n ‘ .. I?» n. . :1) 5|. V C 3.." ,.l 3.4.1:. .. 5., In“ . r u an”. I. glutr... JG...» , .I ‘I‘E...J!4I Q . I J. V 1. the. @3an Pi twat-1V 3 21 0433 This is to certify that the dissertation entitled Virulence of Pseudomonas syringae pv. tomato strain DCBOOO on Arabidopsis thaliana presented by Julie Zwiesler-Vollick has been accepted towards fulfillment of the requirements for the PhD. degree in Genetics 2 Major Professor's Signature /ol v/o-roZ Date MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University 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 c:/CIRC/DateDue.p65-p.15 VIRULENCE OF PSEUDOMONAS SYRINGAE PV. TOMA T0 STRAIN DC3000 ON ARABIDOPSIS THALIANA BY JULIE ZWIESLER-VOLLICK A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 2002 ABSTRACT VIRULENCE OF PSEUDOMONAS SYRINGAE PV. TOMA TO STRAIN DC3000 ON ARA BIDOPSIS THA LIA NA By Julie Zwiesler-Vollick Bacterial diseases are an important cause of economic loss to the agricultural community. The Gram negative plant pathogenic bacterium, Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) has emerged as a model for the study of plant- microbe interactions because it infects the model plant Arabidopsis thaliana. Disease symptoms caused by Pst DC3000 include water-soaking, followed by the development of necrotic spots surrounded by diffuse chlorotic halo. The Pst DC3000-A. thaliana interaction is being used to understand the processes that underlie the complex pathogen- host interactions. My thesis work has focused on how Pst DC3000 causes disease. One virulence system essential for Pst DC3000 pathogenesis is the type 111 protein secretion system. The type III protein secretion system is found in a variety of Gram negative bacterial pathogens, and is thought to deliver bacterial proteins, termed effectors, directly into the host cell cytoplasm. However, the identities and functions of the type III effector proteins of bacterial plant pathogens remain mysterious. My research provides new information on type III effector proteins in Pst DC3000, Pst DC3000 mutants with reduced virulence on A. thaliana, and the effect of transgenic expression of a Pst DC3000 type III effector, Aer, on A. thaliana. In collaboration with other members of the lab, I analyzed the Pst DC3000 genome for the presence of a cis promoter element, the hrp box, which is found in all known Pst DC3000 type III effector genes. The expression of these hrp box-containing genes was then assessed with both microarray and northern blot analyses. hrp box- containing genes which showed expression only in minimal medium were further characterized with an in planta translocation assay. This study revealed Six orthologues of effectors known in other P. syringae pathovars and eight novel candidate effectors, one of which was shown to be secreted via the type 111 protein secretion system. This work has contributed to the knowledge of the effector inventory in Pst DC3000. I also analyzed Pst DC3000 mutants which showed reduced growth in A. thaliana. Reduced virulence mutants with insertions in the PtsP, Uer, and 0er genes were isolated. The PtsP gene encodes a phosphoenolpyruvate protein phosphotransferase, which is involved in sugar uptake and catabolite repression. Uer encodes a DNA helicase II involved in DNA replication and repair. The 0er gene encodes an outer membrane protein F precursor. This protein has been implicated in adaptation to low-osmolarity environments and host cell adhesion. These genes had previously not been implicated in Pst DC3000 virulence. Aer is a type III effector in Pst DC3000. In order to study the affect of Aer on the host plant, I expressed the aer gene in A. thaliana under the control of a DEX- inducible promoter. After induction with DEX, aer transgenic plants developed symptoms which mimicked Pst DC3000 infection and stomatal aperture was affected. In addition, expression of the aer transgene promotes the grth of the non-pathogenic hipH mutant, which cannot deliver any type III effectors. These data suggest that Aer may promote Pst DC3000 pathogenesis in A. thaliana. Copyfightby Julie Zwiesler-Vollick 2002 To my family Acknowledgements I would like to thank my thesis advisor, Dr. Sheng Yang He, for allowing me to do research in his lab. I have thoroughly enjoyed my thesis work, and learned a great deal. I would like to thank Dr. Sheng Yang He for his support, guidance and allowing for our differences in opinion. I would like to thank all of my guidance committee members, Dr. Frans de Bruijn, Dr. Pamela Green, Dr. Jonathan Walton, Dr. Gregg Howe, and Dr. Rebecca Grumet. They have given me wonderful support and guidance over the course of my studies here at MSU. I would like to thank all the members of the He lab, past and present: Suresh Gopalan, Jing Yuan, Wensheng Wei, Anne E. Plovanich-Jones, Wenqi Hu, Mingbo Lu, Yong Bum Kwak, Qiaoling J in, Roger Thilmony, Kinya Nomura, Ola Kolade, Paula Hauck, Sruti Bandyopadhyay, Elena Bray Speth, and Bill Underwood. All of these people have made my time in the He lab unforgettable. We have had many stimulating and interesting scientific discussions as well as sharing both our successes and our fi'ustrations. I thank you all. I gratefully acknowledge all the help that I have received from members of the lab. I would especially like to thank Anne E. Plovanich-J ones, my friend and colleague with whom I had the good fortune to collaborate with on the work described in Chapter 2. I would like to thank Sruti Bandyopadhyay and Kinya Nomura for their work on this project as well. I also acknowledge my collaborators at Washington University in St. Louis, Barbara Kunkel and Vinita J oardar, for providing the hrpL mutant used in Chapter 2. I would like to thank Wensheng Wei and Xin Rong for their contribution to vi the work described in Chapter 3. Finally, I would like to thank three students whom I was fortunate to supervise during their rotations. I thank Elena Bray Speth, Ying Yan, and Guanghui Liu for their efforts on a project not described in this thesis. I would like to thank all the support staff at the PRL and the genetics program. All their help through the years has been instrumental in helping me to successfully complete my degree. I would also like to thank the wonderful undergraduates who have worked in our lab. I hope that they have benefited, as I know I have, from our interactions. I would like to thank all the people in the PRL. The PRL is a wonderful place to do research and I thank everyone for their support and advice over the past years. I feel truly lucky to have been a part of this scientific community. Finally I would like to thank all of my family for their encouragement and support over the course of my graduate work, even if they didn’t really know exactly what I was doing. I would like to thank my parents, Marty and Lynn Zwiesler, for their encouragement and for telling me that I never had any limitations in what I could do. I would like to thank Orion (aka Big) and Andy (aka Bitty) for their love and for sitting on those papers I was trying to read when I really needed a break. I would especially like to thank my husband, Michael, for all of his love, support, and encouragement. Thanks for all the times that you put up with my quirks and put things in perspective. We have completed this thesis work together and I could not have done it without you. A.M.D.G. vii TABLE OF CONTENTS List of Tables ........................................................................................ xi List of Figures ....................................................................................... xii Chapter 1 Literature Review and Introduction ................................................................ 1 Introduction ................................................................................... 2 Virulence mechanisms of plant pathogenic bacteria .................................... 3 T-DNA transfer .............................................................................. 4 Extracellular polysaccharides .............................................................. 5 Cell wall-degrading enzymes .............................................................. 7 Toxins ......................................................................................... 9 Phytotoxins of the phytopathogenic pseudomonads .......................... 10 The type III proteins secretion system ................................................... 13 Type III protein secretion in the phytopathogenic pseudomonads ......... 14 Type III secretion system appendages .......................................... 14 Regulation of the type 111 protein secretion system ........................... 15 Genome-wide inventory of effector proteins .................................. 17 Virulence role of type III effector proteins .................................... 24 Project summary ........................................................................... 27 References .................................................................................. 31 Chapter 2 Identification o f n ovel h m-regulated genes through functional genomic a nalysis o f t he Pseudomonas syringae pv. tomato DC3000 genome .......................................... 41 Abstract ...................................................................................... 42 Introduction ................................................................................. 43 Materials and Methods ..................................................................... 46 Computer analysis ................................................................. 46 Construction of an hrpL mutant of Pst DC3000 .............................. 47 Bacterial DNA isolation and polymerase chain reaction (PCR) amplification .............................................................. 47 Microarray slide preparation and analysis ..................................... 48 Isolation of bacterial RNA ....................................................... 48 Aminoallyl labeling of bacterial RNA .......................................... 49 Northern hybridization ............................................................ 50 Aerpt2 fusion translocation analysis .......................................... 50 Primers used to amplify chp genes for making aerptZ fusions. . . . . . . . ....51 Further genomics searches ....................................................... 52 Results ....................................................................................... 53 Computer-assisted identification of putative ‘hrp box’-containing genes ....................................................................... 53 Gene expression analysis of putative ‘hrp box’-containing genes .......... 56 viii Type III translocation analysis of selected ‘hrp—box’-containing genes ....................................................................... 62 Further computer analysis of Chp proteins .................................... 65 Discussion ................................................................................... 67 References ................................................................................... 74 Chapter 3 A bacterial mutagenesis approach for the discovery of genes required for Pseudomonas syringae pv. tomato strain DC3000 virulence in Arabidopsis ............... 79 Abstract ...................................................................................... 80 Introduction ................................................................................. 81 Materials and Methods ..................................................................... 84 Bacterial culture conditions ...................................................... 84 Transposon mutagenesis ......................................................... 84 Screening of mutants for uidA expression ..................................... 85 Pathogenesis assays ............................................................... 85 Growth assays in liquid media ................................................... 86 Southern blot analysis of bacterial mutants .................................... 86 Cloning and sequencing of transposon-containing fragments ............... 87 UV tolerance assays ............................................................... 88 Complementation of bacterial mutations ....................................... 88 Results ....................................................................................... 90 Isolation of Pst DC3000 mutants with differential uidA expression in LB vs. MM ..................................................................... 90 Screening for mutants with reduced virulence ................................. 93 Identification of the insertionally-inactivated genes .......................... 99 Complementation of mutations ................................................ 102 Discussion ................................................................................. 107 References ................................................................................. 1 12 Chapter 4 Characterization of transgenic Arabidopsis thaliana plants that express the Aer effector of Pseudomonas syringae pv. tomato strain DC3000 ......................................... 116 Abstract .................................................................................... 117 Introduction ................................................................................ 1 18 Materials and Methods ................................................................... 121 Generation of Transgenic plants ............................................... 121 Southern blot analysis ........................................................... 122 Dexamethasone-induction of transgene expression ......................... 123 Northern blot analysis ........................................................... 123 Bacterial culture conditions .................................................... 123 Pathogenesis assays ............................................................. 124 Evaluation of stomatal opening ................................................ 124 Results ...................................................................................... 126 Generation of transgenic plants and examination of expression of ssaer ................................................................ 126 ix ssaer transgenic plants Show two distinct phenotypes .................... 128 DEX-induction of ssaer causes stomatal closure .......................... 130 DEX-induction of ssaer promotes enhanced bacterial grth .................................................................... 135 Discussion ................................................................................. 1 3 8 References ................................................................................. 143 Chapter 5 Conclusions and future perspectives ............................................................ 147 References ................................................................................. 1 59 Appendix A Supplementary material for Chapter 2 .......................................................... 161 Appendix B Supplementary material for Chapter 3 ........................................................... 172 LIST OF TABLES Table 1-1. A list of confirmed and putative type III effector proteins in Pseudomonas syringae ...................................................................................... 22 Table 2-1. Features of ‘hrp box’ sequences and expression analysis of ‘hrp box’- containing genes ............................................................................ 59 Table 2-2. Properties of Chp proteins ............................................................ 66 Table 3-1. Summary of the identification of the insertionally—inactivated genes. . . . . ....101 Table 4-1. DEX—induction of ssaer causes stomatal closure .............................. 132 Table 4-2. Pst DC3000 infection of Col-0 gl causes stomatal closure ..................... 132 Table 4-3. Time course of stomatal response ................................................. 133 Table 4-4. Artificial water-soaking of Col-O g1 leaves causes stomatal opening. . . . . ....133 Table A-1. ‘Hrp box’-containing open-reading frames (HCOS) ........................... 162 xi LIST OF FIGURES Images in this dissertation are presented in color. Fig. 2-1. Consensus ‘hrp-box’ sequence in P. syringae pv. tomato DC3000 ............... 55 Fig. 2-2. Northern blot analysis of eight novel ‘hrp-box’-containing genes in Pst DC3000(WT) and hrpL (L-) and hrpS (S-) mutants ................................... 61 Fig. 2-3. Symptoms on RPSZ and rpsZ Arabidopsis leaves infiltrated with DC3000, DC3000(pUCP19::Aerpt2), or DC3000 (pUCPl9::effector-Aerpt230-255 fusion) ....................................................................................... 64 Fig. 3-1. The Pst DC3000 mutant isolation strategy .......................................... 91 Fig. 3-2. uidA expression of selected mutants .................................................. 92 Fig. 3-3. Symptoms of Pst DC3000 mutants in A. thaliana Col-O g1 plants ............... 95 Fig. 3-4. Bacterial proliferation in A. thaliana Col-O gl plants ............................... 96 Fig. 3-5. Growth of Pst DC3000 mutants in LB medium ..................................... 97 Fig. 3-6. Growth of Pst DC3000 mutants in MM .............................................. 98 Fig. 3-7. Southern blot analysis of the genomic DNA from Pst DC3000 mutants ....... 100 Fig. 3—8. Complementation of in planta growth of the W56 mutant by the 14er gene .................................................................................. 104 Fig. 3—9. Complementation of UV tolerance of the W56 mutant by the :4er gene .................................................................................. 105 Fig. 3-10. Complementation of in planta growth of the X4 mutant by the ptsP gene ................................................................................... 106 Fig. 4-1. Southern blot and northern blot analyses of ssaer transgenic plants .......... 127 Fig. 4-2. Phenotypes of ssaer transgenic plants ............................................. 129 Fig. 4-3. The majority of the stomata in the ssaer plants are closed under high light and humidity .................................................................................... 134 Fig. 4-4. The hrpH mutant is able to proliferate in ssaer transgenic plants ............. 136 xii Fig. 4-5. Bacterial proliferation in A. thaliana Col-O gl and ssaer transgenic plants ....................................................................................... 137 Fig. 8-1. Diagram of the predicted operon structure of the Pst DC3000 ptsP gene region ................................................................................. 173 Fig. B-2. Translation of the Pst DC3000 ptsP genomic region ............................. 174 Fig. B-3. Alignment of the Pst DC3000 ptsP predicted protein with the corresponding protein in Azotobacter vinelandii ....................................................... 177 Fig. B-4. Diagram of the predicted operon structure of the Pst DC3000 uer gene region ....................................................................................... 179 Fig. B-S. Translation of the Pst DC3000 uer genomic region ............................ 180 Fig. B-6. Alignment of the Pst DC3000 uer predicted protein with the corresponding protein in Pseudomonas aeruginosa ...................................................... 183 Fig. B-7. Diagram of the predicted operon structure of the Pst DC3000 oer gene region ....................................................................................... 185 Fig. 8-8. Translation of the Pst DC3000 oer genomic region ............................ 186 Fig. 8-9. Alignment of the Pst DC3000 oer predicted protein with the corresponding protein in Pseudomonas syringae pv. syringae ....................................... 188 xiii Chapter 1 Introduction and Literature review Virulence of bacterial phytopathogens Introduction Plant pathogens are a Significant cause of crop loss worldwide. These losses of major crop species for the years 1988-1990 are estimated at $76.9 billion (US), accounting for approximately 13.3% of the estimated production. A significant proportion of this loss occurs in Asia ($43.8 billion US) where the majority of the world’s population resides and thus the need for a reliable food supply is great (Orke et al., 1994). In addition to concern about naturally occurring plant pathogens, there is now concern in the wake of September 11, 2001 , that plant pathogens could be used as agents of bioterror. Among the variety of organisms which can cause disease are bacterial plant pathogens. These pathogens can be classified into two groups on the basis of their Gram- staining reactions. Gram-staining can be either positive or negative and is based on the properties of the bacterial cell wall. The Gram-positive bacterial plant pathogen genera include Streptomyces and Clavz'bacter. The Gram-negative genera include Ralstom'a, Xanthomonas, Erwim'a, Agrobacterium, Xylella, and Pseudomonas. The bacterial plant pathogens significantly impact agricultural economics around the world. For example, Xylellafastidiosa is a pathogen of grape vines (Hopkins, 1981), and it has recently become a problem for citrus fruits (Simpson et al., 2000). The vineyards of California will have increasing difficulty with the control of Xylella due to importation of the glassy-winged sharpshooter, the preferred insect vector for Xylella (Purcell and Saunders, 1999). In addition, Erwim'a amylovora devastates orchards in the Eastern United States to such an extent that it has virtually eliminated the commercial pear industry from these locations (Jones and Sutton, 1996). It is also important to note that several bacterial pathogens have been used as model systems to further our knowledge of the molecular basis of plant-pathogen interactions. Virulence mechanisms of plant pathogenic bacteria Plant bacterial pathogens utilize a variety of virulence mechanisms to infect their plant hosts. Unlike many intracellular animal bacterial pathogens, most bacterial phytopathogens remain outside the plant cell. This extracellular lifestyle probably requires the bacteria to find ways to release water and nutrients from the host cell into the apoplastic space. In addition, the extracellular space is also thought to contain anti- microbial defense compounds. Plant cells, in addition to the cell membrane of animal cells, contain an additional barrier, the plant cell wall. Plant bacterial pathogens have devised a number of methods to overcome these barriers to modulate plant cell functions: i) injection of bacterial DNA, ii) production of extracellular polysaccharides, iii) degradation of the host cell wall, iv) production of toxins which harm the plant cells, and v) transfer of bacterial effector proteins directly into the plant cell. There may be additional virulence mechanisms which have not yet been discovered. Interestingly, many of the virulence factors described rely upon specific bacterial secretion systems. These bacterial secretion systems have recently reviewed thoroughly (Lee and Schneewind, 2001). I will discuss only those secretion systems shown to pertain to plant- bacterial interactions. The sequencing of a number of plant bacterial pathogen genomes has also begun to aid in our understanding of phytobacterial virulence. These genomes include Xylella fastidiosa, Ralstonia solanacearum, Agrobacterium tumefasciens, Xanthomonas campestris, Xanthomonas axonopodt's, and Pseudomonas syringae pv. tomato. Functional genomic analyses of sequence data is providing new insight into the mechanisms of bacterial pathogenesis. In this review, I will discuss the variety of virulence mechanisms employed by bacterial plant pathogens, focusing on bacteria of the genus Pseudomonas. This genus includes the model pathogen Pseudomonas syringae pv. tomato (Pst) strain DC3000. Pst DC3000 is providing a great deal of new information about bacterial-plant