wild-type plants was divided into two fractions: a root fraction and a shoot fraction containing the stem, shoot apex, and 4th leaf pair. In addition to these two fractions. tissue from the 3rd leaf pair was also analyzed. All three Arabidopsis FtsZ proteins are detected in each of the three tissue fractions. Immunoblot signals for all three FtsZ proteins are strong in the shoot apex and young leaf extracts but the AtFtle -l and AtFtsZ2-1 protein signals in the roots are significantly less. In fact, the signal for the AtFtsZZ-l protein in the root extracts was only observed with very long exposure times and cannot be seen in Figure 4. Samples for the immunoblot analysis were loaded according to equal total protein, but Coomassie Brilliant Blue G250 staining of one gel indicated the amount of total protein in the root extracts was lower than the amount of protein in the extracts from the shoot apex and young leaves (Figure 4B). This loading error is likely due to soil that 135 A E E G D. a "5’ % 8 2’ E Antibody 8 a a 5 3 8 Probe n: to 1: 2 >- .i 7 AtFtsZ1-1 ........ - ‘ AtFtsZZ-1 w 5" AtFtsZ2-2 I” - -I I5 5 B a a 3 V’ 2 1” at g E a, 0 C > MW 8 a r. 3 S a a: in 1:: 2 > .l Figure 4. Immunoblot detection of FtsZ protein in root, stem, shoot apex, and immature leaf extracts. Immunoblot analysis of AtFtle — l. AtFtsZ2— l. or AtFtsZZ-Z proteins was performed on extracts from roots, shoots, and young leaves of l9—day wild—type Arabidopsis plants. Samples were loaded with equal total protein onto four different gels and separated by SDS-PAGE. A, Three of the gels were transferred to PVDF membrane and probed with antibodies specific for AtFtle - l. AtFtsZ2— l. or AtFtsZ2-2. B, the fourth gel was stained with Coomasie Brilliant Blue G250 for visual comparison of the separated proteins. 136 remained on roots following harvest that caused inaccurate determinations of total protein. Because of this loading error. the amount of FtsZ protein signal in the root extracts cannot be accurately compared to the amount of protein in the other two extracts. This is especially significant for the AtFtsZZ-l protein in root extracts and may be the reason the signal was so weak. To obtain a better comparison of the amount of FtsZ protein in root extracts may require the Arabidopsis plants to be grown hydroponically rather than in soil. Hydroponically grown plants are free of the soil that seemed to affect protein assays and may result in more root tissue per plant. An alternative experiment for this section may be immunofluorescence microscopy of root sections with antibodies for each of the FtsZ proteins. Not only would the expression of the FtsZ proteins in roots be determined but their localization patterns could also be analyzed and compared to those in chloroplasts. Relative Expression Levels of the Arabidopsis F tsZ Genes Remain Constant Throughout Leaf Development We sought to compare the relative F tsZ transcript levels at various developmental points using quantitative real-time RT-PCR. For this experiment, tissue from 19-day soil-grown wild-type plants was separated into three pools based on the FtsZ2 promoter- GUS experiments representing tissue with high, intermediate, or low GUS staining. The shoot consisted of the fourth leaf pair, shoot apex, stems, and roots that comprised the high GUS-staining tissue (see Figure2, F and G). The young leaves consisted of the third leaf pair and comprised the intermediate GUS staining tissue (see Figure2, E). The old leaves consisted of the first and second leaf pairs and comprised the low GUS staining 137 tissue (see Figure2, C and D). Total RNA was isolated from each sample and the amount of each FtsZ transcript as well as the EFlatranscript was determined. The entire experiment was repeated four times. The amount of transcript reported in Figure 5 is an average from all four biological replicates. The amount of F tsZ transcript based on equal amounts of total RNA is diagramed in Figure SA. The amount of FtsZ transcript relative to that of EFla'is shown in Figure SB. In Figure 5A. the amount ofAtFtle—l, AtFtsZZ-I, and AtFtsZZ-Z transcript in one intermediate GUS-staining sample was more than two standard deviations away from the average of those in the other three biological replicates and was removed from the analysis. However, this sample was included in Figure SB since the amount of each transcript was not more than two standard deviations from the average of the other three when compared relative to the amount of EFlatranscript. Comparison of transcript levels in each of the tissues indicates the FtsZ genes are expressed at different levels, with AtFtsZZ-I being the most abundant, followed by AtFtsZ1-I and AtFtsZ2-2 (Fig 5; grey, white, and black bars respectively). Furthermore, the ratio of FtsZ] to FtsZZ (combining the amounts of AIF 1522-] and AtFtsZZ-Z) transcript is l to 2.4, 1 to 2.8, and l to 3.4 in the high, intermediate, and low GUS- staining tissue pools, respectively. These data suggest that a constant ratio of about one F tle transcript to three F tsZ2 transcripts is present throughout leaf development. When the concentrations of the AtFtsZI-I , AtFtsZZ-l , and AtFtsZ2-2 transcript were compared among the three tissue pools, each was most abundant in the high GUS- staining pool. Lower concentrations of the three transcripts were observed in the other 138 > T I AlFlSZ‘l -‘1 A1F1822-1 I AlFtSZZ-Z l—+ attomoles/200ng RNA Shoot Young Leaves Old Leaves I AtFtsZ1 -1 AtFtsZZ-1 T I AtFtsZZ-Z 7F —i— Percent of EF1a Shoot Young Leaves Old Leaves Figure 5. Quantification of F tsZ mRNA in various Arabidopsis tissues. The concentration of AtFtsZ1-l, AtFtsZZ-I , and AtFtsZZ-Z transcript was determined by real time RT-PCR in tissue from old leaves (gray bars), young leaves (white bars), and the shoot of the Arabidopsis plant (root, stem, and immature leaves; black bars). These three tissue pools represent plant tissues with low, intermediate, or high GUS staining. A, The reported amount is attomoles of F tsZ transcript per 200 ng of total RNA analyzed. B, The amount of F tsZ transcript is reported as a percentage of the EFI atranscript amount in each sample. 139 pools. The differences between the high and low GUS-staining pools for each transcript are statistically significant. The distribution of the AtFtsZ1-I, AtFtsZZ-l, and AtFtsZZ-Z transcripts correlates with the patterns of GUS expression observed in the transgenic plants with the FtsZZ pramater-GUS constructs. The amount of EFlatranscript was also measured in each of the samples and was to be used for a loading control. The amount of each FtsZ transcript was compared as a percentage of the EFla'transcript (Figure SB). Relative to EFI a. the AtFtsZZ-I transcript is still the most abundant followed by AtFtsZI-I and AtFtsZZ-Z. However, expressed in this way. the data show greater variation between the biological replications, and the differences between transcript amounts are not statistically significant. Only in the old leaf samples is there any significant difference between the amount ofAtFtsZZ-l, AtFtsZ1-l, and AtFtsZZ-Z transcript. When the amount of each FtsZ transcript is compared among the three pools, they are each more abundant in the old leaf extracts than they are in the extract from the shoot apex pool. contrary to the patterns of GUS expression in the F 132 promoter experiments and to the patterns of cell expansion and chloroplast division (Pyke and Leech, 1987; Pyke et al., 1991; Pyke, 1997). However, because of variations between the biological replications, any trends of FtsZ expression between the different tissues are not significant. Expression of EFIain the three tissue pools, and between the biological replications, seems to vary much more than that of the three F tsZ genes. Therefore, EFlaseems to constitute a worse internal control than does basing the comparisons on total RNA. There are reasons why EFlamay not be a good control. One reason may be that EFla'expression may change differently during development than does the 140 expression of the FtsZ genes. Another reason may be the difference in transcript abundance. where EFlais between 10- and 100-fold more abundant than any of the FtsZ transcripts. Instead of EFI (I. the amount of Geranylgeranyl Diphasphute Synthase I (GGPSI ) or either ,6-Irvdrarvlast) l or 2 transcript may be a better internal control because their abundance is more similar to that of the FtsZ transcripts (Eva Collakova, personal communication), assuming they are constitutively expressed throughout development. Discussion Arabidopsis plants express three FtsZ genes that belong to two different families and members of both families are required for chloroplast division (Osteryoung et al.. 1998; McAndrew et al., 2001). To better understand the functional relationship among the three FtsZ genes, we investigated their expression patterns. In the three tissue pools tested, the AtFtsZZ-l transcript was most abundant, followed by the AtFtsZ1-l and AtFtsZ2-2 transcripts. Furthermore. for every FtsZ] transcript, there were approximately three FtsZ2 transcripts. These results indicate that the three FtsZ genes are expressed at a constant ratio throughout leaf development, and are in reasonable agreement with other analyses in our laboratory showing that Ftle and FtsZZ protein levels are maintained at a constant 1:2 ratio throughout leaf development (Chi-Ham et al., 2003; McAndrew et al., 2003). The difference between the transcript and protein ratios may be due to differences in translational regulation or transcript and protein turnover rates. Both transcript and protein distribution patterns suggest that the F tsZ genes are expressed at a constant stoichiometric ratio, which may be important for chloroplast division. 141 Microscopic analyses indicate that Ftle and FtsZ2 proteins co-localize to rings at the chloroplast division site (McAndrew et al., 2001; Vitha et al., 2001; Kuroiwa et al.. 2002). It is unclear whether these rings are composed of separate Ftle and FtsZZ homopolymers or if they represent a heteropolymeric structure (McAndrew et al., 2001; Vitha et al., 2001’). Although there seems to be a stoichiometric balance among the three F [52 transcripts. the ratio of F tsZ l to total F IsZ2 may be all that is important for chloroplast division (i.e., AtFtsZ2—2 may be functionally redundant). Also, the inhibition of chloroplast division does not seem to be a gene dosage effect since neither the transcripts or the proteins are present in a 1:1:1 ratio. Disruption of the stoichiometric ratio among the FtsZ proteins. and possibly other components of the chloroplast division apparatus, seems to be detrimental to chloroplast division (Osteryoung et al., 1998; Stokes et al., 2000; Vitha et al.. 2001). In leaves, the staining patterns in transgenic plants with either of the FtsZ2 promoter-GUS constructs were strongest in young leaves but diminished as the leaves aged. Consistent with this staining pattern, analyses of transcript distribution indicated that F tsZ2 as well as F tsZI levels are highest in young leaves but are reduced in older leaves. This expression pattern correlates with reported patterns of cell expansion and chloroplast division that are reported for Arabidopsis leaves (Pyke et al., 1991; Pyke and Leech, 1992; Pyke, 1997) and is consistent with the role of FtsZ proteins in chloroplast division. Transgenic plants expressing either of the F tsZ2 promoter-GUS constructs exhibited GUS staining in roots. This was true in light-grown and etiolated seedlings as well as in the l9-day-old plants. In addition, immunoblot analysis detected each of the 142 FtsZ proteins in root extracts. Although chloroplasts are not present in roots, non-green plastids are present. Therefore. the expression of the FtsZ genes in root tissue indicates that FtsZ proteins may also have a function in the division of non—green plastids and in the division of all plastids. However, additional experiments are required to determine if the ratio of the FtsZ proteins and their ring structures are identical in all dividing plastids. Staining of transgenic plants expressing the AtFtle—I promoter-GUS construct indicated the GUS transgene was not transcribed ill young seedlings (Figure 3). However, RNA and protein analyses indicate the endogenous AtFtsZ1-I gene is expressed in seedlings and throughout the plant (Figure 2, and 3). Vitha et al. (2001) expressed a GFP-tagged version ofAtFtle-l bearing the same AtFtsZ1-l promoter that was used in our study but containing the entire genomic AtFtsZ1-l sequence. Fluorescence microscopy detected the GFP-tagged protein in leaves as well as in the root and shoot apices of transgenic Arabidopsis plants (Vitha et al., 2001), personal communication). Together, the data described here and those of Vitha et al. (2001) suggest that sequences downstream of the 5‘-UTR are required for proper expression of AtFtsZ1-l since expression is not observed with the promoter alone, but is when the full genomic AtFtsZ1-I sequence is used. There are several reports of other genes that require sequences downstream of the S'-UTR for proper expression (Luehrsen and Walbot. 1991; Curie et al., 1993; Donath et al., 1995; Sieburth and Meyerowitz, 1997: Silverstone et al., 1997; Itoh et al.. 1999; Kim and Guiltinan. 1999; Kloti et al., 1999). For example, proper expression of the GA] gene requires sequences through the first two introns of the coding region (Silverstone et al., 1997). 143 CHAPTER 5 Stokes KD, Osteryoung KW (2003) Early divergence of the F tle and F tsZ2 plastid division gene families in photosynthetic eukaryotes. Gene, In Press 144 Abstract Homologues of the bacterial cell division protein FtsZ are found in higher plants where they function as key components of the chloroplast division complex. In contrast to most bacteria that encode a single FtsZ protein, plants encode multiple proteins that group into two families, FtsZ] and FtsZZ. Using new sequence data from a broad range photosynthetic organisms, we performed a series of analyses to better understand the evolutionary history of the plant FtsZ families. Multiple phylogenetic analyses strongly support the grouping of the plant F tsZ genes and proteins into distinct F ml] and F tsZ2 clades. Protein features representing potentially significant functional differences between Ftle and FtsZ2 are identified. Genomic structure comparisons show that exon length and intron position are conserved within each clade, but differ between the clades except at one position. Our data indicate that the divergence of the Ftle and FtsZZ families occurred long before the evolution of land plants, preceding the emergence of the green algae. The results are consistent with proposals that the two FtsZ families evolved distinct functions during evolution of the chloroplast division apparatus, and indicate that genetic and functional differentiation occurred much earlier than previously hypothesized. 145 Introduction Cell division in bacteria requires the interaction of multiple proteins at the division site, and the most extensively studied among them is FtsZ (Lutkenhaus and Addinall, 1997). FtsZ is a structural homologue of the eukaryotic tubulins, and is probably their evolutionary predecessor (Erickson, 1998). Like tubulin, FtsZ is a GTPase that polymerizes into filaments in vitro (Mukherjee and Lutkenhaus, 1998). In vivo, formation of FtsZ into a cytokinetic ring at the midcell division site is the first step in assembly of the bacterial cell division machinery (Lutkenhaus and Addinall, 1997). Several key residues important for FtsZ enzyme activity, polymerization, and interactions with other cell division proteins have been identified (Lutkenhaus and Addinall, 1997; Lowe, 1998; Lu et al., 2001). Of considerable interest are the interactions between a short, conserved stretch of amino acids in FtsZ called the C-terminal domain, and the two division regulators FtsA and ZipA (Ma and Margolin, 1999; Mosyak et al., 2000). This interaction is essential for cell division in Escherichia coli (Ma and Margolin, 1999), though ZipA and FtsA are less widespread in bacteria than is FtsZ (Rothfield et al., 1999). In most prokaryotes, a single gene encodes F tsZ, but exceptions exist (Margolin and Long, 1994). Chloroplasts are descendants of an ancient cyanobacterial endosymbiont acquired by a eukaryotic host (Martin and Herrmann, 1998; Gray, 1999; McFadden, 1999), and, like prokaryotes, divide by binary fission. The discovery of a nuclear gene in plants encoding a chloroplast—targeted form of FtsZ that is closely related to the FtsZ sequences in cyanobacteria provided compelling evidence for the prokaryotic ancestry of the chloroplast division machinery (Osteryoung and Vierling, 1995). Subsequently, plant 146 FtsZ proteins were shown to be essential for chloroplast division (Osteryoung et al., 1998; Strepp et al.. 1998), indicating that chloroplasts have retained functional components of the prokaryotic cell division machinery during their evolution. In contrast, FtsZ no longer functions in the division of fungal, plant, or animal mitochondria. which descended from an ancient OL-proteobacterium (Gray, 1999). However, the recent discovery of (x-protcobacterial-like. mitochondrial-targeted FtsZ proteins in several primitive unicellular eukaryotes strongly suggests their presence in the endosymbiotic ancestor of mitochondria (Beech et al.. 2000; Takahara et al., 2000; Gilson and Beech, 2001). Phylogenetic analysis has shown that mitochondrial and chloroplastic FtsZ proteins in extant eukaryotes have distinct evolutionary origins that parallel the unique endosymbiotic histories of the two organelles (Beech et al., 2000; Takahara et al., 2000; Gilson and Beech, 2001 ). The chloroplastic FtsZ proteins currently identified, all of which are encoded by nuclear genes, group into three major families: red/brown algae, Ftle, and FtsZZ (Beech et al., 2000; Gilson and Beech, 2001; El-Shami et al., 2002; Wang et al.. 2003). The former are found only in red and brown algae. whereas the latter two have been reported in higher plants and in Chamydamanus rein/rardti. Consistent with the presumed monophyletic origin of chlorOplasts (Gray. 1999). all three FtsZ groups probably arose from a single FtsZ gene present in the cyanobacterial ancestor of chloroplasts, and members of each group have been shown to assemble into mid—plastid rings (Vitha et al., 2001). Genome data suggest that F tle and F tsZ2 sequences are represented in many higher plants. Functional analysis in Arahidapsis has shown that members of both 147 families are required for chloroplast division in this organism (Osteryoung et al., 1998). Ftle and FtsZ2 proteins are targeted to the chloroplast stroma by cleavable transit peptides (Osteryoung and Vierling. 1995; Gaikwad et al., 2000; Fujiwara and Yoshida, 2001; McAndrew et al., 2001) and are tightly colocalized (Vitha et al., 2001), suggesting they may be components of the same ring structure. Ftle and FtsZZ family members are distinguished by conserved differences in their amino acid sequences (Osteryoung and McAndrew, 2001; El-Shami et al., 2002). Especially noteworthy are the presence in FtsZ2, but not Ftle. of a short carboxy—terminal domain important for FtsZ function in bacteria (Osteryoung and McAndrew, 2001), and a single amino acid difference in the highly conserved “tubulin signature motif” (El-Shami et al., 2002). Because FtsZZ proteins share slightly higher similarity with the cyanobacterial FtsZs than do Ftle proteins, it has been suggested that FtsZZ may be the more ancestral form of FtsZ in higher plants (El-Shami et al., 2002). In addition, because both forms of FtsZ had until recently only been identified in vascular plants. it had been postulated that the emergence of the two families may have been coincident with the emergence of this group of land plants (Osteryoung and McAndrew, 2001). However, a recent report showing that FtsZ] and FtsZZ homologues are present in the green alga C. reinhardtii indicates that the two families emerged as distinct clades before the evolution of land plants (Wang et al., 2003). The functional significance of this evolutionary divergence with regard to chloroplast division is not yet understood. In the course of our studies on chloroplast division in higher plants, we have become particularly interested in understanding when during the evolution of chloroplasts the Ftle and FtsZ2 families diverged from one another. Towards this end. we initiated a 148 series of sequence and phylogenetic analyses to reconstruct the evolutionary history of the chloroplast division FtsZs. These studies differ from those previously reported (Beech et al.. 2000; Kiessling et al., 2000; Gilson and Beech, 2001; El-Shami et al., 2002; Wang et al.. 2003) in four respects. First, we have focused solely on the plastid FtsZ lineages rather than on bacterial, mitochondrial and plastid lineages, providing a more focused picture of plastid FtsZ evolution. Second, we have incorporated a larger number of plastid FtsZ sequences from a wider range of photosynthetic eukaryotes, including Oryza sutiva and the green alga C. rein/zardtii, providing a more informative evolutionary perspective than earlier studies. Third, we have examined both protein and cDNA sequences to provide a more thorough analysis of the sequence relationships. Fourth, we have investigated intron positioning, as well as sequence comparisons, as an additional indicator of relatedness among FtsZ genes within and between organisms. The detailed analyses reported here strongly support the emergence of Ftle and FtsZ2 as distinct clades before the split between the charophycean green algae. from which land plants evolved (Graham and Wilcox, 2000), and the chlorophycean green algae. Further, our results, which include analysis of evolutionary rates and testing of constraint trees, provide additional support for the peculiar FtsZ branching order observed previously (Beech and Gilson, 2000; Kiessling et al., 2000; Wang et al., 2003). We discuss possible explanations for this unexpected tree topology. In addition, we have identified conserved differences between the Ftle and FtsZ2 protein families whose further study may help to elucidate why two forms of FtsZ evolved to function in chloroplast division. 