CYCLIC DI-GMP DEPENDENT REGULATION IN THE PLANT PATHOGENIC BACTERIUM ERWINIA AMYLOVORA  By Luisa F. Castiblanco Mosos                  A DISSERTATION   Submitted to Michigan State University in partial fulfillment of the requirements for the degree of  Plant Pathology  Doctor of Philosophy  2016 ABSTRACT  CYCLIC DI-GMP DEPENDENT REGULATION IN THE PLANT PATHOGENIC BACTERIUM ERWINIA AMYLOVORA  By  Luisa F. Castiblanco Mosos  Bacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized intracellularly and degraded by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. This nucleotide signaling molecule is involved in the regulation of many cellular processes in numerous bacterial species, including the transition from motile to sessile lifestyle, virulence, biosynthesis of exopolysaccharides and adhesion structures, and cell differentiation. Signal transduction and phenotypic modulation is determined by binding of c-di-GMP to specific downstream receptor molecules. Erwinia amylovora uses motility and the hypertensive response and pathogenicity- (Hrp-) type III secretion system (T3SS) during initial phases of plant infection. Once inside the xylem, this pathogen aggregates due to the production of exopolysaccharides (EPS) and attachment structures forming a biofilm that ultimately plugs the xylem vessels. Although c-di-GMP has been shown to be an important intracellular signal in several plant pathogenic bacteria, the importance of this molecule in Erwinia amylovora has not been previously investigated. This doctoral research explores the role of c-di-GMP in the genetic regulation of key cellular processes associated with pathogenesis in E. amylovora. Five active DGC enzymes (EdcA,  3 EdcB, EdcC, EdcD and EdcE) were identified in this bacterial pathogen. Phenotypic analyses demonstrated that c-di-GMP positively regulates the biosynthesis of both cellulose and amylovoran, positively regulates biofilm formation, and represses motility. Disease assays demonstrated that c-di-GMP negatively regulates virulence in these infection models. The exopolysaccharides amylovoran and levan and attachment structures such as fimbriae, type IV pili and curli, have been demonstrated to be critical for the formation of a mature biofilm in E. amylovora. The results presented in this work demonstrated that c-di-GMP binds to the receptor protein BcsA, the cellulose synthase subunit, and activates the biosynthesis of cellulose in E. amylovora at a post-translational level. In addition, using SEM and confocal microscopy, it was demonstrated that cellulose is a main component of the biofilms formed by E. amylovora in vitro and in the host. Gene overexpression and site-directed mutagenesis analyses, suggest that BcsZ, an endoglucanase, is also required for cyclic di-GMP activation of cellulose biosynthesis and biofilm formation. Further investigation into the mechanisms of c-di-GMP dependent regulation of virulence, with a special emphasis on the regulation of hrp-T3SS gene expression, revealed that high intracellular levels of c-di-GMP via DGC overexpression lead to a significant reduction in gene expression for hrpL, the hrp alternative sigma factor, and dspE. C-di-GMP binding assays suggest that HrpS, the 54 dependent transcriptional regulator of hrpL, is a candidate receptor of c-di-GMP. Moreover, the results presented in this work indicate that HrpS can bind to other guanosine-containing signaling molecules including the c-di-GMP degradation product pGpG. This work provides an overview of some of the molecular mechanisms for c-di-GMP-dependent regulation on the main pathogenicity determinants in E. amylovora, in order to orchestrate pathogenesis and successfully cause disease.  . iv ACKNOWLEDGEMENTS  I would like to thank my advisor Dr. George Sundin for giving me the opportunity of being part of his team, and his support and guidance. George, thanks for the vote of confidence, I could not ask for a better mentor.  I also would like to acknowledge the members of my committee, Drs. Chris Waters, Frances Trail and Brad Day for their advice and valuable discussions.   I also would like to thank everyone who helped me over these years including former and current fellow Sundinites, and friends. To my family for always being there, to my wonderful husband Alejandro, whose love and support kept me grounded to always see the really important things, and to my babies Gaby and Sammy, thanks for making my life complete.  v TABLE OF CONTENTS   LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix KEY TO ABBREVIATIONS ..................................................................................................... xi Chapter 1: Literature Review ...................................................................................................... 1 I. Fire blight disease and its causal agent Erwinia amylovora .................................................... 2 Fire blight disease cycle .......................................................................................................... 3 Molecular mechanisms of E. amylovora pathogenesis ........................................................... 5 Effector and chaperone proteins. ........................................................................................ 8 Harpin proteins.................................................................................................................. 11 Other secreted proteins ..................................................................................................... 12 T3S regulatory proteins..................................................................................................... 13 Exopolysaccharides........................................................................................................... 14 Adaptations to life within the host ........................................................................................ 15 II. The intracellular secondary messenger molecule cyclic-di-GMP ....................................... 19 III. Rationale and project goals ................................................................................................. 24 Chapter 2:  Cyclic di-GMP modulates the disease progression of Erwinia amylovora ........ 26 I. Abstract .................................................................................................................................. 27 II. Introduction .......................................................................................................................... 28 III. Materials and methods ........................................................................................................ 30 Bacterial strains, plasmids, and growth conditions ............................................................... 30 DNA manipulations .............................................................................................................. 30 Insertional mutagenesis and complementation ..................................................................... 33 Bioinformatics ....................................................................................................................... 34 Determination of intracellular c-di-GMP concentration ....................................................... 34 Motility assays ...................................................................................................................... 35 CPC binding assay for turbidometric quantification of amylovoran production .................. 36 Biofilm formation assay ........................................................................................................ 36 Virulence assays .................................................................................................................... 37 Impact of c-di-GMP on the transcription of type III secretion and ams promoters. ............. 37 IV. Results ................................................................................................................................ 38 Erwinia amylovora encodes five putative diguanylate cyclase enzymes ............................. 38 All of the E. amylovora DGCs synthesize c-di-GMP. .......................................................... 40 C-di-GMP negatively regulates swimming motility in E. amylovora. ................................. 41 C-di-GMP positively regulates amylovoran production. ...................................................... 45 C-di-GMP positively regulates biofilm formation. ............................................................... 48 C-di-GMP inhibits the virulence of E. amylovora in two plant infection models. ............... 50 C-di-GMP regulates transcription of amylovoran synthesis and type III secretion genes.... 53 V. Discussion ............................................................................................................................ 54  vi Chapter 3:  Cellulose production, activated by cyclic di-GMP through BcsA and BcsZ, is a virulence factor and an essential determinant of the three-dimensional architecture of biofilms formed by Erwinia amylovora ..................................................................................... 59 I. Abstract .................................................................................................................................. 60 II. Introduction .......................................................................................................................... 61 III. Materials and Methods ........................................................................................................ 65 Bacterial strains, plasmid and growth conditions ................................................................. 65 Chromosomal mutagenesis and complementation ................................................................ 66 Cellulose biosynthesis assessment ........................................................................................ 67 In vitro biofilm assay ............................................................................................................ 67 Confocal laser scanning microscopy for the visualization of biofilms in a flow cell chamber ............................................................................................................................................... 68 Virulence assay on apple shoots ........................................................................................... 68 Evaluation of in vitro and in planta biofilms using scanning electron microscopy .............. 69 Site-directed residue-replacement mutagenesis .................................................................... 70 IV. Results ................................................................................................................................ 70 Bacterial cellulose synthase operon in Erwinia amylovora .................................................. 70 Cellulose is one of the main component of biofilms formed by E. amylovora in vitro ........ 71 Cellulose is a modulator of biofilm architecture in planta .................................................... 76 C-di-GMP activates cellulose biosynthesis in E. amylovora ................................................ 79 The endoglucanase bcsZ is also required for c-di-GMP regulation of cellulose biosynthesis ............................................................................................................................................... 83 V. Discussion ............................................................................................................................ 86 Chapter 4: Cyclic di-GMP-dependent regulation of virulence in Erwinia amylovora.......... 91 I. Abstract .................................................................................................................................. 92 II. Introduction .......................................................................................................................... 93 III. Materials and Methods ........................................................................................................ 95 Strains, plasmids and growth conditions .............................................................................. 95 RNA extraction ..................................................................................................................... 96 qRT-PCR ............................................................................................................................... 97 Protein expression and purification ...................................................................................... 97 DRaCALA binding assay ..................................................................................................... 98 Protein alignments and tertiary structure modeling .............................................................. 99 IV. Results .............................................................................................................................. 100 C-di-GMP synthesized by four DGCs in E. amylovora negatively regulates the expression of hrp genes at the transcriptional level .............................................................................. 100 Insight into HrpS tertiary structure ..................................................................................... 101 C-di-GMP binds to HrpS .................................................................................................... 104 pGpG is involved in the regulation of hrp genes ................................................................ 107 V. Discussion .......................................................................................................................... 109 Chapter 5: Conclusions and future directions ....................................................................... 114 I. Summary of work ................................................................................................................ 115 II. Future directions ................................................................................................................. 120 APPENDIX ................................................................................................................................ 123  vii LITERATURE CITED ............................................................................................................ 151     viii LIST OF TABLES Table 1.1. Cellular processes regulated by c-di-GMP in plant pathogenic bacteria ..................... 23 Table 2.1. Bacterial strains and plasmids used in Chapter 2 and their relevant characteristics .... 32 Table 3.1. Bacterial strains and plasmids used in Chapter 3 and their relevant characteristics .... 66 Table 4.1. Bacterial strains and plasmids used in Chapter 4 and their relevant characteristics .... 96 Table A.1. Bacterial strains and plasmids used in Appendix A.................................................. 129     ix LIST OF FIGURES Figure 1.1. Disease cycle of fire blight caused by E. amylovora. .................................................. 4 Figure 1.2. Organization of the hrp/dsp gene cluster of E. amylovora. ......................................... 7 Figure 1.3.  Biofilm formation by E. amylovora. ........................................................................ 18 Figure 1.4. Schematic representation of c-di-GMP metabolism. ................................................. 20 Figure 1.5.  Cyclic di-GMP metabolism and associated phenotypes........................................... 22 Figure 2.1. The five putative DGC enzymes present in the genome of Erwinia amylovora Ea1189. ......................................................................................................................................... 40 Figure 2.2. Intracellular c-di-GMP levels in E. amylovora Ea1189 containing edc gene overexpression plasmids. .............................................................................................................. 41 Figure 2.3. Effect of c-di-GMP on flagellar motility in E. amylovora. ....................................... 44 Figure 2.4. Effect of c-di-GMP on the production of amylovoran in E. amylovora. ................... 47 Figure 2.5. Effect of c-di-GMP on biofilm formation of E. amylovora grown under static conditions. ..................................................................................................................................... 49 Figure 2.6. Virulence of E. amylovora DGC mutant strains in an immature-pear infection model........................................................................................................................................................ 51 Figure 2.7. Effect of c-di-GMP on virulence and migration of E. amylovora in an apple shoot infection model. ............................................................................................................................ 52 Figure 2.8. Effect of c-di-GMP on the transcription of genes involved in type III secretion. ..... 54 Figure 3.1. Organization of the bcs operon in E. amylovora. ...................................................... 71 Figure 3.2. Impact of the abrogation of cellulose production on in vitro biofilm formation by E. amylovora Ea1189. ....................................................................................................................... 72 Figure 3.3. Effect of cellulose on the three-dimensional architecture of biofilms produced by E. amylovora Ea1189. ....................................................................................................................... 73 Figure 3.4.  Effect of cellulose on the extracellular matrix organization of E. amylovora Ea1189........................................................................................................................................................ 74 Figure 3.5. Distribution of cellulose in biofilms formed in vitro by E. amylovora Ea1189. ....... 76  x Figure 3.6.  Cellulose is a main component of biofilms formed in planta by E. amylovora Ea1189. ......................................................................................................................................... 77 Figure 3.7. Effect of cellulose production on virulence of E. amylovora Ea1189 in apple shoots........................................................................................................................................................ 78 Figure 3.8. Regulation of biofilm formation and amylovoran biosynthesis by the different DCGs in E. amylovora Ea1189. ............................................................................................................... 80 Figure 3.9. Activation of biofilm formation through cellulose biosynthesis by c-di-GMP in E. amylovora Ea1189. ....................................................................................................................... 82 Figure 3.10. Implication of other genes in the bcs operon in cellulose biosynthesis activation through c-di-GMP in E. amylovora Ea1189. ................................................................................ 85 Figure 4.1. Expression of hrpL in E. amylovora Ea1189 overexpressing individual DGCs. .... 101 Figure 4.2. Structure of HrpS. .................................................................................................... 103 Figure 4.3. C-di-GMP-HrpS binding assays. ............................................................................. 