interactions. Many of the new virulence mechanisms discovered in this bacterium may have broader implications in the study of bacterial pathogenesis of plants. T-DNA transfer One unique virulence mechanism utilized by the bacteria of the genus Agrobacterium is T-DNA transfer (Zupan et al., 2000). The symptoms of Agrobacterium infection include cell proliferation at the site of bacterial infection resulting in galls at the crown of the plant or hairy roots. These bacteria possess the ability to metabolize the plant compounds opines (Dessaux et al., 1993). However, plants do not usually produce large quantities of these compounds. Agrobacteria are able to form a type IV secretion system which delivers a strand of bacterial DNA into plant cells (Winans et al., 1996). This bacterial DNA is encoded on the Ti plasmid and is bordered by two 25-base pair direct repeats, the right and left border T-DNA sequences. The Agrobacterium first identifies the presence of a plant wound via the detection of exudates such as acetosyringone. These exudates activate the transcription of the vir genes which are responsible for the generation of a single strand copy of the T-DNA and the assembly of the T-complex transporter, a type IV secretion system. Accompanying the T-DNA are the VirD2 and VirE2 proteins. The VirE2 protein is a single-stranded DNA binding protein thought to protect the T-DNA from nucleolytic degradation (Christie et al., 1988; Citovsky et al., 1988). In addition, the VirE2 protein contains nuclear localization sequences (N LSs) which promote localization of these proteins to eukaryotic nuclei (Citovsky et al., 1992). The VirD2 protein also contains an NLS (Ballas and Citovsky, 1997). Thus, the NLSS present in both the VirD2 and VirE2 proteins are thought to direct the T-DNA-protein complex to the plant nucleus, where the bacterial T-DNA is then integrated into the genomic DNA of the plant host (Gheysen et al., 1991). The inserted genes include those required for the biosynthesis of opines. In addition, genes for the production of the plant hormones cytokinin and auxin, which promote cell division, are transferred (Akiyoshi et al., 1984; Inze et al., 1984). Thus, the targeted expression of bacterial genes in the plant cell provides an A grobacterium-specific carbon source and niche. Extracellular polysaccharides Many plant bacterial pathogens produce extracellular polysaccharides (EPSS) both in vitro and in planta (Denny, 1995). These polysaccharides can consist of polymers of simple sugar moieties or can have more complex branching structures. The EPSs can be secreted yet anchored to the bacterial membrane via a lipid A moiety forming a polysaccharide capsule around the bacterium. Alternatively, the EPS is secreted and diffuses away from the bacterium as extracellular slime. EPS production has been observed in several plant pathogenic bacteria, including Ralstonia solanacearum, Clavibacter michiganensis, Agrobacterium tumefasciens, Xanthomonas campestris, and Erwinia amylovora (Denny, 1995). Early studies with naturally occurring R. solanacearum (formerly Pseudomonas solanacearum) isolates noted a correlation between virulence and EPS production (Buddenhagen and Kelman, 1964). In these bacteria, the major EPS is acidic and the genes responsible for its biosynthesis comprise the eps operon. Mutants which disrupt the eps operon are unable to trigger the wilt symptoms typical of a successfitl infection of tomato (Denny and Baek, 1991; Kao et al., 1992). A correlation between EPS production and pathogen virulence has also been observed in Erwim'a stewartii (Poetter and Coplin, 1991), Erwinia amylovora (Geier and Geider, 1993), Xanthomonas campestris (Denny, 1995), and Xanthomonas oryzae (Rajeshwari and Sonti, 2000). While Clavibacter michiganensis has been Shown to make EPS, it is not clear whether these molecules contribute to the virulence of this pathogen (Bermpohl et al., 1996; Meletzus et al., 1993; Van Alfen et al., 1987). There is an ongoing debate about the mechanism by which EPS production promotes virulence in plant pathogenic bacteria. EPS-producing bacteria ofien cause similar symptoms in their host plants. These symptoms include wilt and water-soaking (Denny, 1995). One contribution of the EPSS to vascular pathogen virulence is the blockage of the host xylem and disruption of host water relations. If the tissue blocked is leaf vasculature, then the result is water-soaking. If the tissue affected is the stem or root vasculature, the result is wilt. Exogenous application of an EPS from R. solanacearum to tomato shoot cuttings can cause wilt (Buddenhagen and Kelman, 1964). In E. amylovora, infected apple and pear fruits develop an ooze. This ooze is composed of free . EPS molecules in which bacteria are suspended and is thought to aid in the spread of the bacteria (Jones and Sutton, 1996). In addition, the EPS capsule is thought to protect the bacteria from dessication (Denny, 1995). EPSS may even play a role in the attachment of R. solanacearum to its host (Sequeira, 1985). Secreted EPS may form a protective layer around the bacteria isolating them from anti-microbial compounds produced by the plant host or prevent contact with the plant cells and thus impede recognition (Braun, 1990). EPSS have also been shown to be important for the initiation of symbiotic interactions between Rhizobioum meliloti and Medicago sativa (Gonzalez et al., 1996). It is thought that the EPSS are acting as signaling molecules which promote nodule invasion. While EPSS have not yet been implicated in signaling between pathogen and host, the signaling role that EPSS play in symbiotic interactions may indicate that this role should be investigated. Cell wpll-degrading enzymes Several of the bacteria within the genus Erwinia, e. g. E. chrysanthemi and E. carotovora, are collectively referred to as soft-rot bacteria. These bacteria are able to infect many different vegetative tissues from a broad variety of host plants (Beaulieu and Vangijsegem, 1992). These bacteria macerate plant tissues via the destruction of pectins and cellulose. The degradation products are then utilized as a carbon source by the bacterium (Hugouvieux-Cotte-Pattat and Reverchon, 2001). Pectins are important components of the middle lamella which help to maintain the position of individual plant cells with respect to each other and the cuticle. In addition, pectins and celluloses are important components of the plant cell wall. The destruction of these structural plant compounds results in tissue rot. The enzymes responsible for this maceration are the pectate lyases, pectin methylesterases, and the polygalacturonases, collectively called cell wall-degrading enzymes (Barras et al., 1994). These enzymes are secreted from the bacterium via a type II secretion system (general secretory pathway) called the Out pathway (Bouley et al., 2001; He et al., 1991). In E. chrysanthemi, an initial set of five pectate lysases were identified and encoded by the pelABCDE genes. These five enzymes accounted for the majority of pectate lyase activity in vitro. However, deletion of the pelABCDE genes does not cause a complete loss of the ability to elicit soft-rot symptoms on plants (Ried and Collmer, 1987). This prompted researchers to look for pel genes which are induced in planta. Three additional pel genes, pelL, pel/ and pelZ, were identified as genes which were induced in planta (J afra et al., 1999). Mutations in these genes showed that each contributes to the soft-rot ability of E. chifysanthemi on potato tubers. However, it is likely that there are more soft-rot enzymes which all contribute to pathogenesis in a quantitative manner. While the role of pectate lyases has been described most extensively in the soft- rot Erwim'a species where they are required for pathogenicity, evidence suggests that cell wall-degrading enzymes may contribute to the virulence of other plant pathogenic bacteria which do not cause sofi-rot. The burgeoning genomic data available for other bacterial plant pathogens is revealing many pectate lyases in a broad variety of pathogens (da Silva et al., 2002; Wood et al., 2001). The R. solanacearum genome is predicted to contain five genes which are predicted to encode proteins with similarity to known cell wall—degrading enzymes (Salanoubat et al., 2002). Although the complete Pst DC3000 genome sequence has not yet been published, there is evidence for the presence of cell wall-degrading enzymes in this pathogen as well. At least one pectate lyase gene may be co-regulated with other important pathogencity factors in this system (F outs et al., 2002). Early studies with P. syringae indicated that pectate lyase activity is present during the infection process (Bashan et al., 1985). Indeed, the plant cell wall is thought to present a formidable barrier for all bacterial plant pathogens. In bacteria which do not cause soft— rotting, these enzymes may be present in lower quantities or produced and applied to the plant cell wall at specific sites, resulting in small patches of degraded plant cell wall. This would then allow other virulence factors unfettered access to the plant cell membrane without causing total loss of structural integrity of the plant tissues. Toxins Toxins are chemicals produced by pathogens which cause tissue damage. All currently known bacterial toxins are non-specific toxins, having effects on plants that are not hosts for the toxin-producing bacterium. Thaxtomin A is a phytotoxin produced by Streptomyces scabies. Exogenous application of thaxtomin A to potatoes causes necrotic lesions (Lawrence et al., 1990). Mutants of S. scabies which lack thaxtomin A production Show reduced virulence on potatoes (King et al., 1991). Thaxtomin A is a 2,5-dioxopiperazine which acts to trigger necrosis by an unknown mechanism. Glycosylated thaxtomin A is less toxic than unmodified thaxtomin A, and plant hosts may glycosylate thaxtomin A during infection as a method of detoxification (Acuna et al., 2001). The majority of the phytobacterial toxins have been described in the genus Pseudomonas. Phytotoxins of the phytopathogenic Pseudomonads Tabtoxin Tabtoxin is a phytotoxin produced by P. syringae pv. tabaci, P. syringae pv. coronafasciens, and P. syringae. pv. garcae. Spontaneous mutants of these pathogens which lack tabtoxin production remain pathogenic, but do not trigger chlorosis (Kinscherf et al., 1991; Willis et al., 1991). Thus, tabtoxin is considered to be a virulence factor. Tabtoxin is a monocyclic B-lactam which elicits chlorosis after hydrolytic release of the active moiety, the tabtoxinine-B-lactam (Levi and Durbin, 1986). Tabtoxinine-B-lactam inhibits the plant host enzyme glutamine synthetase, resulting in accumulation of toxic ammonia, which is thought to lead to chlorosis and have other deleterious effects on the plant cell (Thomas et al., 1983). Phaseolotoxin Phaseolotoxin is a phytotoxin produced by P. syringae pv. phaseolz'cola and P. syringae pv. actinidiae. Mutants of P. s. pv. phaseolicola which do not produce phaseolotoxin are reduced in virulence (Patil, 1974). This toxin has a tripeptide moiety, consisting of ornithine, alanine, and homoarginine, and an inorganic moiety, an N ’- sulfodiaminophosphinyl (Moore et al., 1984). Phaseolotoxin inhibits the host plant enzyme omithine carbamoyltransferase which is involved in arginine biosynthesis (Mitchell and Bieleski, 1977). This inhibition results in lower levels of arginine which has been implicated in the inhibition of plant growth and development of chlorosis (Patil, 1974) 10 Syringomycin and syringopeptin Syringomycin and syringopeptin are phytotoxins produced by P. syringae pv. syringae. Biosynthetic Ps pv. syringae mutants which produce neither syringomycin or syringopeptin are reduced in virulence (Scholz-Schroeder et al., 2001). Syringomycin and syringopeptin are lipopeptide toxins which can trigger necrosis after exogenous application to plants (Hutchison and Gross, 1997). Syringomycin and syringopeptin cause pore formation in plant plasma membranes (Hutchison and Gross, 1997). Pore formation then leads to electrolyte leakage, cell death, and tissue necrosis. Application of syringopeptin SP22A was also shown to induce stomatal closure (Di Giorgio et al., 1994). Coronatine Coronatine is a phytotoxin produced by P. syringae pvs. morsprunorum, atropurpurea, maculicola, glycinea and tomato (including Pst DC3000). Coronatine is the only known toxin produced by Pst DC3000 (Bender et al., 1999). Coronatine is a polyketide toxin which has two main moieties, coronafacic acid and coronamic acid (Ichihara et al., 1977). The enzymes required for the biosynthesis of coronafacic acid are encoded by the cfa genes. The enzymes required for the biosynthesis of coronamic acid are encoded by the cma genes. The corRS operon encodes a two-component regulatory system which regulates expression of the cfa and cma genes (Bender et al., 1999). The overall structure of coronatine shares some similarities with the phytohorrnone, j asmonic acid (JA). This similarity may be of importance in its role in promoting the virulence of 11 Pst DC3000 (Wastemack and Parthier, 1997). Coronatine-insensitive A. thaliana mutants have been isolated due to the ability of their roots to grow normally in the presence of coronatine (F eys et al., 1994). These coil mutants are also insensitive to the plant hormone JA. They are male sterile due to a defect in pollen development. In addition, they are more resistant to bacterial infection, but less resistant to some fungal pathogens (Thomma et al., 1998). Coronatine causes chlorotic symptoms when applied to tomato leaves, as well as other physiological changes in a broad variety of plants (Gnanamanickam et al., 1982). When genes involved in the biosynthesis of coronatine were mutated, Pst DC3000 became less virulent on tomato (Bender et al., 1987 ). Bacterial multiplication of these coronatine-deficient mutants was decreased relative to wildtype Pst DC3000. In addition, leaves infected with coronatine-deficient mutants of Pst DC3000 showed smaller, less chlorotic lesions. However, the role that coronatine plays in the virulence of Pst DC3000 on A. thaliana is less clear. Work with another coronatine-deficient mutant of Pst DC3000 (DC3661) on A. thaliana showed that it grew to lower levels than wildtype, but only when the plants were infected by dipping the plants in a bacterial suspension and this difference was not seen when plants were infiltrated with bacteria using a needleless syringe (Mittal and Davis, 1995). In addition, this coronatine-deficient mutant induced an increase in the levels of some defense-related mRNAs, relative to wildtype Pst DC3000. This study did not include the standard genetic proof of restoration of virulence by complementation with the wildtype version of the disrupted gene. Subsequent analysis indicated that multiple mutations may have produced the reduction of virulence seen in Pst DC3661 (unpublished data from the He lab). This 12 result, therefore, does not indicate whether or not coronatine plays a role in the virulence of Pst DC3000 on A. thaliana. Recent work focused on the discovery of new effector proteins secreted via the type HI secretion system has yielded some interesting results with respect to the regulation of coronatine production in Pst DC3000. A Hidden Markov Model (HMM) search for hrp box-containing promoters revealed that the corRS operon is a candidate hrp-regulated operon (F outs et al., 2002). This could indicate that the expression of many of the genes required for Pst DC3000 virulence are coordinately regulated. While the hrp-dependent regulation of corRS could not be shown experimentally (by either microarray or northern analysis), two genes required for the biosynthesis of the coronofacic acid moiety of coronatine (cfaI and cfa6) were shown to be expressed in a hrp—dependent manner (F outs et al., 2002). Our laboratory also found that the cfa2 gene is expressed in a hrp-dependent manner. However, a gene required for biosynthesis of the coronamic acid moiety cmaU was not shown to be coordinately regulated with the hrp genes. This lack of regulation implies that the mechanism by which coronatine biosynthesis and the type III secretion system are coordinated is more complicated than hrp-dependent expression of corRS. 1pc tvLe III proteip secretiop sym The type 111 protein secretion system is important for the pathogenicity of many Gram-negative bacterial pathogens (Galan and Collmer, 1999). Many of the animal bacterial pathogens, such as enteropathic Escheria coli, Salmonella typhimurium, and Yersinia pestis, utilize the type 111 protein secretion system during pathogenesis. All 13 known Gram-negative plant bacterial pathogens, with the exception of A. tumefaciens, also rely upon the type 111 protein secretion system to cause disease. The type III secretion system is a protein secretion system which is thought to have the ability to transport bacterial proteins, called type III effectors, directly into the eukaryotic host cell. Type 111 protein secretion in the phytopathogenic pseudomonads The type III secretion system was first described in the plant bacterial pathogen P. s. pv. phaseolicola. Mutants of P. s. pv. phaseolicola were isolated which were unable to elicit the hypersensitive response in the non-host tobacco or disease in the normal host bean. These mutants were classified as hrp mutants (for hypersensitive response and pathogenicity) (Lindgren et al., 1986). Further analysis revealed that other Gram- negative bacterial plant pathogens, including Pst DC3000, also carry the hrp genes. In the genome of a given pseudomonad, the hrp genes are found in a cluster. In Pst DC3000, the hrp gene cluster consists of 27 hrp genes organized into Six operons. The majority of these hrp genes were later found to encode a type 111 protein secretion system, suggesting that the elicitation of both the HR and pathogenicity rely upon the bacterial translocation of effector proteins into the plant cell. Type III secretion system appendages Type III secretion systems of mammalian and plant pathogenic bacteria produce surface appendages. One of the differences between the appendages of plant and animal 14 pathogens are the physical dimensions. Animal bacterial pathogens produce a very short appendage called the needle (Kubori et al., 1998). This appendage has a diameter of 8 nm and has a length which is. less than 100 nm. Plant bacterial pathogens, on the other hand, produce a surface appendage termed the Hrp pilus (Roine et al., 1997). This pilus is 8 nm in diameter, like the needle, but is significantly longer, up to several pm in length. It has been suggested that this increase in length is necessary for plant pathogens to penetrate the plant cell wall (Roine et al., 1997). The HrpA protein is the major structural component of the Hrp pilus. Pst DC3000 hrpA mutants do not produce Hrp pili and are unable to cause disease on hosts or trigger the HR on non-hosts (Roine et al., 1997). The hrpA mutant also does not secrete effector proteins in vitro (Wei et al., 2000). This suggests that the Hrp pilus plays an important role in the type III protein secretion process. Recent work has Shown that some effector proteins are actually secreted from the tip of the Hrp pilus in vitro (J in and He, 2001; Li et al., 2002). This indicates that the Hrp pilus is serving as a conduit for the delivery of type III effector proteins to the host cell. Regulation of the type III protein secretion system The type 111 protein secretion system is regulated tightly at the transcriptional level. As mentioned previously, there is now evidence which suggests that, in Pst DC3000 at least, other virulence factors such as coronatine may also be regulated with the type HI protein secretion system. The type 111 protein secretion system is governed by the hrpRS regulatory system. hrpR and hrpS Show sequence similarity to the NtrC 15 class of response regulators which are 054—dependent enhancer-binding proteins (Grimm et al., 1995; Xiao et al., 1994). These proteins are members of two-component regulatory systems which control transcription in bacterial systems (Stock et al., 2000). hrpR and hrpS are required for the transcription of the hrpL gene. hrpL encodes an alternative sigma factor of the extracytoplasmic fimction (ECF) family (Xiao et al., 1994). HrpL is thought to bind a cis-element present in the promoter sequence of hrpRS regulated genes. This cis-element is called the hrp box and is defined as KGGAACY-N14/15-CCACNNA (K is T or G, Y is C or T) (Innes et al., 1993; Shen and Keen, 1993; Xiao and Hutcheson, 1994). These hrp boxes are found in the promoters of hrp genes as well as genes that encode type III effector proteins. This regulatory system prevents the expression of the genes that encode components of the type III secretion system and the effectors except under specific circumstances, such as in planta and also in hrp-inducing medium, which is deficient in complex nitrogen and is acidic. It is thought to mimic the conditions which are present in the apoplastic space. The sensor for this regulatory system has not yet been identified and it is not yet understood how the bacteria perceive that they are in planta or in hrp- inducing minimal media. While it is not currently known how hrpR and hrpS are regulated at the transcriptional level, the Lon protease has been shown to influence the hrpRS regulatory system at the post-translational level (Bretz et al., 2002). In wildtype Pst DC3000, the HrpR protein has a Shorter half life when in rich media than when in hrp-inducing minimal media. However, if the stability of the HrpR protein is measured in a Ion mutant, the half life remains the same in both types of media. Thus, the Lon protease is 16 required for the differential stability of the HrpR protein under different growth conditions. Increased stability of HrpR under hrp-inducing conditions would favor the expression of the hrp genes and genes which encode effectors. The mechanism by which the Lon protease is able to regulate the stability of HrpR is not yet known. HrpS appears to have a constant stability. There are additional factors that influence hrp gene regulation. The HrpV protein is thought to act as a suppressor of hrp gene regulation. Overexpression of the hrpV gene significantly reduces the mRNA levels of the hrp genes (Preston et al., 1998). However, overexpression of both hrpV and hrpRS results in normal levels of hrp gene transcripts. In addition to encoding the major structural component of the Hrp pilus, the hrpA gene is also thought to play a role in hrp gene regulation. A Pst DC3000 hrpA mutant not only lacks Hrp pili, but also Shows reduced levels of hrp gene transcripts (Wei et al., 2000). Again, the overexpression of hrpRS can compensate for the lack of hrpA. A full understanding of how all these components contribute to the regulation of the hrp genes will require further study. Genome-wide inventory of effector proteins Despite the importance of type III effector proteins in bacterial infection, a complete effector inventory has thus far not been established for any bacterial pathogen. Forward genetic screens for P. syringae mutants that show reduced virulence phenotypes have yielded few effector proteins probably because of apparent functional redundancy. Recently, a multitude of alternative approaches, coupled with the availability of the P. s. 17 pv. tomato DC3000 genome, have proven fruitful for the identification of putative effector proteins and increasing our knowledge of the complete arsenal of effector proteins in P. syringae. One approach that has provided a large number of candidate effectors is the identification of avirulence (avr) genes. Plants have developed a defense mechanism that enables them to recognize specific pathogen strains. If a pathogen harboring an avr gene attempts to infect a plant possessing the corresponding resistance (R) gene, the so-called gene(avr)-for-gene(R) resistance will prevent disease from developing. avr genes have been identified through heterologous expression. In this approach, the genomic library from strain A (which contains one or more avr genes and is therefore avirulent on plant A that contains one or more cognate R genes) was introduced into strain B (which lacks avr genes and is therefore virulent on plant A); the response elicited by the recombinant strain B is then monitored. Any genes from strain A that cause strain B to become avirulent are considered avr genes. The avirulence fimction of several examined P. syringae avr genes is dependent on a functional type III secretion system (Gopalan et al., 1996; Keen et al., 1990; Pirhonen et al., 1996). This observation, coupled with the fact that the action Site of many Avr proteins is inside the plant cell (He, 1997; Kjemtrup et al., 2000), suggests that Avr proteins are effector proteins. It is widely believed now that the original function of the Avr proteins was to promote disease; the avirulence function of these proteins results from the ability of some plants to have evolved recognition capability. Indeed, the virulence function of several Avr proteins has been demonstrated (Kjemtrup et al., 2000; White et al., 2000) and will be discussed later. 