149 Materials and Methods Sequences and Their Alignment Accession numbers for the protein and cDNA sequences used in this study are listed in Table l and most are available from GenBank (http://www.ncbi.nlm.nih.gov). The Syncchacacclls WH8102, Trichadesmium erythraeum. Nastac punctifarme, PrachIaracaccus marinas MED4 and Prachlaracaccus marinas MIT9313 F tsZ sequences were obtained from the DOE Joint Genome Institute (http://www.jgi.doe.gov/JGI_microbial/html/). The Anahuena Sp. PCC7120 and Thermasyncchacaccrm clangatus BP-l sequences are available at the Kazusa DNA Research Institute (http://www.kazusa.or.jp/cyano/cyano.html). Protein sequences were initially aligned with Clustal X (Thompson et al., 1997), but final adjustments were done manually and unaligned regions at the N- and C- termini were removed. The cDNA alignment was done similarly, except the final adjustments were based on the protein alignment. Both alignments are available upon request. Phylogenetic Analyses Neighborjoining, maximum parsimony, quartet puzzling and maximum likelihood analyses were all performed using PAUP 4.0v10b (Swofford, 1998). Ties were randomly broken and, for the cDNA analyses, the 3rd codon position was removed. Parsimony analyses were performed using heuristic searches and tree bisection- reconnection (TBR) swapping. All minimal trees were saved. The “COLLAPSE if maximum length is zero” option was in effect during the searches, and character changes were interpreted under ACCTRAN optimization. Characters were unweighted and 150 Table l. Accession numbers for FtsZ protein and cDNA sequences Accession Number Lettera Organism Name Protein cDN A A Nicatiana tabacum 2—1 CAB89288 A127 1 750 B Nicatiana tabacum 2-2 CAC44257 AJ 31 1847 c Oryza sativa (31.317724;b CLB17724_5b D Gentiana lutea AAF23771 AF205859 E Lilium Iangiflarum BAA96782 ABO42101 F Arabidopsis thaliana 2-1 AAC35987 AF089738 G Arabidopsis thaliana 2—2 AAK63846 AF384167 H 0020 saliva CL005296_338b cr.005296_338b I Physcomitrella patens 2 CAB76386 AJ 249139 J Physcomitrella patens l CABS4558 AJ249138 K Chlamydamonas rein/zardtii AAM22891 AF449446 L C yanidiaschyzan meralae BAA851 l6 ABO32072 M Cyanidium caldarium RK-l BAA82871 AB023962 N Guillardia theta (nucleomorph) CAA07676 AJ007748 O Galdieria sulphuraria BAA82090 ABO22594 P Mallomanas splendcns AAF35433 AF1201 17 Q Galdieria sulphuraria BAA82091 ABO22595 R Trichadesmium erythraeum Genel949c Genel949C S Synechocystis sp. PCC6803 NP_440816 NC_000911 T Anabaena sp. PCC7120 CAA83241° Z3137]c U Nostoc punctifarme Contig 502 Gene 61C Contig 502 Gene 61'C V Thermosynechacaccus elangatus BP-l Gene t112382C Gene tll2382c W Synechacoccus PCC7942 AAC26227 AF076530 X Prachlorococcus sp. CAB56201 M01 1025 Y Prochlaracoccus marinas MED4 Gen61658c Gene 1658C Z Prachlaracaccus marinas ddlb CAB95028 AJ237851 AA Synechocaccus WH8102 Gene 549C Gene 549C BB Prochlorococcus marinas MIT9313 Gene 1 268C Genel268c CC Nicotiana tabacum 1-2 CAB41987 AJ 133453 DD Nicotiana tabacum 1-3 CAB89287 A1271749 EE Tagetes erecta AAF81220 AF251346 FF Arabidopsis thaliana l-l AAA82068 U39877 GG Pisum sativum CAA75603 Y 15383 HH Nicatiana tabacum 1-4 AAF23770 AF205858 II Nicatiana tabacum l-l CAB89286 AJ271748 JJ Orjvza sativa l-l AAK64282 AF383876 KK Chlamydamanas reinhardtii BAB91 150 AB084236 LL C lostridium prapianicum AAC32266 AF067823 MM Escherichia coli 01572H7 P06138 X55034 NN Bacillus subtilis AAA22457 M22630 8Letters to the left correspond to sequences represented in Figures. 1 and 2 and Table 2. t’Sequences derived from genomic sequence. CSequence source given in Materials and Methods 151 unordered, and gaps were treated as missing data. Bootstrapping was performed on trees obtained from each technique. One thousand replications were used for neighborjoining and maximum parsimony bootstrap analyses, as well as for the quartet puzzling frequency. Values from the different analyses indicate that the same sequences were present at similar nodes. When multiple trees were obtained. bootstrapping analysis was performed on the first tree in the list. For the maximum likelihood analysis. the Hierarchical Likelihood Ratio Tests and Akaike Information Criterion models were selected using Modeltest 3.06 (Posada and Crandall, 1998), which also provided the appropriate settings for analysis in PAUP 4.0v10b (Swofford, 1998) [PAUP command lines for each criterion were, respectively: Lset Base=(0.2705 0.2473 0.2146) Nst=6 Rmat=(l.0000 2.9264 1.0000 1.0000 3.4941) Rates=gamma Shape=0.7l44 Pinvar=0.2225; and Lset Base=(0.2629 0.2330 0.2251) Nst=6 Rmat=(2.6l2l 4.5906 1.3185 1.6823 6.0001) Rates=gamma Shape=0.71 l l Pinvar=0.223 l ]. Bayesian Inference was performed using the MRBAYES program (Huelsenbeck and Ronquist, 2001) with parameters set for four chains, 1.5 X 106 generations, and trees saved every 100 generations. The first 3000 trees were not included in the frequency analysis. Bayesian Inference analyses using the Hierarchical Likelihood Ratio Tests and Akaike Information Criterion produced identical trees with nearly identical frequency values. Values from the Hierarchical Likelihood Ratio Test criterion are reported in Figure 2. Testing Constraint Trees Constraint trees were produced using the program MacClade 4.0 (Maddison and Maddison, 2000). Trees were tested against the original maximum likelihood tree using 152 PAUP 4.0v10b (Swofford, 1998) and the Kishino—Hasegawa two-tailed test with a normal test distribution. Results are given as the p-value scores reported by the test. Relative Rate Test The relative rate test was performed using sequences taken from the alignments for the phylogenetic analyses. The analysis was performed using the program Phyltest version 2.0 (ftp://ftp.bio.indiana.edu/molbio/ibmpc). All protein comparisons were done using the proportion-of—differences and poisson correction distance estimation methods. Each cDNA comparison was performed with the proportion-of-differences, Jukes-Cantor, and Kimura 2-parameter distance estimation methods. Settings for all distance estimation methods were provided in the program. Genetic Structure Comparisons Regions of the 0. saliva genome containing possible F tsZ genes were identified in the Torrey Mesa Research Institute rice database (httpzllportal.tmri.org/rice/). Accession numbers for the clones containing FtsZ genes are CL005296_338, CLB17724_5, and CL000716_185. The cDNA and protein sequences of the 0. saliva F tsZ genes were determined using Netstart 1.0 at http://www.cbs.dtu.dk/services/NetStartl, Netgene2 at http://www.cbs.dtu.dk/services/NetGene2/, Genescan at http://genes.mit.edu/GENSCAN.html, and by comparison to known plant F tsZ sequences. Genomic sequences for the Arabidopsis thaliana F tsZ genes were obtained from The Arabidopsis Information Resource database (Huala et al., 2001). The accession 153 numbers for the Physcamitrella patens genomic sequences are AJ249138 and A1249139. The C. rein/lardtii genomic F tsZ sequences are located on scaffold sequences 85 and 262, which were obtained from the DOE Joint Genome Institute (http://genome.jgi- psf.org/chlrel/chlrel.home.html). Comparisons between the genomic and cDNA sequences were used to determine the intron and exon structures. Protein Comparisons The full-length protein sequences reported in Table 1 were aligned using the program Clustal X (Thompson et al., 1997) with default settings. The alignment was adjusted manually to mirror the protein alignment described for the phylogenic analyses, and to align the plant and algal C-terminal peptide sequences with that in E. coli. Regions at the amino termini containing the chloroplast transit peptides, and highly divergent regions near the carboxy termini, were removed. The entire E. coli FtsZ protein sequence was included in the alignment. The final alignment is available upon request. From the final alignment, residues that are identical in all available sequences of each clade were determined. Similar residues were determined using default settings in the Boxshade program at the Biology Workbench 3.2 website (httpzllworkbench.sdsc.edu/'). Results Phylogenetic Analysis of FtsZ Proteins from Photosynthetic Eukaryotes Two recent events have provided critical insights into the plant FtsZ evolutionary origins. First, two FtsZ sequences from a green a1 ga, C. reinhardtii, became available in 154 the database. Second. analysis of the newly sequenced genome of 0. saliva (rice) (Goff et al., 2002) provided a full complement of monocot FtsZ homologues. Focusing on the FtsZ sequences from photosynthetic organisms (Table 1), including the 0. sativa and C. rein/zardtii sequences. we investigated their evolutionary relationships by phylogenetic analysis. Only available sequences that were full-length or near full-length were used to most accurately represent the evolutionary relationships. The outgroup was chosen based on a preliminary phylogenetic analysis by our group and on previous studies (Beech et al., 2000; Gilson and Beech. 2001). and consists of bacterial FtsZ sequences from E. coli, Bacillus subtilis, and C lastridium prapianicum. BLAST searches identified several Bacillus FtsZ sequences as the closest relatives of the cyanobacterial FtsZs. The B. subtilis FtsZ gene. one of these close relatives, was chosen for the outgroup. The extreme amino and carboxy termini of the protein sequences used in this study are highly divergent and could not be aligned; therefore, they were omitted from the alignment. The trimmed protein sequences are about 322 amino acids long and correspond to residues 12 through 304 (Ala to Phe) of the B. subtilis sequence. The protein alignment was analyzed using neighborjoining and maximum parsimony comparative techniques. The neighborjoining tree is shown in Figure 1, along with bootstrap support values for the neighborjoining and maximum parsimony analyses (first two numbers at nodes) and quartet puzzling frequencies (third number). Although six trees were obtained with maximum parsimony (not shown), the strict consensus indicated the differences were within, rather than between, clades. Both neighbor joining and maximum parsimony analyses separate the proteins into four clades: the cyanobacteria. red/brown algae, Ftle, and FtsZ2 families. Strong support is seen N. tabacum 1 N. tabacum B 0. sativa c G. lutea D L. Iongiflorum E A. thaliana FtsZ2 F A. thaliana G 100,100 0. sativa H 97/86 .— P. patens I 1- P. patons J ' K r— C. merolae L rung; __l '— C. caldarium M 88/70! G. cghoul: (nucleiomorph) Red/Brown g ,.. . su p urar a M. splendens Algae P G. sulghuraria G 85167 T. erythraeum R Synechocystis sp. PCC 6803 S r Anabaena sp. PCC 7120 T Baigfil '- N. punctiforme U T. elongatus BP-1 V j Synechococcus sp. PCC 7942 Cyanobacteria W 1“- Prochlorococcus sp. X P. marinus MED4 Y 1001100 P. marinus ddlb Z 99,78 Synechococcus sp. WH8102 AA P. marinas MIT931§ 88 N. ta acum 55 N. tabacum DD T. erecta EE A. thaliana FF P. sativum FtsZ1 GG 100I100 1: N. tabacum HH loomlfl l N. tabacum ll 0. satlva JJ C. reinhargtii KL C. propionicum LL Li I E. coli MM B. subtilis NN 0.05 p-distance Figure 1. Phylogenetic analysis of F tsZ proteins. A neighbor joining tree from analysis of FtsZ protein sequences is shown. Bootstrap support values from the neighbor joining (first number) and maximum parsimony (second number) analyses are shown at selected nodes. Boxed numbers are quartet puzzle frequencies from analysis of the parsimony tree. Nodes with less than 50% support, or not present, are represented by a dash. Letters to the left refer to the sequences listed in Table 1. Alignment is available upon request. 156 for the Ftle (100/97/79) and FtsZ2 (97/86/93) clades, with slightly reduced support for the red/brown algae (88/70/89). The cyanobacteria have the least support (85/53/—) as a single clade; the analyses suggest there may actually be two clades (100/100/94 and 94/55/67). Although resolution of the cyanobacterial proteins into a single clade is weak, both neighbor joining and parsimony trees indicate that all the cyanobacterial sequences are sister to the red/brown algae and FtsZ2 clades. As indicated in previous reports (Osteryoung and Vierling 1995; Osteryoung et al., 1998; Beech et al., 2000), the plant FtsZ protein sequences are more closely related to those in cyanobacteria than in other prokaryotes (not shown). An interesting result of the phylogenetic analyses is that the Ftle clade branches prior to the other three clades rather than after the cyanobacterial clade (Figure 1). This branching order is supported by intermediate to weak bootstrap and frequency scores (85/67/55). The early branching of the Ftle proteins before the cyanobacterial proteins seems inconsistent with the evolution of chloroplasts, as discussed below. Support for branching of the cyanobacterial (71/57/62) from the red/brown and FtsZ2 (71/57/62) clades is less strong, suggesting the differences between these clades are not as pronounced. Significantly, our analyses place all the chloroplastic FtsZ proteins from red/brown algae into a single clade, whereas the sequences from the chlorophycean green alga C. reinhardtii are split between the Ftle and FtsZZ clades. Wang et al. (2003) recently reported a similar result. These findings indicate that the divergence of the Ftle and FtsZ2 families found in plants may have occurred between the divergence of red and green algae. Phylogenetic Analysis of FtsZ cDNA Sequences from Photosynthetic Eukaryotes The phylogenetic relationships among the F tsZ cDNA sequences were analyzed using neighborjoining, maximum parsimony, and maximum likelihood algorithms (Swofford, 1998). The cDNA alignment used in this set of experiments was based on that of the proteins. The third codon position was removed for these analyses due to its variability. The maximum parsimony analysis produced seventeen different trees, but the strict consensus tree indicated that the differences in branching patterns occurred only within clades. Figure 2 shows the maximum likelihood tree with bootstrap support from the neighborjoining and maximum parsimony analyses (first two numbers) and likelihood frequencies from a Bayesian Inference analysis (Huelsenbeck and Ronquist, 2001) (third number) indicated at selected nodes. The cDNA experiments group the sequences into the same four clades as the protein anal yses, namely Ftle, FtsZ2, red/brown algae, and cyanobacteria (Figure 2). Support for each clade is generally strong (100/99/100, 99/97/100, 96/85/100, and 96/— /97, respectively) and the branching order was the same as that obtained for the protein analyses. There is reasonable support in the cDNA analyses for the early branching of the Ftle clade before the cyanobacterial. red/brown algal, and FtsZ2 clades (72/72/94). Like the protein analyses (Figure 1; Wang et al., 2003), the cDNA analyses place distinct C. reinhardtii sequences in the Ftle and FtsZ2 clades. The potential grouping of the cyanobacterial sequences into two clades is supported by bootstrap and frequency values of 93/ 100/ 100 and 100/65/ 100. These two cyanobacterial clades are almost identical to those observed in the protein analyses; the only difference is in the position of the 158 l L. Iongi orum E O. sativa H O. sativa C N. tabacum A N. tabacum B G. lutea FtsZ2 D 100,100,951 A. thaliana F A. thaliana G 99/97/1011 P. patens I P. patens J C. reinhardtii K 465,54 , EC. merolae L _ _ C. caldarium R dIB M r— G. theta (nucleomorph) e rown N 6l85/ G. sulphuraria Algae O M. splendens P G. sulphuraria L 7::172/ Prochlorococcus sp. X _E—l P. marinus MED4 Y P. marinus ddlb Z 4490 93/1001gg£___ Synechococcus sp. WH8102 AA P. marlnus MIT9313 BB 96“ —Synechococcus sp. PCC 7942 Cyanobacteria w 91 .00, Anabaena sp. PCC 7120 r 51 N. punctiforme U T. elongatus BP-1 V 95’1"” T. erythraeum R Synechocystls sp. PCC 6893 §_ N. tabacum CC N. tabacum DD A. thaliana EE P. sativum FF N. tabacum FtsZ1 GG 100,100] N. tabacum HH T. erecta 11 1001991191; 0. sativa JJ 0. rename K.I_<. C. propionicum LL 1 l B. subtilis NN E. coli MM 0.1 p-distance Figure 2. Phylogenetic analysis of F tsZ cDNA sequences. A Maximum likelihood tree using the first two codon positions of the cDNA sequences is shown. Bootstrap support values from neighbor joining (first number) and maximum parsimony (second number) analyses are shown at selected nodes. Bayesian Inference frequencies are underlined. Nodes with less than 50% support, or not present, are represented by a dash. Letters to the left refer to the sequences listed in Table 1. Alignment is available upon request. 159 Synechacaccus sp. PCC7942 sequence. Except for the node at which the red/brown algae and FtsZ2 clades branch, all Bayesian Inference frequencies shown (Figure 2) are greater than 90% and strongly support this as the most likely tree under the selected criteria. We next tested the significance of the maximum likelihood tree from Figure 2 (shown in simplified form in Figure 3A) against two constraint trees in which the branching order among the four major clades was rearranged (Figure 3, B and C; branching within clades was not altered). In one constraint tree (Figure 3B), the positions of the cyanobacterial and Ftle clades have been switched from that shown in Figure 3A, such that the cyanobacterial clade branches first, followed by the Ftle and subsequently the FtsZ2 and red/brown algae clades. In the second constraint tree (Figure 3C) the Ftle clade has been placed as sister to the FtsZ2 clade. with the cyanobacterial clade branching first. These two constraint trees better represent the relationships we expected based on chloroplast evolution theories (Gray, 1999) and on the strong similarity of both the Ftle and FtsZZ proteins to the cyanobacterial FtsZs (Osteryoung et al., 1998; Osteryoung and McAndrew, 2001). When the significance of either constraint tree was tested against the maximum likelihood tree, the p-value was 0.000. This means that, at greater than 99% confidence, the data do not support a branching order that differs from that shown in the maximum likelihood tree (Figure 3A). Therefore, phylogenetic analyses based on both cDNA and protein data support the grouping of FtsZ sequences into four clades, with the Ftle sequences branching first, followed by the cyanobacteria, then the red/brown algae and FtsZ2 sequences. 160 A F622 Red/Brown Algae Cyanobacteria FtsZ1 Outgroup p—value 0% 0.000 B FtsZ2 FtsZ2 C Red/Brown Algae FtsZ1 FtsZ1 Red/Brown Algae Cyanobacteria Cyanobacteria Outgroup Outgroup Figure 3. Testing the maximum likelihood tree against two constraint trees. All trees have been simplified in this diagram. The maximum likelihood tree, A, from Figure 2 is compared to two different constraint trees, B & C, in which the relationships between the clades have been altered. The low p-values indicate the relationships in the two constraint trees are not supported by the data. 161 Testing for Differences in The FtsZ1 and FtsZ2 Evolutionary Rates One possible explanation for the branching order determined from the phylogenetic analyses is that the FtsZ] sequences evolved more rapidly than the FtsZ2 sequences. To test this possibility, we performed a relative rate test on the FtsZ1 and F rsZZ sequences. Only sequences from the same organism were compared in order to minimize the effect of different selective pressures encountered by different organisms. Because of this restriction. only the C. rcinhardtii. 0. sativa, A. thaliana, and Nicatiana tabacum sequences could be tested. Every F tle sequence was tested against every F tsZZ sequence from the same organism, using both protein and cDNA sequences. All Ftle and FtsZZ sequences were compared against each of three outgroups: (l) B. subtilis only, (2) C. prapianicmn only, and (3) C. propionicum, E. coli, and B. subtilis combined. At a 95% confidence limit, no significant difference in the evolutionary rate was detected for any of the sequence combinations tested. Thus, the relative rate test did not detect a significant difference in the rates at which the Ftle and FtsZZ sequences have evolved. Genetic Structure Is Conserved Within the Plant FtsZ Clades The relationship between the FtsZ1 and F tsZ2 sequences was further investigated using genetic structure (intron position/exon length) comparisons. Fujiwara and Yoshida (2001) reported that the intron positions in the two A. thaliana F tsZZ sequences are identical. We analyzed all three A. thaliana FtsZ genes, as well as genes from 0. sativa, P. patens, and C. rein/iardtii (Figure 4). Although relatively few genomic sequences are available for this analysis, especially among members of the Ftle clade, those available represent very diverse groups. Due to high sequence variability in the 5’ and 3’ ends of 3‘33 1 2 3 5"“ 4 '— 5 6 Exon Number _ (294) (100) (230) (168) (234) (276) (Length) AlFtSZl 1 (FF) ATG A Q Q] |A A IntronNumber 1 2 3 4 5 6 (216) (100) (236) (168) (234) (261) 1 2'—7' 3 4 5 6 7 8 9 (221) (133) (138) (156) (65) (161) (153) (93) (320) FtsZ1 FtsZZ I 1 2 3 4 5 6 (633) (300) (91)(125) (93) (195) AiFiSZZ-l (F) ATG A A A $ A 1 2 3 4 5 6 7 (621) (300) (91) (125) (93)(66)(126) AtFtsZZ-Z (G) ATG A A A A AAA 1 2 3 4 5 6 7 (624) (300) (91) (125) (93)(60)(72) OsFtsZZ(H) ATG A & & A M 1 2 3 4 5 6 7 (567) (300) (91) (125) (93)(66)(78) OsFtsZZ (C) Are A A A A AA 1 2 3 4 5 6 7 (603) (300) (91) (125) (93)(66)(123) 13thle (J) ATG A &|& & . I 1 2 3 4 5 6 7 (621) (300) (91) (125) (93)(66)(117) P9151522 1') “G A. Ala 25“ 1 F 3 4 5'__6 7 8 9 10 11 CrFtsZZ (K) ATG (114) (99) (99)(69)(132) (76) (92) (88) (128) (121) (287) Ag“ MIQIA A. as Figure 4. Comparison of F tle and F tsZZ genetic structures. Aligned A. thaliana, P. patens. 0. saliva, and C. rein/zardtii F tsZ cDNA sequences are represented by solid horizontal lines, and drawn to scale. Letters in parentheses correspond to sequences listed in Table l. Intron positions are indicated by numbered triangles. Exon numbers are indicated above the horizontal line; exon lengths in bp are in parentheses. The shaded box indicates an intron positioned similarly in the F ml] and F tsZ2 genes. Hatched boxes indicate C. reinham'tii and plant FtsZ] introns located at equivalent positions. The crosshatched boxes represent intron positions shared only in the two C. reinhardtii sequences. The CrFtsZ] sequence corresponds to the CrFtsZ3 sequence reported by Wang et al. (2003). 