105 Figure 4.4. Expression of hrpL, dspE, hrpN, hrpS, hrpX, hrpY and rpoN in WT strain Ea1189 overexpression the oligoribonuclease (orn) gene and edcD at 6 and 18 h of induction in HrpMM...................................................................................................................................................... 108 Figure 4.5. Working model for c-di-GMP-dependent regulation of hrp-T3SS gene expression...................................................................................................................................................... 112 Figure A.1. Impact of DspF on DspE(1-737)-CyaA translocation. ............................................... 134 Figure A.2. Interactions of T3S chaperones, effector proteins and HrpN in E. amylovora....... 136 Figure A.3. Impact of DspF, Esc1, Esc3 and HrpN for the virulence of E. amylovora. ........... 139 Figure A.4. cAMP accumulation of tobacco leaves mediated by E. amylovora strains expressing fusion proteins. ............................................................................................................................ 142 Figure A.5. Secretion and translocation of Eop1 and Eop3 into tobacco plants........................ 144 Figure A.6. Model for translocation of TTS effectors in E. amylovora. ................................... 150   xi  KEY TO ABBREVIATIONS  2-Fluo-AHC-cdiGMP 2'- O- (6- [Fluoresceinyl]aminohexylcarbamoyl)- cyclic diguanosine monophosphate  AAA+ ATPases Associated with diverse cellular Activities BCA Bicinchoninic acid cAMP Cyclic adenosine monophosphate CBS Chaperone binding site C-di-GMP Bis-(3'-5')-cyclic dimeric guanosine monophosphate CPC Cetylpyridium chloride CV Crystal violet DAPI 4',6-Diamidino-2-phenylindole dihydrochloride DFO Desferrioxamine DGC Diguanylate cyclase DIMP DspE-interacting protein Dpi Days post-inoculation DRaCALA Differential radial capillary action of ligand assay  xii Dsp Disease specific EBP Enhancer-binding protein Edc Erwinia diguanylate cyclase EPEC Enteropathogenic E. coli EPS Exopolysaccharide FRT  Flippase recognition target GFP Green fluorescent protein GMP Guanosine monophosphate GT Glycosyltrasnferase GTP  HA Hemagglutinin HIMP HrpN-interacting protein Hpi Hours post-inoculation Hrc Hypersensitive response conserved Hrp Hypersensitive response and pathogenicity Hrp-MM hrp-inducing minimal medium HTH Helix-turn-helix  xiii IHF Integration host factor IPTG -D-1-thiogalactopyranoside JA Jasmonic acid kb Kilobase kDa Kilodalton LB Luria-Bertani LRR Leucine-rich repeat MCS  Multiple cloning site Ni-NTA Nickel-nitrilotriacetic acid ORF Open reading frame Orn Oligoribonuclease PAI Pathogenicity island PAMP Pathogen-associated molecular pattern  PBS Phosphate-buffered saline PCR Polymerase chain reaction PDE Phosphodiesterase PGA Poly-b-1,6-N-acetyl-D-glucosamine  xiv pGpG 5'- Phosphoguanylyl- (3'-5')- guanosine PMSF Phenylmethylsulfonyl fluoride PS-I Photosystem-I RBS Ribosomal binding site REC Receiver domain RLK Receptor-like serine/threonine kinase ROS Reactive oxygen species SA Salicylic acid SD Synthetic dropout SEM Scanning electron microscopy T3S Type III secretion T3SS Type III secretion system TCTS Two component transduction system TM Transmembrane UAS Upstream activator sequences UDP Uridine diphosphate UPLC-MS-MS Ultra-performance liquid chromatography coupled with tandem mass spectrometry  xv  UPP Unipolar polysaccharide WT Wild type X-gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside    1 Chapter 1: Literature Review    2 I. Fire blight disease and its causal agent Erwinia amylovora   Part I of this chapter has been modified from a publication referenced: Castiblanco, L.F., and G.W. Sundin. 2015. Fire in the orchard: an insight into fire blight disease and its causal agent Erwinia amylovora. In Wang, N., Jones, J.B., Sundin, G.W., White, F., Hogenhout, S., Roper, M.C., De la Fuente, L., and Ham, J.H. (eds.), Virulence Mechanisms of Plant Pathogenic Bacteria.  APS Press, St. Paul, MN, 2015 Erwinia amylovora is a Gram-negative plant pathogenic bacterium that belongs to the Enterobacteriaceae -proteobacteria along with other important human and animal bacterial pathogens such as Salmonella enterica, Yersinia pestis, Escherichia coli, Pseudomonas aeruginosa and Vibrio cholera.  This phytopathogen is the causal agent of fire blight, the first plant disease reported in the 1880s (Thomson, 2000).  Fire blight is a devastating disease that affects plants of the Rosaceae family and is the most important bacterial disease of apple (Malus domestica) and pear (Pyrus communis).  Although the development of fire blight can be controlled when detected early, severe outbreaks can interrupt the production of an orchard for several years (Vanneste, 2000).(Vanneste, 2000).  The first observation of fire blight disease was made in New York in the 18th century.  Since then, the disease has spread to all apple-growing regions of the United States and other countries of North America, New Zealand and Japan in the early 1900s, and Europe and the Middle East in the 1950s with a total of 46 countries around the world that have reported the presence of the disease (Bonn & Van der Zwet, 2000, Van der Zwet, 2006).    3 Fire blight disease cycle E. amylovora infects flowers, where populations as large as 106 cells are established on the stigma surface. From there, the bacteria migrate along the style to the floral cup; dissemination is facilitated by bacterial motility and rainfall or heavy dew (Farkas et al., 2012, Malnoy et al., 2012). Stigma receptivity to bacteria is also dependent on temperature, with 21ºC to 28ºC being the optimal range for epiphytic multiplication of bacteria (Pusey & Curry, 2004).  Flower infection occurs through the nectarthodes which serve as natural opening for bacterial entry.  Symptoms of blossom blight include water-soaking and wilt of the flowers (disease cycle shown in Figure 1.1).  After flower infection, the bacteria migrate systemically to the vascular system of the plant forming biofilms in the xylem and restricting the water movement (Koczan et al., 2009).  In addition, bacterial cells can also emerge from infected plant tissue as bacterial ooze, a hygroscopic polysaccharide matrix embedding bacterial cells, that provides a secondary inoculum for more flower or shoot infections (Thomson, 2000).  Infection of shoots is first visible on young leaves of the shoot tips as necrotic lesions along the central vein and eventually  (Figure 1.1).  Progressive migration of the bacteria downwards, leads to wilting and darkening of the shoot which results in the burning appearance typical of the fire blight disease.  In severe outbreaks, the bacteria can also infect immature fruits producing bacterial ooze.  Infections later in the season often result in the formation of cankers on branches which provide sites for bacterial overwintering and serve as the source of primary inoculum for the next season (Thomson, 2000, Norelli et al., 2003).  The bacteria can also internally migrate to susceptible rootstock or externally infect rootstocks through wounds, killing the tree in one season (Figure 1.1).      4 Figure 1.1. Disease cycle of fire blight caused by E. amylovora.  Solid lines represent bacterial dispersion outside the plant and dashed lines represent bacterial migration inside the plant (Modified from Norelli et al., 2003).     5 Molecular mechanisms of E. amylovora pathogenesis The delivery of effector proteins via the type III secretion system (T3SS) is a critical process for host-pathogen interactions in Gram-negative bacterial pathogens. In E. amylovora, one of the most important pathogenicity factors is the Hrp (hypersensitive response and pathogenicity)-T3SS, which is responsible for the secretion and translocation of effector proteins from the bacterial cytoplasm into the host cell or apoplast (Oh & Beer, 2005a).  The genes encoding the structural components of this secretion system are located in a pathogenicity island (PAI) designated PAI1, along with ca. 51 other genes and an island transfer (IT) region whose presence suggest that this PAI has been a mobile genetic element. hrp genes are contained in a 34-gene cluster within PAI1, called the hrp/dsp (disease specific) gene cluster (Figure 1.2), which includes nine hrc (hypersensitive response conserved) genes, that constitute the structural core of the Hrp-T3SS, four regulatory genes and type three secretion (T3S) effector and chaperone genes (Oh & Beer, 2005a).  Moreover, two additional PAIs (PAI2 and PAI3) containing structural genes for Hrp-T3SSs were identified in the genome sequence of E. amylovora (Bocsanczy et al., 2008a, Sebaihia et al., 2010b).  Interestingly, these PAIs exhibit the same gene organization as the T3SS-encoding islands SSR-1 of the insect endosymbiont Sodalis glossinidius and Ysa of the human pathogen Yersinia enterocolitica (Bocsanczy et al., 2008a).  Phylogenetic analyses, as well as the presence of several genes associated with mobile genetic elements, suggests that these PAIs were acquired by horizontal gene transfer events (Zhao et al., 2011);  however, these T3SSs do not seem to be involved in virulence in plant hosts, as deletion mutants produce symptoms equivalent to the wild-type strain in the immature pear model (Zhao et al., 2009a).   6  Although the structure of the Hrp-T3SS in E. amylovora has not been elucidated, HrpA has been demonstrated to act as the structural protein of the Hrp-T3SS pilus, as it does in Pseudomonas syringae (Jin et al., 2001).  In addition, HrcC encodes an outer membrane protein in the basal body of the Hrp-T3SS (Oh & Beer, 2005a) 7     Figure 1.2. Organization of the hrp/dsp gene cluster of E. amylovora.  This 34-gene cluster contains hrp genes that encode for structural components of the hypersensitive response and pathogenicity type III secretion system (Hrp-T3SS), type III secretion (T3S) effector, and chaperone and regulatory proteins. Different categories of genes are indicated with different colors and textures.  8 Effector and chaperone proteins.   The E. amylovora Hrp-T3SS secretes several proteins in vitro under Hrp-T3SS-inducing conditions including the effectors DspE, Eop1, AvrRpt2Ea/Eop4 and Eop3, the harpin proteins HrpN and HrpW, and HrpJ, a homologue of YopN in Y. pestis (Nissinen et al., 2007b).  DspE, a 198-kilodalton (-kDa) multidomain protein, is a major pathogenicity factor in E. amylovora, because its expression, secretion and translocation are required for symptom development and bacterial multiplication in planta (Gaudriault et al., 1997b, Bogdanove et al., 1998, Triplett et al., 2009b).  DspE belongs to the AvrE family of effector proteins, which includes several conserved effectors from the genera Pseudomonas, Dickeya, Pantoea, Erwinia, and Pectobacterium (DebRoy et al., 2004, Ham et al., 2009).  Although the function of DspE in bacterial pathogenesis remains unclear, several studies have suggested that this effector protein is associated with the alteration of salicylic acid- (SA-) mediated basal defense responses. It suppresses callose deposition, delays the expression of PR1 when transiently inoculated in Nicotiana tabacum, and induces cell death in host and nonhost plants (DebRoy et al., 2004, Boureau et al., 2006b).  Additionally, DspE was demonstrated to interfere with the expression of genes related to jasmonic acid- (JA-) dependent defense responses, as well (Dugé De Bernonville et al., 2012).  Interestingly, another study demonstrated that the nonhost resistance of N. benthamiana to E. amylovora is mediated by NbSGT1, as the silencing of this gene prevents cell death induced by DspE (Oh et al., 2007).  Moreover, DspE induces the activation of reactive oxygen species (ROSs) during the infection of host plants (Venisse et al., 2003).  Yeast two-hybrid and in vitro interaction analyses demonstrated that the N-terminal 4.7-kilobase (-kb) portion of DspE interacts specifically with four proteins in apple. These proteinsnamed DIPM (DspE interacting protein of Malus x domestica) 1 through 4are leucine-rich repeat (LRR),  9 receptor-like serine/threonine kinases (RLKs) that have similar sequences with one another and with the LRRRLKs from other organisms (Meng et al., 2006b).  In addition, the C-terminal fragment of DspE interacts with preferredoxin, an important molecule involved in the electron transfer in photosystem I (PS-I) (Oh & Beer, 2005a), suggesting that DspE may target the chloroplast, disrupting the signaling processes for photosynthesis. DspE interacts with the T3S chaperone DspF, which promotes the secretion and translocation of the effector through the Hrp-T3SS, stabilizes the protein in the cytoplasm and prevents its premature degradation (Gaudriault et al., 2002b).  However, whereas a dspF mutant is greatly reduced in virulence, it retains some pathogenic ability (Gaudriault et al., 2002b, Triplett et al., 2009b), suggesting that a small amount of DspE is translocated into the plant cell in the absence of DspF, and/or that other chaperone proteins may be involved in the translocation process.  Both the chaperone binding site (CBS) for DspF and translocation signal are located within the first 100 residues of the N-terminus DspE (Triplett et al., 2009b, Oh et al., 2010).  The three-dimensional structure of DspF was modeled based on structural similarities with other T3S chaperones, and specific residues interacting with the N-terminal fragment of DspE, were determined (Triplett et al., 2010a).  Additionally, an interaction between DspF with the C terminal portion of DspE was also reported (Oh et al., 2010).  Although the absence of the dspF gene or the deletion of its CBS in DspE do not alter the translocation of N terminal portions of DspE fused with a CyaA reporter (Triplett et al., 2009b), Oh and colleagues (2010) reported that DspF was required for the efficient translocation of a DspE-AvrRpt2 protein fusion, suggesting that the C-terminus of this effector affects the requirement of DspF for its translocation. Another effector protein involved in the virulence of E. amylovora is AvrRpt2Ea (also called Eop4), which shares 70% similarity at the amino acid level, with AvrRpt2 from P.  10 syringae, in the C-terminal region (Zhao et al., 2006).  Despite the low abundance of this protein in supernatants of E. amylovora cultures as suggested by Nissinen and colleagues (2007b), AvrRpt2 was demonstrated to contribute to the virulence of this pathogen since a deletion of the gene resulted in reduction of bacterial growth and symptom development in an immature pear infection model (Zhao et al., 2006).  In addition, AvrRpt2Ea exhibits an avirulence function in Arabidopsis thaliana plants containing the RPS2 resistance gene as it is recognized and elicits an HR response when heterologously expressed from P. syringae (Zhao et al., 2006). Eop1 (formerly EopB or OrfB) is an effector protein that belongs to the YopJ/AvrRXv family of secreted proteins (Oh & Beer, 2005a).  Members of this family, which are present in both animal- and plant-pathogenic bacteria, are characterized by a catalytic triad (i.e., histidine, aspartate/glutamate, and cysteine) that is required for their cysteine protease and/or acetyltransferase activity (Gürlebeck et al., 2006, Mukherjee et al., 2006).  Although the role of this effector in bacterial virulence has not been elucidated, recent studies have characterized Eop1 from Rubus-infecting strains as a host-limiting range factor, because its expression results in virulence reduction of Spiraeoideae-infecting strains of E. amylovora (Asselin et al., 2011).  Moreover, eop1 is located adjacent to the esc1 (also named orfA) gene, encoding a putative T3S chaperone for Eop1; however, this T3S chaperone is not required for in vitro secretion of Eop1 (Asselin et al., 2006b). Other effector proteins that do not seem to have a clear role as virulence factors are HopPtoCEa, whose gene expression was demonstrated to be induced upon infection on immature pears (Zhao et al., 2005b), and Eop2, which shares homology with member of the HopPmaH/HopAK family of effectors and is part of the secretome of E. amylovora (Nissinen et al., 2007b).  In addition, despite that its contribution to virulence remains elusive, the effector  11 protein Eop3 (also called HopX1Ea), was demonstrated to function as an avirulence protein on apple shoots, since its overexpression from an eop3 mutant background significantly reduces symptom development (Bocsanczy et al., 2012).  Harpin proteins  The genome of E. amylovora also harbors the genes hrpN and hrpW, which encode proteins belonging to the harpin family. HrpN, the founding member of this family, was first characterized as a cell-membrane associated protein, capable of HR elicitation when inoculated in the intercellular spaces of tobacco plants (Wei et al., 1992).  Furthermore, this protein has been demonstrated to be required for full virulence of E. amylovora in plants (Barny, 1995). Interestingly, HrpN was shown to be involved in the efficient translocation of DspE, HrpW and itself since a hrpN mutant strain translocates significantly lower levels of these proteins to the plant cell cytoplasm as determined using the CyaA reporter system, (Bocsanczy et al., 2008b).  In addition, HrpN was demonstrated to form ion-conducting pores in liposomes (Engelhardt et al., 2009).  These implications of HrpN in effector translocation and membrane modification properties suggest that this harpin protein might function as a translocator element in E. amylovora, inserting into the plasma membrane of the plant cell, or as a helper protein of pore-forming proteins.  However, although originally HrpN was thought to be secreted only to the apoplast, recent studies suggest that this protein, as well as HrpW, are translocated into the plant cytoplasm along with other effector proteins (Bocsanczy et al., 2008b, Boureau et al., 2011a).  Likewise, HrpN participates in the elicitation of several plant defense responses in host plants such as callose deposition (Boureau et al., 2011a), and activation of the oxidative burst (Venisse et al., 2003).  Additionally, in incompatible interactions, HrpN also induces different defense related responses such as cell death, ROS accumulation and the expression of defense marker  12 genes (Degrave et al., 2008).  A HrpN-interacting protein (HIPM) has been found in apple by yeast two hybrid and in vitro analyses (Oh & Beer, 2007). Interestingly, its ortholog gene in A. thaliana, AtHIPM, is involved in enhanced plant growth in response to treatment with HrpN, and has been postulated as a negative regulator of plant growth (Oh & Beer, 2007). The second harpin protein found in E. amylovora is HrpW. This protein contains a pectate lyase domain in the C-terminal region, indicating that it might be delivered to the plant cell wall after its secretion through the Hrp-T3SS (Gaudriault et al., 1998, Kim & Beer, 1998a).  Whereas the role of HrpW in bacterial virulence remains elusive, at low concentrations, HrpW has been shown to be capable of suppressing HrpN-mediated cell death in A. thaliana, suggesting antagonistic roles for these two harpin proteins (Reboutier et al., 2007). Other secreted proteins  Other proteins identified by secretome analysis are HrpJ and HrpK. HrpJ, which shares some characteristics with YopN from Yersinia spp., belongs to the HrpJ family; members of this family have been found in several plant pathogenic bacteria. In two 2012 studies, researchers demonstrated that HrpJ from P. syringae serves dual functions: being involved in the translocation of effector proteins and exhibiting a role in virulence once it is translocated into the plant cell (Crabill et al., 2012b, Wei & Collmer, 2012a).  