18 Because effector proteins are secreted via the type HI secretion system, another approach used is identification based on protein secretion. This method has been used to identify many effectors of animal pathogens. Yuan and He (1996) used this method to identify the HrpW, HrpZ, and HrpA proteins of Pst DC3000. However, this approach has a limitation; only proteins that are present in abundant quantities can be visualized and then sequenced using this method. Unfortunately, P. syringae and other plant pathogens appear to produce the majority of their effector proteins in quantities too small to be detected by this method. A third approach is based on the fact that all known effector genes are coordinately regulated with hrp genes, which are induced in planta. Therefore, in vivo expression technology (IVET) can be employed to search for effector genes. For example, Boch et al. (2002) fused a Pst DC3000 genomic library to a promoterless hrcC- uidA reporter fusion. These constructs were then introduced into the type III secretion- deficient mutant, AhrcC, of Pst DC3000, and the candidate gene-hrcC—uidA fusion was allowed to recombine into the genome. In principle, any genes that contain the hrp box in the promoter region will be able to trigger the expression of the hrcC-uidA fusion, which would restore the hrcC mutant to grow in planta and elevate the reporter gene activity in hrp-inducing medium, but not in nutrient-rich medium. Several known and suspected type HI effector genes were identified in this study (Boch et al., 2002). Finally, the recent release of the P. s. pv. tomato DC3000 genome sequence has facilitated several genome-wide surveys for type III effector genes based on the presence of the hrp box motif in the promoter, induction of gene expression in hip-inducing minimal medium, and secretion and translocation assays (Fouts et al., 2002; Guttman et 19 al., 2002; Petnicki-chieja et al., 2002; Zwiesler-Vollick et al., 2002). According to these studies, Pst DC3000 alone contains more than 30 putative effector genes (Guttman et al., 2002; Petnicki-chieja et al., 2002). Additional effector genes will likely be found in other P. syringae strains in the future. The large number of effectors revealed by these recent studies has allowed further study of the structure of type III effectors. It is not known how effectors are targeted to the type III protein secretion system within the bacterial cell. Previous studies have supported conflicting theories about the identity of the secretion Signal. Anderson et al (1997, 1999), suggested that the secretion signal resided in the mRNA structure of the effectors of Yersinia enterocolz‘tica. However, Lloyd et al (2001) in work with Yersinia pseudotuberculosis provided evidence that the secretion signal resides in the N-terminal amino acids of the effector proteins. While no specific amino acid motifs are seen in the P. syringae type III effectors, certain biophysical properties do seem to be important. These properties can be summarized as follows: i) the first five amino acids are solvent exposed, ii) acidic amino acids are absent in the first twelve residues, and iii) the subsequent 6 to 50 or more residues are amphipathic. Two recent studies utilized these biophysical properties to search the Pst DC3000 genome. These studies yielded 38 and 28 candidate effectors, respectively (Guttman et al., 2002; Petnicki-chieja et al., 2002). While all of these candidates may not prove to be true effectors, at least one candidate effector from each study was verified to be a secreted effector protein. The observation of these biophysical properties should not only aid in the identification of new effector proteins but should provide more information about the mechanism of type 111 protein secretion. 20 The progress made toward the identification of type III effector proteins in P. syringae is summarized in Table 1-1. Here, we have chosen to use the first published gene name. We have also chosen to maintain the avr name for a gene even if the protein encoded was later directly proven to be secreted. Proposed hop (l_rrp _o_uter protein) names or names of orthologues in other species are shown in the alternative name column. 21 E a; E 99.50.53: 833 Ea $3.3... E a; E 358.. .m : sass 9x3... :. a 8.. mm? 3 sees Name... E a; E .35... . «38...; 828A 38...:er 25...}. E 8> E (55.5 .m: A; x: more}. E a... E 4.5.8.. 8.. :32 assess... 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Alternatively, the presence of a large number of effector genes in the P. syringae genome may reflect the presumably aggressive co—evolution which is taking place between the pathogen and the plant host. It is apparent that plants use type III effectors as a main source of recognition to activate innate defense and turn virulence-intended effector genes into avr genes. To survive, P. syringae must either mutate these avr genes to evade recognition or evolve or acquire additional effector genes to mask the presence of the avr genes. This evolution may have resulted in rapid proliferation of effector genes in the P. syringae genome. Virulence role of type III effector proteins While type III effector proteins have been implicated as having a role in virulence because most are delivered into the host cell, research is only now beginning to elucidate the virulence contributions made by individual effector proteins. Effector proteins can generally be classified into two classes. While most type III effector proteins are thought to be delivered into the host cell, a few are merely secreted outside the bacterial cell. Those that are not thought to enter the host cell are called harpins and include HrpW and HrpZ. The harpins also have the ability to trigger a hypersensitive response (HR) when infiltrated into leaves. Because they are secreted by the type III secretion system, harpins are thought to play a role in virulence. HrpZ has also been shown to be required for the 24 delivery of AvrB into the host cell which then triggers an HR (Gopalan et al., 1996). HrpZ has been shown to associate with plant protoplasts (Lee et al., 2001a) and can trigger pore formation in lipid bilayers in vitro (Lee et al., 2001b). The terminal portion of HrpW shows similarity to pectate lyases and can bind to pectate in vitro, although no enzymatic activity has been shown (Charkowski et al., 1998). Genetic analysis of hrpZ, hrpW, and hrthrpW double mutants showed that loss of these harpins had no effect on virulence. However, the hrthrpW double mutant has a reduced ability to trigger the HR (Charkowski et al., 1998). Taken together, these results suggest that the harpins may aid bacterial grth by interacting with the plant cell wall or membrane and allowing nutrient/water leakage, or by aiding the penetration of the Hrp pilus and the delivery of type III effectors into the host cell. The role that translocated effector proteins play in virulence has also been the subject of intense research. The avr genes are conserved among many bacterial plant pathogens and are coordinately regulated with the hrp genes (Leach and White, 1996). The maintenance in the bacterial genome of these avr genes has been an enigma in the field (Dangl, 1994). Selection should have eliminated avr genes from pathogens, unless selection is also favoring the retention of these genes. It has been suggested that the avr genes, named for their role in the incompatible interaction, may function as virulence factors in the compatible interaction. From the perspective of the plant, the most efficient way to detect a pathogen is to recognize those factors which make the bacterium virulent. There have been several demonstrations that avr genes are virulence factors. The aerme gene of P. s. pv. maculicola was shown to be contribute to virulence on A. thaliana, in the absence of the corresponding R gene, RPM 1 (Ritter and Dangl, 1995). In 25 Xanthomonas campestris, the avrBsZ gene is needed for full virulence on its host (Kearney and Staskawicz, 1990). As mentioned previously, research in this area has been slow due to the apparent redundancy among effector proteins. However, several studies have been able to provide clues about the role that effector proteins play in virulence. Many genes involved in virulence are encoded on plasmids. The bean pathogen P. s. pv. phaseolicola contains such a plasmid. A P. s. pv. plzaseolicola strain which had been cured of this plasmid was shown to have an altered host range (Jackson et al, 1999). This prompted an analysis of the genes encoded on this plasmid. One gene, vierhA, was shown to be required for virulence on some cultivars of bean, and for triggering an HR in others. Another gene encoded on the plasmid, averhC, was shown to act as an avr gene in certain bean cultivars. In addition, the presence of averhC or vierhA was shown to block the recognition of averhF or an unidentified avr gene, respectively. This mechanism would allow virulence determinants which had become targets of the host gene-for—gene resistance system to be maintained without losing the ability to infect these hosts (Tsiamis et al, 2000). While most type III effectors show no similarity to genes which code for proteins of known function, new motif searches are revealing enzymatic motifs in some effectors. The effector AverhB was found to be similar to the Yersinia effector YopT. YopT is a cysteine protease which acts to promote virulence by disrupting the host actin cytoskeleton. AverhB was tested and also has cysteine protease activity (Shao et al., 2002). The investigation of the enzymatic activity of type III effector proteins should reveal more about their role in pathogenesis. 26 Transgenic expression of effector proteins in host plants is also proving to be a method which can shed light on the virulence contribution of these proteins. The effector Aerpt2 has been investigated via transgenic expression in A. thaliana plants which lack the corresponding R gene, RPS2 (Chen et al., 2000). These plants show enhanced growth of Pst DC3000. The expression of AerptZ in these plants also interferes with (1erme- Rpm] mediated resistance. Ectopic expression of aerptZ in Pst DC3000 also allows this pathogen to grow in the cpr5 and coil A. thaliana mutants which are resistant to Pst DC3000. The expression of avrPto from Pst DC3 000 in A. thaliana promotes the growth of the non-pathogenic Pst DC3000 hrpH mutant. These transgenic plants also fail to respond to the hrpH mutant with the formation of callose-containing papillae as is seen with wildtype A. thaliana (P. Hauck, R. Thilmony and S.Y. He, unpublished data). Thus, transgenic expression of effectors in planta is helping to reveal the role that effectors play in pathogenesis. Project Summary Great strides have been made in understanding virulence mechanisms utilized by bacterial plant pathogens. The identification and characterization of the Hrp type 111 protein secretion system has proven to be essential for our understanding of the interaction between bacterial plant pathogens and their hosts. However, research into the modes of action of the type III effectors has been impeded by the presumed functional redundancy among the effectors. Unlike animal bacterial pathogens, the number of effectors present within any given plant bacterial pathogen strain there are estimated to be 27 at least 35, and may be even more (Collmer et al., 2002; Genin and Boucher, 2002; Petnicki-chieja et al., 2002). The proposed redundant functions of these effectors has made forward genetic analysis difficult. Identification of more effector proteins is an essential first step before reverse genetic or other approaches can be undertaken to determine the role of any given effector in virulence. Also, unlike in the study of animal bacterial pathogens, we currently lack a multitude of markers to monitor the process of pathogenesis. The initial work done with a handful of effectors should provide testable hypotheses for further characterization of all effector proteins. The advent of new technologies such as microarray host expression analysis and in planta visualization of bacterial infection should also help to elucidate mechanisms by which effectors contribute to pathogenesis. At the time this project was started the only type III effectors known in Pst DC3000 were AvrPto and Aer. My goal was to further characterize virulence in Pst DC3000. Pst DC3000 is an especially attractive pathogen for study. The type III secretion system has been studied extensively and is amenable to genetic manipulation. In addition, it is a pathogen of the economically important crop plant tomato as well as the model plant A. thaliana. Recent sequencing initiatives have made the Pst DC3000-A. thaliana pathosystem the first where genome sequence for both the host and the bacterial pathogen are available. This interaction can be investigated via the study of the pathogen as well as the host. Chapter 2 reports my research on the identification of novel type III effector proteins of Pst DC3000. A preclosure sequence of the Pst DC3000 genome was used to search for the hrp box motif. The expression patterns of the hrp box-containing open 28 reading frames were then determined by both microarray and northern blot analyses. Six orthologues of effectors known in other systems, but not previously known in Pst DC3000, were identified. In addition, eight new candidate effectors which showed no similarity to any previously-described effectors were identified. An AerptZ based translocation system was used to show that at least one of the effectors is secreted by the type III protein secretion system. Chapter 3 describes my research on the identification of Pst DC3000 mutants which show reduced virulence. Pst DC3000 was mutagenized with a uidA —containing transposon. Mutants were initially screened for minimal medium-induced uidA reporter expression, followed by determination of virulence in A. thaliana. Six mutants were found which showed reduced virulence. These mutations were localized to three genes: The oer gene which encodes the outer membrane protein F precursor, the pstP gene which encodes the enzyme I subunit of the phosphoenolpyruvate phosphotransferase, and uer which encodes a helicase involved in DNA replication and repair. Chapter 4 communicates my work on the Pst DC3000 effector Aer. Although AvrB was shown to be important for virulence in Pst PT23, aer mutants of Pst DC3000 do not show a reduction in virulence. In order to investigate the role that Aer may play in virulence, I expressed the Pst DC3000 aer gene transgenically in A. thaliana under the control of a dexamethasone-(DEX-)inducible promoter. 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Molecular Microbiology 45, 1207-1218. * These authors contributed equally to this work. 41 Abstract Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism employed by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type HI effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC-family protein, and the HrpL alternate sigma factor, which presumably binds to a conserved cis element (called the ‘hrp box’) in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3 000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had a 95% genome coverage. The expression of the 73 genes was analyzed by microarray and northern blot, revealing 24 genes/operons (including 8 novel genes) whose expression was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria- Bertani broth. Expression of all 8 genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An Aerpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins. 42 Introduction Pseudomonas syringae infects a wide range of susceptible plants and causes mainly localized necrosis in infected tissues. A given strain of P. syringae may infect only a few cultivars of a host plant, exhibiting a high degree of specificity. Host specificity is the basis for classifying various P. syringae strains into more than 40 pathovars (Gardan et al., 1999). P. syringae pathovar (pv). tomato strain DC3000 (Pst DC3000 hereafter) infects tomato and Arabidopsis and causes necrotic spots surrounded by diffuse chlorotic haloes (Cuppels, 1986; Whalen et al., 1991; Katagiri et al., 2002). The molecular basis of pathogenicity of Pst DC3000, like that of the majority of plant pathogenic bacteria, is not well understood. An essential weapon in the virulence arsenal of Pst DC3000 is the type IH protein secretion system, which is conserved in many Gram-negative plant and mammalian pathogenic bacteria (He, 1998; Galan and Collmer, 1999; Cornelis and Van Gij segem, 2000; Staskawicz et al., 2001). In plant pathogenic bacteria, the type III secretion system is encoded by hrp (for hypersensitive reaction and pathogenicity) and hrc (h_rp gene conserved) genes (Van Gijsegem et al., 1993; Bonas, 1994; Alfano and Collmer, 1997; Lindgren, 1997; He, 1998; Hutcheson, 1999; Mudgett and Staskawicz, 1998). This secretion system is responsible for the assembly of the Hrp pilus (Roine et al., 1997; Jin and He, 2001) and is thought to deliver virulence effector proteins directly into the host cell. Once there, the effector proteins are believed to modulate the physiology of the host cell to favor pathogenesis. However, different plant cultivars may evolve specific resistance genes to recognize individual bacterial effectors and convert them into elicitors of host defense responses. In such situations, these virulence effector proteins have been named avirulence (Avr) proteins (Leach and White, 1996). 43 The expression of hrc, hrp, and effector genes is tightly controlled in P. syringae. All known hrp, hrc, and type III effector genes are expressed at a low level in standard nutrient-rich media, such as Kings B (King et al., 1954) or Luria-Bertani (LB) medium (Sarnbrook et al., 1989). The expression of hrc/hm genes is induced in plant tissues or in hrp-inducing minimal medium (Huynh et al., 1989; Ralnne et al., 1992; Xiao et al., 1992; Wei et al., 2000) that mimic the in planta conditions. Three regulatory proteins are required for the expression of type III secretion-associated genes in P. syringae: HrpR and HrpS, which belong to the NtrC family of two-component regulatory proteins (Grimm et al., 1995; Xiao et al., 1994; Hutcheson et al., 2001), and HrpL, a member of the ECF family of alternate sigma factors (Xiao et al., 1994). The HrpS, HrpR, and HrpL proteins function as a regulatory cascade in which HrpS and HrpR activate the expression of the hrpL gene in response to an undefined signal in host tissue or in hrp-inducing minimal medium (Xiao et al., 1994; Grimm et al., 1995). HrpL activates the hrp, hrc, and avr genes presumably by binding to a consensus bipartite cis element (‘hrp box’) present in the promoter region of hrp, hrc, and type III effector genes (Innes et al., 1993; Shen and Keen, 1993; Xiao and Hutcheson, 1994). Identification of genes that encode type III effectors is a key step toward a comprehensive understanding of the function of the type III secretion system in plant pathogenesis. In Yersinia pseudotuberculosis, a pathogenic bacterium of mammalian hosts, there are 13 known virulence effectors determined by secretion analysis (Hueck, 1998). Less is known about the number of effectors in plant pathogenic bacteria that employ type III secretion as a virulence system. A recent cDNA-amplified fragment length polymorphism (AF LP) study of Xanthomonas campestris pv vesicatoria hrpG— 44 regulated genes revealed several known hrp gene operons, effector proteins, and unknown genes (Noel et al., 2001). In Pst DC3000, three approaches have been used in the past to discover type III effectors: Cloning based on avirulence phenotype (Ronald et al., 1992; Lorang and Keen, 1995), sequence analysis of extracellular proteins secreted via the Hrp secretion system (Yuan and He, 1996), and characterization of genes located in the so-called Hrp pathogenicity island (Alfano et al., 2000). These approaches have resulted in the identification of four type III-secreted proteins, HrpA, HrpZ, HrpW, and AvrPto, and several putative effector genes (including aer) located in the Hrp pathogenicity island (Alfano et al., 2000). In this study, we used functional genomics approaches to discover new putative type III effector genes in Pst DC3000. We utilized the available Pst DC3000 genome sequence to search for genes that contain the ‘hrp box’-like sequence in their promoters, which is indicative of possible HrpL regulation. The expression of these genes was compared between hrp-repressing and hrp-inducing conditions by microarray and northern blot analyses. Further comparisons of gene expression were made between wild-type bacteria and hrpS and hrpL mutants using northern blot analysis. Then a selected group of Hrp-regulated genes were subjected to an Aerpt2-based type III translocation assay in RPS2+ Arabidopsis plants to identify putative type III effectors. 