163 the coding regions, a cDNA sequence alignment based on that used for the phylogenetic analysis shown in Figure 2 was used for genetic structure comparisons. In the 0. sativa and A. thaliana FtsZ] genes. all five introns (l, 2, 3, 4 and 5) are located at identical positions (Figure 4). The introns vary considerably in sequence and length (Table 2), but the exon lengths are identical. the only exception being a 6 bp insertion in the third exon of the 0. saliva FtsZ] gene. These results indicate that intron locations in the F152] sequences from angiosperms are conserved. The conservation of intron locations among the F 1522 genes is even more striking (Figure 4). The six introns in the A. thaliana, 0. swim, and P. patens F tsZZ genes, though variable in sequence and length (Table 2), are in identical positions, with two exceptions. The first is in the AtFtsZZ—I sequence, which lacks the sixth intron present in the other FIsZZ genes. The second is a six bp shift in the position of the sixth intron of one 0. sativa sequence, possibly due to a deletion. that reduces the length of the sixth exon from 66 to 60 bp. Otherwise, monocot. dicot, and moss F1522 sequences have a conserved genetic structure. Although intron locations within the plant F Ile and F tsZZ genes are conserved, the intron locations between them are significantly different. Only the fourth intron in the F {52] genes and the second intron in the F [522 genes are located at equivalent positions (shaded box in Figure 4). The presence of similarly positioned introns in the F tsZI and F {522 genes from diverse land plants suggests that both clades originated from a common ancestral gene bearing a corresponding intron. Ancient duplication of this ancestral gene presumably allowed the two clades to diverge early and extensively from each other. 164 Table 2. Comparison of FtsZ intron lengths B Gene Intron Number Family Sequence3 1 2 3 4 5 6 7 8 9 10 AtFtsZ1-l (FF) 102 93 89 106 96 NA NA NA NA NA FtsZ] OSFISZI (1]) 357 266 468 68 192 NA NA NA NA NA CrFtsZI (KK) 126 165 198 122 163 173 133 186 NA NA AtFtsZZ—I (F) 165 85 301 202 82 NA NA NA NA NA AtFtsZZ-Z (G) 367 90 377 148 85 106 NA NA NA NA OsFtsZZ (H) 287 474 343 258 143 155 NA NA NA NA FISZZ OsFtsZZ (C) 90 132 500 1006 278 636 NA NA NA NA PthSZI (J) 462 312 204 79 217 276 NA NA NA NA PpFIsZZ (I) 460 338 187 84 225 455 NA NA NA NA CrFtsZZ (K) 74 86 100 371 150 107 119 114 154 181 2]Letters in parentheses correspond to sequences listed in Table 1. bThe intron number corresponds to labeled triangles in Figure 4. NA, not applicable. 165 The genetic structures of the C. reinhardtii F tsZ genes are partially conserved with those of the plant genes. The C. reinhardrii FtsZ] gene shares one intron position with the plant FtsZ1 genes (Figure 4, introns 4 and 3, respectively). Three C. reinhara’tii FtsZZ introns are positioned similarly to those in the plant FtsZ2 genes (introns 4, 7, and 9 versus 1. 2. and 4. respectively). Intron 7 in C. reinluzrdtii F tsZ2 corresponds to the intron that is common to both FtsZ1 and FtsZ2 in plants (shaded box in Figure 4), though this intron is lacking in the C. rein/zardtii FtsZ] gene. Intron 5 in C. reinhardtii F1522 corresponds to introns 4 and 3 in the C. reinhardtii and plant F tsZI genes, respectively (hatched boxes in Figure 4). Finally, intron 2 is shared by both C. reinhardtii genes (crosshatched boxes in Figure 4), but is absent from the plant genes. The positions of the other introns in the two C. reinhardtii F tsZ genes are unique to each gene. FtsZ1 and F tsZZ Protein Comparisons The early divergence of the Ftle and FtsZZ families and their conservation in both plants and C. reinhardri is consistent with the hypothesis that the two proteins evolved to have distinct functions. Because protein function is ultimately defined by amino acid composition, we compared the FtsZ protein sequences in an effort to identify regions that may represent functionally important differences between Ftle and FtsZ2 (Figure 5). Consensus sequences for each clade defined in Figure 1 were aligned, except for the highly divergent chloroplast transit peptides. The alignment included the divergent carboxy termini omitted from the phylogenetic analyses because it contains a region of potential functional significance as described below. A full-length E. coli 166 + + + ++ ++ ++ +¢ + ++ (9 ++ + FtSZL “'*"*"'A‘IKVVGVGGGG'NAVKFMI'SGLQ‘VdFYAiNTD‘QAL"*--**"*l*IG**LTRGLG*GGNP‘LG* FtsZ2 ***‘****'***IKViGVGGGGSNAVN'Mi*S‘m'GVEFWI'NTD'QAm"SPV*'**rl*IG**LTRGLGAGGNPGIG* R/B Algae '*"'**‘“*C'IKViGVGGgU'NAVnRM"***i'VE*"*NTD'QAL'R"**'*****IG‘**'RGLGAGG*P"G' Cydno "'***"'*R*I*VIGVGGGG‘HAVNFM:'S*V*Gv‘f**lXTD'QAL"*‘—'A'"1QlGQ‘LTRGLGAGGXP*IG' E._COli MFEPMELTXDAVIKVIGVGGGGGXAVEHHVRERIEGVEFFAVNTDAQALRKT--AVGQTIQIGSGITKGLGAGAXPEVGR + ++ + + + + + ++ + + + + + + ++ ++ F1521 'AA'ES'e'i"“L*'*DLVFITASMGGGTGSGAAPVVA‘i*Ke'G'LTVGVVTYPF'FEGRkR**QALEaiE'L'"VD FtSZZ 'AA'ES'*‘i"';'*’DHVEVTAGHGfiJTGCGJAPViA"AK'mGILTVGI'TTPF‘FEGR'R**QA**giA‘Lr*‘VD R/B Algae *AAEFS***I*"V**aDLVFVTAGMGGGTGSGAAPVVA**AIE'G‘LTVGVVTKPF‘FEGrrRm*QA**ai**lr'*VD CYRRQ KAAEESr*e;*“L‘*‘DLVF:"GMGJGTGTGRAFVVAEVAK'*G‘LTtiVTkPF*FEGIRR‘*QA*‘G***L*‘*VD E. coli XAADEDRDALRAALEGADMVFlAAGRGGGTGTGAAFVVAEVAKDLGILTVAVVTKPFNFEGKKRMAFAEQGITELSKHVD + +++++ +++ + + + + + + ++++ + ++++ + Ftle tlIVlPNDRLLDi"'***LQ'AF'LADDVL'QGVQGISDIITiPGLVNVDFADVKAVM**SGTAMLGVG**S***RA** FtSZZ TLI*IPN‘kLL*Av****PV*eAF‘lADDiLRQGVIGISdIITVPCLVNVDFADVR'iM**AGSSLMG*G*ATG**rA‘d R/B Algae TLIVVSNDRLL'*VP**T'L"AF"ADdiLRQGV'GISdII'rPGLiNVDFADVRvaa**G‘ALmGIG‘g‘G‘*RA" Cyano TLZ*lPN'*l*"I**‘*‘lQeAF*'ADDVLR'GV‘GISDII'*PGLVNVDFADVR'VM'dAGS*lmGIG"SGkSRA'E E. coli SLITIPNDKLLKVLGRaIS.LDAFGAAXDVLKGAVQG:AELITRPGLMNVDFADVRTVMSEMGYAMMGSGVASGEDRAKE ++ +++ ++++ + + + + + + ++++ +++ + + ++ ++++ FrSZL AA'*AT‘APLI'*S-I'*ATG‘VYNITGGkDiTL‘EVNrVS'VVT‘LADPS'NIIFGAVVDe*Y*GEi‘VTiiATGF‘*S FCSZZ AA *Ai‘SPLLdiG-IERATGiUWNITG***lTL'EVN'AAEvIYDlVDP'ANLIFGaVvD******vSITlIATuF*** R/B Algae AA‘*AISSPLLDFP—I*‘AKG*VFN1'GG‘DM:L*EiN*AA*VIYe‘VD'*ANIIFGAlvD******iSiTVVATGF*** Cyano AA‘*Ai'SPLLE'**f'GAkG‘U'K:rGG'Dth‘ev**A*e‘:Y’V'D"AXII‘GAViD*l**GEi*iTVIATGF*** E._COli ARENA:fiSPLLEDIDLSGARGVLVEITAGFDLRLDEFETVGNTIRAFASDNATVVIGTSLDPDMNDELRVTVVATGIGMD + ++++++ FtSZl F‘fififiLLflfififigiitiififiitttitit!itfitttiifittiii ___________ fittithittitiiitrLi-t $1.972 eiifltififetfiiifitiiitfitittirtviktttfibiiiitifiififi’iflttifiit’piFI‘tkfflfii‘kfflflfl L.\,J ‘ .4 R/B Algae a ______ firidtikiiiittttfiivdkttttit_iiiiiiittt*fitiktfitiii:pDFer'tfitffifififi Pudpo wr*____t-ttwtw*ttttirttt-tttttr'ar _____ tittt‘fltttiitktflTkaLkiriflf*kitt v1 . 4. .h E. coli KRPEITLVTNKQVQQPVMDRYQQHGMAP T,ECKPVAKVVNDNAPQTAKEPDYL‘-T& Figure 5. Comparison of FtsZ proteins from photosynthetic organisms. Consensus sequences are shown for the Ftle , FtsZ2, Red/Brown algae (R/B algae), and cyanobacterial (cyano) clades. For all sequences within a clade, capital letters and small letters indicate identical and similar residues, respectively. Positions where amino acids are not conserved are indicated by asterisks. Gaps in all members of a clade are represented by dashes. Residues shown by structural analysis to either contact GTP (Lowe, 1998) or to be required for regulation of GTPase activity (Erickson, 1998) are underlined in the E. coli protein. Residues located on the side of the E. coli protein that are required for FtsZ function but not for GTPase activity (Lu et al., 2001) are labeled with dots. Residues where the Ftle and FtsZ2 differ are marked at the top with a ‘+’; bold letters indicate residues that are completely conserved within, but that differ between, the Ftle and FtsZ2 clades. The circled ‘+’ is a nucleotide binding residue that was previously recognized to differ between the Ftle and FtsZZ clades (Osteryoung and McAndrew, 2001). 167 protein is included in the alignment as a reference because it is functionally the best characterized of the bacterial FtsZ proteins. FtsZ sequences in all four clades share significant similarity with the E. coli protein. particularly in regions previously shown to contact GTP (L6we, 1998), to be required for GTP activity (Erickson. 1998), or to be important for other aspects of E. coli FtsZ function (Lu et al., 2001) (underscores and dots in Figure 5). With one exception (circled plus), all the residues important for GTPase activity (underscores) are conserved in all the FtsZ proteins analyzed in this study. However, because we are most interested in the features that may functionally distinguish the FtsZ] and FtsZ2 proteins, we used the alignment to highlight the differences between these two clades. The residues indicated in bold represent amino acids that are invariant within the two clades, but that differ between them. None of these conserved differences occur at positions corresponding to the functionally characterized residues in E. coli FtsZ, and most occur downstream of the regions in the E. coli protein associated with GTPase activity (underscores). In addition to these differences, the FtsZ1 and FtsZ2 proteins differ in the presence of a C-terminal peptide similar to a region in E. coli FtsZ (boxed residues in Figure 5) shown to be critical for its interaction with two other bacterial cell division proteins, FtsA and ZipA (Ma and Margolin, 1999; Mosyak et al., 2000). This peptide is identifiable in the FtsZ2 proteins as well as in the cyanobacterial and most of the red/brown algae proteins, but is missing in the FtsZ1 proteins. The absence of this peptide in FtsZ1 and its presence in FtsZ2 suggests the two proteins families may interact with different sets of proteins during chloroplast division, though obvious homologues of ZipA and FtsA are lacking in both plants and cyanobacteria. 168 Discussion Early Divergence of the FtsZ1 and FtsZ2 Families Phylogenetic analyses have defined two major groups of green algae, one containing the charophycean green algae. which are sister to the land plants, and the other containing the chlorophycean green algae. which include C. rein/lardtii (Graham and Wilcox. 2000). Previously. we hypothesized that the emergence of FtsZ1 and FtsZZ as separate clades might have accompanied the evolution of vascular plants (Osteryoung et al., 1998). This conjecture was based primarily on the lack of FtsZ1-like sequences from other organisms in the public databases. However, the grouping of the C. reinhardtii FtsZ genes into the FtsZ] and FtsZ2 clades is well supported by phylogenetic analyses based on both amino acid (Figure 1; Wang et al., 2003) and cDNA alignments (Figure 2). This strongly suggests that the emergence of Ftle and FtsZ2 as separate clades predated not only the split between the charophycean green algae and land plants, but also the split between the two major green algal lineages. To date, FtsZ] sequences have only been reported in angiosperms and C. reinhardtii, but if the two FtsZ families originated as early as the data suggest. then members of both families should eventually be discovered in mosses and other land plants. in charophycean green algae. and in other chlorophycean green algae. Relationship of the FtsZ1 Clade to the Other Three Clades The phylogenetic analyses consistently yielded trees in which the FtsZ1 clade forms a sister group to the clade containing the FtsZZ. red/brown algal, and 169 cyanobacterial FtsZ sequences (Figs. 1 and 2). Although this branching order has also been observed (though not discussed) in previous reports (Beech et al., 2000; Kiessling et al., 2000; Wang et al.. 2003). it is not consistent with the relationships predicted based on the presence of only a single FtsZ gene in all cyanobacterial genomes sequenced to date and on the presumed monophyletic origin of chloroplasts (Gray. 1999). If the Ftle and FtsZ2 sequences shared a common cyanobacterial ancestor, one would expect them to be more closely related to each other than to the cyanobacterial FtsZ sequences. We tested the possibility that the observed branching order might be an artifact of a difference in the rates of Ftle and FtsZ2 evolution. but were unable to detect such a difference. This could be because the portions of the protein sequences aligned in our analysis are functionally constrained. and do not reflect the true evolutionary rates. In fact. the E. coli and Arabidopsis FtsZ protein sequences are about 50% identical in this region, indicating a high degree of conservation among FtsZ proteins from widely divergent organisms. Another possibility for the failure to detect a difference in evolutionary rate could be that the two clades diverged at equal rates. If so. this would suggest that the FtsZ2 sequences evolved similarly to their cyanobacterial counterparts, perhaps due to specific functional constraints, while the FtsZ] sequences evolved to fulfill a new function. In this case. although evolutionary rates would be equal for both clades, the sequence changes would be in different directions. Another potential explanation is that the branching of the F tle and H.922 clades is too deep for the relative rate test to detect a significant difference (Avise. 1994). A related possibility is that the outgroup is too distantly related to the sequences analyzed. However. the B. subtilis FtsZ used in the outgroups for the relative rate test is one of the closest currently available relatives of the 170 cyanobacterial FtsZ proteins based on sequence homology. Regardless of whether the branching order is an artifact of the analyses or reflects the true evolutionary history of the FtsZ gene families, the deep branching of the Ftle and FtsZZ clades supports their early divergence. Comparisons of exon length and intron distribution among the FtsZ genes also support the early divergence of FtsZ] and FtsZ2. Although the intron positions in the angiospenn F tle genes are conserved, they differ from the conserved intron positions in the F1522 genes. except at a single location. Assuming that both clades originated from a single ancestral FtsZ gene, these data indicate that a majority of the introns were inserted into the sequences following an ancestral duplication. The fact that the C. reinhardtii and plant FtsZ genes share partial similarity in their genetic structures is also consistent with the divergence of the FtsZ] and FtsZ2 families prior to the split between the two green algal lineages. However, additional genomic F tsZ sequences from other green algae are needed for a more complete understanding of the relationship between FtsZ evolution and gene structure. Evolutionary Origins of the Chloroplastic FtsZ1 and FtsZ2 Sequences Exactly when the duplication and divergence of the ancestral F tsZ gene occurred remains unknown. but two general scenarios can be envisioned (Figure 6). In the first, the duplication and divergence might have occurred after a single F tsZ gene was transferred from the cyanobacterial endosymbiont to the eukaryotic nucleus, but before separation of the two major green algal lineages (Figure 6A). This scenario would 171 (a? FtsZ gene 08‘ “o A transferred to (:19ng Ancestral C ' nucleus Q ‘50 \ Charophycean Cyanobacteriel E::2:::;ie:: l E \ ‘( Green Algae FtsZ / < \ \XI and Plants Present-day Red/Brown Chlorophycean Cyanobacteria Algae Green Algae ch‘ Q“ @069 FtsZ genes B 40: g transferred to Ancestral T 170 an ob a cteri d "“1“” \ Charophycean Cyanobacterial Ehdosymbiont I \ \ ‘( Green Algae sz ' l \N \X’ and Plants Present-day Red/Brown Chlorophycean . Cyanobacteria Algae Green Algae Figure 6. Two scenarios for the evolutionary origins of the plant FtsZ gene families. Transfer of the F tsZ gene(s) from the cyanobacterial endosymbiont to the eukaryotic nucleus is marked with a vertical line. The evolutionary timeline of the F tsZ gene is represented as a horizontal line. Gray and black horizontal lines represent divergence in the FtsZ genes. A. a single F tsZ gene is transferred from the cyanobacterial endosymbiont to the eukaryotic nucleus. Duplication and divergence of the F tsZ gene occurs before divergence of the two major green algal lineages. B, Duplication and divergence of the FtsZ genes occur in the cyanobacterial ancestor of chloroplasts. Both genes would be transferred to the eukaryotic nucleus. giving rise to separate FtsZ] and F tsZ2 clades. explain why two FtsZ families are present in both chlorophycean green algae (C. reinham’tii) and plants. In a variation. divergence could also have occurred earlier, prior to emergence of the red and brown algae, though at present there are no data to suggest this is the case. Additional genome data from more basal organisms will shed light on the timing of FtsZ duplication and divergence. However, this first scenario (Figure 6A) does not explain the branching of the FtsZ1 clade before the cyanobacterial sequences (Figs. 1 and 2). In the second scenario. duplication of the FtsZ gene might have occurred in the cyanobacterial progenitor of chloroplasts (Figure 6B). In this case, the extant cyanobacteria. which so far bear only a single FtsZ gene, would either have lost one of the duplicated genes or would have descended from the chloroplast progenitor before the duplication occurred. This scenario would explain the relationships we observe in our phylogenetic analyses. but would suggest that. in the red and brown algal lineages, one of the FtsZ families was lost or that a second FtsZ family has yet to be discovered. This scenario would also suggest that a cyanobacterium with both an Ftle -like and an FtsZ2- like gene, more closely related to the original endosymbiont, existed in the past and could still be discovered. Although not a widely accepted idea (Palmer, 2003), it is also conceivable that chloroplasts may be polyphyletic in origin (Stiller et al., 2003), and that the two FtsZ gene families originated from different endosymbiotic events. At present we cannot rule out any of the above scenarios with the currently available information. Addition to the public databases of new F tsZ sequences and genome data from evolutionarily relevant taxa will help to elucidate the history of the chloroplast FtsZ genes, and may shed light on why two forms of FtsZ evolved to function in chloroplast division. 173 Acknowledgements We thank Drs. Jim Smith, Richard Triemer and Eric Linton for advice and instruction on the phylogenetic techniques, Dr. Tao Sang for critical reading of the manuscript and many helpful discussions. and members of our laboratory for their valuable input. This work supported by National Science Foundation grant MCB- 009 2448 . 174 CHAPTER 6 Summaries and Future Directions 175 Summary The major thrust of the work presented in this thesis has been to better understand the function of FtsZ proteins in chloroplast division. The functional. expressional, and evolutionary FtsZ comparisons that have been discussed in this work provide a better understanding of the role FtsZ proteins play in chloroplast division. First, my work has helped us recognize that chloroplast division requires members of two FtsZ families, l Ftle and FtsZ2. Second. my studies have shown that chloroplast division seems to involve a stoichiometric balance of Ftle and FtsZZ proteins. Third, my expression studies have shown that the FtsZ genes are coordinately expressed in many tissues, including tissues in which chloroplast division occurs in a majority of the cells. Also, the F tsZ genes are expressed at different levels, consistent with the hypothesis that a stoichiometeric balance between the FtsZ proteins is required for chloroplast division. Finally, my analyses have shown that sequence and structural characteristics among the Ftle and FtsZ2 families are very conserved. especially in higher plants. This conservation suggests there are differences in functions between Ftle and FtsZ2 proteins. The results discussed in this thesis have laid the foundation for future work on the functions and interactions of the FtsZ proteins, which is central to understanding the complex and important process whereby plastids divide, a process that has only now begun to be explored. 