Likewise, Nissinen and associates (2007b) demonstrated that the deletion of hrpJ from E. amylovora has a negative effect on the disease symptoms in immature pears. Studies from the same research group established that HrpJEa is also involved in the translocation of DspE but in an indirect manner; no interaction between these two proteins was detected in yeast two-hybrid studies, and HrpJEa was shown to facilitate translocation of HrpN to the plant cell (Bocsanczy et al., 2008b).  On the other hand,  13 HrpKEa is secreted by the Hrp-T3SS of E. amylovora (Nissinen et al., 2007b).  This protein is homologous with the HrpK protein of P. syringae pv. tomato, which acts as part of a translocator complex along with other harpin proteins (Petnicki-Ocwieja et al., 2005a). Given this function HrpKEa might be implicated in effector translocation in E. amylovora. However, in several studies, a hrpK mutant was not affected in virulence and its ability to translocate DspE was not impaired (Oh & Beer, 2005a, Bocsanczy et al., 2008b).  T3S regulatory proteins  The expression of the structural components of the Hrp-T3SS and many of the above mentioned genes is controlled by HrpL, an alternate sigma factor that positively regulates the transcription of T3SS-related genes not only in E. amylovora but other plant pathogens such as P. syringae, Pantoea agglomerans and Pectobacterium carotovorum (Wei & Beer, 1995, Chatterjee et al., 2002, Fouts et al., 2002, Nissan et al., 2005).  HrpL recognizes promoters containing a specific sequence, called the hrp-box ranging between 26 and 30 nucleotides. (Oh & Beer, 2005a, McNally et al., 2012).  Recently, the hrp-box sequence in E. amylovora, which was -GGAAC-N(16-20)ACNNC-activities not related to type III secretion, but that might be required for bacterial infection and disease development (McNally et al., 2012).  In fact, Cesbron and associates (2006) demonstrated that HrpL negatively regulates flagellin production and motility under hrp-inducing conditions. The expression of hrpL is modulated by the 54 enhancer-binding protein HrpS, and the two component transduction system (TCTS) HrpX/HrpY (Wei et al., 2000), both encoded in the hrp/dps cluster.   14 Exopolysaccharides  In addition to the Hrp-T3SS and effector proteins, the production of the exopolysaccharide (EPS) amylovoran constitutes an additional pathogenicity factor in E. amylovora.  This EPS was first described in 1974 as a phytotoxin, found in bacterial ooze from infected tissues, and responsible for the wilting symptoms in host plants (Goodman et al., 1974).  Later studies concluded that the wilt-inducing ability of amylovoran is a result of the physical obstruction of the xylem by this EPS, which restricts water movement to aerial tissues (Sjulin & Beer, 1978).  Amylovoran is an acidic polymer, composed of a branched repeating unit of five monosaccharide residues (Nimtz et al., 1996a), and is synthesized by genes encoded in the 12-gene ams operon.  Insertional mutagenesis of these genes results in an amylovoran-deficient, non-pathogenic phenotype (Bugert & Geider, 1995).  Wang and colleagues (2009) demonstrated that the expression of genes in the ams operon is modulated by the RcsCDB phosphorelay system in vitro, and subsequent studies indicated that additional TCTS such as GrrA/GrrS and EnvZ/OmpR are involved in the regulation of amylovoran production, suggesting that the expression of the ams genes is subject to the activation of signaling networks after the perception of environmental signals (Zhao et al., 2009c, Wang et al., 2011).  Recently, another regulatory member of the network controlling amylovoran biosynthesis was described.  As an approach to understanding the role of the orphan gene amyR, Wang and associates demonstrated that amylovoran-biosynthetic genes were highly expressed from an amyR mutant background (Wang et al., 2012).  Interestingly, co-inoculation experiments demonstrated that an ams mutant strain has the ability to complement the loss of pathogenicity of a hrp-T3SS deletion mutant, suggesting the specific timing for the expression of these two pathogenicity factors, where Hrp-T3SS is required  15 in early stages of infection and colonization, and the biosynthesis of amylovoran is necessary for late stages of the disease, when the bacterial populations are established inside the plant vascular tissue (Zhao et al., 2009a).  In addition, amylovoran was demonstrated to be necessary for biofilm formation inside the vascular tissue of host plants (Koczan et al., 2009). A second EPS produced by E. amylovora is levan. This EPS is synthesized by the levan-polymerizing enzyme levansucrase, encoded by the lsc gene, whose expression is regulated by the transcriptional activator RlsA (Gross et al., 1992, Zhang & Geider, 1999). Mutagenesis analyses demonstrated a reduction in bacterial virulence in immature pear for an lsc mutant strain. Similarly, biofilm formation and in vitro cell-to-cell aggregation were reduced in this mutant background indicating that levan also plays an important role in biofilm formation (Koczan et al., 2009). Adaptations to life within the host Nutrient availability in the plant environment is limited, and E. amylovora has evolved several mechanisms of adaptability to this environment that are required for its survival and colonization of the host. Due to the low iron availability in host cells, E. amylovora possesses an iron scavenging system that involves the production of the siderophore desferrioxamines (DFOs) and the expression of the TonB-dependent siderophore receptor FoxR (Expert et al., 2000, Oh & Beer, 2005a).  Deletions on the dfo or foxR genes resulted in reduced virulence on apple flowers compared with the wild-type strain (Dellagi et al., 1998).  In addition, DFOs have also been demonstrated to play an important role in protecting the bacteria against oxidative responses from the host (Venisse et al., 2003).  The importance of siderophores and iron-uptake systems in bacterial virulence and protection against hydrogen peroxide has been reported in other plant pathogenic bacteria such as Dickeya dadantii and Xanthomonas oryzae pv. oryzae  16 (Boughammoura et al., 2008, Pandey & Sonti, 2010, Boughammoura et al., 2012).  The DFO biosynthetic genes, designated dfoJAC, are located in a genetic cluster along with the foxR receptor gene (Smits et al., 2010, Smits & Duffy, 2011).  Adaptation for nutritional availability also includes the srl operon, which encodes the genes required for sorbitol uptake, the major sugar found in rosaceous plant hosts (Oh & Beer, 2005a).  Mutational analysis of this set of genes revealed that they are required for virulence on apple shoot and seedlings (Aldridge et al., 1997). Another important adaptation of pathogenic bacteria to their hosts is the ability to overcome defense responses, which include several mechanisms and components such as efflux pumps and TCTS. In many human pathogenic enterobacteria, the AcrAB efflux pump has been reported as an important mechanism for antibiotic resistance that also involves the action of the outer membrane protein TolC (Fralick, 1996, Okusu et al., 1996, Padilla et al., 2010).  In E. amylovora the multidrug efflux pump AcrAB is also required for tolerance to phytoalexins produced by the host plant and thereby it has an important role for the establishment of the disease (Burse et al., 2004c).  Recently, it was demonstrated that E. amylovora TolC is also required for resistance to phytoalexins and that this activity is mediated by the interaction with the AcrAB efflux pump (Al-Karablieh et al., 2009). On the other hand, TCTS also plays a role in the resistance to antimicrobial compounds.  In E. amylovora, the TCTS PhoPQ is involved in resistance to cecropin A, a lysis-inducing compound used against Gram-negative bacteria (Nakka et al., 2010).  The colonization of the host xylem and the migration of the pathogen throughout the plant is a major virulence trait as well as an evolutionary adaptation in vascular pathogens.  Koczan and associates demonstrated that E. amylovora forms a biofilm in the vascular vessels of  17 the host plant and that the EPSs amylovoran and levan play a significant role for the development of the biofilm (Figure 1.3) (Koczan et al., 2009).  Moreover, attachment structures such as fimbriae, type IV pili, and curli are also required for the formation of a mature biofilm (Koczan et al., 2011a). The formation of biofilms inside the host confers several advantages, such as resistance against defense responses, antibiotics and other external stresses, as well as the access to nutrients on solid surfaces (Koczan et al., 2009).     18 Figure 1.3.  Biofilm formation by E. amylovora.   Scanning electron microscopy view of a longitudinal section of an apple leaf midvein colonized by E. amylovora showing bacterial cells forming a dense biofilm in a xylem vessel.     19 II. The intracellular secondary messenger molecule cyclic-di-GMP  Part II of Chapter 1 has been modified from a publication referenced: Castiblanco, L.F, Sundin G.W. (2015). New insights on molecular regulation of biofilm formation in plant-associated bacteria. J Integr Plant Biol 58: 362372  -GMP or c-di-GMP) has emerged as one of the most important secondary messengers in bacteria since its discovery in 1987 as an allosteric activator of cellulose synthase in Gluconacetobacter xylinum (now Komagataeibacter xylinum) (Ross et al., 1987).  Since then, the presence of genes involved in c-di-GMP biosynthesis and degradation has been reported for bacteria belonging to all of the major bacterial phyla where at least one member has a sequenced genome (Römling et al., 2013).   The intracellular homeostasis of this molecule is controlled by the catalytic action of three protein domains: diguanylate cyclases (DGCs) with the GGDEF domain synthesize c-di-GMP from two GTP molecules, whereas phosphodiesterases (PDE) with either the EAL or the HD-GYP domain, carry out its degradation producing the linear intermediate pGpG or two molecules of GMP, respectively. (Ryjenkov et al., 2005, Schmidt et al., 2005, Ryan et al., 2006b).  Recent studies have demonstrated that after c-di-GMP hydrolysis by EAL- domain containing PDEs, the degradation product pGpG is then processed by the nanoRNA-degrading oligoribonuclease (Orn) generating GMP as a final product (Cohen et al., 2015, Orr et al., 2015) (Figure 1.4). DGCs and PDEs are modular proteins that often harbor a regulatory signal-recognition domain at the N terminal region. These domains function in the sensing of  20 environmental cues in order to activate the enzymatic activity in response (Römling et al., 2013). Environmental signals currently described include light, oxygen and nitric oxide concentration, quorum sensing autoinducer molecules and redox changes, among others (Barends et al., 2009, Qi et al., 2009, Tuckerman et al., 2009, Deng et al., 2012, Plate & Marletta, 2012).  Figure 1.4. Schematic representation of c-di-GMP metabolism.  Biosynthesis of c-di-GMP is carried out by DGC enzymes from two molecules of GTP, whereas enzymes containing EAL or HD-GYP domains, exhibiting PDE activity, degrade c-di-GMP to pGpG or GMP, respectively. pGpG generated by EAL enzymatic activity is then degraded by Orn to GMP.          After the sensing of an environmental cue, c di-GMP binds, or through its degradation is prevented to bind, to diverse receptor molecules including proteins bearing the PilZ domain, several transcription factors, riboswitches, and enzymatically-inactive DGCs or PDEs (Sudarsan et al., 2008, Lee et al., 2010, Tuckerman et al., 2011, Ryan et al., 2012, Chou & Galperin, 2016), which in turn regulate different output behaviors at the transcriptional, post-transcriptional, or post-translational level (Sondermann et al., 2012, Ryan, 2013).    An intriguing characteristic of c-di-GMP signaling systems is that the number of series involved does not exhibit a linear correlation with bacterial genome size, as genomes of different  21 bacterial species can harbor very distinct numbers of c-di-GMP metabolizing enzymes (Römling, 2012). For example whereas Bordeleau and collaborators (2011) reported the presence of at least 37 genes encoding proteins containing GGDEF and EAL domains within 20 genomes of Clostridium difficile, and E. coli K-12 harbors 29 genes encoding c-di-GMP metabolizing proteins (Hengge et al., 2016), Yersinia pestis only produces three enzymes with c-di-GMP synthesis or degradation activity (Bobrov et al., 2011). Moreover the number of GGDEF, EAL, and HD-GYP-containing enzymes is not linearly correlated with the number of c-di-GMP binding proteins encoded in a given genome, which indicates redundancy in signal recognition and the subsequent c-di-GMP metabolism and highlights the complexity of c-di-GMP-dependent regulatory systems for the tight regulation of many cellular processes.    C-di-GMP signaling systems have been historically demonstrated to be involved in the genetic regulation of the transition from a planktonic motile state to aggregation and biofilm formation, where high intracellular c-di-GMP levels activate sessility and low intracellular levels promote motility (Figure 1.5) (Simm et al., 2004, Hengge, 2009, Römling et al., 2013).  However a plethora of different bacterial behaviors have been reported as being subject to c-di-GMP regulation including virulence, cell cycle and differentiation, biosynthesis of EPS and attachment structures (Lamprokostopoulou et al., 2010, Abel et al., 2011, Römling et al., 2013) . Plant pathogenic bacteria are not the exception and research on c-di-GMP-dependent regulation on this group of pathogens has demonstrated a specific regulation of cellular processes on different phytopathogenic bacterial species (Table 1.1).    22 Figure 1.5.  Cyclic di-GMP metabolism and associated phenotypes.  In general, high intracellular levels of c-di-GMP promote sessility and biofilm formation, while low levels of this messenger molecule are associated with activation of motility and T3SS-derived virulence.       23 Table 1.1. Cellular processes regulated by c-di-GMP in plant pathogenic bacteria  Bacterial Species Cellular process(es)  Reference(s) Agrobacterium. tumefaciens Activation of cellulose biosynthesis and production of a unipolar polysaccharide (UPP) adhesin promoting attachment and biofilm formation.  (Xu et al., 2013) Dickeya dadantii Repression of swimming/swarming motility, cell degradation enzymes production and T3SS-related genes; activation of biofilm formation in vitro.  (Yi et al., 2010) Pectobacterium atrocepticum Activation of EPS biosynthesis and biofilm formation   (Pérez-Mendoza et al., 2011) Pseudomonas syringae pv. tomato Repression of flagellin expression and virulence; activation of EPS biosynthesis and biofilm formation in vitro (Engl et al., 2014, Pfeilmeier et al., 2016)  Xanthomonas campestris Repression of virulence and biosynthesis of the EPS xanthan; Activation of aggregation through binding to the Clp receptor, which regulates genes manA and xagA involved in biofilm formation.  (Ryan et al., 2007, Chin et al., 2010, Lu et al., 2012) Xanthomonas oryzae pv. oryzae Repression of virulence, EPS biosynthesis and flagellar motility (Yang et al., 2016)    Xylella fastidiosa Repression of EPS biosynthesis, attachment structures and biofilm formation; activation of virulence  (Chatterjee et al., 2010)      24  III. Rationale and project goals The success in pathogenicity of the vascular enteric plant pathogen E. amylovora depends on the opportune expression of pathogenicity and virulence factors during different infection stages. Flagellar motility, the assembly of the Hrp-T3SS, and the translocation of effector proteins to host cells are required for colonization of flowers, which are host tissue where the primary infection develops in the field (Thomson, 1986, Pester et al., 2012, Bubán & Orosz-Kovács, 2016). At late stages of infection, once the bacterium reaches the vascular tissue, the production of attachment structures and EPS promote the formation of biofilms enabling the establishment of large bacterial populations in the xylem vessels (Koczan et al., 2009, Koczan et al., 2011a). Cellular aggregation in biofilms allows the pathogen not only to evade host immune defenses but also to move systemically to other plant tissues. Ultimately, bacterial cells extruding from host tissues in form of ooze, composed mainly by EPSs and bacterial cells, are a major source of inoculum for the spread of the disease to surrounding plants, by splashing rain and insects (Thomson, 2000). This panoply of expression of pathogenicity factors at different infection stages suggest that a genetic regulatory mechanism(s) that endows the pathogen with an efficient reprogramming of cellular processes upon the recognition of external and internal signals is definitely needed in order to successfully colonize the host.  As stated previously in this chapter, c-di-GMP-dependent regulatory systems have been demonstrated to be expressed in many bacterial species and to act upon multiple bacterial behaviors and processes. However, this regulatory signaling system has not been previously investigated in E. amylovora. Given the ubiquitous nature of c-di-GMP and c-di-GMP-dependent regulation of bacterial cellular processes, and the requirement of timely expression of  25 pathogenicity and virulence factors in order to colonize the host plant and cause disease, we hypothesized that c-di-GMP-dependent regulation is employed by E. amylovora in order to orchestrate pathogenesis. The goals of my doctoral research are 1) to determine if the secondary messenger molecule c-di-GMP is actively produced by E. amylovora by investigating the presence of c-di-GMP metabolizing enzymes encoded in the genome of this plant pathogen, 2) to evaluate the contribution of c-di-GMP-dependent regulation for the activation of repression of the critical cellular processes in the different stages of the fire blight disease, and 3) to develop an inclusive understanding of the specific mechanisms of c-di-GMP-dependent regulation, that include the critical components of specific regulation pathways.    26 Chapter 2:  Cyclic di-GMP modulates the disease progression of Erwinia amylovora  This chapter has been modified from a publication of the same title: Edmunds, A. C.*, Castiblanco, L. F.*, Sundin, G. W. and Waters, C. M. (2013) Cyclic di-GMP modulates the disease progression of Erwinia amylovora. J. Bacteriol., 195, 2155-2165. * These authors contributed equally to this work    27 I. Abstract The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that all of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcB, edcC, edcD and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis     28 II. Introduction  Erwinia amylovora is the causal agent of fire blight and a devastating phytopathogen that infects plant species of the family Rosaceae, most notably apple and pear trees (Eden-Green & Billing, 1974). E. amylovora can infect flowers, fruits, actively growing shoots, and rootstock crowns (Norelli et al., 2003). During the primary infection via the flower (Eden-Green & Billing, 1974), E. amylovora cells multiply rapidly on the stigma. Motility and free moisture are important factors in the subsequent dissemination of cells down the outside of the stigma to nectarthodes, which provide entry into the plant (Wilson et al., 1989, Wilson et al., 1990, Vanneste, 1995).   