45 Materials and methods Computer analysis The Pseudomonas syringae DC3000 genome of approximately 6.2 megabases was sequenced by The Institute for Genomic Research (TIGR; http://www.tigr.org/tdb/mdb/mdbinprogress.html). The 2nd release (January 2001, 95% coverage) was imported into BioNavi gator (http://www.bionavigator.com), a web-based genome analysis tool from Entigen Corporation of Sunnyvale, California. The genome was searched with the FindPattems(GCG) program using the ‘hrp box’ motif (KGGARCY(N){13,20}CCACNNA) derived from the alignment of 12 known ‘hrp-box’- containing genes in Pst DC3000. The first base of the bipartite ‘hrp box,’ is either a T or a G in the 12 hrp-regulated genes. The following three bases, GGA, are invariably conserved in all 12 genes. For the fifth base of our search motif, we chose to use R, representing either A or G, although all 12 genes contain an A at this position. This is because the hrpW gene has a second ‘hrp-box’-1ike sequence at the —1 8 nt position. In this ‘hrp-box’-like sequence, there is a G, instead of an A, in the 5th position. The 6th position is a C in all 12 genes, whereas the 7th position is a C in all genes, except for her and hipP, which have a T. We therefore designated a Y to represent either C or T at this position. The two motifs in the ‘hrp boxes’ of the 12 genes were separated by 15 to 16 nts. However, to investigate the importance of the spacing between the two motifs and to identify a maximum number of candidate genes, we purposely expanded this spacing range to 13 to 20 nts. 46 Construction of an hrpL mutant of Pst DC3000 (by Vinita Joardar and Barbara Kunkel) The strain VJ 202 (hrpL::Q) was constructed using a marker replacement strategy. A 3.2-kb BsaBI fragment containing hrpL and flanking sequences (1.5 kb upstream and 1.2 kb downstream) was cloned into pBluescript II KS+ (Amp’, Stratagene). This construct was moved into Escherichia coli strain GM2163 (dam'; from NEB) to facilitate digestion of internal BsaBI sites in the insert that are sensitive to dam methylation. The hrpL coding region (0.5 kb) in this construct was replaced with the Spr-Q-cassette from pUC4 (Prentki and Krisch, 1984) using the internal BsaBI sites. This insertion/deletion construct was moved into the suicide plasmid pJP5603 (Km'; Penfold and Pemberton, 1992). The pJP5603 construct was conjugated into Pst DC3000 to generate strain VJ 201 (Sp', Km') in which the construct was integrated into the Pst DC3000 genome via a single recombination event. The mutant strain VJ 202 (Sp', Kms) was identified by screening for derivatives of VJ 201 in which a second recombination event resulted in loss of Km’. Strain VJ 202 was confirmed by PCR and by assaying for disease and HR production. Strain VJ202 was unable to cause disease on A. thaliana (Col-0) or elicit an HR on tobacco (Nicotiana tabacum). Complementation of strain VJ 202 with the hrpL gene from Pst DC3000 restored both phenotypes. Bacterial DNA isolation and polymerase chain reaction (PCR) amplification Genomic DNA from DC3000 was prepared as described in Chen and Kuo (Chen and Kuo, 1993). The isolated genomic DNA was used as template for PCR amplification 47 of HCOs. Primers (22mers; see Appendix A) were designed using the Prime3 program from BioNavigator to amplify HCOs (h_rp box-gontaining QRFs). Primers were manufactured by Integrated DNA Technologies (IDT) of Coralville, Iowa. The PCR fragments were verified for correct sizes, purified by ethanol precipitation, and resuspended in 40 ul of 3X SSC. Microarray slide preparation and analysis PCR products for a total of 79 genes (‘hrp-box’-containing genes and 10 spiking controls) were spotted from microtiter plates onto SMA-25 superaldehyde-coated glass slides (Telechem International, Inc). Spotting was done at high density using an Omnigridder robot (Gene Machines, San Carlos, CA) and 16Arraylt chipmaker 1 pin (Telechem). Slides were washed and blocked according to the Telechem protocol. Microarray slides were prepared at the Michigan State University Arabidopsis Functional Genomics Consortium (AF GC) microarray facility. Spiking controls are human cDNA clones: B-cell receptor protein (AF 126021), myosin heavy chain (X13988), myosin reg. light chain 2 (M21812), insulin-like growth factor (X07868), F LJ 10917fis (AK001779), HSPC120 (AF 161469), beta2 microglobulin (NM_004048), phosphoglycerate kinase (pgkl) (NM_000291), tyrosine phosphatase (pac-l) (L11329), and G10 homolog (edg-2) (U11861). Isolation of bacterial RNA RNA was isolated from Pst DC3000, and the hrpL and hrpS (Yuan and He, 1996) mutants of DC3000 following a protocol described by Tao et al. (Tao et al., 1999). 48 Bacteria were grown in Luria-Bertani (LB; Sarnbrook etal., 1989) medium till OD600 reached 0.6, when stop buffer (1.25 ml; 5% acid phenol in EtOH) was added to 10 ml culture and the mixture was centrifuged in Sorvall RC-SB at 7,000 rpm for 10 min. The resulting pellet was frozen at -80°C until being subjected to microarray or northern blot analysis. For RNA isolation from bacteria grown in hrp-inducing medium (Wei et al., 2000), a 10-ml LB culture of OD600=O.6 was centrifuged in a clinical centrifuge at 3,000 rpm for 10 min. The LB medium was removed and bacteria resuspended in 10 ml of hrp- inducing medium. Bacteria were incubated at 20°C for 6 h with shaking. Again, the phenol/ethanol stop buffer was added, samples were centrifuged as above, and RNA pellets frozen at -80°C. Arrrinoallyl labeling of bacterial RNA RNA samples were labeled by Cy3 or Cy5 fluorescent dye, following a protocol by Ben Schroeder, which was modified from Chris Seidel’s protocol (http://www.pangloss.com/seidel/Protocols/amino-allleT.html). Three mg/ml Gibco random hexamers were used in the reverse transcription reaction. The labeled probes were hybridized to a microarray in a total volume of 30 pl of hybridization buffer (3.4 x SSC, 0.32% SDS, and 5 ug yeast tRNA) for 16 h at 60°C. The microarray slide was then washed at room temperature sequentially in 2 x SSC/0.1% SDS for 5 min, 1 x SSC/0.1% SDS for 5 min, and 0.1 x SSC for 15 s. The slide was centrifuged, dried, and scanned with a Scanarray 4000 (GSI Lumonics, Billerica, MA). Each microarray experiment was 49 repeated three times (two technical replicates with the same RNA samples and one biological replicate using RNA isolated from a different culture). Due to the small size of the array, normalization to spiking controls was used. Spike mRNA (0.5 ng each) was added to both RNA samples before labeling. Once the data were obtained, the ratios of the spikes were set to one and from this a normalization constant was determined. Northern hybridization Twenty u g of total bacterial RNA was run on each lane of a denaturing formaldehyde gel and transferred to Millipore Irnmobilon N membrane. 32F probes were made using purified PCR products of 43 ‘hrp-box’-containing genes for which expression was detectable in microarray experiments. Aerpt2 fusion translocation analysis The truncated aerpt230-255 gene, which encodes type III secretion/translocation- incompetent, but biologically active, Aerpt2 (Mudgett etal., 2000) was cloned into the XbaI-HindIII sites of pUCP19 (Schweizer, 1991). Candidate type III effector genes were amplified using appropriate primers (see below) and cloned into the EcoRI- XbaI sites in pUCP19::aerpt230_255 to create in-frarne fusions (5’-effector gene-aerpt230-255-3’). All gene fusions contained the full-length putative effector genes. The recombinant plasmids were then electroporated into Pst DC3000. The tansforrnants were grown in LB to OD600=0.6. Bacteria were collected by centrifugation and resuspended in sterile water to an OD(,00=0.2. The bacterial suspensions were infiltrated into leaves of 6-week-old 50 RPSZ+ or rpsZ' Arabidopsis plants (ecotype Col-0). Tissue collapse was monitored over a 48-h period at room temperature. Primers used to amplify chp genes for making aerpt2 fusions: hrpA : avrPto virPtoA : chp]: chp5 .' chp6: chp 7: chp8: sense primer 5 ’TGAATTCTTGCAAAGACGCTGGAACCG3 ’ antisense primer 5 ’AATCTAGAGTAACTGATACCTTTAGCG3’ sense 5 ’CGAATTCAAGTCAGTGACGCTTTGATG3 ’ antisense 5 ’TGTCTAGATTGCCAGTTACGGTACGGG3 ’ sense 5 ’ GCGAATTCGGGCATGGAAAAATC CTCTTC 3 ’ antisense 5’ GCT CTA GAG GGG ACT ATT CTA AAA GC 3’ sense primer 5 ’CGAATTCTGCCGTGGCGCCGCAACCTG3 ’ antisense primer 5 ’AGTCTAGAGTCAATCACATGCGCTTGG3 ’ sense primer 5 ’CGAATTCAGCTACATCTCTGGTTCGCG3 ’ antisense primer 5 ’AGTCTAGAAGCGGGTAAATTGCCCTGC3 ’ sense primer 5 ’GGAATTCGAACCGGGAGACGGATAGA3 ’ antisense primer 5 ’CATTAAACTACGCGCTCCAGTCTAGACG3 ’ sense primer 5 ’CGAATTCCTATCACTTAACAGACGCTT3 ’ antisense primer 5 ’ AATCTAGACTGCGACGACCTCACAGC C3 ’ sense primer 5 ’CGAATTCTTCGAACTGTCCGACATGCC3 ’ antisense primer 5 ’CCTCTAGAGATACGGCACGCGGCTGCA3 ’ 51 Further genomics searches Search for potential myristoylation signals was done with ExPasy’s scan Prosite (http://ca.expasy.org/tools/scnpsite.htm1). Analysis for protein domains of Chp protein sequences was done by searching Pfam HMMs (http://pfam.wustl.edu/hmmsearch.shtml). Analysis of plant organelle targeting sequences was done using PSORT (http://psort.nibb.ac.jp). 52 Results Computer-assisted identification of putative ‘hrp box’-containing genes Although several ‘hrp box’ cis elements have been described based on promoter regions of hrp and/or avr genes from various P. syringae strains, none were based on strain DC3000 (Innes et al., 1993; Shen and Keen, 1993; Xiao and Hutcheson, 1994). We aligned the promoter regions of 12 known hrp, hrc, and avr genes in Pst DC3000 (Figure 2-1). This alignment was used to generate an initial DC3000-specific consensus bipartite ‘hrp box’ (KGGARCY[N15-16]CCACNNA). Although the 5th base ofthe first motif was an A in all 12 genes, we designated an R in this position because hrpWhas a second hrp-box-like sequence at -18 nts (TGGAGCT[N17]CCACTTA; not shown in Table 2—1), in which the 5th base is a G. In the KGGARCY[N 15-16]CCACNNA sequence, the two highly conserved motifs were separated by 15 to 16 nucleotides (nts). However, to investigate the importance of the spacing between the two motifs and to identify a maximum number of candidate genes, we purposely expanded this spacing range to 13 to 20 nts. We searched the January 2001, release of the Pst DC3000 genome sequence (95% coverage) from The Institute for Genomic Research (TIGR; httgflwww.tigr.org/tdb/mdb/mdbinprogress.html) for ‘hrp box’-containing sequences using FindPattems(GCG) available through BioNavigator (http://www.bionavigator.com). This search produced a total of 73 hits. The region downstream of each ‘hrp box’—like sequence was analyzed for the presence of an open reading frame (ORF) using the BioNavigator ORF finder program. In the majority of DC3000 HrpL-regulated genes, the space between the ‘hrp box’ and the putative ORF start site varied from 31 bases (her) to 306 bases (her). The only exception is the 53 putative type III effector gene orf2 of the EEL, which is located 837 nts downstream of the ‘hrp box’ (Table 2-1). However, upstream of orfl we found another small, predicted ORF (encoding an 82-aa polypeptide) 55 nts downstream of the ‘hrp box.’ We allowed up to 600 bases between the ‘hrp box’-like sequence and the putative ORF start site to maximize the number of candidate genes. The identified ORFs were then used to search for sequence similarity in GenBank. Of the 73 putative hrp-box-gontaining QRFS (HCOs hereafter), we found 11 of the 12 known type III secretion-associated genes/operons shown in Figure 2-1. The 12th gene, or]? of EEL, was not present in the January, 2001, sequence release. We found 6 putative orthologues of type III effectors, 26 HCOs with significant similarity to known bacterial proteins, 25 with similarity to hypothetical proteins in other bacteria, and 5 with no homology to any known protein (Appendix A). 54 hrpA orfl of 11ch avrPto thF aer of thK of orf8 of thP thW of 0:15 of 0:152 of CEL Consensus Figure. 2-1. Consensus ‘hrp box’ sequence in P. syringae pv. tomato DC3000. The ‘hrp box’ regions of 12 Pst DC3000 genes/operons were aligned using the ClustalW program. A consensus sequence is indicated at the bottom. CEL: Conserved effector locus (Alfano et al., 2000). EEL: Exchangeable effector locus (Alfano et al., 2000). K: T or G. R: A or G. Y: C or T. GACGCGCCGTATCGCAGGCTGCT CCCTnGT ‘ ' ‘ GGAAGCCGTAACGGCGA-GCGT CCGTRGG CGC ‘ ‘ GGGAACI‘CICHXGATCCGGGACCGTGACCTCaGC AAGCTTGGAACCGATCCGCTCCCTAT ‘CCACTC G CAT TGGAACCGCTCGGCGGGTTTGCTCCACTC‘ G 55 Gene expression analysis of putative ‘hrp box’-containing genes Primers (see Appendix A) were designed to amplify all 73 HCOs. Of the 73 HCOs, 69 were amplified successfully. The amplified DNA was arrayed onto glass slides for microarray analysis (see Experimental Procedures), or was labeled with 32F for northern blot analysis. In microarray experiments, we also included 10 human cDNAs (see Experimental Procedures) for the purpose of data normalization. Results of microarray expression and northern analysis are summarized in Table 1 and Figure 2-2. Microarray analysis showed that the expression of most of the positive control group (hrp gene operons, avrPto, 0112 of EEL, and 01f], aer, hrp W, 045, and 0717 of CEL) were induced in hrp-inducing medium, with induction ratios close to 2-fold or higher (Table 2-1). The only two exceptions were the her and th operons, which did not show consistent differential expression in LB vs. hrp-inducing medium in the microarray experiments. The expression of the her gene was at background level, giving rise to great variations in microarray experiments (Table 2-1). Also induced were 5 HCOs that show sequence homologies to known avr or vir genes (vierhA, averiB, averiC, averhD, and hopPsyA/hrmA) of other pathovars of P. syringae (Appendix A). Finally, six additional HCO genes (HCOl, 2, 3, 4, 5 and 8) were also differentially expressed (>2 fold) (Table 2-1). The remaining 49 HCOs either were not expressed at a detectable level, were not expressed at a significantly higher level in hrp-inducing medium compared with that in LB, or were expressed at a higher level in LB compared with that in hrp-inducing medium (data not shown). 56 The her operon was previously shown to be induced in hrp-inducing medium in northern blot experiments (Wei et al., 2000). Our inability to detect its induction by microarray assay in this study prompted us to verify expression of the HCOs by northern blotting. Of the 49 HCOs that did not show enhanced expression in hrp-inducing medium in the microarray experiments, 43 were subjected to northern blot analysis. The other 6 HCOs were not analyzed because their expression was not detectable in the microarray assay. As positive controls, we included hrpA, hch (her operon), aer, averiC2, averhD, and hopPsyA/hrmA in northern blot analysis (see Figure 2-2 for hrpA and hch expression). The northern blot results were consistent with the microarray results, except for two HCOs (HCO6 and HCO7; (Table 2-1). These two HCOs were induced in hrp-inducing medium in northern blot assay (Figure 2-2). Thus, with our combined microarray and northern blot analyses, we identified a total of 24 genes/operons that were expressed at higher levels in hrp-inducing medium than in LB (Table 2-1). Of the 24 genes/operons, 11 are known ‘hrp box’-containing genes located in the hrp pathogenicity island, 5 show significant homology to known avr or vir genes of other P. syringae pathovars, and 8 are uncharacterized genes. Because the primary goal of this study was to discover new type III effector genes in Pst DC3000, our subsequent analyses were focused on the eight uncharacterized genes. We determined the dependence of these novel genes on the hrpL and/or hrpRS regulatory genes for expression by northern blot analysis. Of the 8 HCOs, most showed dependence on hrpL and hrpRS for high expression with no detectable level of basal expression (Figure 2-2). The expression of HC08 was clearly dependent on the hrpRS operon, but a high level of expression was observed in the hrpL mutant background, suggesting an alternative 57 regulatory pathway downstream of the hrpRS regulatory step (Figure 2-2). Based on the dependence of their expression on the hrpS and/or hrpL gene, we named the 8 HCO genes chp, for go-regulated with the hrp genes. Although we allowed a 13- to 20-nt gap between the two conserved motifs in the initial ‘hrp-box’ search sequence to identify putative HrpL-dependent genes in our bioinformatic analysis, the genes that were confirmed to be HrpL-dependent by northern blot analysis had either a 15- or 16-nt space between the two motifs of the bipartite ‘hrp box’ sequence. Thus, the 15- to 16-nt space appears to be crucial for HrpS/HrpL regulation in Pst DC3000. Also, in our genomic search we designated a maximum of 600 bases between the ‘hrp box’ and the putative ORF start site. However, the genes that showed HrpL-dependent regulation in this study had spacing that varied from only 28 bases to 125 bases. In comparison, spacing between the ‘hrp box’ and the translational start site in the previously characterized Pst DC3000 hrp-regulated genes varied from 31 (her) to 306 bases (her) (Table 2-1). 58 e: as e; 2.». 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A203 000: as 8288 03 as <08<00 2 005.000 2. 0.0.3 00: as 8288 NS 80 <<00<00 2 005.000 em- 330 802 as 5288 2.0 $0 <<00<00 2 0002000 3. $32 ~00: 8» Beebe 00¢ 20 <0<0<00 2 005.000 21 :53 50: 30% 080330-30 B02 .28 2 20:. 6O chp2 chp3 chp‘ a l... . chp7 : FEB“ WTWTL-RS- LB MM MMMM Figure 2-2. Northern blot analysis of eight novel ‘hrp-box’-containing genes in Pst DC3000 (WT) and hrpL (L-) and hrpS (S—) mutants. The name of each gene is indicated on the right. The rRNA visualized after ethidium bromide staining was used as loading control. LB: Luria-Bertani medium (Sambrook et al., 1989). MM: hrp-inducing medium (Wei et al., 2000). Type III translocation analysis of selected ‘hrp-box’-containing genes The hrpS- and/or hrpL-dependent expression of the eight chp genes suggests that these genes encode type III effector proteins, components of the secretion machinery (e. g., chaperone proteins), or other virulence proteins that function independently of type III secretion. To identify those genes that encode putative type III effectors, we took advantage of the recent finding that when type III secretion/translocation—incompetent, but biologically active, Aerpt280-255 is fused to a protein carrying a type III secretion signal, the fusion protein can be translocated into the plant cell and elicits a hypersensitive response (HR) in Arabidopsis plants carrying a functional copy of the corresponding resistance gene, RPS2 (Mudgett et al., 2000; Guttman and Greenberg, 2001). We attempted to fuse each of the eight hrpS/hrpL-regulated genes (‘hrp box’ + ORF) to aerpt280-255. Five fusion constructs (Chpl, 5, 6, 7 and 8) were made successfully and were mobilized into Pst DC3000. We examined the ability of DC3000 carrying the recombinant fusions to elicit an HR in leaves of RPS2+ and rp52' Arabidopsis plants (ecotype Col-0). We found that the negative control strain DC3000 caused RPSZ-independent, slow disease necrosis visible only after 24 h post-inoculation (Figure 2-3). As positive controls, DC3000 expressing the wild-type Aerpt2 elicited an RPS2-dependent HR at about 9 h post-inoculation and DC3000 expressing the virPtoA (the homologue of P. s. pv. phaseolicola vierhA)-Aerpt280-255 fusion or AvrPto- Aerpt230-255 fusion elicited an RPS2-dependent HR at about 16 h after inoculation (Figure 2-3). Chpl-Aerpt230_255 fusion also caused an RPSZ-dependent HR at about 16 h post-inoculation (Figure 2-3). In contrast, the Chp6 fusion did not elicit an HR (Figure 62 2-3). Chp5, 7, and 8- Aerpt280-255 fusions did not consistently elicit an RPS2-dependent HR (data not shown). 63 AerptZ AvrPto VirPtoA Chp1 Chp6 DC3000 t. V’ 44* ,2 in .. 1 L1 Figure 2-3. Symptoms on RPSZ and rpsZ Arabidopsis leaves infiltrated with DC3000, DC3000 (pUCPl9::Aerpt2), or DC3000 (pUCPl9::effector-Aerpt230-255 fusion). Pictures were taken at 24 h after bacterial infiltration. Note that all rpsZ leaves as well as RPSZ leaves infiltrated with DC3000 or DC3000(pUCP19::Chp6-Aerpt23o-255) did not show HR necrosis (leaves remained green and smooth), whereas RPSZ leaves infiltrated with DC3000(pUCP19::Aerpt2), DC3000(pUCP19::VirPtoA-Aerpt230-255), DC3000(pUCPl 9ZZAVTPtO-AV1’Rpt23o-255), DC3000(pUCP1 922Chp1 -AVI'Rpt230-25 5) showed grey HR necrosis and the infiltrated areas appeared wrinkled. 64 Further computer analysis of Chp proteins We performed further analysis on the eight chp genes (Table 2-2). All eight Chp proteins are hydrophilic. The G+C contents of chp6 (48%) and chp8 (43%) are significantly lower than the G+C content of the Pst DC3000 genome (about 60%; Alfano et al., 2000), indicating the possibility of recent introduction of these genes by horizontal gene transfer. Chpl, Chp3, and Chp4 have no similarity to known proteins, whereas the C-terminus of Chp2 has significant similarity to the DnaJ family of proteins (BLAST score 72; E value 208-12; 72% similar over 63 aa) (Table 2-2). Chp5 has a putative transglycosylase SLT domain (PF 01464) and shares 25% identity with the N-terminus HrpW (BLAST score 64; E value 3.0E—9; 36% similar over 281 aa) (Charkowski et al., 1998). Chp6 shows sequence similarity to Orfl of the averhF locus in P. s. pv. phaseolicola (BLAST score 155; E value 6.0E-38; 75% similar over 130 aa) (Tsiamis et al., 2000). Chp7 shows similarity to proteins of the Apr family (PF 02424) (Pfam score 228; E value 6.0E-61; 93% aligned over 314 aa), some of which are involved in thiamine biosynthesis (Gralnick et al., 2000). Chp8 shows sequence similarity to several hypothetical proteins with the GGDEF domain. The most significant similarity (BLAST score 266; E value le-70; 68% similar over 246 aa) is to a hypothetical 91 .8-kD protein, AGR_L_1027p, of Agrobacterium tumefaciens. Several GGDEF domain (PF00990) proteins may possess diguanylate cyclase activity, modulating cyclic diguanylic acid levels in the cell (Ausmees etal., 2001). Chp8 also contains the Pfam EAL domain (PF00563), which has no known function. No specific plant organelle-targeting sequences are present in these proteins. 65 03000 2 020.» m 5032 05 003 00000000 80008 00,—. .0 2-03 #8821022 2.0.0.0055 23203200.022t01000. 0.0. 0.00 NS. 0.2. 000.0 5.00.0 0.32 2:022 3.2.038 8822530 .2320 08... 02.28%; S 02.. 0% 0.00 0000 2-00.0 0-3: 20.3 2808800 .3 .0 .0 .05 0.0 02 02 0:. 80.0 8-000 0.002005: 000000 0.0 .39: S. 2... 4mm 0% 80.0 - see: «.0 a: 2 0.3 008 - as: 0.0 0.2 N2 0% RE 2-000 : 20:00.2 _.: 0.2 02. 2.8 008 EE0$0~Q 50.000583 JAE 000.2080: H00Q 80008 20000 0000 - 2.8 4.0 0.3 :N 0.00 0&0 00.3 m T0080: 00.000000. 808030 80.0.5 3 A09: 0008 $000 0&00. 0%amha—_E_m cocoavom Douomvvhm guumvoum ©3369:— U+ Q 0\0 can: Ozomv 080008 95 .00 0000085 .N-N 030,—. 