176 Chapter Summaries and Future Directions Function of FtsZ1 and FtsZ2 Proteins in Chloroplast Division Isolation of two Arabidopsis FtsZ genes that belong to different families, FtsZ] and FtsZ2, indicated there is a significant difference between chloroplast and bacterial division. since the latter requires a single FtsZ gene in most cases. To determine the functions of the Ftle and FtsZ2 proteins. plants expressing antisense FtsZ transgenes were generated. When FtsZ1 or FtsZ2 expression is reduced in the transgenic plants. chloroplast division is inhibited, which reduces the number of chloroplasts from about 100 in a normal mesophyll cell to as few as one very large chloroplast. These results indicate that both Ftle and FtsZ2 proteins function in chloroplast division. Although FtsZ1 and FtsZ2 family members are both functional in chloroplast division. Arabidopsis plants actually express two FtsZ2 family members, AtFtsZZ-I and AtFtsZZ-Z. Antisense repression experiments could not differentiate between the functions of the two FtsZZ proteins since expression of both AtFtsZZ-l and AtFtsZZ-Z is repressed in the AtFtsZZ—I antisense lines. Isolation and characterization of AtFtsZZ—l or AtFtsZZ-Z Arabidopsis knockout mutants would greatly benefit studies aimed at determining the function of each FtsZZ protein. Although knockout lines for each of the three Arabidopsis F tsZ genes are sought, AtFtsZZ-Z is the only F tsZ gene for which we have knockout plants in our laboratory (Dr. Deena Kadirjan-Kalbach, unpublished results). The chloroplast phenotype of the AtFtsZ2-2 knockout line is intermediate; it is characterized by about 15 enlarged chloroplasts per mesophyll cell. This phenotype would indicate the efficiency of plastid division is reduced but not inhibited by removal of AtFtsZ2-2 protein. This may reflect the fact that the total FtsZ2 protein level is 177 reduced by only about 20% in this mutant (McAndrew et al., 2003), personal communication) Altered FtsZ1 or F tsZZ Levels Disrupt Chloroplast Division In addition to reducing FtsZ levels. we also asked whether increasing Ftle or FtsZ2 protein levels would affect chloroplast division. For this purpose. I attempted to create transgenic plants overexpressing AtFtsZI-I. AtFtsZZ-I. or AtFtsZZ-Z transgenes. Analysis of the transgenic plants suggests there is a direct correlation between the AtFtsZ1-l overexpression level and the severity of the chloroplast division defect. Because the AtFtsZZ-I cDNA that was used in the overexpression construct was truncated. none of the transgenic plants can be described to be true overexpressors. However, a full-length AtFtsZ2-1-cm_vc transgene was overexpressed by Vitha et al. (2001) who observed that slight increases in AtFtsZZ-I expression did not affect chloroplast division but that large increases did. These results indicate that altering the stoichiometric balance between the Ftle or FtsZ2 proteins themselves, or between FtsZ proteins and other components of the chloroplast division apparatus. disrupts chloroplast division. However, there may be some flexibility in the stoichiometric balance since slight increases in FtsZ levels did not noticeably disrupt chloroplast division. In bacteria, a slight increase in FtsZ protein level induces cell division abnormally at the cell poles because of FtsZ ring assembly at those sites (Ward Jr and Lutkenhaus, 1985). It was hypothesized that increased chloroplast division in plants would be manifested by increased numbers of smaller chloroplasts in mesophyll cells. None of the transgenic plants overexpressing AtF tsZ I -l or AtFtsZZ-I proteins had increased numbers 178 of chloroplasts. However, the experiments do not indicate that overexpression of the plant FtsZ proteins is unable to induce plastid division. One possible explanation for the fact that increased chloroplast division was not observed may be that a very narrow range of FtsZ overexpression is required to induce chloroplast division, and the FtsZ protein levels in the transgenic plants may not have been within that range. Related to this possibility is the use of the 35S promoter in all the overexpressiong constructs, which is typically a very strong promoter for gene expression. It is also possible that overexpression of two of the Arabidopsis FtsZ proteins together, or of all three, may be required before an increase in chloroplast division can be observed, since members of two families are required for chloroplast division. Therefore, increasing the level of only one of the plant FtsZ proteins may be insufficient to increase chloroplast division, no matter what level of overexpression is achieved. Furthermore, preserving the stoichiometric balance between the three FtsZ proteins, and possibly other components of the division apparatus, may also be necessary for the chloroplasts to divide. To address whether FtsZ overexpression could induce chloroplast division, transgenic plants that overexpress combinations of two or even all three F tsZ genes could be generated. The stoichiometric ratios of the FtsZ proteins may be critical for increasing chloroplast division; therefore control of expression may be very important. Expression with the 358 promoter, which was used in the overexpression constructs, may be too strong for these experiments. Also, the strong expression of the F tsZ genes with the 35S promoter may have been a cause for the co-suppression that was observed in many of the transgenic plants. Therefore, it may be better to use the native FtsZ promoters or possibly an inducible promoter for studies of FtsZ function in transgenic plants. 179 FtsZ knockout plants would also benefit studies to determine whether the functions of the FtsZZ proteins are redundant. One experiment might be to overexpress AtFtsZZ—l in the AtFtsZZ-Z knockout line, and vice versa once an AtFtsZZ-l knockout line is found. and see if a normal chloroplast phenotype can be restored. Furthermore, experiments could be performed that investigate whether overexpression of Ftle proteins could substitute for loss of FtsZ2. or vice verse. The results of these experiments would have implications for understanding the roles of FtsZ] and FtsZZ proteins in chloroplast division. FtsZ1 and F tsZZ Genes are Coordinately Expressed Expression of the Arabidopsis FtsZ genes in tissues where chloroplast division is occurring was expected due to the critical role FtsZ proteins have in this process. Histochemical staining of transgenic plants with the FtsZ promoter-GUS constructs, measurements of FtsZ transcript levels by real-time reverse transcription-polymerase chain reaction (RT-PCR), and measurements of protein by immunoblot analysis indicate that the three Arabidopsis FtsZ genes are coordinately expressed, especially in tissues where chloroplast division is occurring in a large proportion of the cell population. Transcript measurements of the F tsZ genes indicate AtFtsZZ-Z is the least abundant, AtFtsZ1-I was intermediate, and AtFtsZZ—l was the most abundant. Furthermore, the ratio of FtsZ] to FtsZ2 transcript was about 1 to 3, which correlates well with the 1 to 2 ratio of Ftle to FtsZ2 protein that was determined by McAndrew et al. (in preparation). The FtsZ transcript ratios seem to be constant throughout leaf expansion, consistent with 180 the hypothesis that the stoichiometric balance between the FtsZ proteins may be important for chloroplast division. One important experiment would be to identify and characterize the sequences that are required for FtsZ expression. This is especially important for AtFtsZI-I since there are probably sequences downstream of the 5‘-UTR that are required for expression, which were not present in our promoter-GUS constructs. Although the expression patterns observed with the AtFtsZZ-l and AtFtsZZ-Z promoters were more defined, there may still be regulatory elements that are missing. An initial step to define the regulatory elements for the FtsZ genes would be to use computer programs to identify known regulatory elements that are involved in gene expression. The results of the computer analysis would allow. at least on a basic level. comparisons of potential regulatory elements among these FtsZ genes. Also, analysis of the sequences surrounding the FtsZ genes with these computer programs would help determine what regions may still be required in the promoter-GUS constructs to accurately represent endogenous F 152 expression. Of course. mutation or deletion analysis would be required to determine the functionality of any identified elements. In addition to computer analysis of the F tsZ promoters. the promoter-GUS plant lines and RNA quantification techniques can be used to determine the factors that affect FtsZ expression. By assaying the amount of GUS enzyme that is expressed with the promoter-GUS constructs, factors that change FtsZ expression in treated plants can be easily assayed. Similarly, changes in FtsZ transcript levels can be assayed using real- time RT-PCR or possibly northern blot analysis. Changes in F {52 transcript amounts are 181 predicted to have an effect on chloroplast division. but the chloroplast phenotypes that result from any treatment will have to be characterized. Some of the factors that should be tested for their effect on FtsZ expression include environmental conditions and growth hormone treatments. Environmental factors might include changes in the light intensity. photoperiod, temperature. nutritional factors, or water availability. There is already some evidence that light induces FtsZ expression and chloroplast division in peas (Gaikwad et al.. 2000) and cucumber (Ullanat and Jayabaskaran. 2002). Although a variety of growth hormones should be tested, cytokinin has been shown to induce both cell expansion and chloroplast division in cultured spinach leaf disks (Possingham and Smith. 1972). Cytokinin treatment also increases transcript levels of one FtsZZ homologue in cucumber cotyledons, whereas auxin treatment did not increase transcript levels (Ullanat and Jayabaskaran, 2002). Although these studies with pea and cucumber FtsZ genes are important. they only investigated one of several FtsZ genes present in these organisms. Expression studies with the FtsZ genes from Arabidopsis would allow for analysis of the entire FtsZ gene complement of the plant. Throughout these studies different mutants could help determine the regulatory factors that are involved in F tsZ expression. For example, studies with mutants defective in light or cytokinin response could help characterize FtsZ regulation or help elucidate other regulatory factors involved in F tsZ expression. During analysis of the F tsZZ promoter-GUS transgenic plants, it was noticed that the older leaves were not stained. However. F tle and FtsZ2 transcripts and protein were detected in the older leaf tissues. indicating they are expressed. Furthermore. immunofluorescence and fluorescence microscopy experiments detected Ftle and FtsZZ 182 protein rings in all the chloroplasts. including those of the older leaf cells (McAndrew et al.. 2001; Vitha et al., 2001). The transcript and protein measurements suggest that FtsZ expression is reduced in the older leaves, and the GUS staining patterns are consistent with reduced FtsZ expression in these tissues. However. the microscopy data suggest there is still enough protein to maintain FtsZ rings in all the chloroplasts. The presence of rings in tissues where expression is reduced may indicate the FtsZ proteins are relatively stable and are not rapidly turned over, at least compared to the GUS protein. Stability could be inherent in the FtsZ proteins alone or a characteristic of the polymerized FtsZ structure. To test the stability of the FtsZ proteins, plants could be generated where an inducible promoter drives expression of an F IsZ transgene fused to a reporter gene or epitope tag. Treatment of transgenic plants with the inducers for only a short period would stimulate expression of the tagged FtsZ protein for a brief period, similar to a pulse labeling experiment. Following removal of the inducers, the stability of the tagged FtsZ proteins could be followed. Mutated FtsZ proteins unable to polymerize could be expressed similarly to determine whether FtsZ protein stability is affected by polymerization. The regulation of AtFtsZZ-Z expression may be different than the regulation of AtFtsZ1-1 or AtFtsZZ-I expression. Three AtFtsZZ-Z transcripts were isolated, all differentially spliced upstream of the start codon. Only a single RNA species was isolated for each of the AtFtsZ1-1 and AtFtsZZ-l genes. The functionality and stability of the three AtFtsZZ-Z species will need to be determined because their presence does not absolutely indicate they are functional. If there is more than one functional transcript, it is possible that they are expressed in different tissues or at different developmental 183 stages. Tissue- or developmental-specific expression could be tested by RT-PCR, in situ hybridization. or in situ RT-PCR. Although tissue- or developmental-specific expression could mean that AtFtsZ2-2 has a regulatory role in chloroplast division. the total ratio of Ftle to FtsZ2 protein may be the most important factor for chloroplast division and the splicing differences may just reflect the evolutionary history of the F152 genes. Fle and FtsZ2 Sequence Comparisons Identify Potential Functional Differences Phylogenetic analyses indicate that the FtsZ] and FtsZ2 families diverged before the split between the chlorophycean and charophycean green algal lineages. Analysis of the intron positions in the F tsZ genes also supports the early divergence of the two families. The reason chloroplast division requires members of two FtsZ families are unclear. but one likely reason is that the functions of the Ftle and FtsZ2 proteins differ. There are several pieces ofevidence that support this hypothesis. First, antisense repression of either FtsZ] or FtsZ2 genes inhibits chloroplast division indicating the proteins in the two families are not functionally redundant, at least at normal levels. However. it is possible that inhibition of chloroplast division in the antisense lines is due to reduction of the total FtsZ protein concentration below critical levels in the chloroplasts. Second. both families seem to be present in all green algae and higher plants. If the two families were functionally redundant, it would be expected that members of one family would be lost in at least one lineage. Third. differences in the FtsZ1 and FtsZ2 sequences also support specific roles for each family. For example, the C—terminal domain. the bacterial counterpart of which interacts with FtsA and ZipA in E. coli (Ma and Margolin. 1999). is present in the FtsZZ but not the Ftle proteins. This 184 sequence difference sugests FtsZZ proteins may interact with a different set of proteins in the chloroplast division apparatus than Ftle. Mutation and deletion experiments would help to determine the functions of this C-terminal domain. as well as the numerous other sequence differences, in the plant FtsZ proteins and help to define the functional differences between the Ftle and FtsZ2 proteins. Phylogenetic analyses indicate that there are two or more FtsZ2 homologues in Arabidopsis. rice. tobacco, and moss. In contrast, the green alga Clzlamydomonas rein/zardtii only has one FtsZ2 homologue. Why do higher plants and moss have two FtsZZ proteins while the green alga has only one? Is the chloroplast division apparatus different in single-celled green algae and multicellular plants? One interesting and important cellular difference between the green alga C. rein/zardtii and Arabidopsis, rice, tobacco. and moss is that the former has only a single chloroplast in each cell, whereas the latter four organisms have numerous chlorOplasts in each mesophyll cell. It is also interesting that only one of the rice FtsZ2 proteins has an identifiable C-terminal domain. Arabidopsis F tsZZ knockout plant experiments could be performed to investigate whether the green alga FtsZ2 protein. either rice FtsZZ protein, or any of the FtsZZ proteins from the various organisms could functionally complement the Arabidopsis FtsZ2 proteins. For instance, in one experiment the C. rein/zardtii FtsZ2 protein could be expressed in AtFtsZZ-I and AtFtsZZ-Z Arabidopsis knockout plants and in a double knockout plant. If AtFtsZZ—l and AtFts22-2 have different functions and expression of the C. reirzlzardtii protein restores chloroplast division in both FtsZ2 Arabidopsis knockout mutant, this would suggest the two FtsZ2 proteins in plants have divergent functions and the divergence occurred in only the plant lineage. Complementation studies where FtsZ 185 genes from different organisms like the archea bacteria, green algae. red algae, and primitive plants are transformed into Arabidopsis F [52 knockout plants would provide valuable information into the functions of the FtsZ proteins as well as the structure of the FtsZ ring. This information may help in determining the reasons chloroplast division in plants evolved to have multiple FtsZ proteins from two families. One weakness in the phylogenetic analyses was insufficient numbers of FtsZ sequences from diverse organisms. Therefore, additional genomic, RNA, and protein FtsZ sequences from a variety of photosynthetic organisms would benefit studies aimed at understanding functionally important regions of the FtsZ proteins, as well as the evolutionary events that led to the two FtsZ families in plants. Of particular interest are the F tsZ sequences from primitive plants like the homworts and liverworts (Araki et al., 2003), which would provide information into the early evolutionary events of the plant FtsZ genes in land plants. Additional algal sequences are also important, especially from green algae since only the C. reinlzardtii sequences have been reported. Adding more cyanobacterial F rsZ sequences to the analysis may help determine whether there are truly two clades for this group and whether those two clades could represent the sources from which the plant Ftle and FtsZ2 clades evolved. Currently. all known cyanobacteria for which FtsZ sequence information is available have only a single FtsZ gene. However, as the genomes of more cyanobacteria are studied, one organism may be isolated with two different FtsZ genes; this could support the hypothesis that the origins of the FtsZ] and FtsZ2 proteins extends back to the cyanobacterial endosymbiont. 186 APPENDICES 187 APPENDIX A FtsZ Protein Alignment The Nexus file with the FtsZ protein alignment that was used in the phylogenetic analyses that are discussed in Chapter 5 and used to produce the tree shown in Figure l of that chapter. Sequences are labeled with the organism initials followed by the accession or gene number. 188 #NEXUS BEGIN DATA; DIMENSIONS NTAX=40 NCHARI322; FORMAT DATATYPE=PROTEIN SYMBOLS = " l 2 3 4" MISSING=? GAP:- INTERLEAVE ; MATRIX [ IO 20 30 40 ] [ l NtCABB9288 AKIKVVGVGGGGSNAVNRMIESSMKGVEFWIVNTDIQAMRMS--- [42] NtCAC44257 AKIKVVGVGGGGSNAVNRMIESSMKGVEFWIVNTDIQAMRMS--- [42] OSCLBl7724_5 AKIKVVGVGGGGSNAVNRMIESSMNGVEFWIVNTDVQAIRMS--- [42] GlAAF2377l AKIKVVGVGGGGSNAVNRMIESAMKGVEFWIVNTDVQAIKMS--- [42] LlBAA96782 AKIKVIGVGGGGSNAVNRMIASSMDGVEFWIVNTDVQAMRMS--- [42] AtAAC35987 ARIKVIGVGGGGSNAVNRMIESEMSGVEFWIVNTDIQAMRMS-—- [42] ACAAK63846 ARIKVIGVGGGGSNAVNRMIESEMIGVEFWIVNTDIQAMRIS—-- [42] OSCL005296_338 PRIKVIGVGGGGSNAVNRMIESDMKGVEFWIVNTDFQAMRMS——— [42] PpCAB76386 AKIKVIGVGGGGSNAVNRMLESEMQGVEFWIVNTDAQAMALS——- [42] PpCAB54558 AKIKVIGVGGGGSNAVNRMLESEMQGVEFWIVNTDAQAMALS——- [42] CrAAM2289l AIIKVLGVGGGGSNAVNNMVNSDVQGVEFWIANTDAQALATS——— [42] CmBAA85116 CLIKVIGVGGGGGNAVNRMADTGISGVEFWAINTDVQALKRS--- [42] CCBAA82871 CLIKVIGVGGGGGNAVNRMADTGISGVEFWAINTDVQALKRS--- [42] GtCAA07676 CVIKVIGVGGGGGNAVNRMVG-GVEGVEFWSINTDAQALSRS--- [41] GSBAA82090 CIIKVVGVGGGGSNAVNRMCE—MVEGVEFWCINTDAQALSRV-—- [41] MSAAF35433 ------------------------- GVELWVVNTDAQALSRS—-— [l7] GSBAA82091 CKIKVVGVGGAGGNAVQRMLESGLQDVEFLCANTDAQALGRFQEV [45] Tel949 AKIKVIGVGGGGGNAVNRMIASEVSGIEFWTVNTDAQALTLS--- [42] SSNP_440816 AKIKVIGVGGGGCNAVNRMIASGVTGIDFWAINTDSQALTNT-—- [42] ASCAA8324I ANIKVIGVGGGGGNAVNRMIESDVSGVEFWSINTDAQALTLA—-- [42] Np6l ANIKVIGVGGGGGNAVNRMIESDVSGVEFWSINTDAQALTLA-—- [42] Tetll2382 ARIKVIGVGGGGGNAVNRMIASNVAGVEFWCVNTDAQAIAQS-—— [42] SyAAC26227 ARIKVIGVGGGGSNGVNRMISSDVSGVEFWALNTDAQALLHS--- [42] PSCABS6201 AKIEVIGVGGGGSNAVNRMIDSDLEGVSFRVLNTDAQALLQS--- [42] Pml658 AKIEVIGVGGGGSNAVNRMIDSDLEGVSFRVLNTDAQALLQS-—— [42] PmCAB95028 ARIEVIGVGGGGSNAVNRMILSDLQGVSYRVLNTDAQALLQS--- [42] Sy549 AKIEVIGVGGGGSNAVNRMILSDLEGVAYRVLNTDAQALIQS--— [42] Pm1268 ARIEVIGVGGGGSNAVNRMILSDLDGVNYRVMNTDAQALLQS--- [42] NtCAB4l987 AKIKVIGVGGGGNNAVNRMIGSGLQGVDFYAINTDAQALLQS——— [42] NCCAB89287 AKIKVIGVGGGGNNAVNRMIGSGLQGVDFYAINTDAQALLQS——— [42] TeAAF81220 AKIKVVGVGGGGNNAVNRMIGSGLQGVDFYAINTDSQALLQS——- [42] AtAAA82068 ARIKVIGVGGGGNNAVNRMISSGLQSVDFYAINTDSQALLQF—-— [42] PSCAA75603 AKIKVVGIGGGGNNAVNRMIGSGLQGVDFYAINTDAQALLHS—-- [42] NCAAF23770 AKIKVVGVGGGGNNAVNRMIGSGLQGVDFYAVNTDAQALLQS-—— [42] NCCAB89286 AKIKVVGVGGGGNNAVNRMIGSGLQGVDFYAVNTDAQALLQS—-- [42] OSAAK64282 ARIKVVGVGGGGNNAVNRMIGSGLQGIEFYAINTDSQALLNS--- [42] CrBAB91150 ACIKVIGVGGGGGNALNRMINSGLQGVEFWAINTDAQALAAH--- [42] CpAAC32266 AQIKVIGVGGGGNNAVDRMIEDGLDGVDFISINTDGQALSKA—-— [42] ECP06138 AVIKVIGVGGGGGNAVEHMVRERIEGVEFFAVNTDAQALRKT--- [42] BSAAA22457 ASIKVIGVGGGGNNAVNRMIENEVQGVEYIAVNTDAQALNLS-—— [42] 189 NtCABB9288 NCCAC44257 OsCLBl7724_5 GIAAF23771 LlBAA96782 AtAAC3S987 AtAAK63846 OSCL005296_338 PpCAB76386 PpCABS4SS8 CrAAM2289l CmBAA85116 CCBAA82871 GtCAAO7676 GSBAA8209O MSAAF35433 GsBAA8209l Tel949 SSNP_440816 ASCAA83241 Np6l Tet112382 SyAAC26227 PsCABS6201 Pml658 PmCAB95028 Sy549 Pm1268 NCCAB41987 NtCAB89287 TeAAF81220 ACAAA82068 PSCAA7S603 NCAAF2377O NtCAB89286 OSAAK64282 CrBAB9llSO CpAAC32266 ECPO6138 BSAAA22457 50 6O 7O 80 90] --PVAAEQRLPIGQELTRGLGAGGNPDIGMNAANESKQAIEEAVY --PVAAEQRLPIGQELTRGLGAGGNPDIGMNAANESKQAIEEAVY --PVLPQNRLQIGOELTRGLGAGGNPDIGMNAAKESVESIQEALY --PVYLENRLQIGQELTRGLGAGGNPDIGMNAAKESKEAIEEAVY -—PVYPENRLQIGQELTRGLGAGGNPDIGMNAAKESKVSIEESVS —-PVLPDNRLQIGKELTRGLGAGGNPEIGMNAARESKEVIEEALY -—PVFPDNRLQIGKELTRGLGAGGNPEIGMNAATESKEAIQEALY ——PIDPDNKLQIGQELTRGLGAGGNPEIGMNAAKESQELVEQAVS -—PVPAQNRLQIGQKLTRGLGAGGNPEIGCSAAEESKAMVEEALR --PVPAQNRLQIGQKLTRGLGAGGNPEIGCSAAEESKAMVEEALR --PVNGKCKVQIGGKLTRGLGAGGNPEIGAKAAEESRDSIAAALQ ——-—AAHHTLSIGNKLTRGLGAGGNPEVGRKAAEESCDQIAEAVR —---AAHHTLGIGNKLTRGLGAGGNPEIGRKAAEESCDQIAEAVR -—-—LAPNTCNIGAKLTRGLGAGGNPEIGRKAAEESRDLIAEAVS —-——KTSNSVTIGSEITRGLGAGGKPEVGRQAAEESQAAISSAVQ ~——-SAKRRLNIGKVLSRGLGAGGNPAIGAKAAEESREEIMAVVK YCQKTHHQVIQIGKQSCRGLGAGGNPEAGRVAAEESKEDIAKALQ -——-RAPKRLQLGQKLTRGLGAGGNPAIGQKAAEESRDEIANALD ———-NAPDCIQIGQKLTRGLGAGGNPAIGQKAAEESRDEIARSLE ———-GAPSRLQIGQKLTRGLGAGGNPAIGQKAAEESRDEIATALE ----GAPSRLQIGQKLTRGLGAGGNPAIGQKAAEESRDEIATALE ———-QAHRCLQIGQKLTRGLGAGGNPAIGQKAAEESREDLAAALK --—-AAPKRMQLGQKLTRGLGAGGNPAIGMKAAEESREELIAALE ———-SADRRVQLGQNLTRGLGAGGNPSIGQKAAEESKDELQQTLE ———-SADRRVQLGQNLTRGLGAGGNPSIGQKAAEESKDELQQTLE ——-—SAENRVQLGQTLTRGLGAGGNPSIGEKAAEESRAELQQALE ——-—QAQHRLQLGQTLTRGLGAGGNPTIGQKAAEESRTDLHDALQ ---—AASNRVQLGQTLTRGLGAGGNPSIGQKAAEESRAELQQALQ -—-—AAENPLQIGELLTRGLGTGGNPLLGEQAAEESKEAIANSLK ————AAENPLQIGELLTRGLGTGGNPLLGEQAAEESKEAIANSLK --—-VAHNPIQIGELLTRGLGTGGNPLLGEQAAEESKEAIGNALK —~——SAENPLQIGELLTRGLGTGGNPLLGEQAAEESKDAIANALK ----AAENPIKIGELLTRGLGTGGNPLLGEQAAEESKEAIANALK -———TVENPIQIGELLTRGLGTGGNPLLGEQAAEESKEHIANALK —--—TVENPIQIGELLTRGLGTGGNPLLGEQAAEESKEHIANALK ——--QAQYPLQIGEQLTRGLGTGGNPNLGEQAAEESKEAIANALK —-~—QALNKVQIGSELTRGLGCGGNPELGRRAAMESEEALRRMVQ —---RSSTKTQIGEKLTKGLGAGGNPEIGEKSVDETQDEIAQALH ----AVGQTIQIGSGITKGLGAGANPEVGRNAADEDRDALRAALE -——-KAEVKMQIGAKLTRGLGAGANPEVGKKAAEESKEQIEEALK 190 [85] [85] [85] [85] [85] [85] [85] [85] [85] [851 [85] [83] [83] [821 [82] [58] [90] [83] [83] [83] [831 [83] [83] [83] 83] 83] 83] 83] [83] 183] [83] [83] [83] [83] [83] [831 [83] [83] [83] [83] r—wr—wr—Ir—u NtCAB89288 NtCAC44257 OsCLBl7724_5 GlAAF2377l LlBAA96782 ACAAC3S987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM2289l CmBAA85116 CCBAA82871 GCCAAO7676 GsBAA8209O MsAAF3S433 GSBAA82091 Te1949 SSNP_440816 AsCAA8324l Np6l Tet112382 SyAAC26227 PSCABS6201 Pml658 PmCAB95028 Sy549 Pm1268 NtCAB4l987 NtCAB89287 TeAAF81220 AtAAA82068 PSCAA75603 NtAAF2377O NtCAB89286 OSAAK64282 CrBAB9llSO