Following flower infection, E. amylovora cells spread systemically through host vascular tissues and cortical parenchyma. The wilting symptoms of fire blight are the result of bacterial invasion, the secretion of extracellular polysaccharide (EPS), and the formation of biofilms within host xylem that plug these tubes, restricting water flow (Koczan et al., 2009, Koczan et al., 2011a). E. amylovora secretes two distinct EPSs, amylovoran and levan, both of which contribute to plant infection (Koczan et al., 2009). Amylovoran is an acidic polysaccharide composed of repeating units of galactose and glucuronic acid (Politis & Goodman, 1980, Nimtz et al., 1996b, Jumel et al., 1997), while levan is a homopolymer of fructose residues synthesized from sucrose by the secreted enzyme levansucrase (Gross et al., 1992). Biofilm formation by E. amylovora is required for effective colonization of host xylem tissues, the exit of pathogen cells from infected leaves into host stems, and systemic spread within trees (Koczan et al., 2009, Koczan et al., 2011a). The impact of biofilm formation on xylem colonization has also been  29 noted for several other plant pathogens, including Clavibacter michiganensis, Pantoea stewartii, and Xylella fastidiosa (Tyson et al., 1985, Koutsoudis et al., 2006, Chalupowicz et al., 2011), and the ability to form biofilms appears to be a common strategy for the survival or transmission of phytopathogens (Koczan et al., 2009, Chatterjee et al., 2010).   The second messenger cyclic di-GMP (c-di-GMP) regulates biofilm formation in the majority of bacteria. In general, a high level of intracellular c-di-GMP positively regulates biofilm formation and negatively regulates swimming motility (Romling et al., 2005, Jenal & Malone, 2006, Cotter & Stibitz, 2007, Hengge, 2009). C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes encoding GGDEF domains and is degraded by phosphodiesterase (PDE) enzymes encoding either an EAL or a HD-GYP domain. C-di-GMP exhibits diverse functions in plant pathogens: it negatively regulates the pathogenesis of Xanthomonas campestris and Dickeya dadantii in plants (Ryan et al., 2007, Yi et al., 2010) but positively influences plant colonization by Xylella fastidiosa (Chatterjee et al., 2010).   Because c-di-GMP is an essential signaling molecule that is necessary for EPS secretion and biofilm formation in many bacteria, and because amylovoran secretion and biofilm formation are critical for E. amylovora virulence (Koczan et al., 2009), we hypothesized that c-di-GMP signaling would positively influence E. amylovora virulence. We systematically determined that four of the five DGCs in E. amylovora, edcB, edcC, edcD and edcE, encode proteins that synthesize c-di-GMP and positively regulate amylovoran production and biofilm formation while negatively regulating flagellum-based motility. Importantly, although biofilm formation and amylovoran secretion levels were reduced in E. amylovora edcC and edcE  30 mutants, these mutants exhibited increased tissue necrosis in two plant infection models. This result could be explained partly by repression of the hypersensitive response and pathogenesis- (Hrp-) type III secretion system (T3SS) gene hrpA by c-di-GMP. Our results suggest that c-di-GMP signaling plays a key role in the establishment and development of plant infections by limiting the virulence of E. amylovora.  III. Materials and methods Bacterial strains, plasmids, and growth conditions The bacterial strains and plasmids used in this study are listed in Table 2.1. Unless otherwise mentioned, E. amylovora strain Ea1189 and Escherichia coli strains were grown in Luria-Bertani (LB) broth and plates at 28°C and 37°C, respectively. Amylovoran secretion assays for wild-type (WT) and mutant strains were conducted in MBMA medium [per liter, 3 g KH2PO4,7g K2HPO4, 1 g (NH4)2SO4, 2 ml glycerol, 0.5 g citric acid, and 0.03 g MgSO4] amended with 1% sorbitol, while those for the overexpression strains were conducted in MBMA-LB (3:1) medium. For biofilm formation assays, WT, mutant, and overexpression strains were grown in 0.5x LB medium. Media were amended with kanamycin (Km; 100 g/ml), ampicillin (Ap; 100 g/ml), chloramphenicol (Cm; 10 g/ml), tetracycline (Tet; 10 g/ml), or gentamicin (Gm; 10 g/ml) as necessary.   DNA manipulations  DNA manipulations were performed using standard techniques (Sambrook et al., 2001). The E. amylovora genome sequence was obtained from GenBank (accession no. FN666575) (Sebaihia et al., 2010b). Native DGCs were amplified from E. amylovora Ea1189 genomic DNA,  31 digested with restriction enzymes, and cloned into plasmid pEVS143 (Dunn et al., 2006) to generate isopropyl-D-1-thiogalactopyranoside (IPTG)-inducible overexpression plasmids.    32 Table 2.1. Bacterial strains and plasmids used in Chapter 2 and their relevant characteristics  Strain or plasmid Relevant characteristicsa Source or reference E. coli S17-pir  (de Lorenzo & Timmis, 1994) E. amylovora Wild type (Burse et al., 2004a) Ea1189 ams Deletion of the ams operon (Zhao et al., 2009a) Ea1189edcC Deletion of Eam_1504, edcC::FRT This Study Ea1189DedcE Deletion of Eam_2435, edcE::FRT This Study Ea1189DedcCE edcC/edcE deletion mutant, CmR This Study Ea1189DedcACE edcA/edcC/edcE deletion mutant, CmR KmR This Study flhC Deletion mutant of flhC, KmR (Zhao et al., 2009c)    Plasmids Contains CmR cassette and flanking FRT sites, CmR (Datsenko & Wanner, 2000) pKD3 pKD4 Contains KmR cassette and flanking FRT sites (Datsenko & Wanner, 2000) pKD46 L-arabinose inducible lambda-red recombinase, ApR (Datsenko & Wanner, 2000) pTL17 IPTG-inducible FLPase, KmR (Long et al., 2009) pTL18 IPTG-inducible FLPase, TetR (Long et al., 2009) pEVS141 pEVS143 with the CmR and GFP removed, vector control, KmR (Dunn et al., 2006) pEVS143 Broad host range cloning vector, inducible CmR and GFP, KmR (Dunn et al., 2006) pCMW75 V. harveyi DGC qrgB, overexpression vector, KmR (Waters et al., 2008) pCMW98 Active site mutant of qrgB in pCMW75 (Waters et al., 2008) pACE-edcA pEVS143 cmR::edcA, overexpression vector, KmR This Study pLFC34 pEVS143 cmR::edcB, overexpression vector, KmR This Study pACE-edcC pEVS143 cmR::edcC, overexpression vector, KmR This Study pLFC42 pEVS143 cmR::edcD, overexpression vector, KmR This Study pACE-edcE pEVS143 cmR::edcE, overexpression vector, KmR This Study pBBR1MCS-1 Broad host range cloning vector, R6K ori, CmR (Kovach et al., 1995) pBBR1MCS-5 Broad host range cloning vector, R6K ori, GmR (Kovach et al., 1995) pLFC19 edcC gene and native promoter in pBBR1MCS-5, GmR This study pLFC13 edcE gene and native promoter in pBBR1MCS-1, CmR This study pACYCDuet-1 Expression vector containing two MCS, P15A ori, CmR Novagen pLFC11 edcC and edcE genes w/ native promoter in pACYCduet-1, CmR This study a MCS, multiple cloning sites   33 Insertional mutagenesis and complementation  Chromosomal mutation of edcA, edcC and edcE was carried out as described previously (Datsenko & Wanner, 2000, Koczan et al., 2011a). Briefly, the 1.1-kb chloramphenicol resistance (CmR ) cassette with flanking identical flippase recognition target (FRT) sites was amplified from plasmid pKD3 (Datsenko & Wanner, 2000) using primers encoding 20 bp of homology to the CmR cassette and 50 bp of homology to the regions immediately upstream and downstream of the target gene. PCR products were purified and electroporated into E. amylovora ,  and Exo recombinase genes from the pKD46 plasmid (Datsenko & Wanner, 2000). After recovery, colonies were selected on LB agar plates amended with the appropriate antibiotics. Cells with the mutation were identified by colony PCR using primers located 500 bp upstream and downstream of the mutation. Mutant colonies containing the Cm or Km resistance cassette were transformed with plasmid pTL17 or pTL18 (Long et al., 2009), each of which encodes an IPTG-inducible site-specific recombinase that triggers recombination between the FRT sites, leading to excision of the antibiotic resistance gene. Isolated colonies were tested for Cm or Km sensitivity, and the loss of antibiotic cassettes was confirmed by colony PCR using the same flanking primers originally used to confirm the insertion. For edcC edcE strains were transformed with plasmids pLFC19 and pLFC13, containing the edcC and edcE genes along with their native promoters, ligated into pBBR1MCS-1 and pBBR1MCS-5 (Kovach et al., 1995)edcCE double mutant was complemented with plasmid pLFC11, which contains the edcC and edcE genes and native promoters, ligated into pACYCDuet-1 (Novagen, Madison WI).     34 Bioinformatics  The search for open reading frames (ORFs) in E. amylovora that contain GGDEF, EAL, and/or HD-GYP domains was carried out using the Motif Alignment and Search Tool (MAST), version 4.6.1 (Bailey & Gribskov, 1998). The presence and organization of conserved protein domains were predicted using Pfam, version 25.0 (Finn et al., 2010), and transmembrane (TM) domains were identified using TMHMM, version 2.0 (Krogh et al., 2001). Amino acid alignment using ClustalW in MEGA5.0 (Tamura et al., 2011) was utilized to examine the conserved sequences of the GGDEF domains.  Determination of intracellular c-di-GMP concentration The procedure for the determination of intracellular c-di-GMP concentrations by use of ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS-MS) has been described in detail elsewhere (Massie et al., 2012). Specifically, for these experiments, overnight cultures were grown in LB medium and were then used to inoculate 7 ml fresh medium in a 25-ml Erlenmeyer flask with a starting optical density at 600 nm (OD600) of 0.05. At an OD600 of about 0.8, corresponding to mid- to late-exponential growth, the CFU counts per milliliter were calculated by serial dilution and colony counts on LB agar plates, and cells were harvested by centrifugation of 5 ml of cells in 35-ml polystyrene centrifuge tubes at 4°C for 10 min at 8,000 x g. The supernatant was removed, and the pellet was resuspended with 1 ml phosphate-buffered saline (PBS) and was transferred to a fresh 1.5-ml polystyrene Eppendorf tube. The cell suspension was centrifuged at 10,000 x g for 1 min, and the PBS was removed by aspiration. The cells were then lysed with 0.1 ml extraction buffer (40% acetonitrile 40% methanol in 0.1 N formic acid), left at 20°C for 20 min, and then centrifuged at  35 4°C for 1 min at 15,000 x g. The debris-free liquid was then analyzed by UPLC-MS-MS. By use of a standard curve of chemically synthesized c-di-GMP (Axxora, San Diego CA), the total amount of c-di-GMP extracted was determined. An estimate of the intracellular c-di-GMP concentration was obtained by dividing the total amount of c-di-GMP extracted by the estimated volume of cytoplasm extracted. The length and width of one cell were quantified using ImageJ software (Schneider et al., 2012a), and cell volume was estimated by using the formula for calculating the volume of a cylinder (volume  · height · radius squared). The average intracellular volume of an E. amylovora cell in LB medium during exponential growth was estimated to be 1.88 x 10-12 ml. The total cellular volume was obtained by multiplying the intracellular volume of one cell by the total number of cells harvested, and the intracellular c-di-GMP concentration was estimated by dividing the total quantity of c-di-GMP by the total intracellular volume.   Motility assays Swimming motility was examined by immersing a plastic tip in overnight bacterial cultures, followed by stab-inoculation onto a 0.3% agar LB plate. The inoculated plates were incubated at 28°C for 20 h. For the overexpression strains, the low-density agar plates were amended with 1.0 mM IPTG and Km. Motility plates were photographed under white light using a Red imaging system (Alpha Innotech), and the files were analyzed with ImageJ software (Schneider et al., 2012a)converted to dark background, and the threshold was adjusted until the area of the colony was roughly translated into pixels. This same technique was used for a reference sticker, and by  36 normalizing the motility pixel area by the 1 cm2 reference sticker, we determined the motility area (in square centimeters). This assay was repeated at least three times.   CPC binding assay for turbidometric quantification of amylovoran production  The concentration of amylovoran in supernatants of bacterial cultures was determined quantitatively as described previously (Bellemann & Geider, 1992, Zhao et al., 2009c). Briefly, E. amylovora strains were grown overnight, pelleted, and washed with 0.5x PBS. Cultures were inoculated into 3 ml of MBMA medium with 1% sorbitol or into a 3:1 mixture of LB-MBMA medium to a starting OD600 of 0.1 and were grown for either 20 or 48 h at 28°C with agitation. The OD600 of the bacterial suspensions was measured, and 1 ml of bacterial suspension was pelleted. A 0.8 ml portion of the supernatant was transferred to a new tube, mixed with 40 µl of 50 mg/ml cetylpyridinium chloride (CPC; Sigma, Carlsbad CA), and incubated at 24°C for 10 min. The amylovoran concentration was determined by measuring the OD600 of the suspensions and was normalized to the OD600 of ams strain, containing a deletion of the entire ams operon, is unable to produce amylovoran (Zhao et al., 2009a) and was thus used as a negative control. This assay was repeated at least three times.  Biofilm formation assay Biofilm formation by the studied strains was quantified by using a previously described method (Koczan et al., 2009). Briefly, 100 µl of equilibrated overnight cultures was added to 2 ml of 0.5x LB medium in individual wells of 24-well plates, each well containing a glass coverslip at a 30° angle. After incubation at 28°C for 48 h, planktonic cells and medium were removed, and coverslips were stained with 10% crystal violet (CV) for 1 h. Stained coverslips  37 were then washed three times with water and were air dried for 1 h. The CV stain was then dissolved in 200 µl 40% methanol10% acetic acid, and the OD600 of the solution was recorded. This experiment was repeated at least three times.   Virulence assays  Virulence assays using immature pears were conducted as described previously (Zhao et al., 2005b). Briefly, immature pears were surface sterilized with 10% bleach, rinsed with sterile distilled water, and dried. Overnight bacterial cultures were adjusted to 1 x 104 CFU/ml in 0.5x PBS. Pears were stab-inoculated with 3 µl of the bacterial suspensions and were incubated at 28°C in a humidified chamber. Calipers were used to quantify the lesion diameter at 4 days post-inoculation (dpi). Each experiment included 10 replicates, and this experiment was repeated at least three times. Apple shoot infection assays were conducted as described previously (Koczan et al., 2011a). Briefly, overnight bacterial cultures were adjusted to 2 x 108 CFU/ml with 0.5x PBS. Two-year-old apple trees (Malus X domestica cv. Gala) on M9 rootstock were inoculated by cutting the youngest leaves of central shoots with scissors previously dipped in the bacterial suspensions. Symptoms were monitored at 4 dpi. This experiment was repeated at least twice, with four replicates for each experiment.   Impact of c-di-GMP on the transcription of type III secretion and ams promoters.  Promoter regions and ribosomal binding sites (RBS) of hrpA, hrpS, and amsG (500-bp fragments upstream of the start codon) were amplified by PCR with primers incorporating BamHI and SalI restriction sites. The hrpA and hrpS PCR products were purified, digested with restriction enzymes, and cloned into the pPROBE-AT plasmid (Miller et al., 2000), which  38 contains the coding region of the gfp reporter gene without the promoter sequence. The amsG PCR product was inserted into pBBRlux-1 (Lenz et al., 2004). Recombinant fusion products were confirmed by PCR and sequencing. Reporter plasmids were electroporated into E. amylovora WT and overexpression strains. To evaluate promoter activity, cultures were grown overnight in LB medium at 28°C, pelleted, and washed twice with 0.5x PBS. For analysis of hrp gene expression, 5 µl of bacterial culture was transferred to 150 µl of hrp-inducing minimal medium (hrpMM) (Huynh et al., 1989) supplemented with Ap, Km, and 1.0 mM IPTG in a 96-well plate. After 9 h of induction, promoter activity was determined by measuring the relative fluorescence of green fluorescent protein (GFP) and was normalized to the OD600 of the corresponding culture by using a Safire plate reader (Tecan, Switzerland). This assay was repeated at least three times with four technical replicates. Analysis of amsG-lux was performed similarly; cultures were grown overnight in 150 µl LB medium supplemented with Cm and Km in a 96-well plate with agitation at 28°C and were then transferred to 150 µl LB medium supplemented with Cm, Km, and 0.1 mM IPTG in a new 96-well plate with a 96-pin replicator tool (V&P Scientific, San Diego CA). Cultures on plates were grown at 28°C with agitation, and maximum luminescence was recorded using a SpectraMax M2 multimode microplate reader at 8 h and was normalized to the OD600 of the culture.  IV. Results Erwinia amylovora encodes five putative diguanylate cyclase enzymes Bioinformatic analysis of the E. amylovora wild-type (WT) strain Ea1189 genome revealed four genes encoding GGDEF domains, one gene encoding both an EAL and a GGDEF domain, and two genes with only EAL domains. No genes with HD-GYP domains were identified. We named the five GGDEF encoding genes edcA to edcE (for Erwinia diguanylate  39 cyclases), and their domain structures are shown in Figure 2.1A. Prediction of conserved domains using the Pfam database revealed that both EdcA and EdcD contain three PAS domains, which have been widely characterized as receptors of different stimuli/signals in Archaea and Bacteria (43). In addition, EdcD also harbors a MASE1 (membrane-associated sensor) domain, usually found in bacterial signaling proteins and associated with GGDEF and EAL domains (Nikolskaya et al., 2003). EdcA also contains an EAL domain, suggesting that this protein could function as either a DGC or a PDE. All of the edc genes, except edcA, are predicted to encode inner membrane proteins based on predicted membrane-spanning domains. Bacteria often encode degenerate DGC enzymes that may function as receptors for c-di-GMP (Ryan et al., 2012). However, based on sequence analysis, all five GGDEF-encoding proteins in E. amylovora are predicted to be enzymatically active, since they contain the residues critical for DGC activity (Figure 2.1B).     40 Figure 2.1. The five putative DGC enzymes present in the genome of Erwinia amylovora Ea1189.  (A) The EAL and GGDEF domains of these proteins are shown with the protein lengths (in amino acids) and gene locus tags. Protein domains are drawn to scale. Membrane-spanning domains are shown as vertical filled bars. (B) The GGDEF domain proteins from E. amylovora were aligned with HmsT, an active DGC from Yersinia pestis. Conserved amino acids (80%) are highlighted in black. Residues required for enzymatic activity from this domain are indicated by black arrows above the amino acid alignment.     All of the E. amylovora DGCs synthesize c-di-GMP.  To determine if the putative DGCs mentioned above can synthesize c-di-GMP, we overexpressed each of the five edc genes in E. amylovora from a plasmid under the control of the Ptac promoter following induction with IPTG (Waters et al., 2008). Metabolites were extracted, and the concentration of c-di-GMP was determined using LC-MS-MS. While intracellular levels of c-di-GMP detected in WT strain containing the vector control appear to be produced at the  41 nanomolar range (59.99 nM), overexpression of the five edc genes found in E. amylovora resulted in significantly higher levels of c-di-GMP (Figure 2.2), showing that c-di-GMP can be synthesized and detected in E. amylovora, and that all of the dgc genes encoded in this phytopathogen encode functional c-di-GMP metabolizing proteins. Interestingly, overexpression of edcA, edcB, and edcC resulted in intracellular levels at the micromolar range (5.72, 43.87 and 8.91 µM, respectively), suggesting that there are the major DGC governing c-di-GMP signaling in E. amylovora.  