66 Discussion The availability of the Pst DC3000 genome makes it possible to conduct global searches for virulence-related genes. Our ‘hrp box’ motif search of the January, 2001, genomic sequence release revealed a large number of genes, including known hrp, hrc, and effector (avr and vir) genes, novel genes, and genes that show sequence similarity to known genes, but that had not been implicated in type III secretion. We took a functional genomics approach to analyze these genes. From a total of more than 73 candidate genes, which resulted from our ‘hrp box’ motif search, we identified eight new hrp-regulated genes (Chpl, 2, 3, 4, 5, 6, 7, and 8). Our search for ‘hrp box’-containing genes has served two purposes. First, it provided a pool of candidate genes for identification of novel putative type III effectors. The efficacy of this method was verified with the identification of hrp/hrc gene operons, known hrp-regulated genes located in the Hrp pathogenicity island, and putative orthologues of several known type III effector (avr and vir) genes identified previously in other P. syringae pathovars. There were a total of 20 known hrp, avr, and putative effector genes/operons in the January, 2002, release of the DC3000 genome (95% genome coverage). The ‘hrp box’ search motif used in our study enabled us to find 17 of these 20 genes/operons, giving a search efficiency of 85%. The three missing genes are orf4 of CEL, and putative orthologues of averhE (Mansfield et al., 1994) and aerps4 (Hinsch and Staskawicz, 1996). The promoter regions of the averhE orthologue and orf4 contain a G, instead of a C, at the 2nd position of the 2nd motif (Figure 2-1). In retrospect, we could have designed the 2nd base of the 2nd motif wobble (G or C) to accommodate genes like orf4 and averhE. However, this putative ‘hrp box’ was not 67 annotated in the literature nor the expression precisely defined in Pst DC3000 at the time we initiated our search. Also, the 5’ end of the averhE orthologue was not available when we conducted our search. Interestingly, when we used a revised ‘hrp box’ sequence, which allows a G or C at the 2nd base of the 2nd motif, to search the January, 2001, release, more than twice as many genes (151 compared to 73) were recovered. That is, even though orf4 and averhE represent a small minority of hrp-regulated genes, one has to screen twice as many candidate genes to find them. We could not find a sequence closely resembling an ‘hrp box’ in the promoter region of the putative aerps4 orthologue presumably because of a lack of a genuine ‘hrp box’ in this gene. Second, we were able to further elucidate the structure/function relationship of the ‘hrp box’ in Pst DC3000. Our gene expression analyses indicate that the spacing of 15 to 16 nucleotides between the two conserved motifs is crucial. Incorporation of this knowledge will aid future analysis of HrpL-regulated genes and the hrpR-hrpS—hrpL signal transduction cascade in Pst DC3000. An important finding of our study is that genes containing the same ‘hrp box’ motif (with the 15- to l6-nt spacing) had very different steady-state transcript levels (Table 1) and many were not even induced in hrp-inducing medium in both microarray and northern blot assays (data not shown). This observation raises the possibility that, at least in hrp-inducing medium, either the ‘hrp box’ is not the only determinant for HrpL- mediated regulation, or different ‘hrp-box’-containing genes produce transcripts with different stabilities that affect an accurate measurement by microarray or northern blot assay. Additionally, there was no correlation between the level of a transcript and the distance of the ‘hrp box’ relative to the start site of the first ORF. Therefore, a major 68 conclusion from this study is that a simple deduction of hrpL-dependent mRNA accumulation based solely on the presence of an ‘hrp-box’-like sequence in the promoter region is not valid. The biological significance of the different expression levels of type IH effector genes is not clear. The different transcript abundances could, however, influence the amounts of effector proteins produced and delivered into the host cells, which may be biologically optimized to alter specific host metabolic and signaling pathways in favor of pathogenesis. The regulation of Chp8 by HrpS, but not by HrpL (Figure 2-2), was not consistent with the current model in which DC3000 senses environmental signals through the HrpR- HrpS-HrpL cascade in a single linear fashion. However, our reproducible demonstration of the HrpS-dependent and HrpL-independent expression of chp8 in hrp-inducing medium strongly suggests that in addition to the HrpL pathway, there is an alternative regulatory circuit downstream of HrpS. This is a very intriguing finding. Future whole genome microarray analysis is needed to determine whether this phenomenon is more prevalent than is revealed in this study. Our analysis has resulted in the identification of a subset of putative type III effector genes that are clearly dependent on hrpS and/or hrpL genes for mRNA accumulation. The actual number of type III effectors in Pst DC3000 is likely larger than that revealed in this study, for two reasons. First, a minority of hrp-regulated genes/operons (e. g., orf4 of CEL, and the putative homologues of averhE, and aerps4) do not appear to have a typical ‘hrp box’ motif as defined in Figure 2-1. It is not known whether these genes/operons are expressed in a HrpL—dependent manner in Pst DC3000, although the orf4 operon of CEL is induced in hrp-inducing medium (Lorang and Keen, 69 1995) and we showed in this study that the averhE orthologue was induced in hrp- inducing medium (Table 2-1). It is likely these putative orthologues are additional type III effectors in Pst DC3000. Second, our microarray, northern blot, and AerptZ-based type III secretion/translocation analyses were performed with the first ORF downstream of the ‘hrp box’ motif. We purposely limited our analysis to the first ORFs to avoid misleading conclusions due to the incomplete annotation of the Pst DC3000 genome used for this study. Some type III effectors could be encoded by additional genes downstream of the first ORFs analyzed in this study (e. g., genes that are part of an operon). Indeed, we found that the second ORF (a putative orthologue of averhF of P. s. pv. phaseolicola) downstream of the chp6 gene, when its was fused with the aerpt280_255, elicited an RPSZ-dependent HR (data not shown). It is important to point out that not all HrpL-dependent genes would encode type III effectors; some may encode accessory secretion functions (e. g., chaperone) or play an important role in pathogenesis other than as type III effector proteins. As the Chp6- Aerpt2 fusion consistently did not elicit an RPS2-dependent HR, chp6 is a candidate for this class of HrpL-regulated genes. Aerpt2 fusions with Chp5, 7, and 8 did not consistently elicit an RPS2- dependent HR (data not shown). It is possible that Chp5, 7, and 8 are normally secreted via the type III secretion system, but are not translocated into the host cell. Alternatively, these Chp-Aerpt230-255 fusion proteins are translocated into Arabidopsis cells, but the Chp portion may interfere with the HR-eliciting function of the AerptZgo- 255, preventing elicitation of an HR in RPS2 plants. It is also possible that fusion of these 70 full-length Chp proteins to Aerpt230-255 interferes with the ability of the Chp and/or Aerpt230-255 portion to be secreted. Previously, the AvrBs3 family of type III effectors in Xanthomonas was shown to carry eukaryotic nuclear localization signals (N LSs) and to function inside the plant nucleus (Yang and Gabriel, 1995 ; Van den Ackerveken et al., 1996; Yang et al., 2000). Furthermore, several P. syringae type III effectors (Aerpml, AvrB, AverhB, and AvrPto) carry fatty acid modification signals that target these effector proteins to the host membrane (Nimchuk et al., 2000; Shan et al., 2000). None of the four putative type III effectors (Chp5, 6, 7, and 8) identified in this study have recognizable NLSS, transmembrane helices, or fatty acid modification signals. However, all four new putative type III effectors share sequence similarity to other proteins. Chp5 has limited similarity to the N-terminal region of another type III effector, HrpW (Charkowski et al., 1998), and contains a putative transglycosylase SLT (soluble lytic transglycosylase) domain. No transglycosylase SLT domain was found in HrpW, but HrpW contains a putative pectate lyase domain at its C-terminus (amino acids 187 to 425), which is involved in plant cell wall binding (Charkowski et al., 1998). Purified HrpW elicits an HR-like necrosis in tobacco (Charkowski et al., 1998). We do not know whether Chp5 also elicits an HR in tobacco. Chp6 shares sequence similarity with Orfl of the AverhF locus (Tsiamis et al., 2000). Finally, Chp8 shares sequence similarity to GGDEF- containing proteins, some of which may be involved in the diguanylate cyclase activity (Ausmees et al., 2001). These homologies will guide fixture investigation of the virulence functions of Chp proteins in the host. 71 In summary, this study illustrates the usefulness of functional genomics approaches, used concomitantly with biological assays, in the successful identification of novel hrp-regulated genes and putative type III effector genes from a large number of candidate genes in Pst DC3000. 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The phytotoxin coronatine and the effector proteins secreted by the type III secretion system are thought to be important for virulence. The genes which are important for production of coronatine and the effectors are transcriptionally regulated such that they are not expressed in rich media. In order to increase our chances of finding reduced-virulence mutants, a Pst DC3000 mutant population was generated with a mini-TnS uidA transposon. Mutants were first examined for uidA expression in minimal, but not rich, media. Those mutants which satisfied these criteria were then assessed for virulence in A. thaliana. Six mutants which demonstrated an apparent reduction in virulence were isolated. Three mutants contained disrupted oer genes which encode the outer membrane porin F precursor protein. These mutants also had a reduced growth rate in minimal media. One of the mutants contained a disrupted ptsP gene which encodes the Enzyme I subunit of the phosphoenolpyruvate protein phosphotransferase system. The final mutant was disrupted in the uer gene which encodes a type 11 DNA helicase involved in DNA replication and repair. Isolation of these mutants provides new tools for the study of virulence of Pst DC3000. 80 Introduction Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) is a bacterial plant pathogen. It can infect both the economically important crop plant tomato as well as the model plant species Arabidopsis thaliana. Pst DC3000 causes disease symptoms typified by water soaking followed by the development of necrotic spots surrounded by diffuse chlorosis. Although there has been a great deal of research, especially in the last 10 years, on the interaction between Pst DC3000 and its model host A. thaliana, there is still little known about how this bacterium invades plant leaves, colonizes the apoplastic space, and causes disease. Although other factors may be involved, it is known that Pst DC3000 virulence is dependent upon both the phytotoxin coronatine (Bender et al., 1999) and the proteins secreted by the type III secretion system (He, 1998). The phytotoxin coronatine has been shown to induce chlorosis in tomato (Palmer and Bender, 1995) but its role in the Pst DC3000 interaction with A. thaliana has not been clearly defined. Coronatine is structurally similar to forms of j asmonic acid (J A), a plant hormone (Wastemack and Parthier, 1997). An A. thaliana mutant, coiI, which is insensitive to coronatine is also impaired in J A signaling. These results suggest that coronatine may act in A. thaliana via perturbation of the IA signaling pathway. Neither the actual mechanism of action of coronatine nor the benefits conferred to Pst DC3000 by coronatine production are known. Virulence in Pst DC3000 is also known to require the type III secretion system. The type 111 protein secretion system is responsible for the delivery of effector proteins into the host cell cytoplasm. Mutations which disrupt the type III secretion system render the mutant bacteria non-pathogenic (Lindgren, 1997; Lindgren et al., 1986). This implies 81 that some effector proteins delivered by a functional type III secretion system are involved in virulence. In several cases, type III effector proteins have been shown to contribute to virulence. The presence of either aerme or aerpt2 allows increased pathogen growth on susceptible hosts (Chen et al., 2000; Ritter and Dangl, 1995). In a few cases, evidence is beginning to suggest the mode of action of these type III effector proteins. These mechanisms include proteolytic activity, suppression of general host defense responses, and interference with host recognition of another avr gene and development of the hypersensitive response (Chen et al., 2000; Guttman and Greenberg, 2001; Shao et al., 2002; Tsiamis et al., 2000). Recent work has revealed that coronatine biosynthesis and type III secretion may be coordinately regulated. Expression of the genes required for coronatine biosynthesis is controlled by a modified two component regulatory system encoded by corRS (U llrich et al., 1995), whereas expression of the type III secretion system is regulated by the hrpRS regulatory system (Xiao et al., 1994). However, recent studies revealed that the corRS operon might be controlled by the hrpRS regulatory system. The transcription of the cfaI, cfa2, and cfa6 genes required for coronatine biosynthesis is dependent on hrpRS (F outs et al., 2002)(Zwiesler-Vollick, Plovanich-Jones and He, unpublished data). In addition, the promoter of the corS gene in Pst DC3000 has a hrp box-like sequence (Fouts et al., 2002). Although the initial characterization of coronatine and the effectors of the type III protein secretion system has begun, many questions remain regarding the role they play in disease development. In addition, other factors may also be required which have not yet been discovered. For example, while we are beginning to understand how pathogens 82 suppress defense responses, we do not yet understand how bacterial pathogens metamorphose from epiphytes to pathogens, enter the apoplastic space, adjust to the environmental conditions in the apoplastic space, attain and maintain their close physical proximity to the plant cells within the apoplastic space, and control the release of water and/or nutrients from the host cell. Coronatine biosynthesis and type III protein secretion appear to be coordinately regulated and both processes occur in minimal media. We mutagenized Pst DC3000 with a mini-TnS transposon containing a promoterless uidA gene and screened for insertions into genes which were induced in minimal media. This may enrich for insertions in pathogenesis related genes. We then further screened for mutants which showed reduced virulence when infiltrated into plants. The genes which had been insertionally-inactivated were identified and the mutants were complemented. 83 Material and methods Bacterial culture conditions Bacterial cultures were grown at 30°C in Luria-Bertani (LB) (Katagiri et al., 2002) medium supplemented with appropriate antibiotics unless otherwise specified. Rifampicin (Rif) was used at the concentration of 100 mg/L. Kanamycin (Km) was used at the concentration of 50 mg/ L. 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X- gluc) was used at the concentration of 50 mg/L. Transposon mutagenesis A population of bacterial mutants was generated by transposon mutagenesis with the mini-TnS uidA transposon in Sml 01p" on the suicide plasmid pGP704 (Taylor et al., 1989). SmlOXpir(mini-Tn5uidA) and Pst DC3000 were grown to log phase in LB medium (Sambrook et al., 1989). Bacteria were then centrifuged in a tabletop centrifiige at 3000 rpm (approx. 2000xg) for 10 min. The supernatant was decanted and the bacterial pellet was resuspended in sterile water to an OD600=0.002 (106 cfu(colony forming units)/ml). SmlOkpir(mini-Tn5uidA) was then mixed with Pst DC3000 in a ratio of 1:5 (V /V ). The mixture was then plated onto non-selective LB plates, allowed to grow for three days and then transferred onto LB plates supplemented with Rif and Km for selection. 84 Screening of mutants for uidA expression Mutants were cultured in liquid LB media with Rif and Km to OD600=0.2 to 0.8. They were then centrifuged in a tabletop centrifuge at 3000 rpm (approx. 2000xg) for 10 min. The supernatant was decanted and the bacterial pellet was resuspended in water to an OD600=2.0 100 uL of the bacterial suspension were plated onto King’s B (KB) agar media (King et al., 1954) supplemented with Km and X-gluc and then replica-plated onto hrp-inducing minimal media (MM) (Wei et al., 2000)supplemented with Km and X-gluc. Colony color on both KB X-gluc and MM X-gluc plates was monitored. Those which were blue, indicating uidA expression, on MM but not KB plates were selected. A second round of differential uidA expression screening was performed to confirm the uidA expression phenotype. Pathogenesis assays A. thaliana (Col-0 g!) plants were grown in growth chambers at 20°C with 70% humidity, a light intensity of 100 uEinsteins, and a 12-hour photoperiod. Bacteria were grown in liquid LB medium with Rif to an OD600=O.4 to 0.8. Bacteria were then centrifuged in a tabletop centrifuge at 3,000 rpm (approx. 2,000xg) for 10 min. The supernatant was decanted and the bacterial pellet was resuspended in water to an OD600=0.002 (106 cfu/ml). Leaves of 6-week-old A. thaliana (Col-0 g1) plants were hand-infiltrated using a needleless syringe. The plants were kept under high humidity at approximately 25°C. After three days the symptoms were recorded. Typical symptoms are characterized by water-soaking at approximately two days post infiltration which then 85 develops into necrotic spots surrounded by chlorotic haloes at about 3 days after infiltration. Quantification of bacterial growth was performed (Katagiri et al., 2002). Growth assays in liquid media Mutants and wildtype Pst DC3000 were grown in LB broth supplemented with Rif overnight to an OD600 of approximately 1.0. For growth assessment in LB broth, the overnight culture was diluted to 10 ml of OD600=0.05 in LB supplemented with Rif. The bacteria were then allowed to grow until OD600=0.8. Cell density was assessed at regular intervals. For growth assessment in liquid minimal media (MM), the overnight LB culture was centrifuged, the supernatant decanted, and the pelleted cells were resuspended in MM to an OD600=0.1. No antibiotic selection was used. The cell density was monitored at various intervals. Two previously characterized Pst DC3000 mutants, hrpH and htpS, which do not form the functional type III secretion system and are therefore not pathogenic, were used as controls (Yuan and He, 1996). Southern blot analysis of bacterial mutants Bacterial DNA was extracted (Chen and Kuo, 1993). The DNA was digested overnight at 37°C with the PstI restriction enzyme (NEB cat #R0140). Ten pg of digested DNA were separated on a 1.0% agarose gel run at 60 mV for approximately 2 hours. The bands of the DNA ladder were identified in the gel under UV light and a Pasteur pipet was used to remove part of each band. The gel was treated with 0.25 M HCl for 10 minutes, and rinsed in water. The DNA was transferred to nitrocellulose membrane via capillary action in 0.4 M NaOH (Sambrook et al., 1989). The lanes of the 86 gel as well as the positions of the bands of the DNA ladder were marked on the membrane. 32P-labelled uidA probe was prepared using approximately 100 ng of polymerase chain reaction (PCR) product (Sambrook et al., 1989) and purified with Bio- Rad columns (CAT #732-6223) according to the manufacturer’s instructions. The approximate size of the fragment which hybridized to the probe was estimated by comparison with the marked bands of the ladder. Cloning and sequencing of transposon-containing fragments Bacterial genomic DNA was extracted from the mutants, digested with PstI and separated with a 0.7% agarose gel as above. DNA fragments of the approximate size of the transposon-containing Pstl fragments (revealed by Southern blot analysis) were cut out of the gel . The DNA was extracted from the gel using the BioRad Prep-A-Gene kit (Cat #732-6012) according to manufacturer’s instructions, except that incubation with the DNA-binding matrix was lengthened to 6 hours. The purified DNA was ligated into the PstI site of pBluescript SK+ treated with calf alkaline phosphatase (NEB Cat #M0290) (Sambrook et al., 1989). E. coli DHSa was transformed with ligation mix and plated on LB Km agar plates, selecting for plasmids containing miniTnSuidA. Km-resistant colonies were selected and cultured in LB Km broth. Plasmid was obtained using the Promega Wizard mini-prep kit (CAT #A7100) and sequenced using the uidA reverse primer 5’ CAGACTGAATGCCCACAGGCC 3’. The DNA sequence obtained was compared to existing sequence available in GenBank using Blastx (Altschul et al., 1990). 87 UV tolerance assays Wildtype Pst DC3000 and mutant W56 were grown in liquid LB broth supplemented with Rif to OD600=0.4 to 0.8. Bacteria were centrifuged at room temperature (2000 x g) for 10 min, the supernatant was decanted, and the bacterial pellet was resuspended in sterile water to OD600=0.002 (106 cfu/ml). Fifty “L of this suspension were plated onto LB Rif plates. The plates were then placed (agar surface down with the lids removed) onto a UV light (UV P dual intensity transilluminator). One half of the plate was protected by aluminum foil while the other half was exposed to UV light on the high setting (365 nm) for 5 seconds. The plates were then incubated at 30°C for two days. Complementation of bacterial mutations The sequence flanking the uidA gene in each insertion was used to search the Pst DC3000 genome sequence available through the TIGR unfinished microbial genome website (http://tigrblast.tigr.org/ufing/) for the full-length genes and/or operons by using the Blastn a1 gorhythm (Altschul et al., 1990). The Primer3 software (available at http://www-genome.wi.mit.edu/cgi-bin/primer/primer3 www.cgi) was used to design primers to amplify the identified genes (Rozen and Skaletsky, 2000). The primers are listed below. (PtsP Forward 5’ ATGCTGGTCAGGTCCTATGG 3’, PtsP Reverse 5’ GGTCGGTACAGCCAATGAAG 3’, PtsP + orfs Forward 5’ GCTCTAGAAAGAAGTCGGGCTTGAACG 3’, PtsP + orfs Reverse 5’ AGAGAGCGAATCTAGCGCTGGAGCTCGC 3’ 88 0er Forward 5’ CACGATATCTGGGGAACGAT 3’ 0er Reverse 5’ CCGCATTCGAGCTGAAAA 3’ Uer Forward 5’ CGAAGCTTGCAGGGTTGTGTCGAAAATC 3’ Uer Reverse 5’ GCTCTAGACCACGCAACGAGGTTATACA 3’). For PCR amplification, Pst DC3000 genomic DNA (Chen and Kuo, 1993) was used as the template. HiFi Platinum Taq (Gibco Cat # 11304-011) was used according to manufacturer’s instructions, and PCR products were purified using Qiagen QIAquick columns (cat # 28104) according to manufacturer’s instructions. The PtsP and 0er genes were cloned into pGEM-T easy and transformed into E coli DHSa. They were subsequently sub-cloned into the EcoRI site of pUCP18 (Schweizer, 1991). All other PCR products were digested with restriction enzymes (PtsP + orfs XbaI (N EB # R0145) and SacI (NEB #R0156), and uer HindIII (NEB # R0104) and XbaI (NEB # R0145)) ligated directly into pUCP18 directly and transformed into E. coli DHSa. Plasmids were extracted as above and the inserts were sequenced using JF L7 and M13 Forward primers. pUCP18 derivatives were electroporated into the corresponding Pst DC3000 mutants (Keen et al., 1990). Transformants were then assessed virulence and bacterial growth on A. thaliana Col-O g1 plants. 89 Results Isolation of Pst DC3000 mutants with differential uidA expression in LB vs. MM The two previously described virulence factors of Pst DC3000, coronatine and the effectors of the type 111 protein secretion system, are transcriptionally regulated and expressed in planta and in minimal medium (MM). In order to enrich the pool of mutants for those genes which are induced in minimal medium, differential uidA expression screening was used. Pst DC3000 was mutagenized with miniTnS-uz’dA Km. The use of this construct permits the identification of the expression pattern of the insertionally—inactivated genes. Mutants which showed more uidA expression on minimal media than on rich (KB) media were selected. A summary of this scheme is shown in Figure 3-1. Approximately 12,000 mutants were screened for uidA expression. One hundred and thirty eight mutants were isolated from the first round of selection. This number was reduced to 96 afier a second round of screening. Figure 3-2 shows the uidA expression phenotypes observed for bacterial mutants that were chosen for further study. The criteria by which these mutants were selected are described in the next section. 90 Mutant Isolation Strategy Pst DC3000::Tn5uidA KB Km X-gluc plate MM Km X-gluc plate Figure 3-1. The Pst DC3000 mutant isolation strategy. Pst DC3000 was mutagenized with mini-TnSuidA which confers Km resistance. Mutants were screened on rich King’s B (KB) medium with X-gluc which confers blue color to colonies which express uidA. Screening was then done on minimal media (MM) plates with X-gluc. Colonies which were not blue on KB but were blue on MM were selected for further study. 91 KB MM DC3000 X4 W27 W38 W56 W62 W127 Figure 3-2. uidA expression of selected mutants. The colonies were allowed to grow for 3 days at 22° C. The pictures on the left are colonies which were plated onto rich King’s B (KB) medium with X-gluc, the substrate for the enzyme B-glucuronidase encoded by the uidA gene. The pictures on the right are colonies which were plated onto minimal medium (MM) with X-gluc. The selected mutants show higher uidA expression in minimal medium than in rich medium. 92 Screening for mutants with reduced virulence Mutants showing reproducible, differential uidA expression in rich vs. MM medium were tested for reduced virulence in A. thaliana (Col-g1). Six mutants were repeatedly shown to cause reduced necrosis and chlorosis compared to wildtype Pst DC3000. These six mutants are X4, W23, W38, W56, W62 and W127. A. thaliana plants infected with the six mutants are shown in Figure 3-3. A. thaliana plants infected with the W56 mutant showed one to two leaves leaves with prominent chlorosis and necrosis while the majority of the sixteen to eighteen leaves showed a significant reduction in chlorosis and no necrosis. A. thaliana infected with the other mutants showed a significant reduction in symptoms, one to three leaves with small chlorotic patches while the majority of the sixteen to eighteen leaves showed no disease symptoms. Multiplication of these mutants within A. thaliana leaves was also assessed quantitatively. Bacterial growth levels after three days of infection are shown in Figure 3-4. The X4 mutant, which caused a significant reduction in symptoms, grew to near wildtype levels in leaves (approximately a lO-fold reduction in growth). The W56 mutant, which showed a moderate reduction in symptoms, showed an approximately 1000-fold reduction in growth when compared to the growth of Pst DC3000. The W23, W3 8, W62 and W127 mutants, which also showed significant reduction in symptoms in A. thaliana, also showed an approximately 1000-fold reduction in growth relative to that of Pst DC3000. Reduction in growth in planta may indicate a more generalized lack of bacterial fitness. To examine this possibility, growth in both MM and LB media was analyzed. 