CpAAC32266 ECP06138 BSAAA22457 100 110 120 130 GADMVFVTAGMGGGTGTGAAPIIAGTAKSMGILTVGIVTTPFSFE GADMVFVTAGMGGGTGTGAAPIIAGTAKSMGILTVGIVTTPFSFE GADMVFVTAGMGGGTGTGGAPVIAGIAKSMGILTVGIVTTPFSFE GADMVFVTAGMGGGTGTGGAPVIAGIAKSMGILTVGIVTTPFSFE GADMVFVTAGMGGGTGTGGAPVIAGVAKSMGILTVGIVTTPFMFE GSDMVFVTAGMGGGTGTGAAPVIAGIAKAMGILTVGIATTPFSFE GSDMVFVTAGMGGGTGTGGAPIIAGVAKAMGILTVGIVTTPFSFE GADMIFVTAGMGGGTGTGGAPVIAGIAKSMGILTVGIVTTPFAFE GADMVFVTAGMGGGTGSGAAPIIAGVAKQLGILTVGIVTTPFAFE GADMVFVTAGMGGGTGSGAAPIIAGVAKQLGILTVGIVTTPFAFE DTDMVFVTAGMGGGTGSGAAPVVAEVARELGILTVGIVTTPFTFE GADLVFVTAGMGGGTGSGAAPVVAEAAREQGCLTVGVVTKPFAFE GADLVFVTAGMGGGTGSGAAPVVAEAAREQGCLTVGVVTKPFAFE AGDLVFVTAGMGGGTGSGAAPIVAEVAKEMGCLTVGVVTKPFAFE GGDLVFVTAGMGGGTGSGAAPIVAKIAKEQGCLTVGVVTKPFSFE NADLVFVTAGMGGGTGSGAAPVVAECAKEAGALTVGVVTKPFGFE GGDLVFVTAGMGGGTGTGAAPIVADVARELGCLTVGVVTKPFAFE HPDLVFITAGMGGGTGTGAAPVIAEIAKEAGSLTVGVVTRPFTFE GTDLVFITAGMGGGTGTGAAPIVAEVAKEMGCLTVGIVTRPFTFE GADLVFITAGMGGGTGTGAAPIVAEVAKEMGALTVGVVTRPFVFE GADLVFITAGMGGGTGTGAAPIVAEVAKEMGALTVGVVTRPFVFE DADLIFITCGMGGGTGTGAAPIVAEVAKEQGALTVAVVTRPFTFE GADLVFITAGMGGGTGTGAAPIVAEVAKEVGALTVGIVTKPFTFE GSDLVFIAAGMGGGTGTGAAPVVAEVAKQSGALTVGIVTKPFSFE GSDLVFIAAGMGGGTGTGAAPVVAEVAKQSGALTVGIVTKPFSFE GADLVFIAAGMGGGTGTGAAPVVAEVAKQSGALTVAIVTKPFSFE GSDLVFIAAGMGGGTGTGAAPVVAEVAREVGALTVGIVTKPFGFE GVDLVFIAVGMGGGTGTGAAPVVAEVAKESGALTVGIVTKPFSFE GSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTYPFSFE GSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTYPFSFE GSDLVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTYPFSFE GSDLVFITAGMGGGTGSGAAPVVAQISKDAGYLTVGVVTYPFSFE GSDLVFITAGMGGGTGSGAAPVVAQISKEAGYLTVGVVTYPFSFE GSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTYPFSFE GSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTYPFSFE DSDLVFITAGMGGGTGSGAAPVVAQISKEAGYLTVGVVTYPFSFE GADLVFITAGMGGGTGTGAAPVVARLSKELGILTVGVVTYPFNFE GSDMVFITAGMGGGTGTGAAPRIAAISKELGILTVGVVTKPFNFE GADMVFIAAGMGGGTGTGAAPVVAEVAKDLGILTVAVVTKPFNFE GADMVFVTAGMGGGTGTGAAPVIAQIAKDLGALTVGVVTRPFTFE 191 [130] [130] [130] [1301 [130] [130] [130] [130] [130] [130] [130] [128] [128] [127] [127] [103] [135] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] [128] NCCABB9288 NCCAC44257 OSCLB17724_5 GlAAF2377l LlBAA96782 ACAAC35987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM2289l CmBAA8Sll6 CcBAA8287l GtCAAO7676 GsBAA8209O MSAAF35433 GSBAA82091 Te1949 SSNP_440816 ASCAA83241 Np6l Tetll2382 SyAAC26227 PsCABS6201 Pml658 PmCAB95028 Sy549 Pm1268 NCCAB41987 NCCAB89287 TeAAF81220 AtAAA82068 PsCAA75603 NtAAF2377O NtCAB89286 OSAAK64282 CrBAB9115O CpAAC32266 ECPO6138 BSAAA22457 140 ISO 160 170 180] -l GRRRAVQ--AQEGIAALRENVDTLIVIPNDKLLTAVSPSTPVTEA GRRRAVQ--AQEGIAALRENVDTLIVIPNDKLLTAVSPSTPVTEA GRRRAVQ--AQEGIAALRNSVDTLIVIPNDKLLSAVSPNTPVTEA GRRRAVQ—-AQEGIAALRDNVDTLIVIPNDKLLTAVSPSTPVTEA GRRRTVQ--AQEGIAALRNNVDTLIVIPNDKLLTAVSPNTPVTEA GRRRTVQ--AQEGLASLRDNVDTLIVIPNDKLLTAVSQSTPVTEA GRRRALQ——AQEGIAALRDNVDTLIVIPNDKLLAAVSQSTPVTEA GRRRALQ—-AQEGIASLRSNVDTLIVIPNDKLLTAVSPNTPVTEA GRRRSVQ-—AHEGIAALKNNVDTLITIPNNKLLTAVAQSTPVTEA GRRRAVQ--AHEGIAALKNNVDTLITIPNNKLLTAVAQSTPVTEA GRQRAQQ--ARSALANLRAAVDTLIVIPNDRLLSAMDSNVPIKDA GRKRMNQ--ALEAIEALRESVDTLIVVSNDKLLQIVPENTPLQDA GRRRMTQ——ALEAIEALRESVDTLIVVSNDKLLQIVPENTPLQDA GKRRMQQ-—ANDAILNLRNKVDTLIVVSNDKLLQIVPDNTPLQDA GRRRMQQ-~AEEAIEALRKEVDTLIVVSNDKLLEIVPENTALEKA GRKRMQQ—-ARNAILEMKDKVDTLIVVSNDKLLKIVPDNTPLTEA GRRRLQQ-—AVEGLANLREKVDTLIVISNDRLLETVPKDTPLTEA GRRRITQ——ADEGITALQTRVDTLIVIPNNRLLSVINDQTPVQEA GRRRAKQ-—AEEGINALQSRVDTLIVIPNNQLLSVIPAETPLQEA GRRRTSQ—-AEQGIEGLKSRVDTLIIIPNNKLLEVIPEQTPVQEA GRRRTSQ——AEQGIEGLKSRVDTLIIIPNNKLLEVIPEQTPVQEA GRRRANQ--ADEGIEALQSRVDTLIVIPNDKILSVISEQTSVQDA GRRRMKQ--AEEGTAALQSSVDTLITIPNDRLLHAISEQTPIQEA GKRRMRQ——AEEGIARLAENVDTLIVIPNDRLKDVIAG—APLQEA GKRRMRQ—-AEEGIARLAENVDTLIVIPNDRLKDVIAG-APLQEA GRRRMRQ——ADEGIAKLTESVDTLIVIPNDRLKDAIAG-APLQEA GRRRMRQ-~ADEGIARLAEHVDTLIVIPNDRLREAIAG-APLQEA GRRRMRQ——AAEGIGRLADHVDTLIVIPNDRIKDVISE-APLQEA GRKRSVQ—-ALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPLQDA GRKRSVQ—-ALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPLQDA GRKRSVQ——ALEAIEKLQKNVDTLIVIPNDRLLDIADENTPLQDA GRKRSLQ-—ALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPLQDA GRKRSLQ—-ALEAIEKLQKNVDTLIVIPNDRLLDIADEQMPLQDA GRKRSLQ--ALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPLQNA GRKRSLQ--ALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPLQNA GRKRSLQASALEALEKLERSVDTLIVIPNDRLLDVVDENTPLQDA GRRRAGQ-—ALEGIEALREAVDSVIVIPNDRLLDVAGASTALQDA GKKRMSN——AEKGIMELKKNVDTLVIIPNQRLLSIIDKKTTLTEA GKKRMAF--AEQGITELSKHVDSLITIPNDKLLKVLGRGISLLDA GRKRQLQ--AAGGISAMKEAVDTLIVIPNDRILEIVDKNTPMLEA 192 in" NtCABB9288 NCCAC44257 OSCLB17724_5 GlAAF23771 LlBAA96782 ACAAC35987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM2289l CmBAA85116 CCBAA82871 GCCAAO7676 GSBAA8209O MSAAF35433 GsBAA8209l Te1949 SSNP_440816 ASCAA83241 Np6l Tet112382 SyAAC26227 PsCABS620l Pml658 PmCAB95028 Sy549 Pm1268 NtCAB4l987 NtCAB89287 TeAAF8l220 AtAAA82068 PsCAA7S603 NCAAF2377O NtCABB9286 OSAAK64282 CrBAB9llSO CpAAC32266 ECPO6138 BSAAA224S7 190 200 210 220 FNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSSLMG FNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSSLMG FNLADDILRQGIRGISDIITVPGLVNVDFADVRAIMQNAGSSLMG FNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSSLMG FNLADDILRQGVRGISDIITVPGLVNVDFADVRAIMANAGSSLMG FNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSSLMG FNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSSLMG FNLADDILRQGVRGISDIITVPGLVNVDFADVRSVMSDAGSSLMG FNLADDILRQGVRGISDIITVPGLVNVDFADVRAIMANAGSSLMG FNLADDILRQGVRGISDIITVPGLVNVDFADVRAIMANAGSSLMG FKIADDVLRQGVKGISEIITVPGLVNVDFADVRAIMAGAGSSLMG FRVADDILRQGVVGISDIIIRPGLINVDFADVRSVMAHAGSALMG FRVADDILRQGVVGISDIIIRPGLINVDFADVRSVMAHAGSALMG FSVADDILRQGVVGISEIIVRPGLINVDFADVRSVMADAGSALMG FSVADDILRQGVVGISEIIVRPGLINVDFADVRSIMADAGSALMG FLVADDILRQGVVGITEIIVKPGLVNVDFADVRTIMGNAGTALMG FIFADEVLRQGVGGISDIITKPGLVNVDFADVRTVMAEKGFALLG FIIADDILRQGIQGISDIITVPGLVNVDFADVRAVMADAGSALMG FRVADDILRQGVQGISDIIIIPGLVNVDFADVRAVMADAGSALMG FRYADDVLRQGVQGISDIITIPGLVNVDFADVRAVMADAGSALMG FRYADDVLRQGVQGISDIITIPGLVNVDFADVRAVMADAGSALMG FRVADDVLRQGVQGISDIINVPGLINVDFADIRSVMADAGSAMMG FRVADDILRQGVQGISDIITIPGLVNVDFADVRAVMADAGSALMG FRNADDVLRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTALLG FRNADDVLRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTALLG FKNADDVLRMGVKGITDIITLPGLVNVDFADVRSVMTEAGTSLLG FRSADDVLRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTALLG FRSADDILRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTALLG FLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTAMLG FLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTAMLG FLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTAMLG FLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTAMLG FRLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTAMLG FLLADDVLCQGVQGISDIITIPGLVNVDFADVKAIMKDSGTAMLG FLLADDVLCQGVQGISDIITIPGLVNVDFADVKAIMKDSGTAMLG FLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKNSGTAMLG FALADDVLRQGVQGISDIITVPGLINVDFADVKAIMSNSGTAMLG FKKADEILRQGVQGIADLISKPGVINLDFADVRTVMANKGIAHMG FGAANDVLKGAVQGIAELITRPGLMNVDFADVRTVMSEMGYAMMG FREADNVLRQGVQGISDLIATPGLINLDFADVKTIMSNKGSALMG 193 [218] [218] [218] [218] [218] [218] [218] [218] [218] [218] [218] [216] [216] [215] [215] [191] [223] [216] [216] [216] [216] [216] [216] [215] [215] [215] [215] [215] [216] [216] [216] [216] [216] [216] [216] [218] [216] [216] [216] [216] NCCABB9288 NCCAC44257 OSCLBI7724_5 GIAAF23771 LIBAA96782 ACAAC35987 AtAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM2289l CmBAA85116 CCBAA82871 GCCAAO7676 GSBAA82O9O MSAAF35433 GSBAA82091 Tel949 SSNP_440816 AsCAA8324l Np6l Tet112382 SyAAC26227 PSCABS6201 Pml658 PmCAB95028 Sy549 Pm1268 NtCAB4l987 NCCABB9287 TeAAF81220 AtAAA82068 PSCAA75603 NtAAF2377O NtCAB89286 OSAAK64282 CrBAB91150 CpAAC32266 ECPO6138 BSAAA22457 230 240 250 260 270] - ] IGTAT ------- GKTRARDAALNAIQSPLLDIG—IERATGIVWNI IGTAT ------- GKTRARDAALNAIQSPLLDIG—IERATGIVWNI IGTAT ------- GKSRARDAALNAIQSPLLDIG-IERATGIVWNI IGTAT ------- GKTRARDAALNAIQSPLLDIG—IERATGIVWNI IGTAT ------- GKTRARDAALNAVQSPLLDIG-IERATGIVWNI IGTAT ------- GKSRARDAALNAIQSPLLDIG—IERATGIVWNI IGTAT ------- GKTRARDAALNAIQSPLLDIG—IERATGIVWNI IGTAT ------- GKTRARDAALNAIQSPLLDIG~IERATGIVWNI IGTAT ------- GKSKAREAALSAIQSPLLDVG—IERATGIVWNI IGTAT ------- GKSRAREAALSAIQSPLLDVG-IERATGIVWNI QGYGS ——————— GPRRASDAALRAISSPLLEVG—IERATGVVWNI IGTGS ------- GKSRAHDAAVAAISSPLLDFP-IERAKGIVFNV IGTGS ------- GKSRAHDAAVAAISSPLLDFP-IERAKGIVFNV IGTGS ------- GKTRAQDAAVAAISSPLLDFP~IEKARGIVFNI IGSGS ------- GKSRAKDAAVAAISSPLLDFP-IERAKGIVFNI IGHGK ——————— GKNRAKDAALSAISSPLLDFP-ITRAKGIVFNI IGTAS ------- GDSRARNAATAAISSPLLDFP-ITSAKGAVFNI IGMGS ------- GKSRAREAANAAISSPLLESS-IEGAKGVVFNI IGVGS ------- GKSRAKEAATAAISSPLLESS-IQGAKGVVFNV IGVSS ------- GKSRAREAAIAAISSPLLECS-IEGARGVVFNI IGVSS ——————— GKSRAREAAIAAISSPLLECS-IEGARGVVFNI IGIAS ------- GKSRATEAALSAISSPLLERS-IEGAKGVVFNI IGSGS ------- GKSRAREAAHAAISSPLLESS-IEGARGVVFNI IGIGS ------- GRSRALEAAQAAMNSPLLEAARIDGAKGCVINI IGIGS ——————— GRSRALEAAQAAMNSPLLEAARIDGAKGCVINI IGIGS ——————— GRSRAAEAAQAAINSPLLEAGRIDGAKGCVVNI IGIGS ------- GRSRAVEAAQAAISSPLLETERIDGAKGCVINI IGEGS ------- GRSRAIEAAQAAISSPLLEAARIDGAKGCVINI VGVSS ------- SKNRAEEAAEQATLAPLIGSS-IQSATGVVYNI VGVSS ------- SKNRAEEAAEQATLAPLIGSS-IQSATGVVYNI VGVSS ------- SKNRAEEAAEQATLAPLIGSS~IQSATGVVYNI VGVSS ——————— SKNRAEEAAEQATLAPLIGSS-IQSATGVVYNI VGVSS ------- GKNRAEEAAEQATLAPLIGSS-IQSATGVVYNI VGVSS ------- SRNRAEEAAEQATLAPLIGSS-IQSATGDVYNI VGVSS ------- SRNRAEEAAEQATLAPLIGLS-IQSATGVVYNI VGVSS ------- SKNRAQEAAEQATLAPLIGSS-IEAATGVVYNI VGAASTATAAPGGPDRAEQAAVAATSAPLIQRS-IEKATGIVYNI IGRAS ------- GENKAEIAAKMAIQSPLLETT-IEGAKSVLINF SGVAS ------- GEDRAEEAAEMAISSPLLEDIDLSGARGVLVNI IGIAT ------- GENRAAEAAKKAISSPLLEAA-IDGAQGVLMNI 194 [255] [255] [255] [255] [255] [255] [255] [255] [255] [255] [255] [253] [253] [252] [252] [228] [260] [2531 [253] [2531 [253] [253] [253] 253] 253] 253] [253] [2531 [253] [253] [253] [253] [253] [253] [253] [255] [260] [253] [254] [2531 F .wr NtCABB9288 NCCAC44257 OSCLBI7724_5 GlAAF23771 LlBAA96782 AtAAC35987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM2289l CmBAA85116 CCBAA82871 GCCAAO7676 GsBAA8209O MSAAF35433 GSBAA82091 Te1949 SSNP_440816 ASCAA83241 Np61 Tet112382 SyAAC26227 PSCABSGZOI Pml658 PmCAB95028 Sy549 Pm1268 NtCAB4l987 NtCAB89287 TeAAF81220 AtAAA82068 PSCAA75603 NCAAF2377O NtCA889286 OSAAK64282 CrBAB91150 CpAAC32266 ECPO6138 BSAAA22457 280 290 300 310 TGGSDLTLFEVNAAAEVIYDLVDPSANLIFGAVIDPSIS-GQVSI TGGSDLTLFEVNAAAEVIYDLVDPSANLIFGAVIDPSIS-GQVSI TGGADMTLFEVNSAAEIIYDLVDPNANLIFGAVIDPSLN-GQVSI TGGSDLTLFEVNAAAEVIYDLVDPSANLIFGAVVDPSLC-GQVSI TGGNDLTLYEVNAAAEVIYDLVDPAANLIFGAVIDPSIS-GQVSI TGGSDLTLFEVNAAAEVIYDLVDPTANLIFGAVVDPALS-GQVSI TGGSDLTLFEVNAAAEVIYDLVDPTANLIFGAVVDPSYS—GQISI TGGNDLTLTEVNAAAEVIYDLVDPGANLIFGSVIDPSYT—GQVSI TGGSDMTLFEVNAAAEVIYDLVDPNANLIFGAVVDEALH-DQISI TGGSDMTLFEVNAAAEVIYDLVDPNANLIFGAVVDEALH-GQVSI TGPPNMTLHEVNEAAEIIYDMVDPNANLIFGAVVDSTLPDDTVSI TGGEDMTLHEINQAAEVIYEAVDPNANIIFGALIDQQME-SEISI TGGEDMTLHEINQAAEVIYEAVDPNANIIFGALVDQQME-SEISI TGGQDMTLHEINSAAEVIYEAVDSNANIIFGALVDDNME—NEISI TGGHDMTLHEINAAAEVIYEAVDLNANIIFGALVDDSME-NELSI VGGSDMSLQEINAAAEVIYENVDQDANIIFGAMVDDKMTSGEVSI TGGTDMTLSEVNQAAQVIYDSVDSDANIIFGAVVDETFK-GKVSV TGGTDLTLHEVNAAAEIIYEVVDPNANIIFGAVIDDKLQ—GEIKI TGGTDLTLHEVNVAAEIIYEVVDADANIIFGAVIDDRLQ-GEMRI TGGSDLTLHEVNAAAETIYEVVDPNANIIFGAVIDDRLQ-GEVRI TGGTDLTLHEVNAAAEAIYEVVDPNANIIFGAVIDDRLQ—GEVRI TGGTDLSLHEVNAAADVIYNVADANANIIFGAVIDPQMQ-GEVQI TGGRDMTLHEVNAAADAIYEVVDPEANIIFGAVIDDRLE-GELRI TGGKDMTLEDMTSASEIIYDVVDPEANIIVGAVIDESME-GEIQV TGGKDMTLEDMTSASEIIYDVVDPEANIIVGAVIDESME—GEIQV TGGKDMTLEDMTSASEVIYDVVDPEANIIVGAVIDEALE—GEVQV SGGRDMTLEDMTTASEVIYDVVDPEANIIVGAVVDEALE-GEIHV SGGRDMTLEDMTSASEVIYDVVDPEANIIVGAVVDEKLE-GEVHV TGGKDITLQEVNRVSQVVTSLADPSANIIFGAVVDERYN—GEIHV TGGKDITLQEVNRVSQVVTSLADPSANIIFGAVVDERYN-GEIHV TGGKDITLQEVNRVSQVVTSLADPSANIIFGAVVDERYN—GEIHV TGGKDITLQEVNRVSQVVTSLADPSANIIFGAVVDDRYT-GEIHV TGGKDITLQEVNRVSQVVTSLADPSANIIFGAVVDDRYT—GEIHV TGGKDITLQEVNKVSQVVTSLADPSANIIFGAVVDERYN-GEIQV TGGKDITLQEVNKVSQVVTSLADPSANIIFGAVVDERYN-GEIQV TGGKDITLQEVNKVSQIVTSLADPSANIIFGAVVDDRYT-GEIHV TGGRDLTLAEVNRVSEVVTALADPSCNIIFGAVVDEQYD-GELHV SGDMNLGLMETEEAADLIREAIDPDAEIIFGTTINEDLN—NEVVV TAGFDLRLDEFETVGNTIRAFASDNATVVIGTSLDPDMN-DELRV TGGTNLSLYEVQEAADIVASASDQDVNMIFGSVINENLK-DEIVV 195 [299] [299] [299] [299] [299] [299] [2991 [299] [2991 [2991 [300] [297] [297] [296] [296] [273] [304] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [297] [299] [304] [297] [298] [297] l 320] [ . l NtCABB9288 TLIATGF [306] NtCAC44257 TLIATGF [306] OSCLBI7724_5 TLIATGF [306] G1AAF23771 TLIATGF [306] L1BAA96782 TLIATGF [306] ACAAC35987 TLIATGF [306] ACAAK63846 TLIATGF [306] OSCL005296_338 TLIATGF [306] PpCAB76386 TLIATGF [306] PpCABS4558 TLIATGF [306] CrAAM2289l TIIATGF [307] CmBAA85ll6 TVVATGF [304] CCBAA82871 TVVATGF [304] GtCAA07676 TVVATGF [303] GSBAA8209O TVIATGF [303] MSAAF35433 TVLATGF [280] GSBAA82091 TVVATGF [311] Te1949 TVIATGF [304] SSNP_440816 TVIATGF [304] ASCAA83241 TVIATGF [304] Np61 TVIATGF [304] Tet112382 TVIATGF [304] SyAAC26227 TVIATGF [304] PSCAB56201 TVIATGF [304] Pml658 TVIATGF [304] PmCAB95028 TVIATGF [304] Sy549 TVIATGF [304] Pm1268 TVIATGF [304] NCCAB41987 TIIATGF [304] NCCAB89287 TIIATGF [304] TeAAF81220 TIVATGF [304] AtAAA82068 TIIATGF [304] PSCAA75603 TIIATGF [304] NCAAF23770 TLIATGF [304] NtCABB9286 TLIATGF [304] OSAAK64282 TIIATGF [306] CrBAB91150 TIIATGF [311] CpAAC32266 TVIATGL [304] ECP06138 TVVATGI [305] BSAAA22457 TVIATGF [304] END; 196 APPENDIX B F tsZ cDNA Alignment The Nexus file with the FtsZ cDNA alignment that was used in the phylogenetic analyses that are discussed in Chapter 5 and used to produce the tree shown in Figure 2 of that chapter. Sequences are labeled with the organism initials followed by the accession or gene number. 197 #NEXUS BEGIN DATA; DIMENSIONS NTAX=40 NCHAR=966; FORMAT DATATYPE=DNA MISSING=? GAP=— INTERLEAVE ; MATRIX [ 10 20 30 40 [ LIABO42101 GCGAAGATCAAGGTTATAGGTGTTGGTGGCGGGGGGTCCAATGCT [45] OSCL005296_338 CCGAGGATCAAAGTCATTGGCGTTGGAGGTGGTGGATCCAATGCT [45] OSCLBl7724_5 GCCAAGATCAAGGTCGTCGGGGTCGGCGGAGGAGGCTCCAATGCC [45] NtAJ271750 GCGAAAATCAAGGTGGTTGGTGTAGGAGGTGGCGGATCGAATGCA [45] NtAJ311847 GCGAAGATCAAGGTGGTTGGTGTAGGAGGTGGCGGATCGAATGCA [45] GIAF205859 GCAAAGATCAAGGTAGTGGGCGTTGGAGGGGGTGGCTCGAATGCA [45] AtAF089738 GCGAGGATTAAGGTTATTGGTGTGGGAGGTGGTGGATCAAATGCT [45] AtAF384167 GCTAGGATTAAAGTTATCGGCGTTGGAGGTGGTGGTTCAAACGCT [45] PpAJ249139 GCGAAAATTAAAGTGATAGGCGTGGGGGGCGGGGGTTCCAACGCC [45] PpAJ249138 GCGAAAATTAAAGTAATAGGGGTCGGAGGTGGGGGTTCCAACGCC [45] CrAF449446 GCCATCATTAAGGTTCTGGGCGTTGGCGGTGGTGGCTCCAATGCC [45] CmABO32072 TGTCTGATTAAAGTTATCGGCGTCGGTGGTGGTGGCGGCAACGCA [45] CCA8023962 TGTTTGATCAAGGTGATTGGTGTCGGCGGCGGCGGTGGCAACGCC [45] GCAJ007748 TGTGTTATTAAAGTTATTGGTGTTGGAGGTGGTGGTGGAAATGCA [45] GSA8022594 TGCATTATTAAAGTTGTTGGAGTCGGAGGAGGTGGAAGTAACGCA [45] MSAF120117 --------------------------------------------- [0] GSA8022595 TGCAAGATAAAGGTAGTTGGGGTAGGAGGAGCAGGAGGGAATGCA [45] PSAJ011025 GCCAAAATTGAAGTAATTGGTGTCGGGGGTGGTGGGAGTAATGCT [45] Pml658 GCCAAAATTGAAGTAATTGGTGTCGGGGGTGGTGGGAGTAATGCT [45] PmAJ237851 GCCCGCATTGAAGTAATTGGCGTAGGTGGTGGTGGCAGCAATGCT [45] Sy549 GCCAAGATTGAGGTCATCGGGGTTGGGGGTGGCGGCAGCAATGCC [45] Ple68 GCTCGCATTGAAGTAATTGGCGTCGGCGGAGGCGGCAGCAATGCC [45] SyAFO76530 GCCCGCATCAAAGTAATTGGCGTTGGCGGTGGCGGCAGCAACGGG [45] ASZ31371 GCCAATATCAAAGTAATTGGTGTTGGCGGTGGTGGTGGTAATGCT [45] Np6l GCCAACATCAAAGTGATTGGTGTCGGTGGCGGCGGTGGTAATGCC [45] Tet112382 GCACGCATCAAAGTGATTGGTGTCGGTGGTGGCGGTGGCAATGCA [45] Te1949 GCAAAAATTAAAGTAATTGGCGTAGGAGGAGGTGGCGGCAATGCT [45] SSNC_000911 GCCAAAATTAAAGTGATCGGCGTTGGGGGAGGCGGTTGCAATGCT [45] NtAJ133453 GCTAAGATTAAGGTTATCGGCGTCGGTGGCGGTGGTAACAATGCC [45] NtAJ27l749 GCTAAGATTAAGGTTATCGGCGTCGGTGGCGGTGGTAACAATGCC [45] TeAF251346 GCAAAAATCAAAGTCGTTGGCGTCGGTGGTGGTGGCAACAATGCC [45] AtU39877 GCGAGAATTAAGGTGATTGGTGTCGGTGGTGGTGGTAACAATGCC [45] PSY15383 GCTAAGATTAAGGTCGTTGGAATTGGGGGTGGTGGTAACAATGCC [45] NtAF205858 GCCAAGATTAAGGTTGTCGGCGTCGGTGGCGGCGGCAACAATGCT [45] NtAJ271748 GCCAAGATTAAGGTTGTCGGCGTCGGTGGCGGCGGCAACAATGCT [45] OSAF383876 GCGAGGATAAAGGTCGTGGGCGTCGGCGGCGGCGGGAACAACGCT [45] CrAB084236 GCCTGCATCAAGGTTATCGGGGTCGGCGGTGGTGGCGGCAATGCC [45] CpAF067823 GCGCAGATAAAAGTAATCGGCGTTGGTGGCGGCGGCAACAACGCT [45] BSM22630 GCATCAATTAAAGTAATCGGAGTAGGAGGCGGCGGTAACAACGCC [45] ECX55034 GCGGTGATTAAAGTCATCGGCGTCGGCGGCGGCGGCGGTAATGCT [45] 198 L1ABO42101 OSCL005296_338 OSCLBI7724_5 NtAJ271750 NtAJ3ll847 G1AF205859 ACAF089738 AtAF384167 PpAJ249l39 PpAJ249138 CrAF449446 CmABO32072 CCA8023962 GCAJOO7748 GSABOZ2594 MSAF120117 GSABOZ2595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ3lB7l Np6l Tet112382 Te1949 SSNC_000911 NCAJ133453 NtAJ27l749 TeAF251346 AtU39877 PsY15383 NtAF205858 NCAJ271748 OsAF383876 CrABOB4236 CpAFO67823 BsM2263O EcX55034 50 6O 7O 8O 90] GTTAATAGGATGATTGCGAGTTCCATGGATGGTGTCGAGTTTTGG GTGAACAGGATGATTGAGAGCGACATGAAGGGGGTGGAATTCTGG GTCAACAGGATGATTGAGAGCTCCATGAACGGCGTCGAGTTCTGG GTAAATCGCATGATTGAGAGTTCGATGAAGGGTGTAGAGTTTTGG GTAAATCGCATGATTGAGAGTTCGATGAAGGGTGTAGAGTTTTGG GTTAATCGGATGATTGAAAGTGCTATGAAAGGCGTAGAGTTTTGG GTGAATCGTATGATAGAGAGTGAAATGTCAGGTGTGGAGTTCTGG GTGAATCGTATGATTGAGAGTGAGATGATTGGTGTGGAGTTTTGG GTCAACCGAATGCTTGAGAGCGAAATGCAAGGTGTGGAATTCTGG GTAAACCGAATGCTTGAGAGCGAGATGCAAGGGGTAGAATTCTGG GTCAACAACATGGTCAATAGCGACGTGCAGGGCGTGGAGTTCTGG GTGAACCGTATGGCGGATACAGGTATTTCGGGTGTGGAGTTCTGG GTCAACCGTATGGCCGACACCGGCATCTCAGGCGTAGAGTTTTGG GTTAATCGAATGGTAGGG--—GGTGTTGAAGGAGTTGAATTTTGG GTGAATCGGATGTGTGAG--—ATGGTTGAAGGAGTTGAATTTTGG ------------------------------ GGCGTCGAACTCTGG GTTCAACGTATGTTGGAGAGTGGTTTACAAGACGTGGAATTTCTA GTAAACAGGATGATTGATAGTGATCTTGAAGGCGTTTCATTCAGA GTAAACAGGATGATTGATAGTGATCTTGAAGGCGTTTCATTCAGA GTAAATCGAATGATTCTTAGTGATCTTCAAGGGGTCTCATACAGA GTCAACAGGATGATCCTCAGTGATCTGGAGGGTGTTGCTTATCGC GTCAACCGCATGATTCTCAGTGACCTCGATGGTGTGAACTATCGG GTCAACCGCATGATTAGCAGCGATGTCAGCGGGGTTGAATTTTGG GTTAACCGCATGATTGAATCTGATGTCTCTGGTGTAGAGTTTTGG GTTAACCGCATGATCGAATCTGATGTCTCTGGTGTAGAGTTTTGG GTAAACCGCATGATTGCCAGTAATGTGGCGGGTGTTGAATTTTGG GTTAACCGAATGATTGCTAGTGAGGTATCTGGTATAGAATTTTGG GTCAACCGTATGATTGCCAGTGGGGTGACGGGCATCGACTTTTGG GTTAACCGTATGATTGGCAGTGGCTTACAGGGTGTTGACTTCTAT GTTAACCGTATGATTGGCAGTGGCTTACAGGGTGTTGACTTCTAT GTTAACCGCATGATTGGTAGCGGCTTACAGGGTGTTGATTTTTAC GTTAACCGGATGATTTCAAGCGGTTTACAGAGTGTTGATTTCTAT GTTAACCGCATGATTGGTAGTGGTTTGCAGGGTGTAGATTTCTAT GTTAACCGTATGATTGGCAGCGGCTTGCAGGGTGTTGACTTTTAT GTTAACCGTATGATTGGCAGCGGCTTGCAGGGTGTTGACTTTTAT GTCAACCGCATGATCGGCAGCGGCCTCCAGGGCATCGAATTTTAT CTAAACCGCATGATCAACAGCGGCCTGCAGGGCGTGGAGTTCTGG GTTGACAGAATGATTGAAGACGGCCTGGACGGCGTTGATTTTATT GTTAACCGAATGATTGAAAATGAAGTGCAAGGAGTAGAGTATATC GTTGAACACATGGTGCGCGAGCGCATTGAAGGTGTTGAATTCTTC 199 [ 100 110 120 130 ] LIABO42101 ATTGTTAACACTGATGTACAGGCGATGAGGATGTCA --------- [126] OSCL005296_338 ATCGTGAATACTGATTTTCAGGCTATGAGAATGTCA --------- [126] OSCLB17724_5 ATTGTGAATACCGATGTGCAGGCGATAAGGATGTCG --------- [126] NCAJ271750 ATTGTGAACACTGATATTCAAGCAATGAGGATGTCA --------- [126] NCAJ311847 ATTGTGAACACTGATATTCAAGCAATGAGGATGTCA --------- [126] G1AF205859 ATTGTGAACACTGATGTTCAAGCCATAAAGATGTCT --------- [126] ACAF089738 ATTGTCAACACTGATATCCAGGCTATGAGAATGTCT --------- [126] AtAF384167 ATTGTGAATACCGATATCCAAGCAATGAGAATATCT --------- [126] PpAJ249l39 ATTGTGAATACGGATGCTCAGGCAATGGCGTTGTCT --------- [126] PpAJ249138 ATCGTGAATACTGATGCGCAGGCTATGGCCTTGTCC --------- [126] CrAF449446 ATTGCCAACACCGACGCTCAGGCCTTGGCAACGTCT --------- [126] CmABO32072 GCGATCAACACTGACGTGCAGGCGCTGAAGCGCTCG --------- [126] CCA8023962 GCGATCAACACGGATGTTCAAGCTCTGAAGCGCTCG --------- [126] GtAJ007748 TCTATTAATACCGATGCTCAAGCACTTTCAAGATCA --------- [123] GSABO22594 TGTATCAACACCGACGCTCAGGCTCTTTCACGTGTG --------- [123] MSAF120117 GTTGTAAATACTGACGCCCAAGCACTGTCAAGAAGC --------- [51] GSABO22595 TGTGCCAATACAGACGCTCAGGCACTAGGCAGATTTCAAGAGGTA [135] PSAJ011025 GTTTTAAATACTGATGCGCAAGCATTATTACAATCT --------- [126] Pm1658 GTTTTAAATACTGATGCGCAAGCATTATTACAATCT --------- [126] PmAJ237851 GTTCTCAATACTGATGCACAAGCTTTGTTGCAATCT --------- [126] Sy549 GTGCTCAACACCGATGCCCAGGCCCTGATCCAATCC ————————— [126] Pm1268 GTCATGAATACGGACGCTCAGGCCCTGCTTCAGTCT --------- [126] SyAF076530 GCCCTCAACACTGATGCTCAAGCTTTGCTCCACTCT --------- [126] ASZ31371 TCAATTAATACTGATGCTCAAGCTTTGACCTTGGCA --------- [126] Np6l TCAATAAATACTGATGCCCAAGCTTTAACTCTGGCA --------- [126] Tetll2382 TGTGTCAATACTGATGCGCAGGCGATCGCCCAATCC --------- [126] Te1949 ACGGTTAATACAGATGCCCAAGCATTAACTCTCTCA --------- [126] SSNC_000911 GCAATTAATACCGATTCCCAGGCATTAACTAATACG --------- [126] NtAJ133453 GCTATAAACACGGATGCTCAAGCACTGCTACAGTCT --------- [126] NCAJ271749 GCTATAAACACGGATGCTCAAGCACTGCTGCAGTCT --------- [126] TeAF251346 GCCATTAACACGGACTCACAAGCGCTTCTGCAATCT ————————— [126] AtU39877 GCGATAAACACGGATTCGCAAGCTCTGTTACAGTTT --------- [126] PSY15383 GCGATAAATACCGATGCTCAAGCGCTATTGCATTCT --------- [126] NCAF205858 GCTGTAAACACGGATGCTCAAGCATTGCTGCAGTCT ————————— [126] NCAJ271748 GCTGTAAACACGGATGCTCAAGCATTGCTGCAGTCT --------- [126] OSAF383876 GCCATAAACACAGATTCCCAGGCGCTTTTGAATTCG --------- [126] CrA8084236 GCCATCAACACCGACGCCCAGGCCCTGGCCGCCCAC --------- [126] CpAF067823 TCAATAAATACAGATGGACAAGCCCTTTCCAAGGCT --------- [126] BSM22630 GCGGTAAACACGGACGCTCAAGCTCTTAACCTGTCA --------- [126] ECX55034 GCGGTAAATACCGATGCACAAGCGCTGCGTAAAACA --------- [126] 200 { LlABO42101 OSCL005296_338 OSCLBI7724_5 NCAJ271750 NtAJ311847 GlAF2OS859 AtAFO89738 AtAF384167 PpAJ249l39 PpAJ249l38 CrAF449446 CmABO32072 CCABOZ3962 GtAJOO7748 GSA8022594 MsAF120117 GSABOZ2595 PsAJOllOZS Pm1658 PmAJ237851 Sy549 Pm1268 SyAFO76530 AsZ3137l Np6l Tet112382 Te1949 SSNC_000911 NtAJ133453 NCAJ271749 TeAF251346 AtU39877 PSY15383 NCAF205858 NtAJ271748 OSAF383876 CrABO84236 CpAFO67823 BsM22630 ECX55034 140 150 160 170 180] .l —————— CCTGTGTATCCTGAGAACCGGTTACAGATTGGGCAGGAG —————— CCCATTGATCCGGATAATAAGTTGCAGATCGGCCAGGAG —————— CCGGTCCTTCCGCAGAACAGGTTGCAGATTGGGCAGGAG —————— CCTGTAGCGGCTGAGCAGCGACTTCCGATAGGTCAAGAA —————— CCTGTAGCGGCTGAGCAACGACTGCCGATAGGTCAAGAA —————— CCTGTATATTTAGAGAACCGGCTGCAAATTGGTCAAGAG ------ CCTGTTTTGCCTGATAATAGGTTACAAATTGGTAAGGAG —————— CCGGTTTTTCCAGACAATAGGTTACAAATTGGTAAGGAG —————— CCGGTTCCGGCTCAGAATCGTCTGCAGATTGGTCAGAAA ------ CCTGTTCCGGCTCAGAATCGTCTGCAGATTGGGCAAAAA —————— CCCGTTAACGGCAAGTGCAAGGTGCAAATTGGAGGCAAG ____________ GCGGCACACCATACGCTGAGCATCGGGAATAAG ____________ GCCGCGCACCACACACTCGGTATCGGCAACAAG ____________ TTAGCACCCAATACATGTAATATTGGTGCTAAA ____________ AAAACAAGCAACTCTGTAACAATAGGGAGCGAA ____________ TCTGCGAAACGGAGGCTGAACATTGGGAAAGTA TATTGCCAGAAAACGCATCATCAAGTTATCCAAATTGGGAAACAA ____________ TCTGCAGATCGTAGAGTTCAACTAGGTCAGAAC ____________ TCTGCAGATCGTAGAGTTCAACTAGGTCAGAAC ____________ TCGGCTGAGAATAGAGTTCAACTTGGTCAAACA ____________ CAGGCTCAGCATCGTCTTCAACTCGGCCAAACC ____________ GCAGCCAGTAACCGCGTGCAGCTGGGTCAGACC ____________ GcAGcCCCCAAGCGGATGCAGTTGGGACAGAAA ____________ GGCGCchTAGTCGTTTACAAATCGGGCAGAAA ____________ GGCGCTCCCAGTAGATTACAAATTGGACAAAAG ____________ CAAGCCCACCGCTGCCTGCAAATTGGCCAGAAA ____________ CGCGCTCCTAAACGCTTACAACTTGGACAAAAG ____________ AACGCCCCGGATTGTATTCAAATTGGCCAAAAA ____________ GCTGCTGAAAACCCGCTTCAAATTGGAGAACTT ____________ GCTGCTGAAAATCCACTTCAAATTGGAGAGCTT ____________ GTTGCACATAACCCTATTCAAATTGGGGAGCTT ____________ TCTGCTGAGAACCCACTTCAAATTGGAGAACTT ____________ GCGGCCGAGAATCCTATTAAAATTGGCGAGCTT ____________ ACGGTTGAGAACCCAATTCAAATTGGAGAACTT ____________ ACGGTTGAGAACCCAATTCAAATTGGAGAACTT ____________ CAAGCGCAATACCCGCTACAAATTGGAGAGCAG ____________ CAGGCTCTCAACAAGGTGCAGATCGGCAGCGAG ____________ AGATCCAGCACAAAAACTCAGATTGGTGAAAAG ____________ AAAGCAGAAGTGAAAATGCAAATCGGCGCAAAG ____________ GCGGTTGGACAGACGATTCAAATCGGTAGCGGT 201 L1ABO42101 OSCL005296_338 OsCLBl7724_5 NCAJ271750 NtAJ311847 GIAF205859 ACAF089738 ACAF384167 PpAJ249l39 PpAJ249l38 CrAF449446 CmABO32072 CCABO23962 GCAJOO7748 GsABO22594 MSAF120117 GSA8022595 PsAJOllO25 Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ3l37l Np6l Tet112382 Te1949 SSNC_000911 NCAJ133453 NCAJ271749 TeAF251346 ACU39877 PSY15383 NCAF205858 NCAJ271748 OSAF383876 CrABO84236 CpAFO67823 BSM2263O ECX55034 190 200 210 220 CTTACGAGAGGTCTTGGTGCTGGCGGGAATCCGGATATTGGTATG CTAACACGAGGTCTTGGTGCTGGTGGGAACCCGGAAATCGGCATG CTGACTCGGGGTCTAGGCGCAGGTGGAAACCCTGACATTGGGATG CTTACAAGGGGACTTGGTGCAGGTGGTAATCCAGATATAGGGATG CTTACAAGGGGACTTGGTGCAGGTGGTAATCCAGATATAGGGATG CTTACCAGAGGACTTGGAGCAGGTGGGAACCCTGATATTGGTATG TTGACTAGGGGTTTAGGTGCTGGAGGAAATCCAGAAATCGGTATG TTGACTAGGGGTTTAGGTGCTGGAGGTAATCCAGAGATTGGGATG TTGACGCGAGGTCTGGGGGCGGGTGGTAATCCGGAAATAGGGTGT TTGACGAGAGGTCTGGGGGCGGGCGGGAATCCAGAAATAGGGTGT CTTACCCGCGGTCTCGGCGCCGGAGGCAACCCTGAGATCGGCGCT CTGACCCGTGGTCTCGGAGCTGGCGGCAATCCCGAAGTGGGTCGC TTGACGCGCGGCCTTGGAGCGGGCGGGAACCCAGAAATTGGACGG TTGACTCGCGGTTTAGGAGCAGGTGGAAATCCTGAAATTGGCAGA ATTACTAGGGGATTGGGTGCTGGAGGAAAACCTGAAGTTGGTAGG CTCAGTCGGGGACTCGGAGCTGGCGGAAATCCAGCCATAGGCGCG AGTTGCAGAGGATTGGGAGCCGGTGGAAATCCGGAAGCTGGTAGA TTAACAAGAGGTCTTGGAGCAGGAGGGAATCCAAGTATTGGTCAA TTAACAAGAGGTCTTGGAGCAGGAGGGAATCCAAGTATTGGTCAA TTAACTCGAGGCCTAGGAGCTGGAGGGAACCCAAGTATTGGAGAG CTCACCCGAGGCCTGGGGGCTGGGGGTAACCCCACCATCGGTCAG CTTACGAGAGGGTTAGGAGCTGGCGGTAATCCCAGCATCGGCCAG CTAACGCGAGGGCTAGGCGCAGGTGGCAACCCTGCGATCGGCATG CTCACGAGGGGTTTAGGTGCAGGTGGTAATCCTGCAATTGGTCAA CTAACGCGGGGCTTAGGAGCAGGTGGTAATCCTGCCATCGGTCAA CTCACCCGTGGTCTTGGCGCAGGTGGCAACCCCGCCATTGGTCAA CTCACAAGAGGTTTGGGGGCAGGGGGTAATCCCGCTATTGGTCAA CTCACCAGGGGTTTGGGGGCCGGTGGTAATCCGGCGATCGGGCAA CTGACTCGTGGGCTTGGTACTGGTGGTAATCCTCTTTTAGGGGAA CTGACTCGTGGGCTTGGTACTGGTGGCAATCCTCTTTTAGGGGAA TTGACTCGTGGATTAGGTACTGGTGGGAACCCGCTTTTGGGAGAA TTAACTCGTGGGCTTGGCACTGGTGGAAACCCGCTTCTTGGAGAA CTGACTCGTGGATTAGGTACCGGTGGGAATCCGCTTTTGGGAGAA CTGACTCGTGGACTTGGTACTGGTGGCAATCCTCTGTTGGGGGAA CTGACTCGTGGACTTGGTACTGGTGGCAATCCTCTGTTGGGGGAA TTAACTCGTGGACTAGGTACTGGTGGAAATCCTAATTTGGGAGAG TTGACCCGCGGCCTGGGCTGCGGCGGCAACCCTGAGCTGGGCCGC CTGACAAAGGGTCTTGGGGCAGGGGGTAATCCTGAAATTGGGGAA CTGACTAGAGGATTGGGAGCAGGTGCGAATCCGGAAGTCGGGAAA ATCACCAAAGGACTGGGCGCTGGCGCTAATCCAGAAGTTGGCCGC 202 [210] [210] [210] [210] [210] [210] [210] [210] [210] [210] [210] [204] [204] [201] [201] [129] [225] [204] [204] [204] [204] [204] [204] [204] [204] [2041 [204] [204] [204] [204] [204] [2041 [204] [204] [204] [204] [204] [204] [204] [204] L1AB042101 OSCL005296_338 OSCLBl7724_5 NtAJ27l750 NtAJ311847 GIAF205859 AtAFO89738 ACAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCABOZ3962 GtAJOO7748 GSA8022594 MSAF120117 GSA8022595 PsAJOllOZS Pm1658 PmAJ237851 Sy549 Pm1268 SyAFO76S3O AsZ3l37l Np6l Tet112382 Te1949 SSNC_OOO911 NtAJ133453 NtAJ27l749 TeAF251346 AtU39877 PSY15383 NCAF205858 NtAJ271748 OSAF383876 CrABOB4236 CpAF067823 BSM2263O ECX55034 230 240 250 260 270] .l AATGCTGCTAAGGAAAGCAAAGTGTCGATAGAGGAGTCGGTTTCT AATGCAGCCAAGGAAAGCCAGGAGTTGGTAGAACAAGCAGTTTCT AATGCGGCGAAAGAAAGTGTCGAGTCCATTCAGGAAGCTCTTTAC AACGCCGCGAACGAAAGCAAGCAGGCGATTGAAGAAGCTGTCTAC AATGCTGCCAACGAAAGCAAGCAGGCGATTGAAGAAGCTGTCTAC AATGCTGCCAAAGAAAGCAAAGAAGCCATCGAGGAAGCAGTTTAC AATGCTGCTAGAGAGAGCAAAGAAGTTATTGAAGAAGCTCTTTAT AATGCTGCTACAGAAAGCAAAGAAGCTATTCAAGAAGCACTTTAT AGTGCCGCGGAAGAGAGCAAAGCTATGGTGGAAGAAGCCTTACGC AGTGCTGCGGAAGAGAGCAAAGCTATGGTGGAAGAAGCCCTACGC AAAGCTGCTGAAGAGAGCCGGGACTCCATCGCCGCGGCACTGCAA AAAGCCGCCGAGGAATCGTGCGACCAAATTGCAGAAGCCGTCCGT AAGGCTGCAGAGGAGTCTTGCGACCAGATTGCCGAAGCGGTGCGT AAAGCAGCAGAAGAAAGTAGAGACTTAATTGCTGAAGCCGTCTCT CAGGCAGCTGAAGAGTCTCAGGCTGCAATAAGTTCTGCAGTTCAA AAAGCTGCCGAGGAAAGTCGGGAAGAAATCATGGCTGTTGTAAAG GTAGCAGCCGAAGAGTCCAAAGAGGATATAGCAAAAGCTCTTCAA AAAGCTGCTGAGGAATCTAAAGATGAATTGCAACAAACCTTAGAG AAAGCTGCTGAGGAATCTAAAGATGAATTGCAACAAACCTTAGAG AAAGCTGCAGAAGAATCTAGAGCAGAGCTTCAACAAGCTTTAGAA AAGGCCGCCGAAGAGTCCCGAACAGACCTCCACGACGCCCTTCAG AAGGCGGCGGAGGAATCTCGAGCAGAACTGCAACAAGCTCTGCAA AAAGCCGCTGAAGAATCGCGGGAAGAACTAATCGCCGCCTTGGAA AAAGCAGCTGAAGAATCACGCGATGAAATTGCTACAGCCTTAGAA AAGGCAGCTGAGGAATCACGAGACGAAATTGCTACAGCTTTAGAG AAGGCGGCCGAAGAATCCCGCGAAGACCTGGCTGCGGCGCTCAAG AAAGCTGCTGAAGAATCTAGAGATGAAATAGCTAATGCTTTAGAC AAAGCGGCGGAGGAATCCCGGGATGAAATTGCCCGTTCCCTTGAG CAGGCAGCGGAGGAGTCGAAGGAAGCCATTGCAAATTCTCTAAAA CAGGCAGCAGAGGAGTCGAAGGAAGCCATTGCAAATTCTCTAAAA CAGGCTGCGGAGGAGTCGAAGGAAGCGATTGGGAATGCGCTTAAA CAAGCTGCAGAAGAATCAAAAGATGCAATTGCTAATGCTCTTAAA CAAGCTGCGGAGGAATCGAAAGAGGCTATTGCTAATGCGCTTAAA CAGGCAGCAGAGGAGTCAAAGGAACACATTGCAAATGCTCTTAAA CAGGCAGCAGAGGAGTCAAAGGAACACATTGCAAATGCTCTTAAA CAAGCTGCTGAGGAATCAAAAGAAGCCATAGCCAATGCCCTGAAG CGCGCTGCTATGGAGAGCGAGGAGGCGCTGCGCCGCATGGTGCAG AAATCCGTTGATGAAACCCAAGACGAAATTGCACAGGCTTTGCAT AAAGCCGCTGAAGAAAGCAAAGAGCAGATTGAAGAAGCACTTAAA AATGCGGCTGATGAGGATCGCGATGCATTGCGTGCGGCGCTGGAA 203 [255] [255] [255] [255] [255] [255] [255] [255] [255] [255] [255] [249] [249] [246] [246] [174] [270] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] [249] LlABO42101 OSCL005296_338 OsCLBl7724_5 NtAJ271750 NtAJ311847 G1AF205859 AtAF089738 AtAF384167 PpAJ249l39 PpAJ249l38 CrAF449446 CmAB032072 CCABOZ3962 GtAJOO7748 GsABO22594 MSAF120117 GSABO22595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ3lB7l Np61 Tet112382 Te1949 SSNC_000911 NtAJ133453 NtAJ27l749 TeAF251346 AtU39877 PsY15383 NCAFZOSBSB NCAJ271748 OSAF383876 CrABOB4236 CpAFO67823 BsM2263O ECXSSO34 280 290 300 310 GGTGCTGACATGGTTTTCGTCACGGCTGGAATGGGTGGGGGTACA GGTGCTGATATGATTTTTGTGACTGCTGGAATGGGAGGAGGGACA GGTGCTGATATGGTTTTTGTCACGGCTGGGATGGGTGGAGGCACT GGCGCAGACATGGTTTTTGTTACTGCTGGAATGGGTGGAGGAACA GGCGCAGACATGGTTTTTGTTACTGCTGGAATGGGTGGAGGAACA GGTGCAGATATGGTTTTTGTAACTGCTGGAATGGGTGGAGGAACA GGCTCAGATATGGTCTTTGTCACAGCTGGAATGGGCGGTGGAACT GGTTCAGATATGGTCTTTGTCACAGCTGGAATGGGTGGTGGAACT GGAGCTGACATGGTTTTCGTTACAGCGGGCATGGGTGGTGGCACT GGAGCTGACATGGTTTTCGTAACGGCGGGTATGGGTGGCGGCACT GATACTGACATGGTATTCGTGACGGCCGGAATGGGCGGCGGCACG GGCGCCGACCTGGTGTTTGTGACTGCCGGAATGGGCGGCGGCACT GGCGCTGACCTCGTCTTTGTGACAGCAGGCATGGGCGGCGGTACC GCAGGTGATCTAGTTTTTGTGACAGCAGGAATGGGAGGAGGTACA GGTGGAGATCTCGTTTTTGTTACAGCCGGTATGGGAGGTGGAACA AACGCAGACCTGGTCTTTGTAACGGCCGGTATGGGTGGTGGCACG GGTGGAGATCTTGTGTTTGTTACTGCTGGTATGGGAGGTGGTACA GGCTCTGACTTGGTTTTTATTGCTGCAGGTATGGGAGGAGGAACT GGCTCTGACTTGGTTTTTATTGCTGCAGGTATGGGAGGAGGAACT GGTGCCGATTTGGTATTTATTGCCGCTGGCATGGGTGGAGGAACA GGTTCCGATCTGGTGTTCATCGCTGCGGGTATGGGTGGCGGAACC GGAGTCGATCTCGTCTTCATCGCCGTTGGCATGGGTGGCGGAACC GGGGCTGACCTCGTCTTTATCACGGCGGGGATGGGCGGTGGAACC GGTGCTGATTTAGTATTTATCACTGCTGGGATGGGAGGTGGTACT GGTGCAGACCTAGTATTTATCACCGCTGGTATGGGTGGCGGTACT GACGCTGATTTGATTTTCATTACCTGTGGCATGGGGGGCGGCACT CATCCAGATCTAGTATTTATTACTGCTGGAATGGGAGGTGGTACA GGTACGGATTTGGTCTTTATTACTGCGGGCATGGGGGGCGGCACT GGTTCAGATATGGTGTTCATAACAGCAGGAATGGGTGGAGGTACA GGTTCAGATATGGTGTTCATAACAGCAGGAATGGGTGGAGGTACA GGGTCGGATCTTGTGTTTATAACAGCAGGTATGGGTGGTGGGACG GGATCAGACCTTGTTTTCATAACTGCTGGTATGGGTGGTGGAACA GGATCGGATTTGGTGTTTATAACAGCTGGGATGGGTGGCGGTACA GGTTCGGATATGGTGTTTATAACAGCAGGCATGGGTGGCGGTACT GGTTCGGATATGGTGTTTATAACAGCAGGCATGGGTGGCGGTACT GATTCTGACCTTGTCTTCATAACGGCTGGAATGGGAGGGGGTACT GGCGCTGATCTGGTGTTCATCACCGCGGGCATGGGCGGCGGTACC GGTTCTGACATGGTATTTATTACTGCCGGAATGGGTGGTGGCACC GGTGCTGACATGGTATTCGTGACAGCTGGTATGGGCGGCGGAACA GGTGCAGACATGGTCTTTATTGCTGCGGGTATGGGTGGTGGTACC 204 [300] [300] [300] [3001 [3001 [300] [300] [300] [300] [300] [300] [294] [294] [291] [291] [219] [315] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] [294] LlABO42101 OSCL005296_338 OSCLBl7724_5 NtAJ27l750 NCAJ311847 GlAF205859 ACAFO89738 AtAF384167 PpAJ249l39 PpAJ249l38 CrAF449446 CmABO32072 CCABOZ3962 GtAJOO7748 GSABO22594 MsAFl20ll7 GSABOZ2595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZBl371 Np6l Tet112382 Te1949 SSNC_000911 NtAJl334S3 NtAJ271749 TeAF251346 ACU39877 PsY15383 NCAF205858 NCAJ271748 OsAF383876 CrABOB4236 CpAFO67823 BsM22630 ECX55034 320 330 340 350 360] .l GGAACTGGTGGTGCTCCTGTAATTGCTGGAGTTGCCAAGTCAATG GGCACAGGCGGGGCCCCAGTTATAGCAGGGATTGCAAAGTCCATG GGAACTGGAGGTGCCCCTGTAATCGCTGGAATTGCCAAGTCCATG GGGACTGGTGCAGCTCCTATAATTGCAGGAACTGCTAAATCAATG GGGACTGGTGCGGCTCCTATAATTGCAGGAACTGCCAAATCAATG GGAACTGGCGGGGCTCCAGTAATTGCGGGAATTGCTAAATCTATG GGCACTGGTGCAGCCCCTGTAATTGCAGGAATTGCCAAGGCGATG GGCACAGGTGGTGCTCCTATAATTGCAGGGGTTGCAAAAGCGATG GGCAGCGGTGCTGCACCAATCATTGCTGGTGTAGCGAAGCAATTG GGCAGCGGTGCAGCACCAATAATTGCGGGTGTGGCGAAGCAGTTG GGCAGTGGCGCCGCGCCCGTCGTGGCGGAGGTGGCGCGTGAATTG GGTTCCGGAGCCGCTCCAGTAGTTGCAGAGGCTGCCCGCGAGCAA GGCTCGGGTGCAGCACCGGTCGTCGCTGAGGCTGCCCGTGAACAG GGATCAGGAGCTGCCCCGATAGTAGCTGAAGTTGCTAAAGAAATG GGCTCAGGTGCTGCTCCAATTGTTGCTAAAATAGCTAAAGAACAA GGATCTGGCGCTGCCCCTGTGGTGGCGGAATGTGCTAAGGAGGCT GGAACAGGAGCAGCTCCAATAGTTGCCGATGTTGCTAGAGAACTG GGGACAGGAGCGGCTCCAGTAGTTGCTGAGGTTGCAAAGCAAAGT GGGACAGGAGCGGCTCCAGTAGTTGCTGAGGTTGCAAAGCAAAGT GGCACTGGAGCAGCACCAGTAGTTGCAGAAGTAGCCAAACAAAGC GGAACGGGTGCCGCCCCCGTGGTTGCGGAGGTCGCCCGTGAGGTC GGCACTGGTGCCGCTCCAGTGGTTGCCGAAGTTGCCAAGGAAAGC GGCACTGGAGCTGCCCCGATCGTGGCAGAAGTCGCCAAAGAAGTG GGTACTGGTGCTGCACCAATTGTTGCGGAAGTGGCAAAGGAAATG GGGACTGGTGCAGCTCCAATCGTAGCAGAAGTAGCCAAAGAAATG GGCACCGGCGCTGCCCCAATTGTGGCGGAAGTGGCCAAGGAACAG GGTACTGGTGCAGCTCCAGTTATAGCTGAAATTGCTAAAGAAGCA GGCACTGGAGCAGCTCCCATTGTGGCCGAGGTGGCCAAAGAAATG GGATCTGGTGCTGCTCCTGTTGTGGCTCAAATAGCAAAAGAAGCA GGATCTGGTGCTGCTCCTGTTGTGGCTCAAATAGCAAAAGAAGCA GGTTCGGGTGCTGCTCCAGTTGTAGCGCAGATAGCGAAAGAAGCA GGGTCTGGTGCTGCACCTGTGGTAGCTCAGATTTCGAAGGATGCT GGGTCCGGTGCTGCCCCAGTTGTGGCTCAAATATCAAAAGAGGCA GGATCTGGTGCTGCTCCTGTTGTTGCTCAAATAGCCAAAGAAGCA GGATCTGGTGCTGCTCCTGTTGTTGCTCAAATAGCCAAAGAAGCA GGATCTGGTGCTGCTCCAGTTGTTGCTCAGATATCAAAGGAAGCC GGCACCGGTGCCGCCCCCGTGGTGGCCCGCCTGTCCAAGGAGTTG GGTACAGGTGCGGCTCCCAGAATTGCCGCAATTTCCAAAGAACTA GGAACAGGTGCCGCACCGGTTATCGCACAAATCGCGAAAGACTTA GGTACAGGTGCGGCACCAGTCGTCGCTGAAGTGGCAAAAGATTTG 205 firm—.1 L1AB042101 OSCL005296_338 OsCLB17724_5 NtAJ271750 NtAJ311847 GlAF205859 ACAFO89738 AtAF384167 PpAJ249139 PpAJ249l38 CrAF449446 CmA8032072 CCABOZ3962 GtAJOO7748 GSA8022594 MSAF120117 GSA8022595 PsAJOllOZS Pm1658 PmAJ237851 Sy549 Pm1268 SyAFO76530 AsZ3l37l Np6l Tet112382 Te1949 SSNC_OOO911 NtAJl33453 NtAJ271749 TeAF251346 AtU39877 PSY15383 NtAF205858 NCAJ271748 OsAF383876 CrABO84236 CpAFO67823 BsM22630 EcX55034 370 380 390 400 GGTATCTTGACTGTAGGCATTGTGACAACGCCATTTATGTTTGAA GGTATATTAACTGTTGGAATTGTGACAACCCCGTTTGCATTTGAG GGTATACTGACAGTTGGCATCGTGACGACACCATTCTCATTTGAA GGTATCTTAACTGTTGGTATTGTTACAACCCCCTTTTCTTTCGAG GGTATCTTAACTGTTGGTATTGTTACAACCCCTTTTTCTTTCGAG GGTATCTTGACCGTTGGTATTGTCACAACACCTTTCTCCTTTGAA GGTATATTGACAGTTGGTATTGCCACTACGCCTTTCTCGTTTGAG GGTATATTAACGGTTGGTATTGTGACAACGCCTTTCTCATTTGAG GGAATTCTTACCGTGGGAATAGTAACTACGCCTTTTGCCTTTGAA GGAATTCTTACTGTAGGAATAGTTACTACTCCTTTCGCCTTTGAA GGCATCCTAACAGTTGGCATCGTCACCACCCCCTTCACCTTCGAG GGCTGCCTAACCGTGGGCGTTGTCACCAAGCCATTCGCGTTTGAA GGCTGCTTGACAGTCGGTGTCGTGACCAAGCCGTTTGCCTTCGAA GGTTGTTTAACTGTTGGAGTTGTAACCAAACCTTTTGCTTTTGAA GGCTGTCTTACTGTTGGAGTTGTGACTAAACCTTTCAGCTTTGAG GGCGCGTTAACCGTAGGCGTAGTCACAAAGCCGTTCGGATTCGAA GGTTGTTTGACAGTTGGTGTTGTTACGAAACCTTTTGCTTTTGAA GGTGCTTTAACTGTTGGGATAGTAACCAAGCCATTTTCATTTGAA GGTGCTTTAACTGTTGGGATAGTAACCAAGCCATTTTCATTTGAA GGAGCCCTTACTGTCGCAATAGTTACTAAACCATTTAGTTTTGAA GGAGCCCTCACCGTGGGAATCGTGACCAAACCCTTTGGTTTTGAA GGTGCGCTCACTGTGGGCATCGTCACCAAACCCTTTAGCTTTGAG GGTGCGCTGACGGTTGGGATTGTCACCAAACCCTTCACCTTCGAA GGCGCTCTTACTGTTGGAGTGGTAACACGTCCTTTTGTTTTTGAA GGCGCTCTCACTGTTGGGGTAGTCACACGTCCATTCGTCTTTGAA GGAGCCCTCACCGTTGCAGTGGTGACCCGCCCCTTTACCTTTGAG GGTTCTTTGACGGTGGGTGTTGTGACTCGCCCTTTTACTTTTGAG GGCTGTTTGACGGTGGGCATTGTCACCCGTCCATTTACCTTTGAA GGCTATTTGACTGTTGGTGTTGTCACATACCCATTCAGCTTTGAA GGCTATTTGACTGTTGGTGTTGTCACATACCCATTCAGCTTTGAA GGGTATTTAACTGTTGGTGTTGTAACGTACCCATTCAGCTTTGAA GGTTATTTGACTGTTGGTGTTGTTACCTATCCGTTTAGCTTTGAA GGTTACTTGACTGTAGGTGTTGTTACATATCCTTTCAGTTTTGAA GGTTATTTGACTGTTGGTGTCGTCACGTATCCATTCAGCTTTGAA GGTTATTTGACTGTTGGTGTCGTCACGTATCCATTCAGCTTTGAA GGTTATCTCACCGTTGGAGTTGTTACCTATCCATTCAGTTTTGAA GGCATCCTGACTGTGGGCGTCGTCACCTACCCCTTCAACTTCGAG GGCATCTTAACGGTAGGTGTTGTGACAAAGCCCTTTAACTTTGAG GGCGCATTAACAGTCGGCGTTGTGACAAGACCGTTTACCTTCGAA GGTATCCTGACCGTTGCTGTCGTCACTAAGCCTTTCAACTTTGAA 206 [390] [390] [390] [390] [390] [390] [390] [390] [390] [390] [390] [384] [384] [381] [381] [309] [405] [384] [384] [384] [3841 [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] [384] L1AB042101 OSCL005296_338 OsCLBl7724_5 NtAJ271750 NCAJ311847 GlAF205859 ACAF089738 AtAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCA8023962 GtAJOO7748 GSA8022594 MsAF120117 GsABOZ2595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O A523137l Np6l Tet112382 Te1949 SSNC_000911 NtAJ133453 NtAJ271749 TeAF251346 AtU39877 PSY15383 NtAF205858 NCAJ271748 OsAF383876 CrA8084236 CpAFO67823 BsM22630 ECX55034 410 420 430 440 450] .1 GGGCGAAGACGAACTGTTCAG ------ GCACAAGAAGGAATTGCA GGAAGGAGGCGTGCACTACAG ------ GCGCAAGAAGGAATTGCG GGGAGGAGACGGGCAGTTCAG —————— GCTCAAGAAGGAATAGCA GGGCGAAGACGAGCAGTTCAA ------ GCCCAAGAAGGTATTGCA GGGCGAAGAAGAGCAGTTCAA ------ GCCCAAGAAGGGATTGCT GGTCGGAGAAGAGCAGTGCAA ------ GCACAAGAAGGCATCGCA GGTCGAAGAAGAACTGTTCAG ------ GCTCAAGAAGGGCTTGCA GGACGGAGAAGAGCACTCCAG ------ GCTCAGGAAGGGATTGCA GGGCGGAGACGATCCGTTCAA ------ GCTCACGAAGGCATCGCG GGGCGGAGACGAGCTGTCCAA —————— GCCCACGAGGGTATTGCA GGCCGCCAGCGCGCGCAGCAG ------ GCTCGCTCTGCTTTAGCC GGCCGGAAACGCATGAATCAG ------ GCGCTGGAGGCTATCGAG GGGCGGAGGCGTATGACGCAG ------ GCGCTCGAGGCTATCGAG GGTAAACGAAGAATGCAACAA ------ GCAAATGACGCAATACTT GGAAGAAGGAGAATGCAACAG ------ GCAGAGGAAGCAATAGAA GGCCGAAAACGAATGCAACAA ------ GCGAGGAACGCTATTCTG GGACGTCGACGTCTGCAGCAA ------ GCAGTCGAAGGATTGGCA GGTAAAAGGAGAATGCGTCAG ------ GCAGAAGAAGGGATTGCA GGTAAAAGGAGAATGCGTCAG ------ GCAGAAGAAGGGATTGCA GGTCGCCGCAGAATGCGTCAA ------ GCAGATGAAGGTATCGCC GGCCGCCGCCGCATGCGCCAG ------ GCCGATGAAGGCATCGCC GGACGTCGTCGCATGCGACAG ------ GCCGCTGAAGGCATTGGC GGGCGTCGCCGAATGAAGCAG ------ GCGGAAGAAGGAACAGCC GGTCGTCGCCGGACTAGTCAG ------ GCAGAACAAGGAATTGAA GGTCGCCGCCGTACCAGCCAA ------ GCCGAACAAGGGATCGAA GGTCGTCGTCGAGCGAACCAA ------ GCCGATGAAGGGATTGAA GGACGACGCCGAATTACTCAG ------ GCTGATGAAGGAATTACT GGCCGACGACGGGCTAAGCAA ------ GCTGAGGAAGGCATTAAT GGACGTAAAAGATCCGTGCAG ------ GCTCTGGAAGCAATTGAA GGACGCAAAAGATCCGTGCAG ------ GCTCTGGAAGCAATTGAA GGCCGTAAAAGATCAGTACAG ------ GCGTTAGAGGCTATTGAG GGACGTAAAAGATCTTTGCAG ------ GCACTGGAAGCTATTGAA GGACGGAAAAGATCCTTGCAG ------ GCACTTGAAGCGATTGAA GGACGTAAAAGATCTTTGCAG —————— GCTTTGGAAGCAATTGAA GGACGTAAAAGATCTTTGCAG ------ GCTTTGGAAGCAATTGAA GGACGCAAGCGCTCTCTTCAGGCAAGTGCGTTGGAAGCATTAGAG GGCCGCCGCCGCGCCGGCCAG —————— GCTCTTGAGGGCATTGAG GGAAAGAAAAGAATGAGTAAT ------ GCCGAAAAAGGTATTATG GGACGCAAAAGACAGCTTCAG —————— GCTGCAGGCGGAATCTCG GGCAAGAAGCGTATGGCATTC ------ GCGGAGCAGGGGATCACT [429] [429] [429] [429] [429] [429] [429] [429] [429] [429] [429] [423] [423] [4201 [420] [348] [444] [423] [423] [423] [4231 [423] [423] [423] [423] [423] [423] [423] [423] [423] [423] [423] [423] [423] [423] [429] [423] [423] [423] [423] Fuo‘. , : L1ABO42101 OsCL005296_338 OsCLBl7724_5 NtAJ27l750 NCAJ311847 G1AF205859 ACAF089738 AtAF384167 PpAJ249139 PpAJ249l38 CrAF449446 CmABO32072 CCABOZ3962 GtAJOO7748 GSABOZ2594 MSAF120117 GSABOZ2595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ3l37l Np6l Tet112382 Te1949 SSNC_000911 NtAJ133453 NtAJ271749 TeAF251346 AtU39877 PSY15383 NtAF205858 NtAJ271748 OSAF383876 CrABO84236 CpAFO67823 BsM22630 ECXSSO34 460 470 480 490 GCTTTAAGAAATAACGTTGACACACTTATTGTCATCCCAAATGAC TCCTTAAGAAGCAATGTTGATACACTGATTGTAATTCCAAATGAC GCCTTGAGAAATAGTGTGGACACCCTCATTGTCATCCCAAATGAC GCTTTGAGAGAAAATGTCGATACTCTAATTGTCATTCCAAATGAC GCTTTGAGAGAAAATGTCGATACTCTAATTGTCATTCCAAATGAC GCACTGAGAGATAATGTCGACACCCTAATTGTGATTCCAAATGAC TCTCTCAGAGACAATGTTGACACTCTCATCGTCATTCCAAATGAC GCCCTCAGAGATAATGTTGACACCCTCATTGTTATTCCAAACGAC GCTCTCAAAAATAATGTTGACACTTTAATTACGATACCAAACAAC GCTCTCAAAAATAACGTGGACACGTTAATTACGATTCCAAACAAC AACTTGCGTGCAGCGGTTGACACGCTCATTGTCATCCCGAACGAC GCCTTGCGCGAGAGCGTGGACACGCTGATTGTCGTGAGCAACGAC GCGCTGCGAGAGAGTGTGGACACGCTGATTGTGGTCAGCAATGAC AACTTGAGAAACAAAGTTGATACACTTATTGTTGTATCTAATGAT GCTTTAAGAAAGGAAGTCGATACTTTGATTGTAGTTTCCAATGAT GAGATGAAGGACAAGGTGGACACGCTCATCGTCGTGTCCAACGAC AATTTGAGAGAAAAGGTCGATACTCTTATTGTTATTTCAAACGAT AGATTAGCAGAAAACGTTGATACGCTTATTGTGATTCCAAATGAT AGATTAGCAGAAAACGTTGATACGCTTATTGTGATTCCAAATGAT AAGCTCACAGAAAGTGTTGACACTTTAATTGTCATCCCTAACGAT CGCTTGGCGGAACACGTGGATACCTTGATTGTGATTCCCAACGAT CGCCTGGCCGATCATGTGGATACCTTGATTGTGATCCCCAACGAC GCACTGCAAAGCTCAGTCGACACTTTGATCACTATTCCTAATGAC GGGCTAAAAAGTAGAGTTGATACTTTAATTATTATTCCTAACAAT GGCTTAAAAAGTAGGGTAGATACACTGATTATTATTCCTAACAAC GCACTGCAAAGTCGCGTGGATACCCTAATCGTGATTCCCAATGAC GCTCTACAAACTAGAGTAGATACTTTAATTGTGATCCCCAATAAT GCTCTCCAATCGCGGGTCGATACCCTAATTGTGATTCCTAATAAC AAACTTCAGAAAAATGTAGATACCCTTATAGTAATTCCCAATGAC AAACTTCAGAAAAATGTCGATACCCTTATAGTAATTCCCAATGAC AAGCTGCAAAAGAACGTTGACACACTTATAGTGATTCCAAATGAC AAGCTCCAAAAGAATGTTGATACCCTTATCGTGATTCCAAATGAT AAGCTTCAGAAAAATGTTGATACGCTTATTGTGATTCCAAATGAT AAACTTCAGAAAAATGTAGATACTCTTATAGTAATTCCCAATGAT AAACTTCAGAAAAATGTAGATACTCTTATAGTAATTCCCAATGAT AAGCTGGAAAGGAGTGTAGACACACTTATTGTTATTCCAAATGAT GCGCTGCGTGAGGCCGTGGACTCCGTGATCGTCATCCCCAACGAC GAATTAAAGAAGAACGTTGATACTTTGGTTATTATTCCAAACCAA GCAATGAAAGAAGCGGTGGATACACTGATCGTGATCCCGAACGAC GAACTGTCCAAGCATGTGAACTCTCTGATCACTATCCCGAACGAC 208 474] 474] 474] 474] [474] [474] [474] [474] [474] [474] [474] [468] [468] [465] [465] [393] [489] [468] [468] [468] [468] [468] [468] [4681 [468] [468] [468] [468] [468] [4681 [468] [468] [468] [468] [468] [474] [468] [468 [468 [468 r—\F—|O—"F—V h—Ae—JH .4! L1ABO42101 OsCLOOS296_338 OsCLBl7724_5 NCAJ271750 NtAJ311847 GlAF205859 ACAF089738 ACAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCABOZB962 GCAJOO7748 GSA8022594 MSAF120117 GSAB022595 PsAJOllOZS Pm1658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ3l37l Np61 Tet112382 Te1949 SSNC_000911 NtAJ133453 NCAJ271749 TeAF251346 ACU39877 PsY15383 NCAF205858 NtAJ271748 OsAF383876 CrABO84236 CpAFO67823 BsM2263O ECX55034 500 510 520 530 540] .l AAGCTATTGACTGCCGTTTCCCCAAATACTCCTGTGACAGAGGCG AAATTGTTGACTGCTGTTTCTCCAAATACTCCTGTGACAGAAGCA AAGCTGTTGTCTGCTGTTTCTCCAAATACTCCAGTAACCGAAGCA AAATTATTGACAGCTGTTTCTCCATCGACCCCGGTAACTGAAGCT AAATTGTTGACAGCTGTTTCTCCATCGACCCCAGTAACTGAAGCT AAATTACTCACTGCAGTTTCCCCATCTACTCCAGTCACAGAAGCA AAGTTGCTTACAGCTGTCTCTCAGTCTACTCCGGTAACAGAAGCA AAGTTACTAGCAGCAGTCTCTCAGTCTACTCCAGTTACAGAAGCA AAGCTTTTGACTGCAGTTGCGCAGTCTACCCCCGTGACGGAAGCA AAACTTTTGACTGCAGTTGCGCAGTCTACCCCAGTGACGGAAGCG CGGCTGCTGTCGGCCATGGACTCCAACGTGCCTATCAAGGACGCC AAGCTGCTTCAGATAGTCCCTGAGAACACACCATTGCAGGACGCA AAGCTGTTACAGATTGTACCAGAGAACACGCCATTGCAGGATGCA AAATTATTACAGATAGTTCCAGATAATACGCCCCTTCAGGATGCA AAGTTACTCGAAATTGTTCCTGAAAATACAGCTTTGGAAAAGGCT AAGCTCTTGAAGATCGTGCCGGACAACACTCCCCTGACGGAAGCC CGACTCTTAGAAACAGTACCGAAAGATACTCCACTGACTGAGGCT CGTTTAAAAGACGTAATTGCAGGA-—-GCTCCACTTCAAGAAGCC CGTTTAAAAGACGTAATTGCAGGA---GCTCCACTTCAAGAAGCC CGCCTTAAAGATGCAATTGCAGGA—--GCGCCCCTTCAAGAAGCA CGTTTGCGGGAAGCGATCGCCGGG---GCTCCGCTTCAGGAGGCC CGCATTAAAGACGTCATCTCCGAA---GCTCCGCTTCAAGAAGCC CGCCTACTCCACGCCATATCTGAGCAGACGCCGATTCAAGAAGCT AAATTATTGGAAGTGATTCCAGAACAAACACCAGTCCAAGAAGCG AAACTACTGGAAGTGATCCCCGAACAAACACCTGTGCAAGAAGCT AAGATTCTCTCGGTCATCTCTGAGCAAACATCGGTTCAGGATGCG CGTTTGCTATCTGTAATTAATGACCAAACTCCAGTACAGGAGGCT CAACTTTTGTCGGTTATTCCCGCCGAAACTCCTCTCCAGGAAGCT CGTCTGCTAGATATTGCTGATGAGCAGACACCACTTCAAGATGCT CGTCTGCTAGATATTGCTGATGAGCAGACACCACTTCAAGATGCT CGTTTGCTGGATATTGCTGATGAAAACACGCCTCTTCAGGATGCT CGTCTGCTAGATATTGCTGATGAACAGACGCCACTTCAGGACGCG CGTCTGCTTGACATAGCTGATGAGCAGATGCCCCTTCAAGATGCT CGTCTTCTGGATATTGCTGATGAGCAGACACCACTTCAAAATGCT CGTCTTCTGGATATTGCTGATGAGCAGACACCACTTCAAAATGCT CGATTATTAGATGTTGTTGATGAAAACACGCCCTTGCAAGATGCG CGCCTGCTGGACGTGGCCGGCGCCAGCACCGCGCTGCAGGATGCC AGATTATTGAGCATTATTGATAAAAAGACGACACTCACCGAAGCT CGTATCCTTGAAATTGTTGATAAAAACACACCGATGCTTGAAGCA AAACTGCTGAAAGTTCTGGGCCGCGGTATCTCCCTGCTGGATGCG 209 LIABO42101 OsCLOOS296_338 OsCLBl7724_5 NtAJ27l750 NtAJ311847 GIAF205859 AtAFO89738 ACAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCABOZ3962 GCAJOO7748 GSA8022594 MSAF120117 GSA8022595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O A523137l Np61 Tet112382 Te1949 SsNC_0009ll NtAJl33453 NtAJ27l749 TeAF251346 AtU39877 PSY15383 NtAF205858 NtAJ271748 OsAF383876 CrABO84236 CpAFO67823 BSM22630 ECX55034 550 560 570 580 TTTAACTTGGCTGATGATATACTTCGACAAGGTGTTCGTGGAATC TTTAATTTGGCAGATGATATACTTCGGCAAGGTGTCCGTGGAATC TTCAACTTGGCTGATGATATTCTTCGACAAGGAATTCGTGGTATC TTTAACCTGGCTGATGATATTCTTCGGCAAGGAGTTCGTGGAATT TTTAACCTGGCTGATGATATTCTTCGGCAAGGAGTTCGTGGTATT TTTAACTTGGCTGATGATATTCTTCGACAAGGAGTTCGTGGAATC TTTAATCTAGCTGATGATATACTCCGTCAGGGGGTTCGTGGGATA TTTAATCTGGCAGATGATATACTTCGTCAAGGTGTCCGTGGAATA TTCAATCTTGCCGATGACATCCTTCGGCAGGGAGTGCGGGGTATT TTCAATCTTGCAGACGACATCCTTCGGCAGGGAGTGCGGGGTATT TTCAAAATTGCGGATGACGTACTGCGGCAGGGCGTAAAGGGCATC TTTCGAGTTGCAGATGACATCCTGCGGCAGGGTGTTGTGGGCATC TTTCGAGTAGCGGATGATATTCTGCGACAAGGTGTCGTTGGGATC TTTTCTGTTGCTGATGATATTCTAAGACAAGGAGTTGTAGGAATA TTTTCTGTAGCGGATGATATTCTCAGACAAGGTGTGGTTGGAATC TTTCTGGTCGCAGACGACATCCTCAGACAGGGCGTGGTGGGCATC TTTATATTTGCGGACGAAGTTTTACGTCAAGGAGTTGGTGGAATT TTTAGAAATGCTGATGATGTTTTAAGGATGGGAGTTAAAGGTATA TTTAGAAATGCTGATGATGTTTTAAGGATGGGAGTTAAAGGTATA TTTAAAAATGCAGATGATGTTTTACGAATGGGAGTGAAAGGCATA TTCCGCAGTGCCGATGACGTGCTTCGGATGGGTGTGAAAGGCATC TTCCGAAGTGCAGATGACATCCTGCGTATGGGTGTTAAAGGTATC TTCCGGGTCGCCGACGATATTCTCCGGCAGGGTGTGCAAGGGATT TTTCGTTATGCAGATGACGTACTACGTCAAGGGGTACAAGGCATT TTTCGCTATGCAGATGACGTGTTGCGTCAAGGGGTGCAAGGTATT TTTCGCGTTGCTGATGATGTGCTGCGCCAAGGGGTTCAGGGGATT TTCATAATTGCAGATGATATCTTACGTCAAGGTATACAGGGAATT TTTCGGGTAGCCGATGATATTCTGCGCCAGGGGGTACAGGGTATT