Figure 2.2. Intracellular c-di-GMP levels in E. amylovora Ea1189 containing edc gene overexpression plasmids. Data represents three biological replicates, and error bars denote the standard error of the mean. Different letters above the bars indicate statistically significant differences (P < 0.05 by t test).       C-di-GMP negatively regulates swimming motility in E. amylovora.  Swimming motility in most plant-pathogenic bacteria, including E. amylovora, is facilitated by the helical rotation of peritrichous flagella (Cesbron et al., 2006). Since c-di-GMP represses flagellar motility in several bacterial species (Simm et al., 2004, Jenal & Malone, 2006,  42 Romling & Amikam, 2006, Merritt et al., 2007, Wolfe & Visick, 2008), we asked whether c-di-GMP inhibits the flagellum-mediated swimming motility of E. amylovora through low-density agar plates. As a positive control, we overexpressed qrgB in WT strain Ea1189, which encodes a DGC from Vibrio harveyi that has been shown to synthesize c-di-GMP in several bacterial species (Waters et al., 2008). Overexpression of QrgB strongly repressed swimming motility, while the WT strain containing the vector control was highly motile (Figure 2.3A). Like the positive control, strains overexpressing EdcB, EdcC, EdcD or EdcE were essentially nonmotile, suggesting that production of c-di-GMP from these enzymes inhibited swimming motility. Surprisingly, EdcA overexpression did not repress motility, even though overexpression of this enzyme in liquid medium generated a higher concentration of c-di-GMP than overexpression of EdcD or EdcE (Figure 2.2). Heterologous expression of DGCs is a powerful approach to determining which enzymes have the potential to contribute to c-di-GMP signaling, because it overrides any native transcriptional control of the corresponding genes. However, overexpression can lead to unnaturally high concentrations of c-di-GMP, which may disrupt signaling specificity mechanisms. To further address the role of the edc genes in E. amylovora motility, we examined the swimming motility of mutants carrying whole-gene deletions of edcA, edcC, and edcE. We hypothesized that mutation of these DGCs would decrease the intracellular c-di-GMP concentration, leading to increased motility. In support of this hypothesis, mutation of edcC and edcE significantly increased motility over that of the WT strain, whereas a double mutation of both edcC and edcE increased motility even further (Figure 2.3B). Deletion of edcA in the edcCE mutant did not alter motility, showing that edcA does not impact flagellar motility under the conditions we examined. The changes in motility in the edcC, edcE, and edcCE mutants were complemented by heterologous expression of the corresponding genes (Figure 2.3B).  43 Complementation resulted in lower motility than that of the WT strain due to the expression of the complementing genes on multicopy plasmids, results similar to those in Figure 2.3A. These experiments revealed that c-di-GMP synthesized by EdcB, EdcC, EdcD and EdcE represses motility in E. amylovora.     44 Figure 2.3. Effect of c-di-GMP on flagellar motility in E. amylovora.  Motility was examined in strain Ea1189 overexpressing DGC genes (A) or in DGC mutant strains (B). Values are normalized to the value for WT strain harboring pEVS141, the empty vector control or to the value for wildtype Ea1189.. Data represent three biological replicates, and error bars indicate the standard errors of the means. Different letters above bars indicate statistically significant differences (P <t test).            A.                 B.            acbdcdde00.20.40.60.811.2Ea1189QrgBEdcAEdcBEdcCEdcDEdcEflhCNormalized Swimming motility area (cm2)dcbaaefdfEa1189edcCedcEedcCEedcACEedcC/pedcCedcE/ pedcEedcCE/pedcC/edcEflhC0.00.51.01.52.02.5Normalized Swimming motility area (cm2) 45 C-di-GMP positively regulates amylovoran production.  Biosynthesis of amylovoran, the major EPS produced by E. amylovora, was quantified in culture supernatants by using the turbidometric cetylpyridinium chloride (CPC) binding assay as described previously (Bellemann et al., 1994). For this experiment, bacteria are typically grown in the glycerol-based MBMA defined medium. However, we observed that the WT strain overexpressing DGCs exhibited slower growth in MBMA medium (data not shown), presumably due to increased levels of c-di-GMP. Growth inhibition caused by c-di-GMP overproduction has been observed in both E. coli and Vibrio cholerae (Ryjenkov et al., 2005, Massie et al., 2012). It should be noted that c-di-GMP impacted the growth of E. amylovora only in MBMA medium, and thus, this finding does not impact the interpretation of the other experiments described here. We determined that medium containing 3 parts MBMA and 1 part LB medium did not exhibit c-di-GMP-dependent growth inhibition upon overexpression of DGCs, and this medium was used for the studies for which results are presented in Figure 2.4A. Overexpression of QrgB, EdcC, EdcD or EdcE led to amylovoran production levels higher than those with the WT harboring the empty vector (Figure 2.4A), whereas overexpression of EdcA and EdcB, did not impact amylovoran secretion compared to that with the vector control. We similarly examined amylovoran production in the edcA, edcC, and edcE deletion mutant strains and observed correlations with our findings from the overexpression studies. For these experiments, the bacteria were grown in complete MBMA medium, which resulted in higher levels of amylovoran production by the WT Ea1189 control. Mutation of edcC or edcE significantly reduced amylovoran production levels, while an edcCE double mutant exhibited a further decrease (Figure 2.4B). Introduction of the edcA mutation into the edcCE double mutant did not significantly alter amylovoran production (Figure 2.4B), suggesting that edcA is not involved in  46 this process. The amylovoran production level was significantly increased in each of the mutants upon complementation with the corresponding edc gene(s) in trans. In these studies, no growth inhibition was observed during complementation, presumably due to removal of the DGCs expressed from the chromosome. Of note, the proedcCE mutant strain remained significaams control, showing that although amylovoran secretion is reduced at low c-di-GMP levels, a significant amount of this EPS is still synthesized (Figure 2.4B). These results show that c-di-GMP positively regulates amylovoran production in E. amylovora and that, EdcC, EdcD and EdcE are the dominant DGCs regulating this process.    47 Figure 2.4. Effect of c-di-GMP on the production of amylovoran in E. amylovora.  Amylovoran biosynthesis was quantified by (A) Overexpression strains and (B) deletion mutants, and corresponding complemented strains. ams was used as a negative control. Data represent three biological replicates, and error bars indicate the standard errors of the means. Different letters above bars indicate statistically significant differences of the means (P < t test)   A.          B.            48 C-di-GMP positively regulates biofilm formation.  To further examine the role of c-di-GMP in E. amylovora, we measured the impact of overexpression and mutation of DGCs on biofilm formation. In agreement with our studies of amylovoran production, overexpression of qrgB, edcC, edcD, or edcE resulted in significant increases in biofilm formation over levels for the WT empty-vector control. Interestingly overexpression of edcB, also induced significantly higher biofilm formation levels than the WT strain, even though amylovoran biosynthesis was not activated by this strain (Figure 2.5A). Mutation of edcE alone, but not edcC, significantly reduced biofilm formation, although the edcCE double mutant formed significantly less biofilm than the edcE mutant (Figure 2.5B). Like flagellar motility and amylovoran production, biofilm formation by a edcACE mutant was indistinguishable from that by a edcCE mutant, suggesting that edcA does not contribute to biofilm formation in E. amylovora. Each of the mutants was complemented with the corresponding gene expressed in trans, and as expected, the ams mutant deficient in amylovoran production exhibited significantly less biofilm formation than all of the other strains examined (Figure 2.5B). Therefore, the regulation of amylovoran production through c-di-GMP synthesis by EdcB, EdcC, EdcD and EdcE positively impacts biofilm formation by E. amylovora.    49 Figure 2.5. Effect of c-di-GMP on biofilm formation of E. amylovora grown under static conditions.   Biofilm formation by DGC overexpression strains (A) and DGC mutants (B) was determined by quantifying crystal violet (CV) binding as described in Materials and Methods. Error bars indicate the standard errors of the means, and different letters above the bars indicate statistically significant differences of the means (P <  t test).    A.           B.          50 C-di-GMP inhibits the virulence of E. amylovora in two plant infection models.  Motility, amylovoran secretion, and biofilm formation are all behaviors of E. amylovora that have been associated with plant infection (Bayot & Ries, 1986, Bellemann & Geider, 1992, Koczan et al., 2009). To determine the impact of c-di-GMP on E. amylovora virulence, we inoculated immature pears with the WT strain Ea1189, edc deletion mutants, or complemented strains and examined the development of tissue necrosis. Inoculation of the WT strain resulted in a necrotic lesion surrounding the inoculation point at 4 days post-infection (dpi) (Figure 2.6B). Based on our previous studies indicating that amylovoran production positively influences E. amylovora virulence for the pear (Koczan et al., 2009), we hypothesized that mutations of active DGCs that reduce amylovoran secretion would lead to decreased disease. However, while single mutations of edcC and edcE did not significantly impact the lesion size, the edcCE double mutant produced a significantly larger area of necrosis (Figure 2.6A and B). In accordance with our results presented above, mutation of edcA had no significant effect: the area of tissue necrosis induced by the edcACE mutant was not significantly different from that with the edcCE mutant. Expression of edcC and edcE in the edcCE mutant exhibited only partial complementation (Figure 2.6). We determined that the complementation plasmids are maintained in E. amylovora during the course of the experiment; however, since overexpression of the edcC and edcE genes from the complementation vector was dependent on IPTG induction, we speculate that the lack of complete complementation is likely due to decreases in the concentrations of IPTG (added only at the inoculation time) in the pears during the 4-day experiment.    51 Figure 2.6. Virulence of E. amylovora DGC mutant strains in an immature-pear infection model. (A) Sizes of lesions on immature pears inoculated with the indicated strains. Lesion size was measured using calipers at 4 dpi. edcCE mutant was complemented with edcC and edcE expressed from a Error bars represent the standard errors. Different letters above the bars indicate statistically significant differences (P <0.05 by t test). (B) Representative pears illustrate symptom development at 4 dpi for Ea1189 and the mutant strains.    To determine if the negative influence of c-di-GMP on virulence and tissue necrosis was specific to the immature-pear assay or relevant to other plant infection models as well, the impact of c-di-GMP on bacterial virulence was also evaluated in apple shoots inoculated with the WT, mutant, and complemented strains. Inoculation with the edcC or edcE mutant resulted in increased tissue necrosis and migration through the central vein of the leaf, in comparison with the lesion elicited by the WT strain (Figure 2.7). This disease phenotype was more pronounced  52 in the edcCE and edcACE double and triple mutants. Complementation of these mutants partially restored WT levels of tissue necrosis and bacterial migration. The results from both the immature pear assay and the apple shoot infection assay indicate that EdcC and EdcE possess DGC activity in both pears and apple shoots and that c-di-GMP negatively modulates the acute virulence of E. amylovora.  Figure 2.7. Effect of c-di-GMP on virulence and migration of E. amylovora in an apple shoot infection model.  Symptom development on apple shoots at 4 dpi is shown. The mutations were complemented     53 C-di-GMP regulates transcription of amylovoran synthesis and type III secretion genes.  C-di-GMP controls bacterial behaviors at multiple levels, including the induction of transcription, posttranscriptional gene regulation, and direct modulation of protein activity (Ryan et al., 2012). The negative effect of c-di-GMP on bacterial virulence in immature pears and apple shoots led us to hypothesize that this second messenger repressed the expression of hrp-type III secretion system (hrp-T3SS)-related genes. To test this hypothesis, the expression of the hrpA gene, which encodes the major structural protein of the Hrp-T3SS pilus in E. amylovora (Jin et al., 2001), was evaluated by using a promoter-gfp transcriptional fusion in the WT and in strains overexpressing qrgB, qrgB* which harbors a mutation of the GGDEF domain, edcC, or edcE. Overexpression of the active DGC qrgB, edcC, or edcE led to levels of promoter activity significantly reduced from those of the WT containing the vector control and the strain expressing qrgB* (Figure 8). We also evaluated the expression of hrpS, a 54-dependent enhancer-binding protein (EBP) that regulates the expression of HrpL, the alternative sigma factor required for the transcription of hrp-T3SS-related genes such as hrpA (Wei et al., 2000). In contrast to the reduced levels of hrpA promoter activity, qrgB and edcE did not significantly affect hrpS promoter activity (Figure 8). Although overexpression of edcC resulted in a statistically significant increase in the level of hrpS promoter activity over that for the WT strain, this result may be not biologically relevant, since this strain synthesizes c-di-GMP at lower levels than the qrgB-overexpressing control strain. Together, these results indicate that c-di-GMP synthesized by EdcC and EdcE negatively regulates the expression of the Hrp-T3SS hrpA gene and that this regulation does not involves changes in the expression levels of the HrpL regulator, HrpS.    54 Figure 2.8. Effect of c-di-GMP on the transcription of genes involved in type III secretion.  The expression of hrpA and hrpS transcriptional fusions to gfp was examined upon expression of the indicated DGCs. gfp is expressed in normalized units (fluorescence units/OD600). Error bars indicate the standard errors. Symbols above the bars indicate statistically significant differences from the vector controls t test (*, P < 0.05).    V. Discussion Our results indicate that c-di-GMP is a positive regulator of amylovoran production and biofilm formation in E. amylovora and a negative regulator of swimming motility, Hrp-T3SS gene expression, and virulence. Using overexpression studies and mutagenesis, we determined that the dominant DGCs in E. amylovora are EdcB, EdcC, EdcD and EdcE. Although overexpression of edcA generated measurable levels of c-di-GMP, we conclude that this DGC does not impact the phenotypes we examined. C-di-GMP signaling has been shown to be a highly specific process whereby different DGC enzymes specifically influence c-di-GMP-mediated behaviors (Kader et al., 2006, Newell et al., 2011, Massie et al., 2012). It is possible  55 that the c-di-GMP generated by EdcA does not control biofilm formation, motility, or virulence as examined here but rather modulates other bacterial behaviors not tested in this study.   Analysis of the regulatory roles of elevated c-di-GMP levels revealed insight into the interplay between biofilm formation, motility, and Hrp-T3SS gene expression in E. amylovora pathogenesis. A previous study demonstrated that biofilm-deficient mutants in which amylovoran production was unaffected, but genes encoding specific attachment factors, such as type I fimbriae, were mutated, exhibited reductions in virulence when inoculated into apple leaves, revealing a distinct role for biofilm formation independent of EPS secretion in plant disease (Koczan et al., 2011a). Moreover, it has been reported previously that the amount of amylovoran produced correlated with virulence in the host (Ayers et al., 1979). Our results contrasted with these conclusions, since we obedcCE and edcACE mutants, which exhibited decreased biofilm formation and amylovoran EPS production, showed an increased virulence phenotype. We conclude that the decreased levels of amylovoran production and biofilm formation at reduced c-di-GMP levels are sufficient to facilitate E. amylovora infection and systemic invasion of leaves, while altered regulation of other behaviors, such as motility and hrp-T3SS gene expression, leads to increased virulence.  edcCE edcACE mutants is likely one behavior responsible for the enhanced virulence of these mutants. Motility is an important virulence factor contributing to the infection of flowers by E. amylovora (Bayot & Ries, 1986, Cesbron et al., 2006) . However, E. amylovora cells are thought to be nonmotile in the apoplast, although these data come from one study in which cells were examined in vitro immediately after recovery from  56 infected stems (Raymundo & Ries, 1981). More recently, E. amylovora strains demonstrating increased swarming motility in vitro were shown to be more virulent than less-motile strains in apple genotypes exhibiting high levels of fire blight resistance (Wang et al., 2010). The edcCE and edcACE mutants exhibited increased swimming motility in vitro due to a lack of c-di-GMP production. We similarly determined that c-di-GMP inhibited the swarming of E. amylovora (data not shown). Thus, it is likely that increased motility in planta following leaf inoculation enabled these mutants to gain more rapid access to host cells over a larger area, leading to the enhanced necrosis phenotype observed.   We determined that, in addition to inhibiting motility, c-di-GMP represses hrp-T3SS-related genes, as has been reported for the plant pathogen Dickeya dadantii and the animal pathogen Salmonella enterica serovar Typhimurium (Lamprokostopoulou et al., 2010, Yi et al., 2010, Ahmad et al., 2011). Our results suggest that this regulation is not associated with a negative effect of c-di-GMP on the expression of HrpS, but it could be associated with downregulation of other key players in the hrp signaling cascade, such as the two-component transduction system HrpX/HrpY, RpoN (54), or with direct negative regulation of HrpL, the alternative sigma factor required for the transcription of hrp genes (Wei & Beer, 1995, Wei et al., 2000).  