93 All mutants grew at rates and to levels similar to those of Pst DC3000 in rich media (Figure 3-5). However, while mutants X4 and W56 grew to near wildtype levels, mutants W23, W38, W62 and W127 showed a significant reduction in growth in MM (Figure 3-6). This result indicates that these mutants are affected in growth in MM. Therefore, reduced growth observed in MM could cause the reduced growth which occurs in planta as MM is thought to mimic conditions within the apoplastic space. 94 Pst DC3000 W23 W56 W127 Figure 3-3. Symptoms of Pst DC3000 mutants in A. thaliana Col-0 g1 plants. Bacteria were syringe-infiltrated into Col—0 gl leaves at 106 cfu/ml. Pictures were taken three days afier infection. I DC3000 ‘ l s . DX4 .. ‘ DW23 E 5 . 3 , uwas “a 8:4j EW56 3 mwez EW127 , Day after infiltration Figure 3-4. Bacterial proliferation in A. thaliana Col-0 gl plants. Bacteria were syringe- infiltrated into Col-0 g1 plants at 106 cfu/ml. Bacterial growth was monitored afier three days. Each bar represents the mean titer of 12 leaf discs from three individual leaves. Error bars were created using standard deviation. 96 10] ‘ + ocsooo + hrpH- hrpS- 00600 _4 _l 0.01 i. , . a . o 2 4 6 8 1o 12 Time (hours) Figure 3-5. Growth of Pst DC3000 mutants in LB medium. Bacteria were grown in LB medium at 30°C for 11 hours. The ODboo was measured at various time points. The data points represent the mean OD600 values from three readings at each time point. Error bars were created using the standard deviation. Pst DC3000 mutants hrpH and hrpS were included as controls. 97 10 00m 0 5 10 15 20 25 time (hours) 4:55.35 ‘1 ‘ +hrpH- l R —t- hrpS- -D— X4 l . —a- W56 . -0- W23 i nts-was \ i .—o—we2 i-o—wur i Figure 3-6. Growth of Pst DC3000 mutants in MM. Bacteria were grown in MM at 22°C for 24 hours. The OD600 values were determined at various time points. The data points represent the mean ODboo values from three readings at each time point. Error bars were created using the standard deviation. Pst DC3000 mutants hrpH and hrpS were included as controls. 98 Identification of the insertionally-inactivated genes To identify genes mutated in the six mutants, genomic DNA was isolated from each mutant and digested with PstI which does not cut within the transposon. Southern blot analysis with the uidA gene as a probe was performed to determine the number of transposon insertions and to estimate the size of the Pstl fragments which contain the transposon insertion. The result of the Southern blot analysis, suggesting a single insertion in each mutant, is shown in Figure 3-7. PstI fragments of estimated size transposon were extracted from an agarose gel, purified, and ligated into pBluescript KS+. In order to isolate these fragments which contained the transposon, the transformants were selected on LB Km plates. Resistance to Km is encoded within the transposon. This method prevents the cloning and sequencing of non-transposon- containing PstI fragments of a similar size. The recombinant plasmids containing the transposon-containing fragments were isolated from Km-resistant transformants, the DNA inserts were sequenced with a reverse uidA prime to obtain the sequence directly upstream of the transposon insertion site. The sequence similarities are shown in Table 3—1. Four of the six mutants (W23, W38, W62, W127) isolated contained transposon insertions in the oer gene. Two of the mutants (W23 and W3 8) contained insertions at the same site within the oer gene. Thus, three independent insertion events in the oer gene were found. Predicted annotation for these Pst DC3000 genes can be found in Appendix B. 99 Strain X4 W23 W38 W56 W62 W127 Kb 7.8 7.6 8.4 10.8 9.6 9.5 Figure 3-7. Southern blot analysis of the genomic DNA fi'om Pst DC3000 mutants. Ten gig of genomic DNA was digested with PstI, which does not out within the transposon. 2P-labelled uidA probe was used in the hybridization. The Pst DC3000 mutant strain from which the genomic DNA was isolated is listed above each lane. The estimated size of the transposon-containing PstI fragments is shown below each lane. 100 Table 3-1. Summary of the identification of the insertionally-inactivated genes. Strain BlastX Blast E- Gene Predicted Protein Score value X4 163 2 e-39 ptsP Enzyme I of the phosphoenolpyruvate protein phosphotransferase system W23 160 2 e-38 oer Outer membrane porin F precursor W38 109 5 e-22 0er Outer membrane porin F precursor W56 180 3 e-64 uer Type II helicase involved in DNA replication and repair W62 78 9 e-l4 oer Outer membrane porin F precursor W127 171 6 e-42 oer Outer membrane porin F precursor Transformants which contained Pst DC3000 mutant genomic DNA which conferred Km resistance were sequenced using a reverse uidA primer. BlastX was then used to determine the identity of the sequences. The BlastX score, expectation (E-value), gene name and putative protein identity are given above. 101 Complementation of mutations The Pst DC3000 genome was used to find the genomic sequence around the putative insertionally-inactivated genes. The region downstream of the oer gene contains a predicted gene in the opposite orientation to the 0er gene. Thus, 0er is probably the last gene in the operon. Analysis of the region surrounding the 14er gene and the PtsP gene revealed that one open-reading frame (ORF) downstream of uer and two ORFs downstream of PtsP were in the same orientation. There was no strong evidence for either a rho-independent terminator or a Shine-Dalgamo sequence in either of these regions. Based on this information, I designed primers to clone the insertionally- inactivated genes with and without the downstream ORFs. The genes were PCR- amplified, cloned into pUCP18, and transformed into the corresponding mutants. Although the oer gene was amplified, it could not be cloned despite repeated attempts. Thus, the putative oer mutants W23, W3 8, W62 and W127 were not complemented. The genomic region containing the uer gene and the putative downstream ORFs could not be PCR-amplified. However, the W56 mutant was complemented by the uer gene alone. The presence of a functional uer gene restores the in planta growth of this mutant to wildtype levels (Figure 3-8). In addition, the uer gene alone could restore the UV tolerance of the W56 mutant. The in vitro growth of Pst DC3000, W56, and W56 with uer after UV exposure is shown in Figure 3-9. The X4 mutant, which shows a ten- fold reduction in growth as well as significant reduction of symptoms in planta, was complemented by the ptsP gene. The ptsP gene is likely present in an operon, however the ptsP gene alone was capable of restoring wildtype levels of in planta growth to the 102 X4 mutant, indicating that the ptsP gene alone is responsible for the mutant phenotype (Figure 3-10). 103 2 ‘ —o—oc pUCP ‘ 0 E . —~o—wse . 0 - X- W56+Uer i 1.E+00 " Vi * ' fi—fi W , #u 7 ,MP. 0 0.5 1 1.5 2 2.5 3 3.5 day post inoculation Figure 3-8. Complementation of in planta growth of the W56 mutant by the :4er gene. Bacteria were syringe-infiltrated into Col-0 gl plants at 106 cfu/ml. Bacterial grth was monitored over three days. Each datum point represents the mean titer of 12 leaf discs from three individual leaves. Error bars were created using standard deviation. DC+pUCP represents Pst DC3000 with the empty vector pUCP18. W56 represents the Pst DC3000 mutant W56. W56+uer represents the Pst DC3000 W56 mutant with pUCP18 with the uer gene. 104 \‘t. Pst DC3000 W56 + uer Figure 3-9. Complementation of UV tolerance of the W56 mutant by the uer gene. Bacteria were plated onto LB Rif Ap plates with 106 cfu/ml. One half of each plate was exposed to 5 second of UV light. The plates were then allowed to grow at 30°C for 2 days. Pst DC3000 represents the wildtype bacteria. W56 represents the W56 mutant. W56 + uer represents the Pst DC3000 W56 mutant with pUCP18 with the 14er gene. 105 —-- oc pUCP “' -A-X4 E l % +X4+ptsP l l - o- X4+pBP+O 1.E+01 1.E+00 —— . . _- .. . . - -_ __ _ . 0 0.5 1 1.5 2 2.5 3 3.5 day post inoculation Figure 3-10. Complementation of the X4 mutant by the ptsP gene. Bacteria were syringe-infiltrated into Col-0 g1 plants at 10° cfu/ml. Bacterial growth was monitored over three days. Each datum point represents the mean titer of 12 leaf discs from three individual leaves. Error bars were created using standard deviation. DC+pUCP represents Pst DC3000 with the empty vector pUCP18. X4 represents the X4 mutant. X4+EI represents the X4 mutant with pUCP18 with the ptsP gene. X4+EI+O represents the Pst DC3000 X4 mutant with pUCP18 with the ptsP gene and the downstream ORFs. 106 Discussion The intended purpose of this study was the identification of novel virulence genes, especially type III effector genes, which would be expected to be expressed in MM. Therefore, it is surprising that no mutations in the previously described hrp genes were found. These genes are known to be hrp-regulated and should be expressed in hrp- inducing minimal media but not rich King’s B media. In addition, mutations in many of the hip genes show a loss of pathogenicity. However, the overall expression level of these genes may be low compared to the genes identified in this study. Perhaps the visual screening that we used in this study was not sensitive enough to discern the subtle uidA expression which would result from a mini-Tn5 uidA Km insertion into a hrp gene. Despite the fact that no novel type III effector genes were found in this study, the mutants identified still provide insight into the process of Pst DC3000 pathogenesis. There were three independent insertion events into the oer gene (W23/W 3 8, W62 and W127). The oer gene encodes the precursor of the outer membrane protein F. This protein has been of interest in the study of mammalian bacterial pathogens because it is an outer membrane protein and may be a target for recognition by the host immune system (Knapp et al., 1999). The ability of the W23/W38, W62 and W127 mutants to be complemented by the DC3000 oer gene could not be determined because positive transformants could not be obtained in E. coli. A literature search indicates that this is not an uncommon problem. Other studies which have complemented oer mutants have had to clone the oer gene with a weakened promoter or a mutated promoter to reduce the expression of the oer gene (Brinkman et al., 1999; Rawling et al., 1998). 107 Overexpression of the oer gene is apparently lethal. Despite the fact that the W23/W3 8, W62 and W127 mutants could not be complemented, the presence of three independent mutations in this gene does reinforce the idea that this gene is important for virulence. Studies of oer mutants of a related bacteria, Pseudomonas aeruginosa, hint at the role that Oer may play in Pst DC3000 virulence. P. aeruginosa is a Gram—negative bacterial pathogen which can infect the lungs of immune-compromised individuals and cystic fibrosis patients (Oliver et al., 2000). P. aeruginosa oer mutants have an altered cell morphology. In addition, these mutants have a reduced ability to grow in media with low osmolarity (Rawling et al., 1998). This is intriguing especially in light of the observed inability of the W23/W 38, W56 and W127 mutants to grow in MM while their grth in rich media is comparable to that of Pst DC3000. While the environmental conditions in the apoplastic space where Pst DC3000 resides during infection are unknown for the most part, it has been suggested that this environment may not be rich in nutrients and/or water. The reduced virulence phenotype observed in the W23/W 3 8, W56 and W127 mutants suggests that the apoplastic space may be a low osmolarity environment, similar to MM. There has also been a study which shows that P. aeruginosa oer mutants exhibit a reduced ability to bind to human cells in vitro (Azghani et al., 2002). The Oer protein of P. fluorescens has been shown to bind to plant roots (DeMot et al., 1992). The mechanism by which Pst DC3000 binds to host plant cells within the apoplastic space is not known. The Hrp pilus of the type 111 protein secretion system may play a role in this process, but this has not been proven. The reduced virulence of the W23/W 38, W56 and W127 mutants in A. thaliana leaves provides an alternative hypothesis. The development of new microscopic techniques with fluorescent-labeled bacteria now allows for the 108 visualization of bacteria within the apoplastic space (Badel et al., 2002). These techniques could be used to observe the course of infection with the oer and hrp mutants, compared to that of wildtype Pst DC3000, and determine if the 0er or hrp mutants are unable to adhere to host plant cells. Another mutant isolated in this screen was the W56 mutant which lacks a functional 14er gene. The 14er gene encodes a type 11 DNA helicase which is required for unwinding the DNA helix during DNA replication and repair. Bacterial mutants which lack the uer gene have a higher mutation rate than wildtype bacteria, especially in response to mutagens such as UV light (Oliver et a1, 2002). The Pst DC3000 W56 mutant also showed an increased sensitivity to UV light. In mammalian pathogens, the mutation rate has been shown to play an important role in pathogenesis. In P. aeruginosa, the loss of the mutS, mutL and uer genes (which enable DNA repair functions) confers an advantage in virulence over time, on a population level (Oliver et al., 2002; Oliver et al., 2000). The advantage accorded by a higher mutation rate is thought to be the result of co-evolution between pathogens and host. The mammalian immune system is able to recognize certain antigenic pathogen proteins. A higher mutation rate confers an advantage to a pathogen because it allows the pathogen’s surface antigens to change and evade immune recognition. However, in our case the W56 mutant showed reduced virulence in a three day period and we have not followed the W56 mutant through many generations or cycles of infection. There is evidence to suggest that UV tolerance is important for the epiphytic phase of P. syringae growth. As the bacteria exist on the surface of leaves, they face an onslaught of UV light (Sundin and Jacobs, 1999). rulA mutants of P. syringae showed 109 reduced tolerance to UV light and are not as competitive as wildtype bacteria in the phyllosphere (Sundin et al., 2000). The rul operon is often located on plasmids of the pPT23A family which frequently encode genes involved in host-pathogen interactions (Sundin et al., 2000). The inoculation methods used in this study essentially bypassed phyllospheric growth because the bacteria were infiltrated directly into the apoplastic space. It is possible however, that some UV light is permeating the leaf and entering the apoplast. This would then impact the growth of the pathogen. Another possibility is that this mutant is less able to handle stresses such as those caused by exposure to plant defense compounds present in the apoplastic space. Preliminary evidence indicates that the W56 mutant grows to a higher level in the NahG transgenic host (data not shown), which lacks salicylic acid-mediated host defenses (Hunt et al., 1996). Further experiments are needed to determine the role that uer plays in Pst DC3000 virulence in planta. Finally, the X4 mutant contains an insertion in the ptsP gene which encodes a component of the phosphoenolpyruvate protein phosphotransferase system (PTS). The PTS is a sugar uptake system which has been well characterized in E. coli (Ginsburg and Peterkofsky, 2002; Postma et al., 1993). The ptsP gene encodes the Enzyme I subunit of the PTS. Enzyme I is biologically active as a dimer. In the presence of Mg, it is able to self-phosphorylate. This phosphoryl moiety can then be transferred to the HPr carrier protein, then transferred to the Enzyme II subunit, and finally transferred to the sugar as it is transported into the bacterial cell. Several Enzyme II subunits have been characterized and they seem to be designed for specific sugars such as glucose, mannose, mannitol and cellobiose. The PTS has also been implicated in processes other than sugar uptake, such 110 as catabolite repression and chemotaxis. In two other bacterial pathogens, mutations in the ptsP gene have led to a reduced virulence phenotype. In a multi-host strain of P. aeruginosa, the loss of the ptsP gene leads to a loss of virulence on Caenorhabditis elegans and a loss of pathogenicity on burnt mice (Tan et al., 1999). Legionella pneumophila ptsP mutants are also impaired in pathogenecity (Hi ga and Edelstein, 2001). In Azotobacter vinelandii, the ptsP gene has been shown to be required for the production of 8-polyhydroxy butyrate (Segura and Espin, 1998). The mechanisms by which nutrients are released from the plant host or obtained by the bacterimn are not currently known. The reduced virulence of the X4 mutant may indicate that the PTS plays a role in nutrient uptake by bacteria. In addition, the reduction in virulence may stem fi'om a loss of catabolite repression or chemotaxis which could be required for pathogen growth within the apoplastic space. Overall, this study has identified three novel Pst DC3000 genes, mutations in which cause a reduction in virulence on A. thaliana. 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(1996). The Pseudomonas syringae hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. Journal of Bacteriology 1 78, 6399-6402. 115 Chapter 4 Characterization of transgenic Arabidopsis thaliana plants that express the Aer effector of Pseudomonas syringae pv. tomato strain DC3000 I would like to gratefirlly acknowledge Elena Bray Speth, Guanghui Liu, and Ying Yang, who worked with me during their rotations. However, their projects are not included in this chapter. 116 Abstract Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) is the causal agent of bacterial speck disease of tomato and Arabidopsis thaliana. Effector proteins secreted by the type IH protein secretion system have been implicated in Pst DC3000 virulence. Aer is one effector produced by Pst DC3000. Bacterial genetic studies have failed to determine the role that Aer plays in Pst DC3000 virulence, purportedly due to functional redundancy among effector proteins. We utilized an inducible promoter system to express aer with the PRl-b tobacco signal sequence in A. thaliana. These transgenic plants displayed symptoms similar those caused by Pst DC3000 infection after transgene induction. These symptoms included water-soaking followed by the development of necrotic spots surrounded by chlorotic haloes. The water-soaking phenotype was correlated with stomatal closure. These transgenic plants supported increased growth of the normally non-pathogenic hrpH mutant of Pst DC3000. This study provides evidence that Aer does contribute to the virulence of Pst DC3000. 117 Introduction Plant pathogens have a significant impact on agriculture. Therefore, a great deal of study regarding plant-pathogen interactions has focused on understanding plant resistance. One important resistance mechanism is gene-for-gene resistance (Flor, 1971), which occurs when a pathogen harboring a given avr gene attempts to infect a plant expressing the corresponding R gene. A hypersensitive response (HR) will then occur. The HR is a form of programmed cell death which is thought to limit the spread of the bacterial pathogen, although how this happens mechanistically is not understood. Our lab focuses on understanding the virulence role of Avr proteins in Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). This bacterial pathogen is the causal agent of bacterial speck in both Arabidopsis thaliana and tomato. In this study, we have chosen to determine the role of Aer in virulence of Pst DC3000 on A. thaliana. A Pst DC3000 mutant in which the aer gene has been replaced with an antibiotic marker cassette shows no reduction in virulence on A. thaliana (He, unpublished data). However, it has been proposed that the effectors of Pst DC3000 are functionally redundant. Therefore, traditional bacterial mutagenesis would not be amenable for determining the role that a given effector plays in virulence. If the only role of an avr gene was to trigger host resistance, avr genes should not be maintained in the bacterial genome. This suggests that the avr genes play a role in promoting bacterial fitness. In support of this speculation, many Avr proteins have now been shown to be secreted from bacteria via the type III secretion system (Guttman et al., 2002; Petnicki-chieja et al., 2002), which is often essential for bacterial pathogenesis (He, 1998; Galan and Collmer, 1999). Furthermore, several bacterial avr genes have 118 been shown to be required for pathogen growth in susceptible hosts (Chen et al., 2000; Kearney and Staskawicz, 1990; Ritter and Dangl, 1995). While there is no direct evidence that Aer promotes Pst DC3000 virulence, there is correlative evidence. aer is required for full virulence in Pst strain PT23 on tomato (Lorang et al., 1994). Loss of DspE, an effector which shares sequence similarity with Aer, abolishes pathogenicity of E. amylovora on apple and pear (Bogdanove et al., 1998; Gaudriault et al., 1997 ; Tharaud et al., 1994). Aer is a type III effector protein. The promoter of the aer gene contains a classic canonical hrp-box motif and it is co- regulated with the hrp genes in a hrpS- and hrpL-dependent manner (Fouts et al., 2002; Lorang and Keen, 1995; Zwiesler-Vollick et al., 2002). It is secreted by the type 111 protein secretion system in vitro and is capable of promoting the translocation of secretion-incompetent but biologically-active Aerpt2 (Guttman et al., 2002). In Pst DC3000, aer is linked to the hrp gene cluster, in a locus known as the conserved effector locus (CEL). A mutant which lacks four of the effectors from this region, including Aer, shows a significant reduction in virulence on both A. thaliana and tomato hosts (Alfano et al., 2000). Several recent studies have focused on the identification of new phytobacterial type III effector proteins (Boch et al., 2002; Fouts et al., 2002; Guttman et al., 2002; Petnicki-chiej a et al., 2002; Zwiesler-Vollick et al., 2002). These studies indicate that there are many more putative effectors than previously predicted among the phytopathogenic Pseudomonads. Even within an individual subspecies, such as Pst DC3000, there are a remarkable number of putative effectors, at least 36 (Collmer et al., 2002). If some of these effectors are, as previously suggested, functionally redundant, 119 few bacterial studies will be able to demonstrate the subtle advantage conferred by any specific effector protein. For this reason we have begun to assess the impact of production of a single effector in planta. In this study, we present further evidence that Aer contributes to Pst DC3000 virulence. If Pst DC3000 contains another virulence factor which is able to perform the same function as Aer, bacterial mutational studies would be unlikely to yield information about the role of Aer. Thus, we expressed the Aer protein transgenically in A. thaliana. This approach investigated the effect of Aer within the host plant cell, independent of the role that other effectors might play. We were therefore able to examine the effect of a single effector, Aer, on A. thaliana health, appearance, and ability to sustain growth of a normally non-pathogenic bacteria, the Pst DC3000 hrpH mutant. 