TTTCTTCTTGCTGATGATGTATTACGCCAAGGTGTCCAAGGAATT TTTCTTCTTGCTGATGATGTATTACGTCAAGGTGTCCAAGGAATT TTTCTTCTTGCTGATGATGTACTCCGCCAAGGAGTTCAAGGAATC TTTCTTCTTGCAGATGATGTTTTACGCCAAGGAGTACAAGGAATC TTTCGTCTTGCAGATGATGTTTTACGCCAAGGAGTTCAGGGAATA TTTCTTCTTGCTGATGATGTACTTTGTCAAGGCGTCCAAGGAATA TTTCTTCTTGCTGATGATGTACTTTGTCAAGGCGTCCAAGGAATA TTTCTTCTTGCAGATGATGTTCTTCGTCAAGGTGTCCAAGGAATA TTCGCGTTGGCAGACGATGTGCTGCGCCAGGGTGTGCAGGGTATC TTCAAAAAGGCGGACGAAATTTTACGCCAAGGTGTTCAGGGTATT TTCCGCGAAGCGGATAACGTACTTCGCCAAGGGGTTCAAGGTATT TTTGGCGCAGCGAACGATGTACTGAAAGGCGCTGTGCAAGGTATC 210 [564] [564] [564] [564] [564] [564] [564] [564] [564] [564] [564] [558] [558] [555] [555] [483] [579] [555] [555] [555] [555] [555] [5581 [558] [5581 [558] [558] [558] [558] [558] [558] [558] [558] [558] [558] [564] [558] [558] [558] [SS8] L1AB042101 OSCL005296_338 OSCLB17724_5 NtAJ271750 NCAJ311847 G1AF205859 AtAF089738 ACAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCABOZ3962 GCAJOO7748 GSABO22594 MsAFlZOll? GSA8022595 PSAJ011025 Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ31371 Np6l Tet112382 Te1949 SSNC_OOO911 NtAJ133453 NtAJ271749 TeAF251346 AtU39877 PSY15383 NtAF205858 NtAJ27l748 OSAF383876 CrABO84236 CpAFO67823 BsM2263O ECX55034 590 600 610 620 630] .l TCTGATATAATCACGGTTCCTGGTCTAGTTAATGTTGATTTCGCT TCAGATATAATCACTGTGCCAGGTTTGGTCAATGTTGACTTTGCT TCTGATATTATCACGGTTCCTGGGTTGGTTAATGTTGATTTTGCT TCTGATATTATTACGATTCCTGGGCTAGTAAATGTGGATTTTGCT TCTGATATAATTACGATTCCTGGGCTAGTAAATGTGGATTTTGCT TCTGATATAATTACGATCCCTGGGCTAGTAAATGTGGACTTTGCT TCTGATATCATTACGATTCCTGGTTTGGTGAATGTGGATTTTGCT TCTGATATTATTACGATTCCTGGATTGGTCAATGTGGATTTTGCT TCAGATATTATCACTGTTCCTGGTCTCGTTAACGTGGACTTTGCG TCAGATATTATCACGGTCCCTGGGCTGGTTAACGTAGATTTTGCC AGCGAAATTATCACAGTGCCCGGCCTAGTCAACGTGGACTTCGCG AGCGATATTATCATCCGCCCTGGTCTCATTAACGTAGACTTTGCC AGCGACATCATCATCCGCCCTGGACTGATTAATGTTGATTTTGCT TCCGAGATTATTGTAAGACCAGGTTTAATTAATGTTGATTTTGCC TCAGAAATCATTGTTCGTCCAGGTCTGATTAATGTTGACTTTGCG ACCGAAATCATTGTGAAGCCAGGGCTCGTGAATGTTGATTTCGCT TCCGATATTATTACTAAACCGGGCTTAGTCAACGTAGATTTTGCA AGTGACATAATTACATGCCCTGGATTAGTTAACGTTGATTTTGCT AGTGACATAATTACATGCCCTGGATTAGTTAACGTTGATTTTGCT ACCGACATAATCACTTTGCCTGGTCTTGTAAATGTGGACTTTGCG AGCGACATCATCACGTGCCCCGGTTTGGTGAACGTCGACTTCGCT AGTGACATCATCACCTGCCCTGGCCTTGTCAATGTGGACTTCGCC TCTGACATCATCACGATCCCAGGTCTGGTCAACGTCGACTTTGCC TCCGACATCATTACTATTCCTGGTTTGGTAAATGTAGACTTTGCT TCTGATATCATCACAATTCCCGGTTTGGTAAATGTTGACTTTGCT TCCGACATTATCAACGTGCCGGGGCTGATTAACGTGGATTTTGCC TCAGATATTATTACTGTACCTGGATTAGTAAATGTTGACTTTGCT TCCGACATTATCATCATCCCCGGTTTGGTGAATGTGGACTTTGCG TCCGATATAATTACTATACCTGGGCTTGTAAATGTGGATTTTGCC TCCGATATAATTACTATACCTGGGCTTGTAAATGTGGATTTTGCC TCAGATATAATTACAATACCTGGGCTGGTAAATGTGGACTTTGCA TCAGATATTATTACTATACCTGGACTAGTCAATGTGGATTTTGCA TCGGACATTATAACAATACCTGGACTTGTAAATGTGGATTTTGCT TCTGATATAATCACTATACCTGGGCTGGTAAATGTGGATTTTGCA TCTGATATAATCACTATACCTGGGCTGGTAAATGTGGATTTTGCA TCAGATATTATTACAATACCTGGACTTGTCAATGTTGATTTTGCT TCAGACATCATCACCGTGCCCGGCCTCATCAACGTTGACTTTGCG GCTGATTTGATTTCCAAGCCCGGCGTAATTAACTTGGACTTTGCC TCTGACTTGATTGCTACACCTGGTCTTATCAACCTTGACTTTGCT GCTGAACTGATTACTCGTCCGGGTTTGATGAACGTGGACTTTGCA 211 [609] [609] [609] [609] [609] [609] [6091 [609] [609] [609] [609] [6031 [603 [600 [600 [528 [624 [600 [600 [600] [600] [6001 [603] [603] [603] [603] [603] [603] [603] [603] [603] [603 [603 [603 [603] [609] [603] [603] [603] [603] L—JS—JL—J LlAB042101 OSCL005296_338 OSCLBI7724_5 NCAJ271750 NtAJ311847 GIAF205859 ACAF089738 AtAF384l67 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCABO23962 GtAJOO7748 GSABO22594 MSAF120117 GSAB022595 PsAJOllOZS Pm1658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ31371 Np6l Tet112382 Te1949 SSNC_OOO911 NtAJ133453 NtAJ271749 TeAF251346 AtU39877 PSY15383 NCAF205858 NtAJ27l748 OSAF383876 CrABOB4236 CpAFO67823 BsM2263O ECX55034 640 650 660 670 GATGTTAGAGCAATTATGGCAAATGCAGGCTCATCCCTAATGGGT GATGTCCGATCAGTTATGTCGGATGCAGGGTCATCTTTGATGGGC GATGTTCGAGCCATCATGCAAAATGCAGGCTCATCCTTGATGGGT GACGTGCGTGCTATTATGGCAAATGCTGGTTCTTCTTTAATGGGA GACGTGCGTGCTATTATGGCAAATGCTGGTTCCTCTTTAATGGGA GATGTGCGGGCTATAATGGCCAATGCTGGGTCTTCCTTAATGGGC GATGTGAGAGCTATAATGGCAAATGCGGGGTCTTCATTGATGGGA GATGTGAGGGCGATAATGGCAAATGCAGGTTCTTCATTGATGGGA GATGTGCGGGCGATCATGGCCAATGCAGGATCATCTTTGATGGGA GACGTGCGGGCGATCATGGCTAATGCAGGATCATCTTTGATGGGC GACGTTCGTGCCATCATGGCGGGCGCCGGTAGCTCGCTCATGGGG GACGTCCGAAGTGTCATGGCACATGCGGGATCGGCCTTAATGGGC GACGTGCGGAGTGTTATGGCGCACGCAGGATCCGCGCTTATGGGC GATGTAAGATCTGTCATGGCAGATGCGGGAAGCGCACTTATGGGT GATGTTCGTTCTATTATGGCAGATGCTGGTTCAGCTTTGATGGGC GACGTGCGGACAATCATGGGCAACGCAGGCACGGCCTTGATGGGC GATGTTCGTACGGTTATGGCAGAAAAAGGTTTTGCTTTGTTAGGC GATGTTAGGTCTGTAATGACTGAAGCTGGCACTGCCCTGCTTGGT GATGTTAGGTCTGTAATGACTGAAGCTGGCACTGCCCTGCTTGGT GACGTTCGCTCTGTAATGACTGAAGCTGGAACATCATTACTTGGA GACGTGCGTTCCGTGATGACGGAAGCAGGAACAGCATTGCTAGGC GACGTGCGTTCGGTGATGACCGAAGCCGGCACGGCCCTGCTAGGG GACGTTCGCGCCGTCATGGCCGATGCTGGATCAGCCCTGATGGGC GACGTGCGGGCTGTCATGGCCGATGCGGGATCAGCATTGATGGGT GATGTCCGGGCTGTGATGGCAGATGCAGGATCGGCATTGATGGGA GATATTCGCTCGGTGATGGCTGATGCCGGTTCTGCCATGATGGGC GATGTTAGAGCAGTTATGGCCGATGCTGGTTCGGCATTGATGGGA GACGTGCGGGCGGTGATGGCCGATGCTGGCTCCGCATTAATGGGC GATGTAAAGGCAGTGATGAAAGATTCTGGAACTGCTATGCTTGGA GATGTAAAGGCAGTGATGAAAGATTCTGGAACTGCTATGCTTGGA GACGTTAAAGCAGTCATGAAAGATTCTGGAACTGCAATGCTTGGT GATGTGAAGGCAGTCATGAAAGATTCTGGAACTGCAATGCTCGGG GATGTAAAAGCTGTGATGAAAGACTCTGGTACCGCAATGCTCGGA GATGTTAAGGCAATCATGAAAGATTCTGGAACTGCTATGCTCGGA GATGTTAAGGCAATCATGAAAGATTCTGGAACTGCTATGCTCGGA GATGTGAAAGCTGTTATGAAAAACTCTGGAACTGCAATGCTTGGT GATGTCAAGGCCATCATGAGCAACAGCGGCACGGCCATGCTGGGT GATGTACGTACAGTAATGGCAAATAAAGGTATTGCCCATATGGGT GATGTGAAAACAATCATGTCAAACAAAGGATCTGCTTTGATGGGT GACGTACGCACCGTAATGTCTGAGATGGGCCACGCAATGATGGGT 212 [654] [654] [654] [654] [654] [654] [654] [654] [654] [654] [654] [648] [648] [645] [645] [573] [669] [645] [645] [645] [645] [645] [648] [648] [648] [648] [648] [648] [648] [648] [648] [648] [648] [648] [648] [654] [648] [648] [648] [648] L1ABO42101 OSCL005296_338 OSCLB17724_5 NtAJ27l750 NCAJ311847 G1AF205859 ACAFO89738 ACAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCABOZ3962 GtAJOO7748 GSA8022594 MsAF120117 GSABOZ2595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO76530 AsZ31371 Np6l Tet112382 Te1949 SSNC_000911 NtAJl33453 NtAJ271749 TeAF251346 AtU39877 PsYlS383 NtAF205858 NtAJ271748 OsAF383876 CrABOB4236 CpAFO67823 BsM2263O ECX55034 680 690 700 710 720] .l ATAGGAACTGCAACA --------------------- GGTAAGACA ATTGGAACTGCAACA --------------------- GGTAAGACA ATTGGAACGGCTACA --------------------- GGGAAGTCA ATAGGAACTGCTACT --------------------- GGAAAGACC ATAGGAACCGCTACG --------------------- GGGAAGACC ATTGGGACAGCCACA --------------------- GGGAAAACC ATAGGAACTGCGACA --------------------- GGAAAGAGT ATAGGAACTGCAACA --------------------- GGAAAGACC ATTGGAACCGCTACA --------------------- GGGAAGTCA ATAGGGACCGCCACA --------------------- GGTAAGTCA CAGGGCTATGGATCC --------------------- GGTCCGCGG ATCGGCACCGGCAGT --------------------- GGCAAGTCC ATCGGCACCGGCAGT ------------ * -------- GGCAAGTCC ATAGGAACAGGATCT --------------------- GGAAAAACT ATTGGAAGCGGTTCG --------------------- GGAAAATCC ATCGGCCACGGCAAG ---------------------- GGAAAGAAC ATTGGAACTGCAAGT --------------------- GGAGATTCG ATAGGTATTGGTTCT --------------------- GGTAGATCT ATAGGTATTGGTTCT --------------------- GGTAGATCT ATAGGCATTGGATCT ————————————————————— GGTCGTTCT ATCGGCATTGGATCC --------------------- GGCCGCTCA ATTGGTGAAGGTTCA --------------------- GGACGTTCC ATCGGTAGCGGCTCT --------------------- GGCAAGTCC ATTGGTGTGAGTTCA --------------------- GGAAAATCT ATTGGCGTTAGTTCT ————————————————————— GGAAAATCT ATTGGTATTGCCTCT --------------------- GGAAAGTCA ATTGGTATGGGGTCT --------------------- GGTAAGTCT ATTGGGGTGGGTTCC --------------------- GGCAAGTCC GTTGGGGTTTCATCA --------------------- AGCAAGAAC GTTGGGGTTTCATCA --------------------- AGCAAGAAC GTCGGTGTTTCCTCA --------------------- AGTAAAAAC GTAGGTGTTTCTTCC --------------------- AGCAAAAAC GTAGGTGTTTCATCC --------------------- GGTAAAAAC GTTGGGGTTTCTTCC --------------------- AGTAGGAAC GTTGGGGTTTCTTCC --------------------- AGTAGGAAC GTTGGTGTTTCTTCC --------------------- AGCAAAAAT GTGGGCGCTGCCTCCACAGCCACCGCCGCCCCCGGCGGCCCCGAC ATCGGTCGTGCCAGT --------------------- GGCGAAAAT ATCGGTATTGCTACT --------------------- GGGGAAAAT TCTGGCGTGGCGAGC ————————————————————— GGTGAAGAC 213 ‘15-. .11 L1ABO42101 OSCL005296_338 OsCLBl7724_5 NtAJ271750 NtAJ311847 G1AF205859 AtAFO89738 AtAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCABO23962 GtAJOO7748 GSA8022594 MSAF120117 GSA8022595 PsAJOllOZS Pm1658 PmAJ237851 Sy549 Pm1268 SyAFO76530 AsZ3l37l Np6l Tet112382 Te1949 SSNC_000911 NtAJl33453 NtAJ27l749 TeAF251346 AtU39877 PsY15383 NtAF205858 NtAJ271748 OSAF383876 CrABOB4236 CpAFO67823 BsM2263O ECX55034 730 740 750 760 CGGGCAAGAGATGCTGCGTTAAATGCTGTTCAGTCACCATTACTG CGTGCCAGGGATGCTGCGCTTAATGCTATACAGTCTCCTCTTCTT AGAGCAAGAGATGCTGCTCTTAATGCCATTCAGTCACCACTGCTA AGAGCCAGAGATGCAGCATTGAACGCCATTCAATCTCCTTTACTG AGAGCCAGAGATGCAGCGTTGAACGCTATTCAATCTCCTTTACTG AGGGCCAGAGATGCTGCCTTAAATGCGATCCAATCTCCCTTGCTA CGGGCAAGAGATGCTGCGCTAAATGCAATCCAATCCCCTTTGTTA CGAGCAAGAGATGCTGCATTAAACGCAATCCAATCCCCTTTATTA AAAGCTAGAGAGGCAGCATTGAGTGCCATTCAGTCTCCATTGTTG AGAGCTAGAGAAGCAGCATTGAGCGCAATCCAATCTCCTCTATTG CGTGCCTCTGACGCCGCGCTGCGCGCCATCAGCTCGCCGCTGCTG CGGGCCCATGACGCCGCCGTGGCAGCGATTTCCTCACCGCTGCTG CGAGCGCACGATGCTGCTGTCGCTGCCATCTCTTCTCCGCTCTTA CGAGCACAAGATGCAGCAGTTGCAGCTATAAGTTCTCCTTTACTT CGTGCCAAGGATGCCGCAGTTGCTGCAATTTCCTCGCCATTACTT AGAGCCAAGGACGCGGCGCTGTCGGCCATCTCCTCCCCGCTGCTG AGAGCTCGAAATGCAGCAACTGCGGCTATTTCATCACCTCTTTTA AGAGCATTAGAGGCTGCTCAAGCCGCAATGAATAGTCCTTTACTA AGAGCATTAGAGGCTGCTCAAGCCGCAATGAATAGTCCTTTACTA AGAGCCGCCGAAGCCGCTCAAGCAGCAATAAACAGTCCTTTATTA AGGGCGGTTGAGGCCGCGCAAGCTGCCATCAGCAGCCCGTTACTT AGGGCGATAGAAGCAGCCCAGGCTGCCATCAGCAGTCCGCTACTG CGCGCTCGGGAAGCCGCTCATGCAGCCATTAGCTCACCGCTGCTG AGAGCCAGAGAAGCTGCGATCGCCGCTATATCTTCACCACTGCTA AGAGCCAGAGAAGCTGCGATCGCAGCTATTTCTTCACCGTTACTA CGGGCCACTGAAGCTGCCCTCAGCGCCATTTCTTCACCTCTACTG AGGGCAAGGGAAGCAGCAAATGCGGCAATTTCTTCTCCTTTGCTT CGGGCCAAGGAGGCGGCCACGGCGGCCATTTCCTCTCCTTTGTTG CGTGCTGAAGAAGCAGCCGAACAAGCAACTCTTGCCCCTCTAATT CGTGCTGAAGAAGCAGCCGAACAAGCAACTCTTGCCCCTCTAATT CGAGCTGAAGAAGCAGCTGAACAAGCAACTCTTGCTCCTTTGATT CGGGCAGAAGAAGCAGCTGAACAAGCAACTTTGGCTCCATTGATC CGAGCAGAAGAAGCAGCAGAACAGGCTACCTTGGCTCCTTTAATC CGTGCTGAGGAAGCAGCTGAACAAGCAACTCTGGCCCCTCTCATT CGTGCTGAGGAAGCAGCTGAACAAGCAACTCTGGCCCCTCTCATT CGGGCCCAAGAAGCTGCAGAGCAGGCAACACTTGCTCCTTTAATC CGCGCGGAGCAGGCGGCCGTGGCCGCCACATCGGCACCGCTCATC AAAGCTGAAATTGCAGCGAAAATGGCAATTCAGAGCCCTCTTTTG CGCGCGGCAGAGGCAGCAAAAAAAGCAATTTCCAGCCCGCTTCTT CGTGCGGAAGAAGCTGCTGAAATGGCTATCTCTTCTCCGCTGCTG 214 [723] [723] [723] [723] [723] [723] [723] [723] [723] [723] [723] [717] [717] [714] [714] [642] [738] [714] [714] [714] [714] [714] [717] [717] [717] [717] [717] [717] [717] [717] [717] [717] [717] [717] [717] [723] [738] [717] [717] [717] ruu--q LlABO42101 OSCL005296_338 OSCLB17724_5 NtAJ27l7SO NCAJ311847 G1AF205859 ACAF089738 ACAF384167 PpAJ249l39 PpAJ249l38 CrAF449446 CmABO32072 CCABOZ3962 GCAJOO7748 GSABOZ2594 MsAFl20ll7 GSA8022595 PsAJ011025 Pml658 PmAJ237851 Sy549 Pml268 SyAFO7653O AsZ31371 Np6l Tet112382 Te1949 SSNC_000911 NtAJl33453 NtAJ27l749 TeAF251346 ACU39877 PsY15383 NtAF205858 NCAJ271748 OsAF383876 CrABOB4236 CpAFO67823 BsM22630 ECX55034 770 780 790 800 810] .l GATATTGGT---ATTGAAAGAGCTACTGGAATAGTATGGAATATA GATATTGGC---ATTGAAAGAGCAACAGGAATTGTATGGAATATC GACATTGGA---ATTGAAAGAGCTACAGGCATTGTGTGGAATATC GACATTGGT—--ATAGAGAGGGCTACTGGAATTGTGTGGAATATA GATATTGGT---ATAGAGAGGGCTACTGGAATTGTGTGGAATATA GATATCGGT—-—ATCGAGAGAGCTACTGGTATTGTGTGGAATATT GATATTGGG-——ATTGAGAGAGCCACTGGAATTGTTTGGAACATT GATATTGGC——-ATTGAGAGAGCCACTGGAATAGTTTGGAACATA GATGTGGGT-—-ATTGAGCGAGCCACAGGGATCGTTTGGAATATT GATGTGGGT-—-ATTGAGCGAGCCACAGGGATAGTCTGGAATATC GAGGTGGGC—-—ATTGAGCGCGCCACTGGCGTGGTGTGGAACATC GATTTCCCT---ATCGAGCGTGCCAAAGGTATTGTTTTCAACGTC GATTTCCCT—u—ATAGAACGCGCCAAGGGCATCGTCTTCAATGTC GATTTTCCA---ATCGAAAAAGCCAGAGGAATTGTATTTAATATC GACTTTCCT———ATTGAGCGTGCAAAAGGAATTGTTTTTAACATT GACTTCCCC——-ATCACCCGCGCCAAAGGCATCGTTTTCAACATT GATTTTCCT-—-ATAACATCTGCGAAAGGTGCCGTTTTTAATATT GAAGCGGCAAGAATTGATGGAGCTAAAGGTTGTGTGATAAATATT GAAGCGGCAAGAATTGATGGAGCTAAAGGTTGTGTGATAAATATT GAAGCTGGTCGTATAGATGGAGCAAAAGGCTGCGTAGTAAATATT GAAACCGAGCGAATCGATGGTGCCAAGGGCTGTGTGATCAACATC GAAGCGGCCCGCATCGACGGAGCCAAAGGTTGCGTCATCAACATC GAGTCTTCG---ATCGAAGGGGCGCGCGGCGTTGTCTTCAACATC GAATGTTCT-——ATCGAAGGTGCTAGAGGTGTTGTTTTTAACATC GAATGTTCT--—ATTGAAGGTGCTAGAGGGGTTGTATTTAATATT GAGCGGTCT---ATCGAGGGTGCCAAGGGCGTTGTCTTTAACATT GAGTCTTCC—-—ATTGAAGGAGCAAAAGGGGTTGTATTTAATATT GAATCTTCT-—-ATCCAGGGAGCTAAAGGAGTCGTATTTAATGTC GGATCGTCC-——ATTCAATCTGCAACTGGGGTAGTATACAACATT GGATCGTCT—--ATTCAATCTGCAACTGGGGTAGTATATAACATT GGATCATCA--—ATTCAATCTGCTACAGGTGTTGTTTATAATATT GGATCATCC-——ATACAATCAGCTACTGGTGTCGTCTACAACATC GGATCATCT--—ATTCAATCAGCCACGGGAGTAGTGTATAATATC GGATCATCA—--ATTCAATCTGCAACTGGTGATGTATATAACATT GGATTATCA---ATTCAATCTGCAACTGGTGTTGTATATAACATT GGGTCGTCT--—ATTGAGGCGGCTACTGGTGTTGTGTACAATATC CAGCGCAGC--—ATCGAGAAGGCTACCGGCATTGTGTACAACATC GAAACTACC--—ATTGAGGGAGCTAAGAGCGTATTGATTAATTTC GAAGCGGCC--—ATTGACGGTGCGCAAGGCGTCCTCATGAACATC GAAGATATCGACCTGTCTGGCGCGCGCGGCGTGCTGGTTAACATC 215 [765] [7651 [765] [765] [765] [765] [765] [765] [765] [765] [7651 [759] [759] [756] [756] [684] [780] [759] [759] [759] [759] [759] [759] [759] [7591 [759] [7591 [759] [759] [759] [759] [759] [759] [759] [759] [765] [7801 [759] [7591 [762] den;«4rd.JadaJnJthJnJ4.4.11.9pD/4A94444444444444. .A...1.a.~.. 1.1.41.1..«44‘PV «J «J CV22AU «.9 fiU 11¢ flu fluOOfiU GU Au CV AU ad AU «5.52.2CQCRCRCRCRCBRCQJQJRCRCAI8888888888889C888 A T T A A T G A A A C T T A A G A A A A T G G C A A T T T T T A CCCCCCCGC CCCCCC C C C C CCC C C C C C C C C C C G G G G G G G G G G G G G G G G GT? TTTG G G G G G TTT T 3 T A T. A A T T _. A A G C T A C C T. T. T -. C A A C C C T G G G T. A .3 C C C C C C C C C C C C C C C C C C C C C C C C C C C C T T T T .3 . G G G G G G G G G G G G G G G G G G G G G G G G G G G G G GGG TATTCATTTTG G GTCCG A ATCCA T ATT T. GGG A CCCC CC CC C C A A A C CCA CCCCCC C CCCTG G G G A3 A; _ . cC as he a; ”G n; he he AC AC F . cC no AC 4. C. A. .A ... no no no no no .u .A .A .A AC TTTTTTTTTTCCfimTTCCACAPWMCxxCTTLTCTTnmC 1. Z. A. L. L. L. A. L. A. l. L. A. . L. l. l. .. p C . l. 4 L. .. 4 a 4 4 ‘ ‘. Am 1“ AW 1“ 1“ Am A“ L” A” A“ L“ Am A“ I“ A“ A” Am Z“ A“ L“ A“ A“ A” ‘M ‘nm ‘m .M in am .m A” 4“” Ac .c n; .r .r AC .r .r AC .r no AC .. AC .r AC ha nu no no nu nu nC .r as nC m. a. nu nu nC nu A. ......_...m._.*.*.m.m.m.m.a.~.m._.m.m...m.m.m.m.m.m.n.n.m.n.m1m. 4. G G G G G G 3 G G S G A A A A A G A A A A A G G GGGGGGGG :: . GGGGGGG 3;“ G3 L.G GEL. ATT CC C GA A:“ GAL“ AA 1“ A” A” l” l” A” A” l” l” l. A” A“ L. L“ L“ L. A. L. l“ .n in A“ A” .. L. A. L. .n L. L. .r L. L. G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G Tel..._._.TC?TCnettinefzmzmA3 GTTTTTCGGAG ._uny~._..._.......~...n .nluluLHLHCZ.._.1.LU.A Zulu lulu ZULU L“ L“ L. A“ TATTTTTTTTC CCCCCTG G G G G C CCC CCCC C C A; A; A; A” A” n; a; n; n. Av a; a; at A” A” as 1“ A” L“ A” A-» _. ha 1” L” n; A” Aid Pd flu Lu av _.....~...m....._.~..._.~...~.T.m._..........1.._.._..~._....-._.m.—._. .. _.if???TCCCCCCTCCTTTTCCCCTCTCTTCT 1. TC... A A A A A C TC CG A GTC A A A C C CTTT -. C T.TTT 2. . GGGCGCGGGGCG CGGGGC C CC: C C C G CC C C C C .m.A.A.m.m.A.A.A.A.A.A.A.A.A.A...A.A.A.A.A.A.A.A.A.A.A.A;“;“;mA l. L. n; L. 1. no AG L. n; h; PC PC CV no PC no n...» he PO F; P0 PC PC a» .. by L. Pu L. L. L. L. TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-. TC A C CT TTA A A A A A A A A A A A A A A C CCTCA A A A TTTTTTCC CCCCCTTCTTTCCCTCTTT: CCCC ‘HHLH‘SLH Lulu LHLH‘S‘AH‘m‘h‘nL“‘n.n1A.h‘n;“‘n‘n‘n‘.‘m‘n‘h‘n.h‘ngnlu GGGGGGGGGGL.GGGGGGGGGGGGGGGGGGGGG 3 TTTTTTTTC C L. A A ATCG.......TCCTCCTCA. AG A 2 A A C G G G G G G G C A a. ... A C C .. .. Ls. G G G G C C C C 41.1.... e: . L. L. G A A A A L“ L“ A C G G CCT A. ..L.CCCA A A A ASL. A A. A G A T T A A P A A G A T A A C A G G G C T C T T C T P A A G A GGGGGGGGGGCGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGCGGGGGGGGGGGGGGGGGGGGG GAGTTCCTGGTTCTTCATTCCCCTCGTTAAAT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGG.GGGGGGGGGGGGGGGGGG TTTTTT.TCT.TCCTPAGAAAACCATAAT.T.AACT CCCCCCCCCCCCCCCTCCCCGGCCCCCCCCCC. A A A A A A A A A A A A A A A G A A A A A A A A A A A A A A A A 8 3 3.3 _ _ l 1,04379QC7935r022347iPDPD «L Q «1396 Aug?“ ,3 Au. r33r03nj 1.3 H/ r0 43911964 PD 3 2 9quaQ l.m4778874111.aa nJ. 037.3 15 AJ no .31 do nu4737 2.37 11.591. 9992 37 232 1. 7 57 3 0311.7 43v 1.. a! «.nu n5 .5 «,4. 4. a». 1) 7“ AJ «.4 «.4 «4.183 3713 A/HOJAUAJA/pDB quad 37.3?“AJQJUE21.36.32363151.755.495.601. «14. _1.../~29 RD TH ..~ .0 vu H: r. S. TU wu T. “.3 BTU as T. BTV r0 Try 4.1 A/u an. 14 11. «-LQ/Cviu Tu r. «(a A CC A A A A A A A A A A A A A A A 1. A 5 1 A Z 6 21.?” A A A U .1 s S C :1. C t 0.0.: u C t s s s s m r Y a Y s p e e S t t e C [it LOO}.NGAAD.PCCCGGMGD.D.D.S .SANTTSNNTA [804] [804] :1 C,‘ ‘| f‘ mu 'GTCC u-y-s AAGC ‘9 GGAAGCGGCT I". ‘ 'GAATAAGG AG m & AC GG ‘ _ ‘ h 00:1 A b C . .C‘ 'Jx— A . \J IV A \J I Kr--- p-q .. .wa—A -. -V..V\—\.- '0'. --vv-- LA Wfi‘ r‘hn—f‘ M;mm\ fua-a-w’v! CC CC ACC .‘ r‘ ‘ .Mc.” G ‘ GAA CG- .0 y ”1"“ AA “‘f“ ‘3 HM .. GG GG.‘ b!“ m orum- TGGCCGTG . .‘ AH. ‘ I". TGG cc- . GG A M CGGCGGGC y ACTGG A ACTGG AC AC LlABO4210l OSCL005296_338 OsCLBl7724_5 NCAJ271750 NtAJ311847 GlAF205859 ACAF089738 AtAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmA8032072 CCABO23962 GCAJOO7748 GSA8022594 MsAF120117 GSABOZ2595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pm1268 SyAFO7653O AsZ3l37l Np61 Tetll2382 Te1949 SsNC_OOO9ll NtAJ133453 NtAJ27l749 TeAF251346 ACU39877 PSY15383 NCAF205858 NCAJ271748 OsAF383876 CrABOB4236 CpAFO67823 BsM2263O ECX55034 860 870 880 890 900] . ] GAAGTGATCTATGACCTTGTCGATCCGGCTGCAAACTTAATATTT GAAGTGATATACGATCTTGTTGATCCTGGTGCAAATCTCATTTTT GAGATCATCTATGACCTTGTTGATCCAAATGCTAATCTGATATTT GAGGTTATATATGACCTTGTGGATCCTAGTGCGAACCTTATTTTC GAGGTTATATATGACCTTGTGGATCCTAGTGCGAACCTTATTTTC GAAGTTATATATGATCTGGTAGATCCAAGTGCCAACTTAATTTTT GAAGTAATATATGATCTTGTCGATCCAACTGCCAATCTTATATTC GAAGTGATTTACGACCTCGTTGATCCAACAGCGAATCTTATATTT GAGGTAATCTATGATTTGGTGGATCCTAACGCAAATCTTATTTTC GAGGTGATTTATGATTTGGTCGATCCCAACGCAAATCTTATTTTT GAAATTATCTACGATATGGTGGACCCCAACGCCAACCTTATCTTT GAGGTAATCTACGAAGCAGTCGACCCCAACGCGAATATCATCTTC GAGGTGATCTACGAAGCGGTAGACCCGAATGCAAACATCATATTC GAAGTAATTTATGAAGCAGTAGATTCTAATGCAAATATAATATTT GAAGTTATTTACGAAGCCGTGGATTTGAATGCCAATATAATCTTC GAGGTCATTTACGAGAACGTGGATCAGGACGCGAATATCATATTC CAAGTTATTTATGATAGTGTAGATTCTGATGCAAATATTATTTTT GAAATTATTTATGATGTTGTAGATCCAGAAGCAAACATAATAGTA GAAATTATTTATGATGTTGTAGATCCAGAAGCAAACATAATAGTA GAGGTTATTTACGATGTTGTAGACCCAGAAGCAAATATTATTGTT GAAGTGATCTACGACGTGGTGGACCCAGAAGCCAACATCATTGTT GAAGTGATCTACGACGTGGTCGATCCAGAAGCCAACATCATTGTT GATGCGATTTACGAAGTCGTCGATCCTGAAGCCAATATCATTTTC GAAACGATTTATGAAGTAGTTGATCCCAACGCCAACATTATTTTT GAAGCAATCTATGAAGTAGTTGATCCCAACGCCAATATTATTTTT GACGTCATTTACAATGTGGCCGATGCCAACGCCAATATCATCTTT GAAATTATCTATGAAGTTGTAGATCCTAATGCTAATATTATTTTT GAAATTATCTATGAAGTGGTGGATGCCGATGCCAACATCATCTTT CAGGTTGTTACCAGTCTGGCTGATCCCTCCGCTAACATCATATTT CAGGTTGTTACCAGTCTGGCTGATCCCTCCGCTAACATCATATTT CAGGTGGTAACAAGTTTGGCAGATCCATCAGCAAACATTATATTC CAGGTCGTGACAAGTTTGGCAGACCCATCGGCCAACATCATATTT CAGGTTGTGACTAGTTTGGCCGATCCTTCTGCCAATATTATATTT CAGGTTGTCACAAGCTTGGCTGATCCATCCGCCAACATCATATTC CAGGTTGTCACAAGCTTGGCTGATCCATCCGCCAACATCATATTC CAGATTGTGACAAGCTTGGCCGATCCTTCTGCAAATATAATTTTC GAGGTGGTGACCGCCCTGGCCGACCCCTCATGCAACATTATCTTT GATTTAATCAGAGAGGCTATTGATCCTGATGCAGAAATCATCTTT GACATTGTCGCTTCGGCGTCTGATCAAGACGTAAACATGATTTTC AACACCATCCGTGCATTTGCTTCCGACAACGCGACTGTGGTTATC 217 [855] [855] [855] [855] [855] [855] [855] [855] [855] [855] [855] [849] [849] [846] [846] [774] [870] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [849] [855] [870] [849] [849] [852] LlABO42101 OSCL005296_338 OSCLBl7724_5 NCAJ271750 NtAJ311847 GlAF205859 ACAF089738 ACAF384167 PpAJ249l39 PpAJ249138 CrAF449446 CmA8032072 CCABOZ3962 GtAJOO7748 GSABO22594 MsAF120117 GSABOZ2595 PsAJOllOZS Pml658 PmAJ237851 Sy549 Pml268 SyAFO7653O AsZ3l37l Np6l Tet112382 Te1949 SsNC_OOO9ll NtAJl33453 NtAJ271749 TeAF251346 AtU39877 PSY15383 NCAF205858 NCAJ271748 OSAF383876 CrABO84236 CpAFO67823 BSM22630 ECX55034 910 920 930 940 GGAGCAGTGATTGATCCGTCGATTAGT-—-GGTCAAGTTAGCATA GGCTCTGTTATTGATCCATCGTATACT-~—GGTCAAGTGAGCATA GGTGCTGTCATAGACCCATCACTCAAT---GGCCAAGTGAGCATA GGGGCCGTGATAGACCCATCAATAAGT—--GGACAGGTCAGCATA GGGGCGGTGATAGACCCATCAATAAGT-——GGACAGGTCAGCATA GGAGCTGTAGTAGATCCATCACTGTGT—-—GGTCAAGTCAGTATA GGTGCTGTTGTAGATCCAGCCCTCAGC---GGTCAAGTAAGCATA GGTGCTGTGGTAGATCCATCTTATAGT--~GGTCAAATAAGTATT GGAGCCGTAGTAGACGAAGCACTTCAT-——GACCAAATTAGCATA GGAGCCGTAGTAGACGAAGCACTTCAT---GGCCAAGTTAGTATA GGAGCCGTGGTGGACTCTACCCTGCCCGACGACACGGTGTCCATC GGTGCCCTTATTGATCAGCAAATGGAA—-—AGCGAGATATCCATA GGCGCCCTCGTTGACCAACAAATGGAG--—AGCGAGATATCGATT GGAGCGCTTGTTGATGATAATATGGAA---AATGAAATTTCAATT GGTGCTTTGGTCGATGATAGTATGGAA———AATGAATTATCCATT GGGGCGATGGTGGACGACAAGATGACCTCTGGAGAGGTGTCCATC GGTGCAGTTGTAGATGAGACATTCAAA~-—GGAAAAGTTTCGGTT GGTGCTGTTATAGATGAATCAATGGAA———GGCGAAATACAGGTA GGTGCTGTTATAGATGAATCAATGGAA———GGCGAAATACAGGTA GGCGCAGTTATTGATGAAGCTCTTGAA---GGGGAAGTTCAAGTA GGTGCTGTGGTGGATGAAGCCCTAGAA———GGCGAAATCCACGTC GGAGCTGTTGTAGACGAGAAACTCGAA———GGTGAAGTTCACGTC GGCGCCGTGATTGACGATCGATTGGAA——-GGAGAGCTGCGGATC GGTGCCGTGATTGATGATAGGTTGCAA~—-GGAGAGGTGCGAATT GGGGCTGTAATTGATGACAGACTCCAA———GGTGAGGTCAGAATT GGTGCTGTCATTGATCCGCAAATGCAG———GGGGAAGTTCAAATC GGGGCTGTAATTGATGATAAACTTCAG—--GGAGAAATTAAAATC GGAGCGGTGATTGACGATCGCCTGCAG—--GGAGAAATGAGAATT GGTGCTGTTGTGGATGAGCGCTACAAT~—-GGCGAAATACACGTG GGTGCTGTTGTGGATGAGCGCTACAAT——-GGCGAAATACACGTG GGGGCAGTGGTAGATGAGAGATACAAC---GGGGAGATTCATGTG GGAGCTGTTGTGGATGATCGCTACACC-——GGAGAGATTCATGTA GGAGCTGTAGTTGATGATCGTTACACC-—-GGAGAGATTCACGTG GGTGCTGTTGTGGATGAGCGCTATAAT-—-GGAGAGATCCAGGTG GGTGCTGTTGTGGATGAGCGCTATAAT---GGAGAGATCCAGGTG GGGGCTGTTGTTGATGACCGGTACACT—-—GGTGAGATTCATGTG GGCGCCGTTGTGGACGAGCAGTACGAC——-GGCGAGCTGCACGTG GGTACAACCATTAACGAAGATCTCAAT---AATGAGGTTGTTGTT GGTTCTGTTATTAATGAAAATCTAAAA—--GATGAGATTGTGGTG GGTACTTCTCTTGACCCGGATATGAAT—--GACGAGCTGCGCGTA 218 LlABO4210l OSCL005296_338 OSCLBl7724_5 NCAJ271750 NtAJ311847 GlAF205859 AtAFO89738 ACAF384167 PpAJ249139 PpAJ249138 CrAF449446 CmABO32072 CCA8023962 GCAJOO7748 GSA8022594 MSAF120117 GSABOZ2595 PsAJOllOZS Pm1658 PmAJ237851 Sy549 Pm1268 SyAFO7653O A523137l Np6l Tet112382 Te1949 SSNC_OOO911 NLAJ133453 NCAJ271749 TeAF251346 ACU39877 PSY15383 NCAF205858 NCAJ271748 OSAF383876 CrABO84236 CpAFO67823 BSM22630 ECX55034 I END; 950 960 ACTCTGATTGCTACGGGATTC ACCCTGATTGCAACTGGATTC ACCTTGATTGCCACTGGCTTC ACCCTAATCGCCACTGGTTTT ACCCTAATCGCCACTGGTTTT ACCCTTATAGCCACGGGTTTT ACCCTGATAGCTACGGGTTTC ACCCTGATAGCAACTGGCTTC ACCTTGATAGCAACAGGGTTT ACTTTGATAGCAACAGGATTT ACCATCATTGCCACAGGCTTC ACGGTGGTTGCCACAGGCTTC ACAGTTGTTGCCACGGGCTTT ACAGTTGTGGCAACAGGTTTT ACTGTCATTGCTACTGGTTTT ACAGTCCTGGCCACGGGCTTT ACCGTAGTTGCCACAGGCTTT ACTGTTATTGCAACAGGTTTC ACTGTTATTGCAACAGGTTTC ACTGTAATTGCAACTGGTTTT ACCGTAATCGCCACGGGATTT ACCGTGATCGCCACAGGCTTT ACCGTGATCGCCACGGGCTTC ACCGTCATTGCTACTGGATTT ACTGTAATTGCCACTGGGTTT ACCGTTATCGCCACTGGCTTT ACTGTCATAGCTACTGGTTTT ACCGTCATTGCCACGGGCTTC ACCATAATTGCAACTGGTTTT ACCATAATTGCAACTGGTTTT ACCATTGTTGCTACTGGCTTT ACGATAATCGCCACAGGCTTC ACTATCATCGCAACTGGCTTC ACTCTAATTGCAACTGGTTTC ACTCTAATTGCAACTGGTTTC ACGATCATTGCCACAGGGTTT ACCATCATCGCTACCGGCTTC ACTGTAATTGCAACAGGGCTT ACAGTGATTGCAACCGGCTTT ACCGTTGTTGCGACAGGTATC 219 '37me! APPENDIX C Conserved Residues of the FtsZ Proteins The Nexus file with the FtsZ protein alignment that was used to determine the conserved residues in each FtsZ family that is discussed in Chapter 5 and diagramed in Figure 5 of that chapter. Sequences are labeled with the organism initials followed by the accession or gene number. 