Although the regulation of the transition between infection stages in E. amylovora is poorly understood, recent studies have demonstrated that coinfection of a strain lacking the entire ams operon with a Hrp-T3SS deletion mutant leads to full virulence (Zhao et al., 2009b). This finding suggests that E. amylovora specifically controls the expression of pathogenicity  57 determinants, among which Hrp-T3SS is required in host colonization and the early stages of infection while the biosynthesis of amylovoran is required for establishment in the host vascular tissues during late stages of the disease. Moreover, regulatory elements of Hrp-T3SS expression negatively control the production of amylovoran, since a T3SS deletion mutant produced 3- to 4-fold-larger amounts of this EPS than the WT strain (Zhao et al., 2009b). In addition, HrpL regulates the expression of non-Hrp-T3SS related virulence factors (McNally et al., 2012). Our results suggest that c-di-GMP is involved in the orchestration of bacterial pathogenesis, since hrp-T3SS-related genes and amylovoran production are inversely modulated by this second messenger. However, further studies are needed to elucidate the specific role of c-di-GMP in the coordination of expression of pathogenesis-related traits.  C-di-GMP has now been implicated in the ability of a number of plant pathogens to cause infections. Our results suggest that E. amylovora appears to most closely resemble D. dadantii, in which mutation of two PDE enzymes, which presumably increased c-di-GMP levels, enhanced biofilm formation while reducing plant virulence (Yi et al., 2010). The decreased virulence of D. dadantii was attributed to downregulation of T3SS-related genes and decreased secretion of pectate lyase by c-di-GMP (Yi et al., 2010). Similarly, a recent study demonstrated that high levels of c-di-GMP promote the activation of a biofilm-forming phenotype in Pectobacterium atrosepticum and positively regulate the expression of poly--1,6-N-acetyl-D-glucosamine (PGA), an EPS essential for biofilm formation (Pérez-Mendoza et al., 2011). In Xanthomonas campestris pv. campestris, increased levels of c-di-GMP similarly inhibit plant virulence and increase biofilm formation (Ryan et al., 2007). However, c-di-GMP negatively influences biosynthesis of the EPS xanthan, which is necessary for plant disease, in X.  58 campestris pv. campestris, whereas we determined that c-di-GMP positively regulated amylovoran biosynthesis in E. amylovora. From the perspective of c-di-GMP signaling, Xylella fastidiosa appears to be the most distinct from other bacterial plant pathogens; in this organism, c-di-GMP positively influences the expression of secreted virulence factors and type IV pili while negatively impacting biofilm formation (Chatterjee et al., 2010). Therefore, each of these phytopathogens has integrated c-di-GMP as a central regulator in the control of virulence, EPS secretion, and biofilm formation in unique ways presumably optimally adapted to their different disease progressions.  In summary, we have identified five genes that potentially encode DGC enzymes in the plant pathogen E. amylovora. Analysis of these DGCs through overexpression or deletion indicated that c-di-GMP synthesized by four of them, EdcB, EdcC, EdcD and EdcE, regulate motility, amylovoran production, biofilm formation, expression of the Hrp-T3SS, and plant virulence. E. amylovora is a powerful model system for the study of c-di-GMP signaling in a bacterial plant pathogen, since its pathway is relatively simple, encoding only seven DGCs and PDEs. Moreover, E. amylovora allows the determination of the role of c-di-GMP in the pathogenesis of a bacterial pathogen during the infection of its native host. The coordination of pathogenesis by E. amylovora and the transition between infection stages that require motility, Hrp-T3SS, amylovoran production, or biofilm formation suggest that continued investigation into the regulatory networks controlled by c-di-GMP will be essential to full understanding of the pathogenesis of E. amylovora.     59 Chapter 3:  Cellulose production, activated by cyclic di-GMP through BcsA and BcsZ, is a virulence factor and an essential determinant of the three-dimensional architecture of biofilms formed by Erwinia amylovora  This chapter has been modified from a manuscript of the same title submitted to Molecular Plant Pathology: Castiblanco, L. F. and Sundin, G. W. (2016) Cellulose production, activated by cyclic di-GMP through BcsA and BcsZ, is a virulence factor and an essential determinant of the three-dimensional architecture of biofilms formed by Erwinia amylovora.  60 I. Abstract Multicellular aggregates in bacterial biofilms are encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein, and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. Production of the EPSs amylovoran and levan are critical for the formation of a mature biofilm. Additionally, cyclic dimeric GMP (c-di-GMP) was reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora. In this study, we demonstrated that cellulose is synthesized by E. amylovora, is a major modulator of the three-dimensional characteristics of biofilms formed by this bacterium, and contributes to virulence during systemic host invasion. Additionally, we demonstrated that cellulose biosynthesis activation in E. amylovora is a c-di-GMP dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We reported that the endoglucanase BcsZ is a key player in c-di-GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.   61 II. Introduction  Biofilm formation is one of the most extensively studied processes in bacteria as it represents a major adaptation to life in the different environments that these prokaryotic organisms colonize.  The formation of biofilms, defined as the assembly of multicellular aggregates on solid surfaces, depends on the production of a self-synthesized matrix mainly composed of exopolysaccharides (EPSs), proteins, lipids and extracellular DNA (Karatan & Watnick, 2009, Flemming & Wingender, 2010).  Although the actual matrix composition of a biofilm is often a species-specific feature and its EPS content is quite variable, some EPSs are frequently produced by multiple bacterial species; moreover, the combination of different EPS has been reported as an important structural component of biofilm matrices produced by several species such as Pseudomonas and Escherichia coli (Zogaj et al., 2001, Karatan & Watnick, 2009, Mann & Wozniak, 2012).  In addition to their role as modulators of biofilm architecture, EPSs are essential in all of the developmental stages of biofilm formation, from initial attachment to solid surfaces to final detachment and dispersion (Flemming & Wingender, 2010, Petrova & Sauer, 2016). Moreover, EPSs provides the multicellular aggregates with matrix cohesiveness, protection from antimicrobial compounds, host defense responses and harsh environmental conditions, enhanced water retention and accumulation of nutrients from the environment (Flemming & Wingender, 2010).  In bacterial plant pathogens, the formation of biofilms is an important virulence factor for xylem-dwelling organisms such as Erwinia amylovora, Xylella fastidiosa, and Ralstonia solanacearum that form biofilms inside the host vasculature impairing water and nutrient transport in the plant. Biofilms are important components of ecological fitness in epiphytic  62 bacteria on plant surfaces and for bacteria colonizing the plant rhizosphere (reviewed in (Danhorn & Fuqua, 2007).  The genetic regulation of biofilm formation includes several layers of regulatory steps that involves regulation of cooperative behaviors with population-based regulatory strategies such as quorum sensing and other intracellular regulators including -cyclic dimeric guanylic acid (c-di-GMP) and other nucleotide messengers, non-coding RNAs and RNA binding proteins (Ng & Bassler, 2009, Castiblanco & Sundin, 2015)  Erwinia amylovora, the causal agent of fire blight on rosaceous plants, is a highly virulent pathogen that infects flowers and actively-growing shoots and moves systemically either inside the vascular tissue or through the cortical parenchyma of its host. This pathogen produces at least two EPSs, amylovoran and levan, each of which play a critical role in the formation of biofilms inside the host xylem, resulting in vessel blockage and water transport restriction and consequently, wilt and necrosis of the infected tissues (Koczan et al., 2009). Moreover, biofilm formation and amylovoran biosynthesis have been demostrated to be under the regulation of c-di-GMP, as high intracellular levels of this secondary messenger molecule induce the activation of these two cellular processes (Edmunds et al., 2013).  Cyclic di-GMP is a ubiquitous secondary messenger in bacteria that is involved in the regulation of a plethora of cellular processes and bacterial behaviors. In most cases, low intracellular levels of c-di-GMP are associated with a planktonic, highly motile state whereas high levels of this molecule are associated with EPS biosynthesis, and cellular aggregation and biofilm formation. Additionally, c-di-GMP signaling is also involved in the regulation of many other cellular functions such as the expression of virulence factors, cell cycle and differentiation  63 and RNA processing (reviewed in (Hengge, 2009, Mills et al., 2011, Römling, 2012, Römling et al., 2013). Intracellular homeostasis of this messenger molecule is modulated by the antagonistic action of diguanylate cyclases (DGC) harboring a GGDEF active domain and phosphodiesterases (PDE) with either an EAL or HD-GYP active domain. After biosynthesis or degradation, c-di-GMP allosterically binds, or is prevented from binding, to specific receptor molecules (effectors), modifying their structure and altering their output function which ultimately results in the activation or repression of a given cellular process (reviewed in (Chou & Galperin, 2016). These receptors include proteins containing a PilZ domain, DGCs or PDEs with inactive catalytic domains but that have retained the ability to bind to c-di-GMP, transcriptional factors that do not share a predictable c-di-GMP-binding domain (Ryan et al., 2012, Römling et al., 2013, Chou & Galperin, 2016), the polynucleotide phosphorylase (PNPase) in E. coli (Tuckerman et al., 2011) and several riboswitches (Sudarsan et al., 2008, Lee et al., 2010). The diversity of c-di-GMP catalytic enzymes and receptors explains the global effects that this signaling system exerts in many aspects of the bacterial lifestyle.   PilZ domain-containing proteins are the best-characterized type of c-di-GMP effector, due to their predictability by bioinformatics analyses. Ryjenkov and collaborators demonstrated that the PilZ domain in the cellulose catalytic subunit BcsA from Gluconacetobacter xylinus (now Komagataeibacter xylinus) binds specifically to c-di-GMP, similar to YgcR in E. coli, another PilZ domain-containing protein that is involved in the repression of motility in this bacterial species (Ryjenkov et al., 2006). Since then, several PilZ domain-containing proteins have been characterized as c-di-GMP receptor proteins not only as important players in the regulation of EPS biosynthesis, biofilm formation and motility but also as regulators of bacterial  64 virulence (Merighi et al., 2007, Pratt et al., 2007, McCarthy et al., 2008, Wilksch et al., 2011, Zorraquino et al., 2013, Martínez-Granero et al., 2014, Yang et al., 2014). BcsA is one of the components of the membrane-integrated bacteria cellulose synthase multiprotein complex along with at least two other proteins, BscB and BcsC, whose genes are encoded in the bcs operon. BcsA, the catalytic subunit, is located in the inner membrane with a C-terminal cytoplasmic region where the c-di-GMP binding PilZ domain is located. Along with BcsB, a periplasmic protein anchored to the inner membrane, BcsA forms a catalytic active complex that has been reported to be sufficient for cellulose synthesis and translocation (Omadjela et al., 2013, McNamara et al., 2015, Römling & Galperin, 2015). BcsC is a predicted periplasmic porin -barrel in the outer membrane, allowing the export of the nascent -glucan chain out of the cell (Whitney & Howell, 2013). Although the biosynthesis of cellulose is performed by many bacterial species, the organization and gene content of bcs operons across different bacterial species is quite diverse; Römling and Galperin (2015) classified all charactized bcs operons in four different catergories, containing at least two subcategories each.   Allosteric binding of c-di-GMP to the PilZ domain in the cytoplasm post-translationally activates the biosynthesis of cellulose by a  recently-described mechanism: it promotes a conformational change in the gating loop of the BcsA PilZ domain, that allows the glycosyltransferase domain (GT) to be available for the entrance of the substrate, UDP-glucose and the posterior exiting of the UDP secondary reaction product (Morgan et al., 2014). Bioysnthesis of cellulose has been demostrated to be a critical process for the formation of biofilms, as this EPS is one of the main components of the extracellular matrix produced by  65 many enteric bacterial species including Salmonella, E. coli, and Dickeya dadantii (Zogaj et al., 2001, Solano et al., 2002, Jahn et al., 2011, Serra et al., 2013).  In this study, we investigated cellulose biosynthesis in E. amylovora and established that, in addition to amylovoran and levan, cellulose is a critical component of  biofilms formed in vitro and in the apple host. Moreover, we demonstrated that, as reported for other bacterial species, this cellular process is also regulated at the post-translational level by the secondary messenger c-di-GMP through the cellulose synthase subunit BcsA and through the endoglucanase BcsZ .   III. Materials and Methods Bacterial strains, plasmid and growth conditions The bacterial strains and plasmid used in this study are listed in Table 3.1. Unless otherwise noted, E. coli strains were grown at 37°C and E. amylovora strains at 28°C in Luria-Bertani liquid and solid medium, amended with ampicillin (Ap; 100 µg mL-1), kanamycin (Km; 50 µg mL-1), chloramphenicol (Cm; 10 µg mL-1), tetracycline (Tc; 10 µg mL-1) or gentamicin (Gm; 10 µg mL-1), as required.     66 Table 3.1. Bacterial strains and plasmids used in Chapter 3 and their relevant characteristics Strain or plasmid Characteristics a Source or reference Escherichia coli strain    F-80dlacZ M15 (lacZYA-argF)U169 endA1 recA1 hsdR17 (rk-mk+)deoR thi-1 supE44 gyrA96 relA1 - Invitrogen, Carlsbad, CA, USA    Erwinia amylovora strains   Ea1189 Wild type (Burse et al., 2004c) bcsA bcsA deletion mutant in Ea1189 This study bcsD bcsD deletion mutant in Ea1189 This study bcsE bcsE deletion mutant in Ea1189 This study bcsZ bcsZ deletion mutant in Ea1189 This study    Plasmids   pKD46 Arabinose-inducible lambda red recombinase, ApR (Datsenko & Wanner, 2000) pKD3 CmR cassette flanked by FRT sites (Datsenko & Wanner, 2000) pBBRMCS-5 Broad-host-range cloning vector, GmR (Kovach et al., 1994) pEVS143 Broad-host-range cloning vector, KmR (Dunn et al., 2006) pLFC45 pBBR1MCS-5 expressing bcsA under native promoter, GmR This study pLFC51 pBBR1MCS-5 expressing bcsAR556D under native promoter, GmR This study pLFC34 pEVS143:edcB overexpression vector, KmR This study pACE-edcC pEVS143:edcC overexpression vector, KmR (Edmunds et al., 2013) pMP2444 pBBR1MCS-5 expressing gfp under lac promoter, GmR (Stuurman et al., 2000) (a) KmR, CmR, GmR and ApR indicate resistance to kanamycin, chloramphenicol, gentamicin and ampicillin respectively.   Chromosomal mutagenesis and complementation PCR, restriction digestions, gene cloning and gel electrophoresis were performed according to standard methods (Sambrook et al., 2001). Chromosomal mutant strains were constructed using a modification of the lambda red recombination technique as previously described (Datsenko & Wanner, 2000, Koczan et al., 2009). Briefly, the Cm resistance cassette in template plasmid pKD3 was amplified with primers containing 50-bp overhangs, homologous to the first and last 50 nt of the gene of interest. Purified PCR product was transformed into E. amylovora wild type strain Ea1189 expressing the genes of redexo recombinases from pKD46. Mutant  67 strains were transformed with pTL18 (Long et al., 2009) encoding an IPTG-inducible flippase and plated in solid media amended with 1mM IPTG, which led to the chromosomal excision of the antibiotic cassette. Insertion and posterior excision of the antibiotic resistance cassette were verified by PCR and sequencing.  For gene complementation, bcsA was transformed with pLFC45, which contained 500bp of the native bcs operon promoter linked to the bcsA gene by overlap extension PCR (OE-PCR) (Higuchi et al., 1988), cloned into pBBRMCS-5 (Kovach et al., 1994).  Cellulose biosynthesis assessment To evaluate the activation of cellulose biosynthesis in vitro, bacterial strains were spotted onto LB plates lacking NaCl, and amended with Congo Red (40 µg mL-1) and Coomassie Brilliant Blue (20 µg mL-1). Plates were incubated 24 h at 28°C. Red colonies are indicative of Congo Red binding to cellulose.  In vitro biofilm assay Biofilm formation on abiotic surfaces was evaluated as previously described (Koczan et al., 2009). Briefly, 1.5 mL of 0.5x LB broth emended with the appropriate antibiotic were inoculated with 150 µl of equilibrated overnight cultures in individual wells of 24-well plates containing a glass coverslip placed at a ~30° angle. After 48 h of incubation at 24°C, planktonic cells were removed and the coverslips were stained with 10% crystal violet (CV) for 1 h, washed three times with distilled water, and air-dried. CV was solubilized with 200 µl of 40% methanol / 10% acetic acid and the absorbance at 595 nm was recorded. To determine the location of cellulose in the biofilms, glass cover slips were placed in test tubes containing 10 mL of 0.5x LB liquid at  68 24°C. After 48 h, coverslips were dipped in a Calcofluor solution (50 µg mL-1) for 10 min, washed three times with distilled water, and visualized using a Leica DM5000 B microscope equipped with the Leica DFC450 digital microscope camera using the DAPI filter for fluorescence (Leica; Buffalo Grove IL).   Confocal laser scanning microscopy for the visualization of biofilms in a flow cell chamber The topology of the biofilms produced by E. amylovora strains expressing gfp from pMP2444 was studied using a flow cell chamber as previously described (Koczan et al., 2009). Briefly, flow cell chambers (Stovall Life Sciences; Greensboro NC) were inoculated with WT Ea1189 bcsA, and fresh 0.5x LB medium was allowed to pass continuously through the chamber for 48 h at 24°C. Biofilms formed inside the flow cell chamber were visualized using a Zeiss 510 Meta ConfoCor3 LSM confocal laser scanning microscope (Carl Zeiss Microimaging; Germany). Z-stacks of the bacterial aggregates were acquired, and the fluorescence intensity was employed to obtain a three-dimensional representation of the biofilms using the 2.5-D setting in the LSM browser.  Virulence assay on apple shoots Virulence of WT and mutant strains was evaluated on apple shoots as previously described (Zhao et al., 2005b). Briefly, shoots of 2-year old potted apple trees (cv. Gala on M9 rootstock) were inoculated with a bacterial suspension of ca. 5 x 108 CFU mL-1, derived from an overnight culture, by cutting the tip of the youngest leaf of growing shoots with scissors previously dipped in the bacterial suspension. Symptoms were recorded at 5 dpi. This experiment was performed three times with five replicates per strain.  