120 Materials and Methods Generation of Transgenic plants Pst DC3000 genomic DNA was extracted (Chen and Kuo, 1993). Polymerase chain reaction was used to amplify aer using eLONGase polymerase (Gibco Cat # 10480028) according to manufacturer’s instructions. The primers used were: ssaer Forward 5’ GCGGATCCCAGTCACCATCGATCCACCG 3’ aer Forward 5’ CCGCTCGAGACCATGGAGTCACCATCGATCCACCG 3’ aer Reverse 5’GACTAGTTTCGTTATTAGCTCTTCAGTTCG 3’. The aer gene of Pst DC3000 was cloned into the pTA7002 vector (Aoyarna and Chua, 1997; McNellis et al., 1998) with and without the tobacco PRl-b signal sequence (ss) (PRl-b Forward 5’ CCGCTCGAGACCATGGGATTTTTTCTCTTTTCACAAATGCCCTCATTTTTTCTT GTCTCTACACTTCTC 3’ PRl-b Reverse 5’ CGGGATCCAGAGTTTTGGGCATGAGAAGAGTGAGATATTATTAGGAATAAGA GAAGTGTAGAGACAAG 3’). pTA7002 allows for inducible expression of transgenes after application of the animal glucocorticoid hormone, dexamethasone (DEX). The recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3850 by electroporation (Keen et al., 1990). Four pots of Arabidopsis thaliana Col-0 g1 plants were transformed with A. tumefaciens carrying pTA7002-aer or pTA7002-ssaer via vacuum infiltration (Bechtold et al., 1993). Seeds collected from the pots were kept separate to ensure that independently transformed lines could be isolated. Seeds were vapor-sterilized by incubation in a dessicator for 16 hours with 100ml of bleach mixed 121 with 3ml of concentrated HCl. Transformants were selected on Murashige-Skoog (MS) plates supplemented with 1x vitamins and 40 units/ml hygromycin B (hyg) (Calbiochem Cat # 400051). Southern blot analysis Genomic DNA from Col-0 g1 and putative transgenic plants was extracted by grinding a single leaf in 400 pl extraction buffer (100mM Tris pH 8.0, 10 mM EDTA, and 1.0% SDS) followed by phenolzchloroformzisoamyl alcohol (25:24:1) extraction, chloroform extraction and ethanol precipitation (Sambrook et al., 1989). DNA was digested with BamHI (N EB Cat # R0136) with overnight incubation at 37°C. Ten pg of digested DNA were separated on a 1.0% agarose gel, at 60 mV for approximately 2 hours. The bands of the DNA ladder were identified in the gel under UV light and a Pasteur pipet was used to remove part of each band. The gel was then treated with 0.25 M HCl for 10 minutes, and rinsed in water. The digested DNA was then transferred to nitrocellulose membrane via capillary action in 0.4 M NaOH (Sambrook et al., 1989). The lanes of the gel, as well as the bands of the DNA ladder, were marked on the membrane. 32P-labelled probe was prepared (Sambrook et al., 1989) with approximately 100 ng of an internal aer PCR product (Intra aer Forward 5’ GCCCCCGCCACCCACCGCCGC 3’ and Intra aer Reverse 5’ CGGAGGCCTTCCCCCGGACTC 3’) and purified with Bio-Rad columns (Cat #732-6223) according to manufacturer’s instruction. The approximate size of the fragment which hybridized to the probe was estimated by comparison with the marked bands of the ladder. 122 Dexamethasone-induction of transgene expression A 30mM stock of dexamethasone (Sigma Aldrich Cat # D1756) (DEX) was made up in 100% ethanol. This stock solution was diluted 1000-fold in water to make 30pM DEX. The transgene was induced by either infiltrating the leaves with a needleless syringe or by spraying the leaves with the DEX solution. Northern blot analysis A. thaliana Col-0 gl and transgenic plants were sprayed with 30pM DEX. Tissue was collected at selected time points between 0 hours and 36 hours and snap-frozen in liquid N2. A. thaliana RNA was isolated using the Promega RNAgents kit (Cat # Z5110) according to manufacturer’s instructions. Twenty pg RNA were denatured with two volumes of loading buffer (500 pl formamide, 170 pl formaldehyde, 100 p1 10X MOPS buffer, and 10 pl of 1 mg/ml ethidium bromide) for ten minutes at 65°C. The RNA was separated on a formaldehyde agarose gel and transferred to nitrocellulose membrane via capillary transfer (Sambrook et al., 1989). Probe labeling and hybridization were performed as above. 32P-labeled eIF 4a (eukaryotic initiation factor 43) probe was used to assess the amount of sample loaded. Bacterial culture conditions Bacterial cultures were cultured at 30°C in Luria-Bertani (LB) medium (Katagiri et al., 2002) supplemented with appropriate antibiotics. Rifampicin (Rif) was used at a concentration of 100 mg/L. Ampicillin (Ap) was used at a concentration of 100 mg/L. 123 Pathogenesis assays A. thaliana (Col-0 g1 and transgenic) plants were grown in growth chambers at 20°C with 70% humidity, a light intensity of 100 pEinsteins, and a l2-hour photoperiod. Bacteria were grown to approximately OD600=0.4 to 0.8. Bacteria were then centrifuged in a tabletop centrifuge at 3,000 rpm (approximately 2,000xg) for 10 min. The supernatant was decanted and the bacterial pellet was resuspended in sterile water to OD600=0.002 (106 cfu(colony forming units)/ml). The leaves of 6-week-old A. thaliana (Col-0 g1) plants were infiltrated with the bacterial suspension using a needless syringe. Infected plants were kept under high humidity at approximately 25°C. After three days the symptoms were recorded. Typical symptoms were characterized by water-soaking, at approximately two days post infiltration followed by the development of necrotic spots surrounded by chlorotic haloes at about 3 days after infiltration. Quantification of bacterial growth was performed as in Katagiri et al. (2002). The bacterial strains used in this study were Pst DC3000, the 12er mutant, and Pst DC3000 expressing the aerpt2 gene. The hrpH mutant was previously described by Yuan and He (1996). The Pst DC3000 expressing the aerptZ gene was previously described (Zwiesler-Vollick et al 2002) Evaluation of stomatal opening A. thaliana Col-0 g1 and ssaer plants were kept under a high humidity dome. Leaves were observed at various time points. Leaves were removed from the plants and 124 sliced in half with a razor blade to remove the mid-rib vein. The leaf halves were placed ventral side up on a microscope slide. A bead of water was added to each leaf and a cover slip was place on the leaf halves. Stomata were observed with a Reichert Microstar IV microscope at 40X magnification. At least 100 stomata from one leaf were examined and rated as either open or closed. Col-o gl stomata which were less than completely open were evaluated as closed. ssaer stomata which were showed any space between the two guard cells were evaluated as open. This stringent evaluation method would underestimate the stomatal closure seen in the ssaer transgenic plants. The entire process, from the removal of the leaf from the plant to the completion of the counting, took less than 15 minutes. 125 Results Generation of transgenic plants and examination of expression of ssaer To ascertain the function of the Aer protein in Pst DC3000 virulence, aer was transformed into susceptible A. thaliana plants with and without the tobacco PR-lb signal sequence (55). Transformants were selected on MS medium supplemented with hyg. Despite four independent transformation attempts, no transgenic plants containing aer without the signal peptide could be obtained. In contrast, 21 ssaer transgenic lines were obtained. Three independent ssaer transgenic lines (2-9, 3-1, and 4-5) were chosen for further study. Results from studies with the representative line ssaer 2-9 will be presented in this chapter. Southern blot analysis with BamHI-digested DNA indicated that these plants contain aer hybridizing bands (See Figure 4-1). Northern blot analysis showed that the ssaer mRNA was produced afier exposure of plants to DEX. A time course showed that the ssaer mRNA could be detected as early as 12 hours after 30 pM DEX application and remained detectable until 36 hours after 30 pM DEX application (see Figure 4-1). The ssaer mRNA levels before 12 hours and beyond 36 hours after 30 pM DEX exposure were not determined. 126 . 12 Kb 0» w 7.5 Kb Col-0 ssaer 9' , 2-9 j aer I_ O 12 01211518 212436 hpd Figure 4-1. Southern blot and northern blot analyses of ssaer transgenic plants. A. Southern blot analysis of BamHI digested ssaer 2-9 genomic DNA hybridized with 32P- labeled aer. The approximate size of the hybridizing bands is indicated. B. Northern blot analysis of total RNA extracted from Col-0 gl and ssaer 2-9 transgenic plants. The plants were sprayed with 30pM DEX. hpd indicates the hour post-DEX application when the tissue was collected. aer indicates that the blot was hybridized with 32P- labeled aer. eIF 4 indicates that the blot was hybridized with 32P-labeled eIF 4. 127 ssaer transgenic plants show two distinct phenotypes The ssaer transgenic plants were slightly smaller than the parent plants but otherwise showed no obvious morphological differences from their Col-0 g1 parents in the absence of DEX (see Figure 4-2). After treatment with 30 pM DEX, the ssaer plants displayed a distinct phenotype. If the plants were treated with DEX and allowed to remain at ambient humidity, the leaves began to show signs of chlorosis by 36 hours after spraying. By 48 to 72 hours the leaves which were chlorotic had necrosed and collapsed (Figure 4-2). If, however, the plants were placed under a high humidity dome after exposure to 30 pM DEX, the phenotype was slightly altered. These plants showed water- soaking at 6 to 12 hours after DEX exposure. Then at 36 to 48 hours after DEX exposure, chlorosis with necrotic spots began to develop (see Figure 4-2). This phenotype mirrors the symptoms caused by Pst DC3000 infection of A. thaliana. 128 Col-0 gl ssaer 2-9 No DEX DEX low humidity DEX high humidity Figure 4-2. Phenotypes of ssaer transgenic plants. Experiments were conducted when plants were approximately 6-weeks-old. The plants shown in the panel labeled “No DEX” were not treated with DEX. The plants shown in the panel labeled “DEX low humidity” were sprayed with 30 pM DEX and left uncovered for two days before pictures were taken. The plants shown in the panel labeled “DEX high humidity” were sprayed with 30 pM DEX and covered with a humidity dome for three days before pictures were taken. 129 DEX-induction of ssaer causes stomatal closure Six hours after DEX-induction, ssaer transgenic plant leaves could not be vacuum-infiltrated efficiently with bacterial suspension containing surfactant L-77 nor with water and surfactant L-77. In contrast, DEX-induced Col-0 g1 plants were able to be efficiently vacuum-infiltrated with either solution. Because vacuum-infiltration relies upon open stomata as the sole point of bacterial entry into the apoplastic space, I decided to look at the stomatal aperture after DEX-induction in Col-0 g1 and ssaer transgenic plants. Stomata] counting was completed within fifteen minutes after the leaves were removed from the high humidity environment to prevent the stomata from reacting to the change in environmental conditions. For each plant and treatment, 100 stomata per leaf were counted and evaluated as open or closed. The data collected are shown in Table 4- 1. A sample picture showing the difference between DEX-induced Col-0 gl and ssaer transgenic plants is shown in Figure 4-3. While the majority of stomata in the DEX- treated Col-0 gl leaves kept under high humidity were open, the majority of stomata in DEX-treated ssaer leaves were closed. This phenotype is dependent on the application of DEX. Both the Col-0 gl and ssaer transgenic plants have the majority of their stomata open if plants are kept under high humidity after spraying with water. The DEX- induced closure of stomata could only be observed in a high humidity environment. Under low humidity, most of the stomata of Col-0 g1 plants were closed. To determine if this phenomenon is also produced by Pst DC3000 infection, Col-0 g1 leaves which had been infiltrated with Pst DC3 000 or the hrpH mutant were examined under high humidity. Two days after infection, water-soaking could be observed in the Pst DC3000 130 infected plants, but not in the hrpH infected plants. At this time point, approximately half of the stomata in the hrpH infected plants are closed, while approximately 75% of the stomata in Pst DC3000 infected plants are closed (Table 4-2). ssaer expression in planta results in a phenotype which is reminiscent of symptoms triggered by Pst DC3000 infection. Because both water-soaking and stomatal closure may affect the water relations within the leaves, I checked to see if the stomatal closure preceded the appearance of visible water-soaking under high humidity. Three hours after spraying with DEX, no visible water-soaking could be seen. However, the majority of the stomata in the ssaer transgenic plants were closed at this time point, in contrast to the Col-0 gl plants in which only half the stomata were closed (Table 4-3). Finally, I wanted to determine how stomata react to the presence of excess water in the apoplastic space. For this purpose, Col-0 gl leaves were infiltrated with water and kept under high humidity. The leaves were then monitored for an hour. After an hour, almost all of the stomata of these leaves were open (Table 4-4). 131 Table 4-1. DEX-induction of ssaer causes stomatal closure Experiment Treatment Plant Open stomata Closed Stomata 1 Mock Col-0 gl 64% 36% 1 Mock ssaer 2-9 62% 38% 1 30 pM DEX Col-0 gl 67% 33% 1 30 pM DEX ssaer 2-9 30% 70% 2 30 pM DEX Col-0 gl 73% 27% 2 30 pM DEX ssaer 2-9 26% 74% 3 30 pM DEX Col-0 gl 43% 57% 3 30 pM DEX ssaer 2-9 24% 76% The plants were sprayed with 30 pM DEX or sprayed with water (mock) as indicated. The plants were kept under humidity domes and each row represents at least 100 stomata from one leaf observed at six hours after treatment. Three experiments were conducted with separate sets of sprayed plants on different days. Table 4-2. Pst DC3000 infection of Col-0 g1 causes stomatal closure Experiment Bacteria Open Stomata Closed Stomata 1 hrpH 5 8% 42% l hrpH 46% 54% 1 Pst DC3000 33% 67% 1 Pst DC3000 34% 66% 2 hrpH 49% 5 1% 2 Pst DC3000 19% 81% The plants were syringe-infiltrated with either the non-pathogenic hrpH mutant or Pst DC3000 at 106 cfu/ml. The observations were made two days after bacterial inoculation when the Pst DC3 000 plants showed water-soaking symptoms. Each row represents at least 100 stomata from one leaf. Two experiments were conducted with separate sets of infiltrated plants on different days. 132 Table 4-3. Time course of stomatal response. Time (hours) Plant Open Stomata Closed Stomata 0 Col-0 gl 25% 75% 0 ssaer 2-9 34% 66% 3 Col-0 gl 44% 56% 3 3.90er 2-9 24% 7 6% 6 Col-0 gl 54% 46% 6 ssaer 2-9 30% 70% The plants were sprayed with 30 pM DEX and stomata were observed at 0, 3, and 6 hours after DEX exposure. Each row represents at least 100 stomata from one leaf. Table 4-4. Artificial water-soaking of Col-0 gl leaves causes stomatal opening. Time (minutes) Open Stomata Closed Stomata 0 72% 28% 0 64% 36% 15 89% 11% 15 88% 12% 30 93% 7% 30 91% 9% 45 96% 4% 45 95% 5% The plants were syringe-infiltrated with sterile water. The stomata were observed at 0, 15, 30, and 45 minutes after infiltration. Two leaves were used for each time point. 133 Figure 4—3. The majority of the stomata in the ssaer plants are closed under high light and humidity. Pictures were taken eight hours after spraying with 30 pM DEX. Plants were kept in the light under a high humidity dome. A. Col-0 g1 plants treated with 30 pM DEX. B. ssaer 2-9 plants treated with 30 pM DEX. Black arrows indicate closed stomata. White arrows indicate open stomata. 134 DEX-induction of ssaer promotes enhanced bacterial growth In order to ascertain the virulence contribution of Aer in the absence of other type III effector proteins, the transgenic ssaer plants were used. I examined the multiplication of the hrpH mutant in Col-0 gl and ssaer transgenic plants. The Pst DC3000 hrpH mutant is unable to form a functional type III protein secretion system and is thus incapable of secreting Aer as well as all other type III effector proteins. The plants were treated with DEX six hours before bacterial inoculation and daily during the course of the experiment to ensure that Aer was present in the plants. The bacterial multiplication in these plants is shown in Figure 4-4. The hrpH mutant was unable to multiply beyond the inoculation level in DEX-treated Col-0 gl plants, but was able to grow to levels similar to Pst DC3000 in DEX—treated ssaer transgenic plants. To determine if the benefit to the hrpH mutant conferred by ssaer expression was specific to those bacteria which lack Aer and other type III effector proteins, the grth of Pst DC3000 carrying the aerpt2 gene was assessed. aerpt2 is not a naturally occurring gene in the genome of Pst DC3000, but is present in other P. syringae strains. Because the Col-0 gl plants contain the cognate R gene, RPS2, Pst DC3000 carrying a plasmid- bome copy of aerpt2 are avirulent and unable to infect Col-0 gl (Kunkel et al., 1993; Yu et al., 1993). However, as seen in Figure 4-5, the DEX-treated ssaer plants (which also contain the RPSZ gene) are able to promote the growth of Pst DC3000 carrying aerptZ to levels similar to those of Pst DC3000. The growth of wildtype Pst DC3000 was unaffected in the ssaer transgenic plants. 135 1 E+08 l 1 E10? 1 [ 1 E+06 . I ‘ _Lr- ___. 1 E+05 J ' -o—Coi DC3000 ‘g r +ssE DC3000 ‘3’ 1 51-04 “‘3 I I -‘- COI hrpH. ________________ x . 1 E+03 T i —O- 355 hrpI-l- . 1 E+02 i 1 E+01 j l 1 E+00 i —- -——-5— - - . . — — _— 0 0.5 1 1.5 2 2.5 3 3.5 day post inoculation Figure 4-4. The hrpH mutant is able to proliferate in ssaer transgenic plants. Plants were sprayed with 30 pM DEX six hours prior to bacterial infiltration and daily during the course of the experiment. Bacteria were syringe-infiltrated into plants at 106 cfu/ml. Bacterial growth was monitored over three days. Each datum point represents the mean titer of 12 leaf discs from three individual leaves. Error bars were created using standard deviation. Col represents Col-0 g1 plants. ssE represents ssaer transgenic plants. DC3000 represents Pst DC3000. hrpH- represents the hrpH mutant. 136 1.E+08 - 17c; 1.E+07 DC3000 ElssE 1.E+06 003000 ‘ ‘ DCol I 1.E+05 hrpl-l- ; "E 1 g _ + , ‘ZssE % 1E 04 hrp“ 1.E+03 == 5°” . = j, aerptZ} 154.02 E .EssE E ‘ aerpt2| 1.E+01 E 1.E+00 » AER Day post inoculation Figure 4-5. Bacterial proliferation in A. thaliana Col-O gl and ssaer transgenic plants. Plants were sprayed with 30 pM DEX six hours prior to bacterial infiltration and daily during the course of the experiment. Bacteria were syringe-infiltrated into plants at 106 cfu/ml. Bacterial growth was monitored after three days. Each bar represents the mean titer of 12 leaf discs from three individual leaves. Error bars were created using standard deviation. Col represents Col-0 g1 plants. ssE represents ssaer transgenic plants. DC3000 represents Pst DC3000. H- represents the hrpH mutant. aerpt2 represents Pst DC3000 expressing the aerpt2 gene. 137 Discussion The objective of this study was the identification of the in planta function of the Pst DC3000 type III effector, Aer. Despite four independent transformation attempts, I was unable to obtain A. thaliana Col-0 gl plants that expressed the aer transgene under the control of the DEX-inducible promoter. We hypothesize that Aer may be toxic to the plant cell, even at levels produced in the absence of DEX. Embryonic lethality might account for the inability to obtain aer transgenic plants. While no ssaer mRNA could be seen before induction with northern blot analysis, this technique may not be sensitive enough to detect very low transcript levels. This presumed toxicity may be related to the necrosis which develops during Pst DC3 000 infection or it may be related to overexpression of the Aer protein in the transgenic plants. I was able to obtain transgenic plants which express aer-tobacco PR1-b signal sequence fusion. These plants could be viable due to the removal of the majority of the fusion protein from the cytoplasm of the host plant cell, as speculated for other fusion proteins (Lund and Dunsmuir, 1992; Gopalan et al., 1996). Thus the tobacco PR1-b signal sequence may help to reduce toxicity due to expression of foreign proteins. Because both the ssavrB (Gopalan et al., 1996) and ssaer transgenic plants have necrosis phenotypes, this could suggest that the expression of a foreign bacterial gene fused to the signal sequence would cause a cell death phenotype. However, I have created other transgenic plants which express the Pst DC3 000 hrpZ and hrpW genes fused to the tobacco PR1-b signal sequence under the control of the DEX-inducible promoter. These plants do not show an ssaer- or ssavrB—like cell death phenotype after 138 DEX treatment (data not shown). Therefore, the necrosis is not likely due to the presence of the tobacco PR1-b signal sequence which might block the plant general secretory pathway. Members of our lab are now developing an Agrobacterium-based system for the transient expression of transgenes in the leaves of A. thaliana. This system may allow for the transient expression of aer without the tobacco PR1-b signal sequence. This assay would address whether aer expression affects the plants in a manner similar to ssaer expression. The experiments described here suggest that Aer in the plant cell is altering the plant cell physiology. The expression of ssaer triggers chlorosis followed by wide- spread necrosis under low humidity. Symptoms reminiscent of Pst DC3000 infection, including water-soaking, chlorosis and localized necrosis, occur under high humidity. The role that symptom development plays in the Pst DC3000 infection process has not been determined. The symptoms may be triggered to help the bacteria reach high population levels within the leaf. Alternatively, the symptoms may be due to the high levels of bacteria present within the leaf. Our study demonstrated that the in planta expression of a single type III effector, Aer, caused symptom development. Because the expression of ssaer also promotes the growth of the normally non-pathogenic Pst DC3000 hrpH mutant, we hypothesize that symptoms may contribute to allowing pathogen growth. It is tempting to conclude that because the expression of ssaer in planta can restore hrpH mutant bacterial growth to near wildtype levels that the Aer effector is sufficient to cause the hrpH mutant to regain its ability to be pathogenic. However, 139 several factors caution against this. First, in the transgenic plants Aer was present at the beginning of the hrpH mutant growth. This would not be the case during normal pathogen growth. Second, the amount of Aer produced in planta by the DEX-inducible system may be different than that produced by Pst DC3 000 timing the course of an infection. The DEX-induced Aer may not be physiologically relevant. Finally, the promotion of growth seems to apply to Pst DC3000 expressing aerptZ as well as the hrpH mutant. The modes by which these two bacteria are prevented from establishing successful infections are distinct. The hrpH mutant is unable to secrete any type III effectors, whereas Pst DC3000 with aerptZ expresses an extra type III effector which confers recognition by the gene-for— gene resistance system. It is possible that Aer is a multifirnctional effector which is capable of promoting the growth of the hrpH mutant by one mechanism, while interfering with RPS2-mediated recognition of Pst DC3000 with aerpt2 by another mechanism. However, it is also possible that Aer is preventing recognition of both of these bacterial strains by one mechanism. One mode of action by which Aer could accomplish the growth promotion of both the hrpH mutant and Pst DC300 expressing aerpt2 would be to prevent the bacteria from contacting the plant cell while also allowing nutrients to be made available for growth. It is possible that water-soaking would promote these conditions. There may be nutrients present in the apoplastic space, but these nutrients could be unavailable to the bacteria. The nutrients may be concentrated and localized until water-soaking aids in nutrient dispersal. The water could solubilize the nutrients, convert them into a form which is accessible to the bacteria, and spread the nutrients evenly throughout the apoplast. In addition, it is thought that secreted plant defense compounds are also present 140 in the apoplastic space (Osbourn, 1999). The release of water might dilute these compounds to a non-toxic level. Alternatively, these plant defense compounds may be water-insoluble. The plant defense compounds may be localized to specific locations within the apoplastic space, such as the cell wall. Thus, the release of water would separate the bacteria from these toxic plant defense compounds. In either case, water- soaking could act to protect the bacteria from the plant defense compounds. The observation that ssaer transgenic plants develop water soaking after transgene induction suggests that Aer may promote the release of water during Pst DC3000 infection. Stomata] closure is also affected in these transgenic plants. The regulation of stomatal aperture is one way that plants control the loss of water during photosynthetically active time periods (Schroeder et al., 2001). Active photosynthesis requires gas exchange in the leaf for maximum efficiency. Open stomata promote gas exchange. However, open stomata also allow for water loss via evaporation. Thus, under dry or drought conditions, fewer stomata are open. This reduces the efficiency of photosynthesis, but also reduces water loss. We have observed a correlation between ssaer expression, water-soaking and stomatal closure. However, whether this relationship is causal is not currently known. One hypothesis is that the AvrB-induced stomatal closure is a cause of water-soaking. Experimental evidence suggests that stomatal closure precedes visible water-soaking. However, there may be microscopic, localized water-soaking which occurs before both stomatal closure and visual water- soaking. However, stomatal closure may not be the cause of water-soaking. Water-soaking could be caused by loss of water from plant cells. This water loss could be perceived as 141 water stress and could then trigger stomatal closure. Stomata do not respond to artificially water-soaked leaves with closure. This artificial water-soaking was created by infiltration of water into the apoplast with a needleless syringe. Little is known about the microscopic characteristics of pathogen-induced water-soaking, but even visual assessment with the naked eye indicates that these two types of water-soaking are morphologically distinct. Artificially created water-soaking saturates the entire leaf apoplast with water. Pathogen-induced water-soaking is not homogeneous. There are patches of water soaking present within a leaf. Thus, stomatal closure may be a natural plant response to pathogen- or AvrB-induced water-soaking and might not play a part in the promotion of pathogen growth. It is also possible that the relationship between water- soaking and stomatal closure is merely correlative. This study provides evidence that transgenic production of Aer promotes pathogen infection. The observations made in this study could not have been made using bacterial studies with the aer mutant. This study indicates the utility of in planta expression for the study of Pst DC3000 virulence. 