220 In. . #NEXUS BEGIN DATA; DIMENSIONS NTAX=38 NCHAR=397; FORMAT DATATYPE=PROTEIN SYMBOLS = " l 2 3 4" MISSING=- GAP=# INTERLEAVE MATRIX [ 10 20 3O 4O [ NtCAB89288 DSSRSNNFNEAKIKVVGVGGGGSNAVNRMIESSMKGVEFWIVNTD [45] NtCAC44257 DSSRSNNFNEAKIKVVGVGGGGSNAVNRMIESSMKGVEFWIVNTD [45] OSCLBl7724_5 APPDHCDYDGAKIKVVGVGGGGSNAVNRMIESSMNGVEFWIVNTD [45] GlAAF2377l DSSSSNNYSEAKIKVVGVGGGGSNAVNRMIESAMKGVEFWIVNTD [45] LIBAA96782 SSVTSSDYNGAKIKVIGVGGGGSNAVNRMIASSMDGVEFWIVNTD [45] AtAAC35987 EPSAPSNYNEARIKVIGVGGGGSNAVNRMIESEMSGVEFWIVNTD [45] AtAAK63846 ELSTPNTYNEARIKVIGVGGGGSNAVNRMIESEMIGVEFWIVNTD [45] OSCL005296_338 DVSASHRYSEPRIKVIGVGGGGSNAVNRMIESDMKGVEFWIVNTD [45] PpCAB76386 NGDEYESSNEAKIKVIGVGGGGSNAVNRMLESEMQGVEFWIVNTD [45] PpCABS4558 SGDDTGSYNEAKIKVIGVGGGGSNAVNRMLESEMQGVEFWIVNTD [45] CrAAM2289l LVRLYASSDQAIIKVLGVGGGGSNAVNNMVNSDVQGVEFWIANTD [45] CmBAA85116 PLSSDSSAPPCLIKVIGVGGGGGNAVNRMADTGISGVEFWAINTD [45] CCBAA8287I PMSSDSSAPPCLIKVIGVGGGGGNAVNRMADTGISGVEFWAINTD [45] GCCAAO7676 FFNQEISSSPCVIKVIGVGGGGGNAVNRMVG-GVEGVEFWSINTD [4S] GSBAA8209O FVSSGGAVNPCIIKVVGVGGGGSNAVNRMCE-MVEGVEFWCINTD [45] MSAAF35433 ----------------------------------- GVELWVVNTD [45] GSBAA82091 QSSTNLPQQQCKIKVVGVGGAGGNAVQRMLESGLQDVEFLCANTD [45] Te1949 RSDDIVPSNTAKIKVIGVGGGGGNAVNRMIASEVSGIEFWTVNTD [45] SSNP_440816 KRDQIVPSNIAKIKVIGVGGGGCNAVNRMIASGVTGIDFWAINTD [45] ASCAA83241 RIGEIVPGRVANIKVIGVGGGGGNAVNRMIESDVSGVEFWSINTD [45] Np6l RIGEIVPGRVANIKVIGVGGGGGNAVNRMIESDVSGVEFWSINTD [4S] Tet112382 SYDKLVETSAARIKVIGVGGGGGNAVNRMIASNVAGVEFWCVNTD [45] SyAAC26227 PEELIIPSSVARIKVIGVGGGGSNGVNRMISSDVSGVEFWALNTD [4S] PSCABS62OI QSKDILPSQNAKIEVIGVGGGGSNAVNRMIDSDLEGVSFRVLNTD [45] Pml658 QSKDILPSQNAKIEVIGVGGGGSNAVNRMIDSDLEGVSFRVLNTD [45] PmCAB95028 RSESIQPSQNARIEVIGVGGGGSNAVNRMILSDLQGVSYRVLNTD [45] Sy549 DATGIQPSQNAKIEVIGVGGGGSNAVNRMILSDLEGVAYRVLNTD [45] Ple68 ETAGILPSQSARIEVIGVGGGGSNAVNRMILSDLDGVNYRVMNTD [45] NCCAB41987 ISSSFTPFDSAKIKVIGVGGGGNNAVNRMIGSGLQGVDFYAINTD [45] NtCAB89287 ISSSFTPFDSAKIKVIGVGGGGNNAVNRMIGSGLQGVDFYAINTD [45] TeAAF8122O VCCSFASLDSAKIKVVGVGGGGNNAVNRMIGSGLQGVDFYAINTD [45] AtAAA82068 LRCSFSPMESARIKVIGVGGGGNNAVNRMISSGLQSVDFYAINTD [45] PSCAA75603 VRCSLAYVDNAKIKVVGIGGGGNNAVNRMIGSGLQGVDFYAINTD [4S] NCAAF2377O RPSICSSLSSAKIKVVGVGGGGNNAVNRMIGSGLQGVDFYAVNTD [4S] NtCA889286 RRSICSSLSSAKIKVVGVGGGGNNAVNRMIGSGLQGVDFYAVNTD [45] OSAAK64282 VRCSFAPVETARIKVVGVGGGGNNAVNRMIGSGLQGIEFYAINTD [4S] CrBAB91150 STGYIPFGGDACIKVIGVGGGGGNALNRMINSGLQGVEFWAINTD [45] ECPO6138 MFEPMELTNDAVIKVIGVGGGGGNAVEHMVRERIEGVEFFAVNTD [45] 221 NtCA889288 NtCAC44257 OSCLBl7724_S GlAAF2377l LIBAA96782 ACAAC3S987 ACAAK63846 OSCLOOS296_338 PpCAB76386 PpCABS4558 CrAAM2289l CmBAA85116 CCBAA82871 GtCAAO7676 GSBAA8209O MSAAF3S433 GSBAA82091 Te1949 SSNP_440816 AsCAA8324l Np61 Tet112382 SyAAC26227 PSCABS6201 Pml658 PmCAB95028 Sy549 Pm1268 NtCAB4l987 NtCABB9287 TeAAF8l220 AtAAA82068 PsCAA7S603 NCAAF2377O NtCABB9286 OSAAK64282 CrBAB9llSO ECPO6138 50 6O 7O 8O 90] IQAMRMSPVAAEQRLPIGQELTRGLGAGGNPDIGMNAANESKQAI IQAMRMSPVAAEQRLPIGQELTRGLGAGGNPDIGMNAANESKQAI VQAIRMSPVLPQNRLQIGQELTRGLGAGGNPDIGMNAAKESVESI VQAIKMSPVYLENRLQIGQELTRGLGAGGNPDIGMNAAKESKEAI VQAMRMSPVYPENRLQIGQELTRGLGAGGNPDIGMNAAKESKVSI IQAMRMSPVLPDNRLQIGKELTRGLGAGGNPEIGMNAARESKEVI IQAMRISPVFPDNRLQIGKELTRGLGAGGNPEIGMNAATESKEAI FQAMRMSPIDPDNKLQIGQELTRGLGAGGNPEIGMNAAKESQELV AQAMALSPVPAQNRLQIGQKLTRGLGAGGNPEIGCSAAEESKAMV AQAMALSPVPAQNRLQIGQKLTRGLGAGGNPEIGCSAAEESKAMV AQALATSPVNGKCKVQIGGKLTRGLGAGGNPEIGAKAAEESRDSI VQALKRS-—AAHHTLSIGNKLTRGLGAGGNPEVGRKAAEESCDQI VQALKRS——AAHHTLGIGNKLTRGLGAGGNPEIGRKAAEESCDQI AQALSRS——LAPNTCNIGAKLTRGLGAGGNPEIGRKAAEESRDLI AQALSRV——KTSNSVTIGSEITRGLGAGGKPEVGRQAAEESQAAI AQALSRS-—SAKRRLNIGKVLSRGLGAGGNPAIGAKAAEESREEI AQALGRFQKTHHQVIQIGKQSCRGLGAGGNPEAGRVAAEESKEDI AQALTLS-—RAPKRLQLGQKLTRGLGAGGNPAIGQKAAEESRDEI SQALTNT—-NAPDCIQIGQKLTRGLGAGGNPAIGQKAAEESRDEI AQALTLA—~GAPSRLQIGQKLTRGLGAGGNPAIGQKAAEESRDEI AQALTLA—-GAPSRLQIGQKLTRGLGAGGNPAIGQKAAEESRDEI AQAIAQS--QAHRCLQIGQKLTRGLGAGGNPAIGQKAAEESREDL AQALLHS-—AAPKRMQLGQKLTRGLGAGGNPAIGMKAAEESREEL AQALLQS-—SADRRVQLGQNLTRGLGAGGNPSIGQKAAEESKDEL AQALLQS—-SADRRVQLGQNLTRGLGAGGNPSIGQKAAEESKDEL AQALLQS-—SAENRVQLGQTLTRGLGAGGNPSIGEKAAEESRAEL AQALIQS—-QAQHRLQLGQTLTRGLGAGGNPTIGQKAAEESRTDL AQALLQS-~AASNRVQLGQTLTRGLGAGGNPSIGQKAAEESRAEL AQALLQS--AAENPLQIGELLTRGLGTGGNPLLGEQAAEESKEAI AQALLQS——AAENPLQIGELLTRGLGTGGNPLLGEQAAEESKEAI SQALLQS-—VAHNPIQIGELLTRGLGTGGNPLLGEQAAEESKEAI SQALLQF——SAENPLQIGELLTRGLGTGGNPLLGEQAAEESKDAI AQALLHS——AAENPIKIGELLTRGLGTGGNPLLGEQAAEESKEAI AQALLQS—-TVENPIQIGELLTRGLGTGGNPLLGEQAAEESKEHI AQALLQS-—TVENPIQIGELLTRGLGTGGNPLLGEQAAEESKEHI SQALLNS-—QAQYPLQIGEQLTRGLGTGGNPNLGEQAAEESKEAI AQALAAHn-QALNKVQIGSELTRGLGCGGNPELGRRAAMESEEAL AQALRKT——AVGQTIQIGSGITKGLGAGANPEVGRNAADEDRDAL 222 [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] [90] gwmm" 1 NCCA889288 NCCAC44257 OSCLBl7724_5 G1AAF23771 LIBAA96782 ACAAC35987 ACAAK63846 OSCLOO5296_338 PpCAB76386 PpCABS4558 CrAAM22891 CmBAA85116 CCBAA82871 GtCAAO7676 GsBAA8209O MSAAF35433 GSBAA82091 Te1949 SSNP_440816 AsCAA8324l Np61 Tet112382 SyAAC26227 PsCABS6201 Pm1658 PmCAB95028 Sy549 Pm1268 NCCAB41987 NtCABB9287 TeAAF8122O ACAAA82068 PSCAA75603 NCAAF2377O NtCA889286 OSAAK64282 CrBAB9115O ECPO6138 100 110 120 130 EEAVYGADMVFVTAGMGGGTGTGAAPIIAGTAKSMGILTVGIVTT EEAVYGADMVFVTAGMGGGTGTGAAPIIAGTAKSMGILTVGIVTT QEALYGADMVFVTAGMGGGTGTGGAPVIAGIAKSMGILTVGIVTT EEAVYGADMVFVTAGMGGGTGTGGAPVIAGIAKSMGILTVGIVTT EESVSGADMVFVTAGMGGGTGTGGAPVIAGVAKSMGILTVGIVTT EEALYGSDMVFVTAGMGGGTGTGAAPVIAGIAKAMGILTVGIATT QEALYGSDMVFVTAGMGGGTGTGGAPIIAGVAKAMGILTVGIVTT EQAVSGADMIFVTAGMGGGTGTGGAPVIAGIAKSMGILTVGIVTT EEALRGADMVFVTAGMGGGTGSGAAPIIAGVAKQLGILTVGIVTT EEALRGADMVFVTAGMGGGTGSGAAPIIAGVAKQLGILTVGIVTT AAALQDTDMVFVTAGMGGGTGSGAAPVVAEVARELGILTVGIVTT AEAVRGADLVFVTAGMGGGTGSGAAPVVAEAAREQGCLTVGVVTK AEAVRGADLVFVTAGMGGGTGSGAAPVVAEAAREQGCLTVGVVTK AEAVSAGDLVFVTAGMGGGTGSGAAPIVAEVAKEMGCLTVGVVTK SSAVQGGDLVFVTAGMGGGTGSGAAPIVAKIAKEQGCLTVGVVTK MAVVKNADLVFVTAGMGGGTGSGAAPVVAECAKEAGALTVGVVTK AKALQGGDLVFVTAGMGGGTGTGAAPIVADVARELGCLTVGVVTK ANALDHPDLVFITAGMGGGTGTGAAPVIAEIAKEAGSLTVGVVTR ARSLEGTDLVFITAGMGGGTGTGAAPIVAEVAKEMGCLTVGIVTR ATALEGADLVFITAGMGGGTGTGAAPIVAEVAKEMGALTVGVVTR ATALEGADLVFITAGMGGGTGTGAAPIVAEVAKEMGALTVGVVTR AAALKDADLIFITCGMGGGTGTGAAPIVAEVAKEQGALTVAVVTR IAALEGADLVFITAGMGGGTGTGAAPIVAEVAKEVGALTVGIVTK QQTLEGSDLVFIAAGMGGGTGTGAAPVVAEVAKQSGALTVGIVTK QQTLEGSDLVFIAAGMGGGTGTGAAPVVAEVAKQSGALTVGIVTK QQALEGADLVFIAAGMGGGTGTGAAPVVAEVAKQSGALTVAIVTK HDALQGSDLVFIAAGMGGGTGTGAAPVVAEVAREVGALTVGIVTK QQALQGVDLVFIAVGMGGGTGTGAAPVVAEVAKESGALTVGIVTK ANSLKGSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTY ANSLKGSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTY GNALKGSDLVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTY ANALKGSDLVFITAGMGGGTGSGAAPVVAQISKDAGYLTVGVVTY ANALKGSDLVFITAGMGGGTGSGAAPVVAQISKEAGYLTVGVVTY ANALKGSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTY ANALKGSDMVFITAGMGGGTGSGAAPVVAQIAKEAGYLTVGVVTY ANALKDSDLVFITAGMGGGTGSGAAPVVAQISKEAGYLTVGVVTY RRMVQGADLVFITAGMGGGTGTGAAPVVARLSKELGILTVGVVTY RAALEGADMVFIAAGMGGGTGTGAAPVVAEVAKDLGILTVAVVTK 223 [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] [135] r- NCCABB9288 NCCAC44257 OsCLBl7724_5 G1AAF23771 LlBAA96782 AtAAC35987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM22891 CmBAA85116 CCBAA82871 GCCAAO7676 GsBAA8209O MsAAF35433 GsBAA8209l Te1949 SSNP_440816 ASCAA83241 Np61 Tet112382 SyAAC26227 PsCABS6201 Pm1658 PmCAB95028 Sy549 Pm1268 NtCAB41987 NCCABB9287 TeAAF81220 ACAAA82068 PSCAA75603 NCAAF2377O NCCAB89286 OSAAK64282 CrBAB9llSO ECPO6138 140 150 160 170 180] . ] PFSFEGRRRAVQAQEGIAALRENVDTLIVIPNDKLLTAVSPSTPV PFSFEGRRRAVQAQEGIAALRENVDTLIVIPNDKLLTAVSPSTPV PFSFEGRRRAVQAQEGIAALRNSVDTLIVIPNDKLLSAVSPNTPV PFSFEGRRRAVQAQEGIAALRDNVDTLIVIPNDKLLTAVSPSTPV PFMFEGRRRTVQAQEGIAALRNNVDTLIVIPNDKLLTAVSPNTPV PFSFEGRRRTVQAQEGLASLRDNVDTLIVIPNDKLLTAVSQSTPV PFSFEGRRRALQAQEGIAALRDNVDTLIVIPNDKLLAAVSQSTPV PFAFEGRRRALQAQEGIASLRSNVDTLIVIPNDKLLTAVSPNTPV PFAFEGRRRSVQAHEGIAALKNNVDTLITIPNNKLLTAVAQSTPV PFAFEGRRRAVQAHEGIAALKNNVDTLITIPNNKLLTAVAQSTPV PFTFEGRQRAQQARSALANLRAAVDTLIVIPNDRLLSAMDSNVPI PFAFEGRKRMNQALEAIEALRESVDTLIVVSNDKLLQIVPENTPL PFAFEGRRRMTQALEAIEALRESVDTLIVVSNDKLLQIVPENTPL PFAFEGKRRMQQANDAILNLRNKVDTLIVVSNDKLLQIVPDNTPL PFSFEGRRRMQQAEEAIEALRKEVDTLIVVSNDKLLEIVPENTAL PFGFEGRKRMQQARNAILEMKDKVDTLIVVSNDKLLKIVPDNTPL PFAFEGRRRLQQAVEGLANLREKVDTLIVISNDRLLETVPKDTPL PFTFEGRRRITQADEGITALQTRVDTLIVIPNNRLLSVINDQTPV PFTFEGRRRAKQAEEGINALQSRVDTLIVIPNNQLLSVIPAETPL PFVFEGRRRTSQAEQGIEGLKSRVDTLIIIPNNKLLEVIPEQTPV PFVFEGRRRTSQAEQGIEGLKSRVDTLIIIPNNKLLEVIPEQTPV PFTFEGRRRANQADEGIEALQSRVDTLIVIPNDKILSVISEQTSV PFTFEGRRRMKQAEEGTAALQSSVDTLITIPNDRLLHAISEQTPI PFSFEGKRRMRQAEEGIARLAENVDTLIVIPNDRLKDVIAG-APL PFSFEGKRRMRQAEEGIARLAENVDTLIVIPNDRLKDVIAG-APL PFSFEGRRRMRQADEGIAKLTESVDTLIVIPNDRLKDAIAG-APL PFGFEGRRRMRQADEGIARLAEHVDTLIVIPNDRLREAIAG—APL PFSFEGRRRMRQAAEGIGRLADHVDTLIVIPNDRIKDVISE—APL PFSFEGRKRSVQALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPL PFSFEGRKRSVQALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPL PFSFEGRKRSVQALEAIEKLQKNVDTLIVIPNDRLLDIADENTPL PFSFEGRKRSLQALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPL PFSFEGRKRSLQALEAIEKLQKNVDTLIVIPNDRLLDIADEQMPL PFSFEGRKRSLQALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPL PFSFEGRKRSLQALEAIEKLQKNVDTLIVIPNDRLLDIADEQTPL PFSFEGRKRSLQALEALEKLERSVDTLIVIPNDRLLDVVDENTPL PFNFEGRRRAGQALEGIEALREAVDSVIVIPNDRLLDVAGASTAL PFNFEGKKRMAFAEQGITELSKHVDSLITIPNDKLLKVLGRGISL 224 [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] [180] NCCAB89288 NCCAC44257 OSCLBl7724_5 GlAAF23771 LIBAA96782 ACAAC35987 ACAAK63846 OSCL005296_338 ppCAB76386 PpCABS4558 CrAAM22891 CmBAA85116 CCBAA82871 GtCAAO7676 GSBAA82O9O MSAAF35433 GSBAA82091 Te1949 SSNP_440816 ASCAA83241 Np61 Tet112382 SyAAC26227 PSCA856201 Pm1658 PmCAB95028 Sy549 Pm1268 NCCAB41987 NCCA889287 TeAAF81220 ACAAA82068 PSCAA75603 NtAAF2377O NtCABB9286 OSAAK64282 CrBAB91150 ECPO6138 190 200 210 220 TEAFNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSS TEAFNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSS TEAFNLADDILRQGIRGISDIITVPGLVNVDFADVRAIMQNAGSS TEAFNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSS TEAFNLADDILRQGVRGISDIITVPGLVNVDFADVRAIMANAGSS TEAFNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSS TEAFNLADDILRQGVRGISDIITIPGLVNVDFADVRAIMANAGSS TEAFNLADDILRQGVRGISDIITVPGLVNVDFADVRSVMSDAGSS TEAFNLADDILRQGVRGISDIITVPGLVNVDFADVRAIMANAGSS TEAFNLADDILRQGVRGISDIITVPGLVNVDFADVRAIMANAGSS KDAFKIADDVLRQGVKGISEIITVPGLVNVDFADVRAIMAGAGSS QDAFRVADDILRQGVVGISDIIIRPGLINVDFADVRSVMAHAGSA QDAFRVADDILRQGVVGISDIIIRPGLINVDFADVRSVMAHAGSA QDAFSVADDILRQGVVGISEIIVRPGLINVDFADVRSVMADAGSA EKAFSVADDILRQGVVGISEIIVRPGLINVDFADVRSIMADAGSA TEAFLVADDILRQGVVGITEIIVKPGLVNVDFADVRTIMGNAGTA TEAFIFADEVLRQGVGGISDIITKPGLVNVDFADVRTVMAEKGFA QEAFIIADDILRQGIQGISDIITVPGLVNVDFADVRAVMADAGSA QEAFRVADDILRQGVQGISDIIIIPGLVNVDFADVRAVMADAGSA QEAFRYADDVLRQGVQGISDIITIPGLVNVDFADVRAVMADAGSA QEAFRYADDVLRQGVQGISDIITIPGLVNVDFADVRAVMADAGSA QDAFRVADDVLRQGVQGISDIINVPGLINVDFADIRSVMADAGSA QEAFRVADDILRQGVQGISDIITIPGLVNVDFADVRAVMADAGSA QEAFRNADDVLRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTA QEAFRNADDVLRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTA QEAFKNADDVLRMGVKGITDIITLPGLVNVDFADVRSVMTEAGTS QEAFRSADDVLRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTA QEAFRSADDILRMGVKGISDIITCPGLVNVDFADVRSVMTEAGTA QDAFLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTA QDAFLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTA QDAFLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTA QDAFLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTA QDAFRLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKDSGTA QNAFLLADDVLCQGVQGISDIITIPGLVNVDFADVKAIMKDSGTA QNAFLLADDVLCQGVQGISDIITIPGLVNVDFADVKAIMKDSGTA QDAFLLADDVLRQGVQGISDIITIPGLVNVDFADVKAVMKNSGTA QDAFALADDVLRQGVQGISDIITVPGLINVDFADVKAIMSNSGTA LDAFGAANDVLKGAVQGIAELITRPGLMNVDFADVRTVMSEMGYA 225 [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] [225] NCCA889288 NCCAC44257 OSCLBl7724_5 G1AAF23771 L18AA96782 ACAAC35987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM22891 CmBAA85116 CCBAA82871 GtCAAO7676 GSBAA8209O MSAAF35433 GSBAA82O91 Te1949 SSNP_440816 AsCAA83241 Np61 Tet112382 SyAAC26227 PSCABS6201 Pm1658 PmCAB95028 Sy549 Pm1268 NCCAB41987 NtCA889287 TeAAF81220 AtAAA82068 PsCAA7S603 NCAAF2377O NCCAB89286 OSAAK64282 CrBAB9115O ECPO6138 230 240 250 260 270] .] LMGIGTATGKTRARDAALNAIQSPLLDIG-IERATGIVWNITGGS LMGIGTATGKTRARDAALNAIQSPLLDIG-IERATGIVWNITGGS LMGIGTATGKSRARDAALNAIQSPLLDIG—IERATGIVWNITGGA LMGIGTATGKTRARDAALNAIQSPLLDIG-IERATGIVWNITGGS LMGIGTATGKTRARDAALNAVQSPLLDIG-IERATGIVWNITGGN LMGIGTATGKSRARDAALNAIQSPLLDIG—IERATGIVWNITGGS LMGIGTATGKTRARDAALNAIQSPLLDIG—IERATGIVWNITGGS LMGIGTATGKTRARDAALNAIQSPLLDIG-IERATGIVWNITGGN LMGIGTATGKSKAREAALSAIQSPLLDVG~IERATGIVWNITGGS LMGIGTATGKSRAREAALSAIQSPLLDVG-IERATGIVWNITGGS LMGQGYGSGPRRASDAALRAISSPLLEVG-IERATGVVWNITGPP LMGIGTGSGKSRAHDAAVAAISSPLLDFP-IERAKGIVFNVTGGE LMGIGTGSGKSRAHDAAVAAISSPLLDFP-IERAKGIVFNVTGGE LMGIGTGSGKTRAQDAAVAAISSPLLDFP-IEKARGIVFNITGGQ LMGIGSGSGKSRAKDAAVAAISSPLLDFP-IERAKGIVFNITGGH LMGIGHGKGKNRAKDAALSAISSPLLDFP-ITRAKGIVFNIVGGS LLGIGTASGDSRARNAATAAISSPLLDFP-ITSAKGAVFNITGGT LMGIGMGSGKSRAREAANAAISSPLLESS-IEGAKGVVFNITGGT LMGIGVGSGKSRAKEAATAAISSPLLESS-IQGAKGVVFNVTGGT LMGIGVSSGKSRAREAAIAAISSPLLECS-IEGARGVVFNITGGS LMGIGVSSGKSRAREAAIAAISSPLLECS-IEGARGVVFNITGGT MMGIGIASGKSRATEAALSAISSPLLERS-IEGAKGVVFNITGGT LMGIGSGSGKSRAREAAHAAISSPLLESS—IEGARGVVFNITGGR LLGIGIGSGRSRALEAAQAAMNSPLLEAARIDGAKGCVINITGGK LLGIGIGSGRSRALEAAQAAMNSPLLEAARIDGAKGCVINITGGK LLGIGIGSGRSRAAEAAQAAINSPLLEAGRIDGAKGCVVNITGGK LLGIGIGSGRSRAVEAAQAAISSPLLETERIDGAKGCVINISGGR LLGIGEGSGRSRAIEAAQAAISSPLLEAARIDGAKGCVINISGGR MLGVGVSSSKNRAEEAAEQATLAPLIGSS-IQSATGVVYNITGGK MLGVGVSSSKNRAEEAAEQATLAPLIGSS-IQSATGVVYNITGGK MLGVGVSSSKNRAEEAAEQATLAPLIGSS-IQSATGVVYNITGGK MLGVGVSSSKNRAEEAAEQATLAPLIGSS—IQSATGVVYNITGGK MLGVGVSSGKNRAEEAAEQATLAPLIGSS-IQSATGVVYNITGGK MLGVGVSSSRNRAEEAAEQATLAPLIGSS—IQSATGDVYNITGGK MLGVGVSSSRNRAEEAAEQATLAPLIGLS-IQSATGVVYNITGGK MLGVGVSSSKNRAQEAAEQATLAPLIGSS~IEAATGVVYNITGGK MLGVGAASTADRAEQAAVAATSAPLIQRS-IEKATGIVYNITGGR MMGSGVASGEDRAEEAAEMAISSPLLEDIDLSGARGVLVNITAGF 226 [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] [270] 270] 270] 270] [270] [270] [270] [270] [270] [270] [270] l—\r-'\F—'Q NCCA889288 NCCAC44257 OSCLBl7724_5 GlAAF23771 LIBAA96782 AtAAC35987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCA854558 CrAAM22891 CmBAA85116 CCBAA82871 GtCAAO7676 GsBAA8209O MsAAF35433 GSBAA82091 Te1949 SSNP_440816 ASCAA83241 Np61 Tet112382 SyAAC26227 PSCABS6201 Pm1658 PmCAB95028 Sy549 Pm1268 NCCAB41987 NtCA889287 TeAAF81220 ACAAA82068 PSCAA756O3 NCAAF2377O NtCA889286 OSAAK64282 CrBAB91150 ECPO6138 280 290 300 310 DLTLFEVNAAAEVIYDLVDPSANLIFGAVIDPSISGQVSITLIAT DLTLFEVNAAAEVIYDLVDPSANLIFGAVIDPSISGQVSITLIAT DMTLFEVNSAAEIIYDLVDPNANLIFGAVIDPSLNGQVSITLIAT DLTLFEVNAAAEVIYDLVDPSANLIFGAVVDPSLCGQVSITLIAT DLTLYEVNAAAEVIYDLVDPAANLIFGAVIDPSISGQVSITLIAT DLTLFEVNAAAEVIYDLVDPTANLIFGAVVDPALSGQVSITLIAT DLTLFEVNAAAEVIYDLVDPTANLIFGAVVDPSYSGQISITLIAT DLTLTEVNAAAEVIYDLVDPGANLIFGSVIDPSYTGQVSITLIAT DMTLFEVNAAAEVIYDLVDPNANLIFGAVVDEALHDQISITLIAT DMTLFEVNAAAEVIYDLVDPNANLIFGAVVDEALHGQVSITLIAT NMTLHEVNEAAEIIYDMVDPNANLIFGAVVDSTLPDTVSITIIAT DMTLHEINQAAEVIYEAVDPNANIIFGALIDQQMESEISITVVAT DMTLHEINQAAEVIYEAVDPNANIIFGALVDQQMESEISITVVAT DMTLHEINSAAEVIYEAVDSNANIIFGALVDDNMENEISITVVAT DMTLHEINAAAEVIYEAVDLNANIIFGALVDDSMENELSITVIAT DMSLQEINAAAEVIYENVDQDANIIFGAMVDDKMTSEVSITVLAT DMTLSEVNQAAQVIYDSVDSDANIIFGAVVDETFKGKVSVTVVAT DLTLHEVNAAAEIIYEVVDPNANIIFGAVIDDKLQGEIKITVIAT DLTLHEVNVAAEIIYEVVDADANIIFGAVIDDRLQGEMRITVIAT DLTLHEVNAAAETIYEVVDPNANIIFGAVIDDRLQGEVRITVIAT DLTLHEVNAAAEAIYEVVDPNANIIFGAVIDDRLQGEVRITVIAT DLSLHEVNAAADVIYNVADANANIIFGAVIDPQMQGEVQITVIAT DMTLHEVNAAADAIYEVVDPEANIIFGAVIDDRLEGELRITVIAT DMTLEDMTSASEIIYDVVDPEANIIVGAVIDESMEGEIQVTVIAT DMTLEDMTSASEIIYDVVDPEANIIVGAVIDESMEGEIQVTVIAT DMTLEDMTSASEVIYDVVDPEANIIVGAVIDEALEGEVQVTVIAT DMTLEDMTTASEVIYDVVDPEANIIVGAVVDEALEGEIHVTVIAT DMTLEDMTSASEVIYDVVDPEANIIVGAVVDEKLEGEVHVTVIAT DITLQEVNRVSQVVTSLADPSANIIFGAVVDERYNGEIHVTIIAT DITLQEVNRVSQVVTSLADPSANIIFGAVVDERYNGEIHVTIIAT DITLQEVNRVSQVVTSLADPSANIIFGAVVDERYNGEIHVTIVAT DITLQEVNRVSQVVTSLADPSANIIFGAVVDDRYTGEIHVTIIAT DITLQEVNRVSQVVTSLADPSANIIFGAVVDDRYTGEIHVTIIAT DITLQEVNKVSQVVTSLADPSANIIFGAVVDERYNGEIQVTLIAT DITLQEVNKVSQVVTSLADPSANIIFGAVVDERYNGEIQVTLIAT DITLQEVNKVSQIVTSLADPSANIIFGAVVDDRYTGEIHVTIIAT DLTLAEVNRVSEVVTALADPSCNIIFGAVVDEQYDGELHVTIIAT DLRLDEFETVGNTIRAFASDNATVVIGTSLDPDMNDELRVTVVAT 227 [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] [315] Fr NCCABB9288 NCCAC44257 OSCLBl7724_5 G1AAF23771 LIBAA96782 ACAAC35987 ACAAK63846 OSCL005296_338 PpCAB76386 PpCABS4558 CrAAM22891 CmBAA85116 CCBAA82871 GCCAAO7676 GSBAA8209O MSAAF35433 GSBAA82091 Te1949 SSNP_440816 ASCAA83241 Np61 Tet112382 SyAAC26227 PSCABS6201 Pm1658 PmCAB95028 Sy549 Pm1268 NCCAB41987 NtCA889287 TeAAF81220 AtAAA82068 PSCAA75603 NCAAF2377O NCCAB89286 OSAAK64282 CrBAB9115O ECPO6138 320 330 340 350 360] -] GFKRQE ——————— ESDGRPLQ—GNQLTQGDVS —————————— LGN GFKRQE ——————— ESDGRPLQ—GNQLTQGDAS —————————— Les GFKRQD ------- EPEGRTTK ________________________ GFKRQE ------- ESDKRSIQAGGQLAPGDAN ---------- QGI GFKRQD ——————— ETEGQKSQ———GTQLGLGGN ————————— LGI GFKRQE ------- EGEGRTVQ~—-—MVQADAAS --------- VGA GFKRQE ------- EGEGRPLQ——--ATQADAS —————————— MGA GFKRQE ——————— EAESRQEE ________________________ GFSSQD ——————— DPDARSMQYASRVLEGQAG ———————————— R GFSSQD ——————— EPDARSMQNVSRILDGQAG ———————————— R GFGHVEPELGALADRGSRAAAAASPRVAAAANAPAAAAAVPPVTT GFPQ ————————— PNESANSGGSSSTLNATANEFYQAG—PPNRQV GFPQ ————————— PNESASNGGTSSTLNATASDFYQAG—PSARPA GFTQ --------- PNDSK --------------- FFSTK ------- GFPQ ————————— PSDSPSSS ———————————— MTQTP ------- GFSTDY ------ FSNDGSGLENLPPNRLSPPKTVGSAK—KPKG-— GFS ------------------------------------------ GFSGEVQT—-—-QPIQEKVQP ———————— RRPVPNPTQN ------ GFNGEKEK-—-—PQAKTSSKPVLSGPPAGVETVPSTTTP ------ GFTGEIQA----APQQNAANARVVSAPPKRTPTQTPLTN ----- s GFTGEVQA————AVQQSVASVRVAPNTSKRPTTQQPAVNPSSTPT GFSGEPMS—-——RTRATTKTTPLTN——--RPLATTSPPP —————— GFSTDRPN————LNTISTSTS ————————— QPTSQPSVS ------ GFETNQPL----KQQRIKNR ——————————— LSNQPLYN ______ GFETNQPL--——KQQRIKNR ——————————— LSNQPLYN ______ GFDGNQPY——-—TKQKAGAK ——————————— LSPQSLYR ______ GFDQGQQY——--RSDRSSASG —————————— LPVQPQRS ______ GFEGNQPY-———RSERSINK ——————————— IASQSIYS ...... GFTQSFQKTLLSDPRGAKLADKGPVIQESMASPVTLRS ------- GFTQSFQKTLLSDPRGAKLADKGPVIQESMASPVTLRS ------- GFAQSFQKSLLADPKGAKLVDRNQEPTQPLTSARSLTT ------- GFSQSFQKTLLTDPRAAKLLDKMGSSGQQENKGMSLPHQ ------ GFSQSFQKKLLTDPRAAKLLDKVAEGKESKTVPPPLKSS ------ GFAQSFQNSLLTDPRGAKLVDKSKGTTERTVSPDTLRS ------- GFAQSFQNSLLTDPRGAKLVDKSKGTTERTVSPDTLRS ------- GFPQSFQKSLLADPKGARIMEAKEKAANLTYKAVAAAT ------- GFAPTYENELLNGGNAQQQQARAARRASNQATAASLAPNPAVQPT GIGMDKRPEITLVTNKQVQQPVMDRYQQHGMAPLTQEQKPVAKVV 228 [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] [360] 5‘ [ 370 380 390 ] NCCAB89288 NRRPAS—FLEGGSVEIPEFLRKKGRSRYPRA —————— [397] NCCAC44257 NRRPAS—FLEGGSVEIPEFLRKKGRSRYPRA ------ [397] OSCLBl7724_5 VQKLCVPMIGEIESGITVTIQRQRI ------------ [397] GIAAF23771 NRRPSS—FSESGSVEIPEFLRKKGRSRYPRA ------ [397] LlBAA96782 NRRPSSSMTMGGIVEIPHFLRKKAGSRNPRA ------ [397] ACAAC35987 TRRPSSSFRESGSVEIPEFLKKKGSSRYPRV ------ [397] ACAAK63846 TRRPSSSFTEGSSIEIPEFLKKKGRSRYPRL ------ [397] OSCL005296_338 ---------- GPTLQIPEFLQRKGRSGFSRG ------ [397] PpCAB76386 SSMASSRGGNSSTINIPNFLRKRGQR ——————————— [397] PpCAB54558 SPTGLSQGSNGSAINIPSFLRKRGQTRH --------- [397] CrAAM22891 AAPETPGGASSSGVEIPAFLRRRRVQGK --------- [397] CmBAA85116 TPPPPQPGPSRTIGNIPDFLRRFQK ------------ [397] CCBAA82871 SQPPSQTGPSRSIGSIPDFLRRFQK ------------ [397] GtCAAO7676 ----------------- DLWKKFY ------------- [397] GSBAA8209O -------------- DIPDFLRRFQQENK --------- [397] MSAAF35433 ------------- GGFRGFIKRLFS ------------ [397] GSBAA82091 ------------------------------------- [397] Te1949 PNSTPEPQRKLPGLDIPDFLQRRRNPSNK -------- [397] SSNP_440816 EDPLGEIPMA-PELDIPDFLQKRRFPRR --------- [397] ASCAA83241 PAPTPEPKEK—SGLDIPDFLQRRRPPKN --------- [397] Np61 PTPTPEPKEK-PGLDIPDFLRNRRTPRN --------- [397] Tet112382 EAPAPEVEAK-PKLDIPEFLQRRRPTP ---------- [397] SyAAC26227 PNPASAPPASGGGLDIPAFLQRKIQNRP --------- [397] PSCAB56201 ----- ISDNKDTGTNIPEFLRLRQNKKDIE ------- [397] Pm1658 ----- ISDNKDTGTNIPEFLRLRQNKKDIE ------- [397] PmCAB95028 ————— QTPNKEPGASIPEFLRLRQLRRDQ -------- [397] Sy549 ----- AIE--ENGARIPEFLRQRQQQTNDPT ------ [397] Pm1268 ----- QPEANESGARIPEFLRKRQPRNDNEI ------ [397] NCCAB41987 ----------- *—-~STSPSTTSRTPTRRLFF ------ [397] NtCABB9287 —————————————— STSPSTTSRTPTRRLFF —————— [397] TeAAF8122O --------------- PSPAPSR---SRKLFF ------ [397] ACAAA82068 ———————————— KQSPSTISTKSSSPRRLFF —————— [397] PSCAA75603 ———————————— NFS—SKVESRPPPPRKLFF ------ [397] NCAAF2377O -------------- SESPSTKPRPATRRLFF ------ [397] NtCABB9286 —————————————— SESPSTKPRPAARRLFF ------ [397] OSAAK64282 ---------------- VQPAPAATWSRRLFS ------ [397] CrBAB9115O T ----------- PPANPAAPWSRPNRAKLDFLGRSIL [397] ECPO6138 NDNAPQTAKEPDYLDIPAFLRKQAD ------------ [397] END; 229 LITERATURE CITED Addinall SG, Cao C, Lutkenhaus J (1997) Temperature shift experiments with an ftsZ84(Ts) strain reveal rapid dynamics of FtsZ localization and indicate that the Z ring is required throughout septation and cannot reoccupy division sites once constriction has initiated. 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