69  Evaluation of in vitro and in planta biofilms using scanning electron microscopy The youngest leaves of actively growing apple shoots were inoculated as described above, collected and sectioned in 0.5 cm fragments at 2 dpi. Samples were fixed with 2.5% paraformaldehyde / 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (Electron Microscopy Sciences; Hatfield PA) for 24 h at 24°C and dehydrated with 25, 50, 75 and 90% ethanol for 30 min each and with 100% ethanol three times for 30 min each. After critical point drying in a Leica Microsystems model EM CPD300 critical point dryer (Leica Microsystems; Buffalo Grove, IL), samples were sectioned longitudinally and mounted on aluminum studs with high vacuum carbon tabs. Samples were coated with platinum in a Quorum Technologies/Electron Microscopy Sciences Q150T Turbo Pumped Coater (Quorum Technologies; Guelph ON). Samples were imaged on a JEOL JSM-7500F (cold field emission electron emitter) scanning electron microscope (Japan Electron Optics Laboratory Ltd; Tokyo Japan). To visualize biofilms formed on abiotic surfaces, 10 µL of equilibrated bacterial cultures were added to 100 µL of 0.5X LB broth in individual wells of a 96-well plate containing a 300 mesh gold grid (Electron Microscopy Sciences; Hatfield PA). After incubation at 24°C for 48 h, 100 µL of 2.5% paraformaldehyde / 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer was added to each well for fixation and plates were incubated for 1 h. Grids were successively dehydrated with ethanol at increasing concentrations, critical point dried, osmium coated and imaged as described previously for the apple tissue.    70 Site-directed residue-replacement mutagenesis  Replacement of the critical amino acid residues for c-di-GMP binding in BcsA was carried out using the Quikchange II site-directed mutagenesis system (Agilent Technologies; Santa Clara CA) with some modifications. The entire pLFC45 expressing the bcsA gene under its native promoter was amplified with Phusion Taq polymerase (New England Biolabs; Ipswich, MA) with complementary mutagenic primers, under the following conditions: 16 cycles of 98°C for 1 min, 68°C for 1 min and 72°C for 7 min. Two µL of DpnI were added to the PCR reaction which was incubated at 37°C for 1 h. One µL of the digestion product was transformed in competent cells of E. coli mutated bcsA sequence on plasmid pLFC49 was confirmed by sequencing. pLFCbcsA.  IV. Results Bacterial cellulose synthase operon in Erwinia amylovora Examination of the gene content of the bcs operon encoded by E. amylovora showed that this organism harbors a Type Ib bcs operon, in accordance with the recent classification of bcs operons by Römling & Galperin (2015); in addition to the three core genes bcsA, bcsB and bcsC, the E. amylovora bcs operon contains bcsO, encoding a protein of unknown function, bcsQ, encoding a predicted ATPase, bcsD, which encodes a predicted periplasmic protein that is the distinguishing feature of type I bcs operons, and bcsZ, encoding a putative endoglucanase (Fig 3.1). Interestingly, immediately downstream of the bcs operon, the genome of E. amylovora harbors a bcsE gene, usually found in type II operons along with other two genes, bcsF and bcsG which constitute a second cellulose biosynthetic cluster in other bacterial species (Römling & Galperin, 2015). bcsE encodes a protein harboring a DUF2819 domain, recently described to  71 bind c-di-GMP and enhance cellulose biosynthesis in other enterobacterial species (Fang et al., 2014).  Figure 3.1. Organization of the bcs operon in E. amylovora.    Cellulose is one of the main component of biofilms formed by E. amylovora in vitro  Since cellulose has been reported as a critical component for the formation of biofilms in many bacterial species, we hypothesized that this EPS could have an important role for the formation of multicellular aggregates by E. amylovora in vitro and inside the host. Analysis of biofilm formation in vitro on glass coverslips at the air-bcsA mutant strain was significantly reduced in its ability to form biofilms in vitro, compared with the Ea1189 WT strain (Fig 3.2). The mutanbcsA was complemented with the bcsA gene under the control of the native bcs promoter in trans.    72 Figure 3.2. Impact of the abrogation of cellulose production on in vitro biofilm formation by E. amylovora Ea1189.  bcsA abcsA complemented strain after 48 h of incubation at 28°C. Data represents three biological replicates. Error bars indicate standard error of the mean. Letters above the bars indicate statistically significant differences (P< 0.05 by Tukey HSD test)  To further evaluate the characteristics of the biofilm produced by E. amylovora when cellulose biosynthesis has been abrogated, we used a flow cell system and confocal microscopy bcsA strains expressing gfp from plasmid pMP2444. This method allowed us to generate a three-dimensional image of the biofilm developed inside the chamber and to visualize the topology of the bacterial aggregates. In bcsA mutant strain formed only small aggregates that were sparsely distributed throughout the chamber and that did not transition to mature macro-colonies (Fig 3.3). This result contrasts with the biofilm observed for the Ea1189 WT strain which presents a consistent pattern of large aggregates with increased bcsA, and evidenced by an increased fluorescence intensity throughout the biofilm (Fig 3.3).  73 Figure 3.3. Effect of cellulose on the three-dimensional architecture of biofilms produced by E. amylovora Ea1189.  (A) Confocal laser scanning microscopy of biofilms developed inside a flow cell chamber after 24 h of incubation at 24°C bcsA strain. Images were obtained using a 20X objective. (B) Intensity mapping of the topology of biofilms formed inside a flow chamber. Axes x and y indicate biofilm formation area and z indicates intensity.     74 Figure 3.4.  Effect of cellulose on the extracellular matrix organization of E. amylovora Ea1189.  SEM observations of biofilms formed on 300-mesh gold grids after 48 h of incubation at 28°C bcsA, bcsA complemented strain and (D) WT strain overexpressing edcB. Gold grids were examined at 22,000X magnification.    75 In addition to our confocal microscopy studies, the impact of deficiency in cellulose production on the formation of biofilms in vitro was also examined using scanning electron microscopy (SEM) for bacterial strains forming aggregates on 300-mesh gold grids. After 48 h of incubation, a very dense, highly branched network of fibrillar material, with irregular strands linking bacterial cells inwardly and to the solid surface was observed for the WT Ea1189 strain bcsA mutant strain, microfibers connecting cells were visualized, but at a much less dense proportion than the fibrillar matrix formed by the WT strain, with more regular and organized strands (FbcsA with the native bcsA gene partially restored the complexity of the matrix structure and the branching of the microfibers (Fig 3.4C).  To visualize the overall contribution of cellulose for the formation of biofilms by E. amylovora in vitro, biofilms formed at the air-liquid interface on glass coverslips were stained -glucans and enables us to visually assess the amount of cellulose (Fig 3.5). After 48h, WT biofilms strongly bound to calcofluor which is indicative of active cellulose biosynthesis. Conversely, calcofluor binding to the biofilm formed bcsA was extremely poor, with sparsely distributed foci and lower fluorescence intensities, indicating the lack of cellulose biosynthesis in this mutant background. This phenotype was partially restored with the in trans expression of bcsA in the bcsA mutant background.  Overall, these results suggest that cellulose is one of the main components of the biofilms formed by E. amylovora in vitro, as abrogation of cellulose biosynthesis significantly reduced the ability of this pathogen to form multi-cellular aggregations and mature biofilms. Moreover, these data suggest that the fibrillar material in the biofilm extracellular matrix is composed of cellulose and at least one additional polymer, most likely amylovoran.  76 Figure 3.5. Distribution of cellulose in biofilms formed in vitro by E. amylovora Ea1189.  Biofilms formed on glass coverslips were stained with calcofluor to assess the location of bcsA and complemented strain after 48 h of incubation at 24°C. Images were obtained using the 40X objective and the DAPI filter for fluorescence.  Cellulose is a modulator of biofilm architecture in planta  In order to assess the impact of cellulose biosynthesis on the formation of biofilms inside the host vascular system, longitudinal sections of the main vein of leaves inoculated with E. amylovora bcsA mutant strains were examined using SEM at 2 days after inoculation. Multicellular aggregates were visualized inside xylem vessels from WT inoculated leaves. Moreover, the production of an extracellular material similar to polymeric microfibers emerging from the bacterial cells and linking cells to the xylem walls was also observed for this strain (Fig 3.6). On the other hand, although extensive cell growth with the appearance of cellular bcsA, we were not able to detect the same fibrillar material emerging from the bacterial cells as observed with the WT strain.   77 Figure 3.6.  Cellulose is a main component of biofilms formed in planta by E. amylovora Ea1189.  bcsA strains at 2 days after inoculation in xylem vessel of apple shoots. Images were examined at 12,000X magnification.     To determine if the differences in the extracellular matrix produced inside the vascular bcsA mutant strain had an impact in the virulence of E. amylovora, bcsA strain in apple shoots were assessed at 5 days after inoculation. While all of the studied strains were capable of colonizing the leaf vascular system and produced necrotic lesions along the main vein of the leaf, a delay in symptom migration bcsA mutant when compared with shoots inoculated with the WT and complemented strain (Fig 3.7). Bacterial population analyses determined that this difference in lesion length was not due to a bcsA background (data not shown). These data suggest that cellulose biosynthesis plays an important role in the architecture and maturation of biofilms inside the xylem vessels of the plant host and that it has a positive impact in the internal dispersion of the pathogen inside the vascular system of the apple host.     78 Figure 3.7. Effect of cellulose production on virulence of E. amylovora Ea1189 in apple shoots.  (A) Symptom development on an apple shoots at 5 days after inoculation with Ea1189 WT, bcsA, bcsA-complemented strain. The youngest leaf was inoculated with scissors previously dipped in a bacterial suspension (5 x 108 CFU mL-1). Arrows indicate the migration front of the necrotic lesion. (B) Average lesion length on apple shoots inoculated with the same bacterial strains. Data represents three biological replicates. Error bars indicate standard error of the mean. Letters above the bars indicate statistically significant differences (P< 0.05 by Tukey HSD test).    79 C-di-GMP activates cellulose biosynthesis in E. amylovora  We have previously reported the activation of amylovoran biosynthesis and a hyper-biofilm forming phenotype in E. amylovora when high intracellular concentrations of c-di-GMP are induced with the overexpression of the DGC-encoding genes edcC and edcE (Edmunds et al., 2013). In an attempt to dissect the role of individual DGCs on the complete set of c-di-GMP biosynthetic proteins in this pathogen, the modulation of EPS biosynthesis and biofilm formation of two additional DGC genes, edcB and edcD, was evaluated. Overexpression of both genes induced a significant increase in biofilm formation compared with the Ea1189 wild type (WT) strain, with the edcD overexpressing strain exhibiting an intermediate phenotype (Fig 3.8A). Interestingly, overexpression of edcB did not have any effect on amylovoran biosynthesis whereas high c-di-GMP levels induced via edcD overexpression resulted in activation of biosynthesis of this EPS at the same level as that with edcC and edcE overexpression (Fig 3.8B). These intriguing results suggest that, in addition to amylovoran, another critical unknown component for the formation of biofilms by E. amylovora is also regulated in an independent fashion by c-di-GMP synthesized by EdcB.  Since the cellulose catalytic subunit BcsAEa (GenBank accession number WP_042327657) was identified to contain a canonical PilZ domain, we hypothesized that cellulose is the other critical component of biofilms, regulated by c-di-GMP.   80 Figure 3.8. Regulation of biofilm formation and amylovoran biosynthesis by the different DCGs in E. amylovora Ea1189.  (A) Biofilm formation on glass coverslips and (B) amylovoran biosynthesis were quantified for Ea1189 WT and strains overexpressing individual DGCs. An ams operon deletion mutant was used as negative control for the amylovoran quantification assay.  Data represents three biological replicates. Error bars indicate standard error of the mean. Letters above the bars indicate statistically significant differences (P< 0.05 by Tukey HSD test).      81 In order to determine if cellulose biosynthesis, in addition to amylovoran biosynthesis, is regulated by c-di-GMP to modulate the activation of biofilm formation, cellulose production was qualitatively evaluated for the Ea1189 WT and Ea1189 strains overexpressing either edcB or edcC, on plates containing Congo Red, a red dye that binds to cellulose leading to a red colony morphotype. Whereas the WT strain exhibited a pink colony, strains overexpressing DGCs developed bright red colonies, indicative of active cellulose production when intracellular levels of c-di-GMP are high (Fig 3.9A). In order to investigate if the activation of cellulose biosynthesis is induced by allosteric binding of c-di-GMP to the cytoplasmic PilZ domain in BcsAEa, a plasmid containing the bcs native promoter plus the bcsA gene with a point mutation of the c-di-GMP binding residues in the PilZ domain (RxxxR to RxxxD) was generated (bcsAR556DbcsA mutant strain with or without overexpression of edcB or edcC, which allowed us to modulate the intracellular c-di-GMP concentration. Although overexpression of DGCs induced high levels of c-di-bcsA background, no activation of cellulose was evidenced for these strains as they developed pink colonies on the CR-amended medium (Fig 3.9B). The red colony morphotype was restored when bcsA was complemented with the native bcs promoter and bcsA gene from a plasmid, and DGCs were overexpressed (Fig 3.9C). Furthermore, when this mutant was complemented with a R556D), the activation of cellulose biosynthesis was abolished even in the presence of high c-di-GMP concentrations (Fig 3.9D). These results suggest that cellulose biosynthesis in E. amylovora is positively regulated by c-di-GMP, and activated by binding of this messenger molecule to the RxxxR residues in the PilZ domain of BcsA.   82 Figure 3.9. Activation of biofilm formation through cellulose biosynthesis by c-di-GMP in E. amylovora Ea1189.  Colony morphology was analyzed on plates containing Congo Red + 1 mM IPTG after 24 h of incubation at 28°C bcsA bcsA + bcsA bcsA + bcsAR556D, overexpressing either edcB or edcC. Red colony morphotype is indicative of active cellulose biosynthesis. (E) Quantification of biofilm formation on glass coverslips for Ea1189 bcsA strains overexpressing either edcB or edcC, after 48 h of incubation at 24°C. Data represents three biological replicates. Error bars indicate standard error of the mean. Letters above the bars indicate statistically significant differences (P< 0.05 by Tukey HSD test).    83  To further examine the role of c-di-GMP and cellulose biosynthesis in the formation of biofilms in vitro, we measured the biofilm formed bcsA in the presence of high c-di-GMP levels due to the overexpression of edcB or edcC. In agreement with the lack of cellulose bcsA strain, even when high c-di-GMP concentrations were induced via overexpression of DGCs in this mutant background (Fig 3.9E). These results confirm that c-di-GMP positively impacts the formation of biofilms, not only through the activation of amylovoran biosynthesis but also through the positive regulation of cellulose biosynthesis in E. amylovora. Interestingly, when edcB is overexpressed in the WT strain, and therefore active cellulose production is induced, the sizes along the fibrillar material in the biofilm matrix was observed with SEM (Fig 3.4D). Because overexpression of edcB activates cellulose production but does not activates the biosynthesis of amylovoran, the additional major component of the biofilm matrix in E. amylovora, we hypothesize that these ornaments on the biofilm microfibers are made of cellulose.   The endoglucanase bcsZ is also required for c-di-GMP regulation of cellulose biosynthesis  We further investigated whether the homologs of bcsD, bcsZ and bcsE in the bcs operon of E. amylovora had an impact in the c-di-GMP regulation of cellulose activation. We studied these genes because bcsD and bcsZ had been reported as required for maximal cellulose biosynthesis in K. xylinus (Saxena et al., 1994, Standal et al., 1994) and because bcsE from E. coli, Salmonella and other enterobacteria was demonstrated to be an additional c-di-GMP receptor modulating the activation of cellulose biosynthesis (Fang et al., 2014). Mutant strains of  84 bcsD, bcsZ, and bcsE were constructed and qualitatively assessed for cellulose production on Congo Red amended medium when high c-di-GMP levels were induced via DGC overexpression. Deletion of bcsD and bcsE did not have any evident effect on cellulose biosynthesis activation evidenced by a bright red colony morphotype when DGCs are overexpressed. However, high intracellular levels of c-di-GMP due to edcB or edcC overexpression in a bcsZ mutant background did not activate cellulose biosynthesis, and these strains exhibited a pink colony morphotype in both cases, similar to the phenotype exhibited by bcsA (Fig 3.10). These data suggest that in addition to BcsA, the endoglucanase BcsZ is involved in the c-di-GMP regulatory pathway to control the production of cellulose. However, bcsZ expression levels did not significantly change when a high c-di-GMP concentration was induced in the WT strain (data not shown), suggesting that c-di-GMP regulation through BcsZ is not carried out at the transcriptional level.   85 Figure 3.10. Implication of other genes in the bcs operon in cellulose biosynthesis activation through c-di-GMP in E. amylovora Ea1189.  Qualitative assessment of cellulose production on plates containing Congo Red + 1 mM IPTG after 24 h of incubation at 28°C for Ea1189 WT, bcsD, bcsZ, bcsE, overexpressing either edcB or edcC. Red colony morphotype is indicative of active cellulose biosynthesis.     86 V. Discussion  The formation of biofilms inside the xylem vessels of leaves is a critical virulence factor and is one of the most important adaptations of E. amylovora to life within the host plant. The biofilm not only enables bacteria to more effectively acquire nutrients from the xylem vessels but also endows the pathogen with a higher resilience to antimicrobial compounds and host defense responses (Koczan et al., 2009).  At early stages of infection, E. amylovora is highly motile and requires the expression of hrp-Type III secretion system genes (hrp-T3SS) to infect apple flowers, after cells gain ingress through the nectaries (Malnoy et al., 2012, Pester et al., 2012). After migration to and establishment within the vascular tissue, E. amylovora forms biofilms inside the xylem that allow for large increases in population size and facilitate systemic spread of the pathogen within host trees (Koczan et al., 2009, Koczan et al., 2011a).  