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Microbiology-UR 140, 659-669. Yu, G. L., Katagiri, F. and Ausubel, F. M. 1993. Arabidopsis mutations at the RPS2 locus result in loss of resistance to Pseudomonas syringae strains expressing the avirulence gene aerptZ. Molecular Plant-Microbe Interactions 6: 434-443. Yuan, J ., and He, S. Y. (1996). The Pseudomonas syringae hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. Journal of Bacteriology 178, 6399-6402. Zwiesler-Vollick, J ., Plovanich-Jones, A. E., Nomura, K., Bandyopadhyay, S., Joardar, V., Kunkel, B. N., and He, S. Y. (2002). Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome. Molecular Microbiology 45, 1207-1218. 146 Chapter 5 Conclusions and Future Perspectives 147 The study of plant-pathogen interactions should allow researchers to help farmers reduce economic losses due to pathogen infection. While the mechanisms utilized by plants for defense are being discovered and elucidated, little is known about the processes which underlie disease. A widely studied plant-pathogen system consists of the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) and its model host, Arabidopsis thaliana. The focus of my thesis work has been to gain a greater understanding of virulence in Pst DC3000. It is known that Pst DC3000 produces the phytotoxin coronatine, but the mechanism by which this toxin promotes disease is not yet known. Pst DC3000 also utilizes a type 111 protein secretion system during pathogenesis. This protein secretion system is thought to secrete bacterial proteins, collectively referred to as effectors, directly into the plant host cytoplasm. Once they are translocated into the plant host cell, the effectors are thought to promote disease by suppressing host defense responses and promoting the release of nutrients for utilization by the bacterial pathogen. At the time this thesis was started, only two putative effectors were known to be present in Pst DC3000, AvrPto and Aer. However, avrPto and aer bacterial mutants did not show a reduction in virulence. This lack of phenotype was hypothesized to be due to functional redundancy with other unidentified Pst DC3000 effectors. In order to begin to understand the role that type III effectors play in virulence, we needed to learn more about the number and types of effectors encoded in the Pst DC3000 genome. A bioinforrnatics search was conducted followed by functional genomic characterization of putative type III effectors of Pst DC3000. This work is described in Chapter 2. In our search we found the eleven known and/or suggested hrp-regulated genes previously described and present in the available release of the Pst DC3000 genome sequence. We 148 identified six orthologues of avirulence and virulence genes which had been identified in other bacterial plant pathogens. Finally, we identified eight novel putative effectors which were shown to have hrpS-dependent transcription. One of these putative effectors was shown to be translocated into plant cells with an Aerpt2-based translocation assay. This study and others have revealed that there are many putative type III effectors encoded in the Pst DC3000 genome. A different study has also identified a large number of putative type III effectors in another plant bacterial pathogen, Pseudomonas syringae pv. maculicola (Guttman et al., 2002). Therefore, the presence of a large number of effectors is not a characteristic unique to in Pst DC3000, but is more broadly representative of the P. syringae pathovars, and possibly other plant bacterial pathogens. It is currently estimated that there are at least 36 effectors produced by Pst DC3000 (Collmer et al., 2002). This number exceeds that of identified type IH effectors in any animal bacterial pathogen. While the reason for this large number of effectors is not known, it has been suggested that it may enable the pathogenic pseudomonads to have a broad host range. Now that many putative effectors have been identified, we must begin to characterize the role that they play in Pst DC3000 virulence. Efforts are currently underway to create mutations in each individual effector gene. However, due to the large number of effectors and the likelihood of functional redundancy, this approach has not yet led to any new knowledge of effector function. Information about the identity of many candidate effectors could be used to construct multi-effector knockouts which might then show a reduction in virulence. One multi-effector knockout, the delta CEL mutant, is reduced in virulence and this cannot be phenocopied by mutation of any single 149 effector within the CEL region (Alfano et al., 2000; J. Badel, K. Nomura, S. Bandyopadhyay, A. Collmer and S.Y. He, in prep). However, the sheer magnitude of predicted effectors suggests that this approach would be labor intensive. The development of virulence assays which could be used to quickly test all putative effectors for a variety of functions would also help to determine the role that putative effectors may play in virulence. One assay that is being developed is the expression of candidate effectors in non- pathogenic bacteria, such as Pseudomonasfluorescens, which heterologously express a hrp gene cluster-encoded type III protein secretion system (P. Hauck, R. Thilmony, and S.Y. He, unpublished). P. fluorescens which contains hrp cluster is unable to attain pathogenic levels of growth within plants. These bacteria could then be used to assess if the additional effector imparts greater bacterial growth in planta. If increased growth is observed, it could indicate that the effector is contributing to bacterial growth. Alternatively, this system could also be used to evaluate the ability of putative effectors to interfere with gene-for-gene mediated resistance. P. fluorescens expressing the hrp cluster and a known avr gene for the host being infiltrated should elicit a hypersensitive response (HR). If however, P. fluorescens expressing the hrp cluster, a known avr gene for the host being infiltrated, and an effector that interferes with the function of this avr gene, no HR would be observed. This approach does have a potential drawback. Guttman et al (2002) have indicated that putative type III effectors can be found in P. fluorescens even though it lacks a native functional type III secretion system and is non- pathogenic. There is evidence to suggest that there may be complex interactions among the effectors of a pathogen. The presence of unknown native putative effectors of P. 150 fluorescens could interfere with interpretation of the results of these heterologous expression studies. Another assay being used is the expression of putative effectors in planta, either transiently or stably. Stable transgenic lines are currently being used to elucidate the role that individual candidate effectors play in virulence. A. thaliana plants which express aerpt2 (and lack the corresponding R gene RPS2) show increased susceptibility to Pst DC3000 (Chen et al., 2000). Expression of avrPto in A. thaliana promotes the growth of the non-pathogenic Pst DC3000 hrpH mutant (Yuan and He, 1996) and suppresses the formation of callose-containing papillae (P. Hauck, R. Thilmony, and S.Y. He, unpublished). In addition, expression of ssaer in A. thaliana induces stomatal closure and also promotes the growth of the hrpH mutant. One disadvantage of this approach is that the generation of stable transgenic A. thaliana plants which express putative effectors requires a great deal of labor, space, and time. Transient-expression analysis would allow candidate effectors to be screened relatively quickly. However, this technique has not yet been perfected in A. thaliana. For now, transient expression is attainable in Nicotiana benthamiana. This system could be used to screen for phenotypes after transient expression of candidate effectors. Care must be taken because N. benthamtana is not a host for Pst DC3000. While some candidate effectors may elicit a similar response in A. thaliana and N. benthamiana, others may elicit a hypersensitive response (HR) in N. benthamiana which would not happen in A. thaliana. In addition, some putative effectors would trigger changes in the A. thaliana host but may not affect N. benthamiana. 151 Overall, the identification of many new effectors and candidate effectors which are thought to contribute to phytobacterial virulence will aid in future study of plant pathogen-interactions. Our field is now poised to contribute to greater understanding of pathogenesis. Another goal of my thesis work was the identification of reduced-virulence bacterial mutants which would aid in our understanding of Pst DC3000 virulence. Because the previously described virulence factors in Pst DC3000 are transcriptionally induced in minimal medium, a transposon with a promoterless reporter gene was used to generate a population of mutagenized bacteria and subsequently determine the expression pattern of the insertionally—inactivated genes in minimal versus rich medium. Mutants disrupted in the ptsP, uer, and oer genes were isolated in this way, as described in Chapter 3. The ptsP gene encodes the Enzyme 1 subunit of the phosphoenolpyruvate protein phosphotransferase system. In concert with other subunits of this system, Enzyme I mediates the uptake of sugar into the bacterial cell. However, these systems have also been implicated in other processes such as catabolite repression and chemotaxis (Postma et al., 1993). Interestingly, ptsP mutants of other bacteria also show reduced virulence (Higa and Edelstein, 2001; Tan et al., 1999). The specific role that the phosphoenolpyruvate protein phosphotransferase system plays in virulence has not yet been determined in any bacterial system. Loss of virulence in the Pst DC3000 ptsP mutant could be due to the inability of these mutants to acquire sugar. Little is known about the environment present in the plant apoplastic space. If indeed a defect in sugar uptake is the cause of the ptsP mutant phenotype, this mutant 152 could be used to augment our knowledge of the conditions present in the apoplastic space. Feeding experiments could be done with l4C-labeled sugars to determine which sugars can/cannot be taken up by the ptsP mutant in vitro. These experiments might reveal whether certain sugars, which can be taken up by Pst DC3000, cannot be taken up by the ptsP mutant. These sugars might be present in the apoplastic space during infection, and this could account for the loss of virulence of the ptsP mutant. These experiments could be followed up with chemical composition analysis of A. thaliana apoplast wash fluid to determine if these sugars are indeed present. An alternative hypothesis is that chemotaxis is required within the apoplastic space before the bacteria adhere to the host cells and that this process is disrupted in the ptsP mutant. Chemotaxis could allow the bacteria to find areas which contain nutrients or to avoid areas with plant defense compounds. Badel et al. (2002) have developed a green fluorescent protein (GFP)-based system for the observation of Pst DC3000 in the apoplastic space. This technology could be used to determine if the ptsP mutant bacteria fail to disperse throughout the apoplastic space to the same extent as wildtype Pst DC3000. Three independent mutants which contain a disrupted oer gene were identified. The oer gene encodes an outer membrane porin F precursor protein. P. fluorescens mutants which lack the oer gene are unable to grow in medium with low osmolarity (Rawling et al., 1998). The Pst DC3000 oer mutants also show a reduced growth rate in hrp-inducing minimal medium. Taken together, these results suggest that the Pst DC3000 oer gene may be required for growth in low-osmolarity conditions such as hrp- inducing minimal medium and the apoplastic space. The protein encoded by the oer 153 gene has also been implicated in adherence to host cells (Azghani et al., 2002; DeMot et al., 1992). The mechanism by which Pst DC3000 adheres to host cells in the apoplastic space is not currently known. The above mentioned studies (Azghani et al., 2002; DeMot et al., 1992) have tested the ability of bacteria to remain adhered to host cells after perturbation by washing. To examine the adherence of Pst DC300 to A. thaliana cells, leaves which had been previously infiltrated with bacteria (either Pst DC3000 or the oer mutant) could be infiltrated again with water, followed by centrifugation. This could be used to wash away bacteria which had not adhered to the plant cells. Conditions and timing would have to be determined such that bacteria that had been infiltrated would have begun to adhere to host cells. However, water-infiltration would also have to be done before high levels of multiplication had occurred because the growth differences in planta between Pst DC3000 and the oer mutants could mask the effect of differences in adhesion. If more oer bacteria than wildtype Pst DC3000 were recovered from the apoplastic wash fluid, and assessment of the bacterial levels remaining in the washed leaves indicated higher levels of Pst DC3000 than the oer mutant, this might indicate that Oer plays a role in the adhesion of Pst DC3000 to A. thaliana cells within the apoplastic space. The uer mutant is also reduced in virulence. The uer gene encodes a type 11 DNA helicase which is important for DNA replication and repair, especially after exposure to UV light. The Pst DC3000 :4er mutant is more sensitive to UV light. While this sensitivity has been shown to be important during the epiphytic growth of plant pathogenic bacteria (Sundin and Jacobs, 1999), the Pst DC3000 uer mutants never grew epiphytically during my study because they were infiltrated directly into the 154 apoplastic space. It is possible that UV light could impact bacterial growth within the apoplastic space, but this would require UV light to penetrate through the leaf. An alternative hypothesis is that the uer gene is required for in planta growth because it plays a role in tolerance to plant defense compounds present in the apoplastic space. Secreted compounds have been demonstrated to play a role in defense against plant pathogens in other systems (Osbourn, 1999). Unfortunately, there is still little known about the defense compounds produced by A. thaliana which are effective against bacterial pathogens. This lack of knowledge prevents in vitro studies which could compare the tolerance of wildtype Pst DC3000 and the uer mutant to known A. thaliana defense compounds. As indicated in Chapter 3, preliminary evidence suggests that the uer mutant is able to grow to a level similar to that of wildtype Pst DC3000 in NahG transgenic plants. The NahG transgenic plant expresses the bacterial napthalene hydroxylase gene (nahG) which converts salicylic acid to catechol (Delaney et al., 1994; Gaffney et al., 1993). However, there is evidence to suggest that this transgenic plant lacks not only salicylic acid but other defense compounds derived from the phenylpropanoid pathway (Hunt at al., 1996). If this phenomenon is reproducible, it may indicate that the uer mutant is less tolerant to plant secondary metabolite defense compounds which originate from the phenylpropanoid pathway. Chapter 4 describes my efforts to characterize the role that a specific type III effector, Aer, plays in Pst DC3000 virulence. Earlier studies with the Pst DC3000 aer mutant failed to indicate a role for this effector in virulence, probably due to functional redundancy (S.Y. He, unpublished). To avoid the problems of effector redundancy, aer fused to the tobacco PR1-b signal sequence (ss) was expressed in A. 155 thaliana under the control of a dexamethasone (DEX)-inducible promoter. After induction, the ssaer transgenic plants exhibited water-soaking followed by the development of chlorosis and necrosis. The occurrence of water-soaking was correlated with stomatal closure. Expression of ssaer also permitted the growth of the normally non-pathogenic Pst DC3000 hrpH mutant. In addition, Pst DC3000 with the aerptZ gene was able to grow to higher levels in ssaer-expressing plants. One mechanism that could cause an increased level of bacterial growth would be the prevention of contact with the plant cells in the AvrB-expressing plants. Prevention of plant-bacterial contact would prevent the translocation of Aerpt2 and thus gene-for- gene resistance would not be triggered. In addition, this might prevent the hrpH mutant from contacting plant defense compounds which are localized in the plant cell wall. A previous study showed that infiltration of a bacterial suspension in 0.01% agarose was able to prevent gene-for—gene resistance. The agarose was hypothesized to prevent bacterial contact with the host cells (Stall and Cook, 1979). Disruption of contact between bacterial and host cells could be accomplished by the release of water into the apoplastic space. A. thaliana leaves infected with Pst DC3000 develop water-soaking symptoms two days after bacterial infiltration. Although no specific role in virulence has yet been attributed to water-soaking, it might serve to protect the bacteria from the cell wall-localized defense compounds. This hypothesis provides a role for AvrB-induced water-soaking during the course of infection. Water-soaking in ssaer transgenic plants is correlated with the closure of stomata. This relationship may be mere correlation or it may be causal. Water-soaking may cause stomatal closure, stomatal closure may cause water-soaking, or both may 156 caused by Aer independently. In leaves which were artificially water-soaked, nearly all the stomata were open. While this does suggest that water-soaking is not the cause of stomatal closure, it is important to acknowledge that this artificial water-soaking is apparently different from pathogen-induced water-soaking. The two treatments are visually different. Artificially induced water-soaking results in leaves which are thoroughly saturated with water. The pathogen-induced water soaking is more mottled in appearance. The complete saturation of the artificially water-soaked leaves probably results in an anaerobic environment within the apoplastic space. This anaerobic environment could account for the open stomata observed in the artificially water-soaked leaves. I have observed that stomatal closure occurs during Pst DC300 pathogenesis and is correlated with water-soaking, however, this observation still does not address the nature of the relationship. Because I was unable to create a true reproduction of pathogen-induced water- soaking, I was unable to determine if pathogen-induced water-soaking causes the observed stomatal closure. We could, however, use chemicals which artificially alter stomatal aperture to determine if stomatal closure is sufficient to cause water-soaking. 1- (5-lsoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, has been shown to inhibit stomatal opening in the light and enhance stomatal closure in the dark in Commelina communis (Lee and Assmann, 1991). H-7 could be used to artificially trigger stomatal closure. The leaves could then be observed to determine if water-soaking occurs. In addition we could utilize a synthetic diacyl glycerol such as 1,2- dioctanoylglycerol which has been shown to inhibit dark-induced stomatal closure and enhance light-induced stomatal opening (Lee and Assmann, 1991). This diacylglycerol 157 could then be used to determine if artificial opening of the stomata could prevent ssaer- induced water-soaking. Additional observations could determine the role that water- soaking plays in further symptom development and enhanced growth of the hrpH mutant. Pst DC3000 infected plants could also be treated with this diacylglycerol to attempt to inhibit pathogen-induced water-soaking and determine if bacterial growth is altered under these conditions. These experiments could further understanding of the relationship between stomatal aperture, water-soaking, and Pst DC3000 virulence. Overall, my thesis work has provided new information about Pst DC3000 virulence. The effectors and bacterial mutants identified, as well as the transgenic plants generated, should be useful for further examination of the infection process. *"a'l 158 References Alfano, J. R., Charkowski, A. O., Deng, W. L., Badel, J. L., Petnicki-chieja, T., van Dijk, K., and Collmer, A. (2000). The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants. Proceedings of the National Academy of Sciences of the United States of America 97, 4856-4861. Azghani, A. O., Idell, S., Bains, M., and Hancock, R. E. W. (2002). Pseudomonas aeruginosa outer membrane protein F is an adhesin in bacterial binding to lung epithelial cells in culture. Microbial Pathogenesis 33, 109-114. Badel, J. L., Charkowski, A. O., Deng, W. L., and Colhner, A. (2002). 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Journal of Bacteriology 178, 6399-6402. 160 Appendix A Supplementary material for Chapter 2 This appendix contains supplementary material for Chapter 2. Appendix table 1 represents the collection of hrp box containing ORFs (HCOS) found in Pst DC3000 in Chapter 2. This table provides information about the primers used to amplify the HCOs for inclusion on the microarray slide. It also contains the top blastX hit and expectation value. 161 2 8+m88 v8.0.8”? amok? m 8.88.8 «8392 amok? 8+m88 888%. 58:8 8+m8.o 883.? it: 8+m8.o 8882 m3 $8283 on??? 385%:me _m-moo.v .5085 ooeo_E_>< Slag 8+m88 .8292 8.3 8+m88 882%. :03 8+m88 888%.. :5 8+m88 4828... SE 888.8 5882.... on: 4.2.5:: 5:832: .8823 «9.96% .5821 9.qu..— o=_a> m 535%.... .5895 CD: bin—2:? 35.5% .mAmOOIV 92:9ngsmorcomolmgcfiacoouxon BI. .T< 038. UO<<<5 003-2.? 8205333l 658550 5 25 3.55.5 H8883: 58:03.51 45 80.80559. m0=0£0pzmml .5805 @5558. 58558 .55 .3 03.2.2.8 n0=050wammn~ .5228 8283. 00.55.25,. w0=0§0~§mmn~ .2555. 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