The secondary messenger dinucleotide c-di-GMP plays a key role in the regulation of the transition from motile, planktonic state to the establishment of bacterial aggregates and biofilm formation. Our previous studies indicated that c-di-GMP orchestrates the transition between early and late infection stages in E. amylovora, as it inversely regulates flagella motility and the expression of hrp-T3SS-related genes, and the biosynthesis of the EPS amylovoran and biofilm formation in vitro (Edmunds et al., 2013). In this study, we have shown for the first time 1) that cellulose is produced by E. amylovora, 2) that in addition to the main EPS produced by this pathogen, amylovoran, it constitutes a major component of the biofilms formed in vitro and inside the vasculature of the apple host and 3) that, similar to amylovoran biosynthesis, activation of cellulose production is a process positively regulated by c-di-GMP.  87 Cellulose has been reported as a key component of biofilms in several Gram-negative bacteria including E. coli, Salmonella, Vibrio fisherii, and D. dadantii among others (Zogaj et al., 2001, Darnell et al., 2008, Jahn et al., 2011). More specifically, studies on the structure and complexity of biofilms formed by E. coli have demonstrated that cellulose is the major generator of the particular architecture of a macrocolony and the three-dimensional structure of biofilms (Hung et al., 2013, Serra et al., 2013). In E. coli, -model the extracellular matrix, provide cohesion and elasticity to the biofilm (Serra et al., 2013). Moreover, mutant strains of uropathogenic E. coli deficient in cellulose production exhibited the formation of a fibrous matrix encasing bacterial cells but had a much looser appearance than the extracellular matrix formed by the WT strain, suggesting that cellulose is essential to maintaining the integrity and stability of biofilms in this pathogen (Hung et al., 2013). Our results suggest that cellulose plays a similar role for the biofilms formed by E. amylovora both in vitro and in planta. We determined that abrogation of cellulose biosynthesis led to the formation of a fibrillar matrix exhibiting lower complexity around bacterial cells when we examined biofilms formed at the air-liquid interface with SEM. Moreover, it was not possible to detect any fibrillar material in biofilms formed inside the vasculature of apple plants by cellulose-deficient strains. It is possible that cellulose in the extracellular matrix of biofilms produced by E. amylovora provides the structural cohesiveness and strength necessary to endure the continuous flow of water and nutrients inside the xylem vessel environment. Along the same lines, it is likely that the immature aggregations formed by cellulose production-impaired strains are more easily disturbed by the counter-flowing xylem sap, explaining the delay in symptom migration, following inoculation of E. amylovora on apple shoots.  88  Synthase-dependent production of many exopolysaccharides in gram-negative bacteria share two distinguishing features: 1) the requirement of an inner membrane bound synthase, a periplasmic scaffold subunit and an outer membrane porin, and 2) the post-translational regulation of c-di-GMP by allosteric binding to the synthase subunit (reviewed in (Whitney & Howell, 2013, McNamara et al., 2015, Römling & Galperin, 2015). For this reason, we hypothesized that cellulose production in E. amylovora would share these same features. It is interesting that this process is regulated by c-di-EdcC and EdcE, which also regulate amylovoran biosynthesis at the transcriptional level, and a more specialized DGC such as EdcB, which does not have a regulatory impact in amylovoran production. These observations confirm that the c-di-GMP regulatory network in E. amylovora has several levels of regulation to modulate the correct timing and input of a specific phenotype or cellular process, in order to orchestrate bacterial pathogenesis.  The plant pathogenic enterobacterium D. dadantii, closely related to E. amylovora, produces a conspicuous biofilm matrix composed of cellulose nanofibers decorated with microbeads and nanofibers made of a cellulase-resistant material (Jahn et al., 2011). This fact and the intriguing formation of irregular beads along the microfibers in the biofilm matrix of cellulose overexpression strains of E. amylovora suggests that these ornaments have a cellulosic origin rather than another polymer. However, it also possible that the formation of beads is a result of the overexpression of an additional component of the E. amylovora extracellular matrix when artificial levels of c-di-GMP are induced.  Regarding organization and gene content, the bcs operon in E. amylovora appears to have a high resemblance to the bcs operon in D. dadantii (Jahn et al., 2011), as both of them harbor one of the two clusters involved in cellulose synthesis in bacteria (bcsOQABCDZ  and bcsEFG).  89 However, the presence of only the gene bcsE downstream of the bcsOQABCDZ cluster in both organisms suggests that this gene is a remnant of a bcsEGF cluster present in the Enterobacteriaceae ancestor but lost in many recent clades within the phylogeny of this bacterial group with the exception of the human pathogens such as E.coli, Shigella, and Salmonella (Hauben et al., 1998). It has been demonstrated that the presence of the bcsE gene confers optimal cellulose biosynthesis and additional levels of regulation by c-di-GMP binding since it acts as a second c-di-GMP receptor involved in regulation of cellulose production in Salmonella Typhimurium and Klebsiella pneumoniae (Fang et al., 2014). Interestingly, our results suggest that BcsE from E. amylovora is not involved in the regulation of cellulose biosynthesis as a c-di-GMP receptor, as bright red colonies, indicative of active cellulose production, were obtained when high levels of c-di-GMP were introduced in a bcsE mutant background. Surprisingly, our studies demonstrated that cellulose biosynthesis was abolished in a bcsZ mutant strain even when an elevated c-di-GMP intracellular concentration was induced via DGC overexpression, indicating that BcsZ plays a critical role in the regulation of this cellular process by c-di-GMP. -endo-1,4-glucanase activity that it is thought to carry out degradation of accumulating polymer in the periplasm or has a role in the -glucans before export to assemble the microfibers (Mazur & Zimmer, 2011). Although several studies have demonstrated that the endoglucanase encoded by bcsZ is required for maximal cellulose production in K. xylinum (Standal et al., 1994, Koo et al., 1998, Nakai et al., 2013), to the best of our knowledge, there is no previous evidence of BcsZ being necessary for c-di-GMP regulation of cellulose biosynthesis. Even though we did not detect any changes in the transcriptional regulation of bcsZ at high intracellular c-di-GMP concentrations, regulation of cellulose production through BcsZ at a post-transcriptional level (i.e. acting as a c-di-GMP receptor) needs  90 further investigation. Moreover, an indirect role of BcsZ in the c-di-GMP modulation of cellulose biosynthesis, such as regulation of the bcsA gene or modification of BcsA functionality is also plausible. The results presented in this study provide an overview of cellulose biosynthesis in E. amylovora, a process that has not been previously investigated in this plant pathogenic bacterium. Furthermore, we demonstrated that cellulose biosynthesis is another component of the c-di-GMP regulatory network, which is timely controlled by this pathogen and allows it to successfully transition between infection stages, colonize its plant host, cause disease, and be disseminated to new hosts.    91 Chapter 4: Cyclic di-GMP-dependent regulation of virulence in Erwinia amylovora This chapter is a research in progress    92 I. Abstract The widespread signaling molecule cyclic dimeric GMP (c-di-GMP) exerts regulatory activity over a plethora of cellular processes in bacteria including motility, biofilm formation and virulence among others. In Erwinia amylovora, the causal agent of fire blight disease of rosaceous plants, c-di-GMP has been demonstrated to inversely regulate virulence via repression of hrp-Type III secretion system (hrp-T3SS) genes, and biofilm formation and exopolysaccharide biosynthesis. However, the molecular mechanisms of such regulation remain 54-dependent enhancer binding protein (EBP) HrpS binds to c-di-GMP. This result led us to postulate that HrpS functions as the receptor protein responsible for c-di-GMP-dependent regulation of the Hrp-T3SS. Moreover, the linear nucleotide signaling molecule 5'- phosphoguanylyl- - guanosine (pGpG), which is an intermediate degradation product of c-di-GMP turnover, also appears to have regulatory functions not only over HrpS but also over other hrp regulatory genes, suggesting a multi-tiered control of the expression of pathogenicity factors in E. amylovora.    93 II. Introduction The secondary messenger dinucleotide cyclic di GMP (c-di-GMP) is a nearly ubiquitous signaling molecule in bacteria that has been implicated in the global regulation of many cellular processes including the transition from a motile planktonic lifestyle to multicellular aggregation and biofilm formation, virulence, RNA processing, and cell cycle and differentiation (Römling et al., 2013, Ryan, 2013). The intracellular levels of c-di-GMP are controlled by the catalytic action of diguanylate cyclases (DGCs) containing a GGDEF domain which synthesize c-di-GMP from two molecules of GTP, and phosphodiesterases (PDEs) with either an EAL or HD-GYP domain, which hydrolyze c-di-GMP to pGpG or GMP, respectively (Paul et al., 2004, Schmidt et al., 2005, Ryan et al., 2006a). Many of these proteins contain additional sensory input domains in their N-terminal region suggesting that they are activated after the recognition of cellular or environmental cues (Römling et al., 2013). Changes in intracellular c-di-GMP concentration are then detected by downstream receptor molecules which are then functionally activated or inactivated upon binding to the secondary messenger molecule (Sondermann et al., 2012, Chou & Galperin, 2016).  Although the enzymatic activities of DGCs and PDEs, and their structural characteristics have been extensively studied in multiple bacterial species, the identification of the activating signals and the downstream receptor molecules and the deciphering of the complexity c-di-GMP signaling systems have become major goals for researchers in the field. Compared with the vast amount of described enzymes involved in c-di-GMP turnover, only few types of receptor molecular have been described so far which include bioinformatically predictable proteins containing the PilZ domain, and inactive GGDEF, EAL or HD-GYP containing proteins, and  94 less predictable riboswitches, transcription factors and other proteins with diverse functions (Sudarsan et al., 2008, Lee et al., 2010, Tuckerman et al., 2011, Ryan et al., 2012, Römling et al., 2013, Chou & Galperin, 2016). Interestingly, three c-di-GMP binding transcription factors 54-dependent enhancer binding proteins (EBP): FleQ from Pseudomonas aeruginosa, and VpsR and FlrA from Vibrio cholerae (Hickman & Harwood, 2008, Srivastava et al., 2011, Srivastava et al., 2013)54-54-dependent transcription, as their interaction with upstream activator sequences (UAS) in the target promoter region, followed by hydrolysis of ATP, promotes the remodeling of the RNA polymerase- 54 holoenzyme and the formation of an open complex, necessary to initiate transcription (Bush & Dixon, 2012). The proteins belonging to this family typically contain three domain: a cis-acting regulatory domain located at the N-terminus, a catalytic AAA+ central domain that carries out the ATPase activity and is indispensable for their transcriptional activation activity, and a helix-turn-helix (HTH) DNA-binding domain in the C-terminal region (Studholme & Dixon, 2003).  Our previous studies demonstrated that elevated levels of c-di-GMP in the plant pathogenic bacterium Erwinia amylovora, activate the synthesis of the exopolysaccharides (EPS) amylovoran and cellulose and the formation of biofilms, while having a negative impact in motility and virulence (Edmunds et al., 2013); Castiblanco and Sundin, unpublished). In this pathogen, regulatory and structural genes of the Hrp type III secretion system (Hrp-T3SS), one of the main pathogenicity factors, are encoded in the hrp/dsp gene cluster including hrpL, which encodes the alternative sigma factor responsible for the expression of the structural genes of the Hrp-T3SS component (Wei & Beer, 1995, McNally et al., 2012). In turn, transcription of hrpL  95 54 RpoN and its modulator protein YhbH, the integration host factor 54-dependent EBP transcriptional activator HrpS (Wei et al., 2000, Ancona et al., 2014, Lee & Zhao, 2015). Analyses using a transcriptional fusion of the promoter of hrpA encoding the Hrp-T3SS pilin, and green fluorescent protein (GFP), revealed that the c-di-GMP negative regulation of virulence in E. amylovora is exerted at the transcriptional level, on hrp-T3SS structural genes regulated by HrpL (Edmunds et al., 2013).  However, the specific c-di-GMP receptor molecule modulating the expression of hrp-T3SS genes and virulence in this pathogen remains unknown.   54-depedent EBP family of transcription activators, we hypothesized that HrpS could act as a c-di-GMP binding receptor protein. In consequence, the main goal of this study was to investigate the role of HrpS in the c-di-GMP-dependent expression of hrp-T3SS structural genes and virulence in E. amylovora. Results presented in this work suggest that HrpS binds with strong affinity not only to c-di-GMP but also to the c-di-GMP degradation product pGpG. Moreover, our results indicated that pGpG also exerts a regulatory role on both hrp-T3SS regulatory and structural genes.  III. Materials and Methods Strains, plasmids and growth conditions Bacterial strains and plasmids used in this study are listed in Table 4.1. E. amylovora and E. coli strains were grown in Luria- Bertani (LB) liquid and solid media at 28°C and 37°C, respectively unless otherwise indicated. Growth media was supplemented with kanamycin (Km; 50 µg mL-1), as required.  96 Table 4.1. Bacterial strains and plasmids used in Chapter 4 and their relevant characteristics Strain or plasmid Characteristics a Source or reference Escherichia coli strains    F-80dlacZ M15 (lacZYA-argF)U169 endA1 recA1 hsdR17 (rk-mk+)deoR thi-1 supE44 gyrA96 relA1 - Invitrogen, Carlsbad, CA BL21 DE3 fhuA2 [lon] ompT dcmhsdS  DE3 =  -B int::(lacI::PlacUV5nin5 Novagen, Madison, WI    Erwinia amylovora strain   Ea1189 Wild type (Burse et al., 2004c)    Plasmids   pET-28b Expression vector, N and C-terminal His tag, KmR Novagen, Madison, WI pLFC40 pET-28b:hrpS expression vector, KmR This study pLFC53 pET-28b:hrpSR63H expression vector, KmR This study pEVS143 Broad-host-range cloning vector, KmR (Dunn et al., 2006) pACE-edcA pEVS143:edcA overexpression vector, KmR (Edmunds et al., 2013) pACE-edcC pEVS143:edcC overexpression vector, KmR (Edmunds et al., 2013) pACE-edcE pEVS143:edcE overexpression vector, KmR (Edmunds et al., 2013) pLFC34 pEVS143:edcB overexpression vector, KmR This study pLFC42 pEVS143:edcD overexpression vector, KmR This study (a) KmR indicate resistance to kanamycin.   RNA extraction   For the evaluation of hrp gene expression, 1 mL of bacterial cells grown overnight in LB medium was collected and washed twice in hrp- inducing minimal medium (Hrp-MM) (Huynh et al., 1989) amended with 10% galactose, and resuspended in 2 mL of the same liquid medium amended with 1 mM i-D-1-thiogalactopyranoside (IPTG) and the appropriate antibiotic. After 6 and 18h of incubation at 24°C, 1 mL of RNA protect (Qiagen, Valencia CA) was added to 0.5 mL of induced culture to stabilize cells. RNA was extracted using the miRNeasy Mini kit (Qiagen) with an on column DNAse treatment (Qiagen) during the extraction. The quality of isolated RNA was assessed using a NanoDrop 1000 spectophotometer (NanoDrop Technologies Inc, Wilmington DE).  97  qRT-PCR  cDNA was synthesized from 1 µg of purified RNA using TaqMan® reverse transcription (RT) reagents (Applied Biosystems, Foster City, CA) according to the manufacturer instructions. Quantitative PCR (qPCR) was performed using the SYBR® green master mix (Applied Biosystems) on a CFX96 Real-Time PCR detection system (BioRad laboratories, Hercules CA). Thermal cycling conditions included one initial step of 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min and a final melting curve analysis step. recA was used as endogenous gene control. Gene expression levels were quantitatively analyzed via the relative (Pfaffl et al., 2002).     Protein expression and purification The hrpS gene was amplified from genomic DNA of the E. amylovora wild type (WT) strain Ea1189 using Phusion® high-fidelity polymerase (New England Biolabs, Ipswich, MA) and cloned into pET28-b, generating pLFC40, which was transformed into E. coli strain BL21 DE3. Replacement of the arginine at position 63 to histidine in HrpS was performed using the Quikchange mutagenesis system (Agilent technologies, Santa Clara CA). Briefly, the entire pLFC40 plasmid was amplified using mutagenic primers followed by DpnI digestion. The PCR product was then transformed into strain BL21 DE3. The generated plasmid containing the mutation was sequenced to confirm the introduction of the specific mutation and to rule out frameshifts or other sequence alterations. The resultant mutant construct hrpSR63H:pET-28b eas designated as pLFC53. Strains containing the expression vectors were grown in LB to  98 exponential phase (O.D. = 0.5) and 1 mM IPTG was added to the cultures. After 24 h of induction at 24°C, cells were collected and resuspended in binding buffer (20 mM sodium -mercaptoethanol). Cells were disrupted by sonication, pelleted, and the supernatants were loaded onto a column previously packed with a nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen). Columns were washed with wash buffer (binding buffer pH 6.0), and purified proteins were eluted with elution buffer (wash buffer amended with 50 mM, 100 mM, 200 mM or 300 mM imidazole). Samples were dialyzed using a Slide-A-Lyzer Dialysis cassette (10K cut off; Thermofisher scientific) against binding buffer with 250 mM NaCl, as per manufacturer instructions. Protein concentration was assessed using the Pierce BCA protein assay kit (Thermofisher scientific)  DRaCALA binding assay  DRaCALA binding assays using 32P-c-di-GMP were performed by Geoffrey Severin (Waters Lab, Microbiology and Molecular genetics department, MSU), using a modification of a previously described method (Roelofs et al., 2011). Cultures of the E. coli strains harboring the expression plasmids were grown in LB to the exponential phase, and 1 mM IPTG was added to the cultures. After 4 h of induction at 24°C, cells were collected by centrifugation and resuspended in c-di-GMP binding buffer (100 mM KCl, 5 mM MgCl2, 100 mM TRIS pH 8.0 and 100 µM PMSF). Samples were subjected to three freeze-thaw cycles and stored at -80°C. Forty µL of cells were thawed and incubated with 3 µL of 42 nM 32P-c-di-GMP for 5 min at 24°C. Two µl of the mixture was spotted onto nitrocellulose membranes. After air-drying, membranes were exposed for 15, 20, 25 and 30 min prior to imaging.  For binding assays with -fluo-aminohexylcarbamoyl-c-di--AHC-cdiGMP; Biolog, Germany) 50 µM  99 -AHC-cdiGMP and spotted onto a nitrocellulose membrane, that was scanned for fluorescence using a ChemiDoc MP system (BioRad).  For competition experiments, unlabeled nucleotide signaling molecules were added to the 32P-c-di-GMP-HrpS mix at concentrations ranging from 0.16 µM to 100 µM. After 30 min of incubation at 24°C, 2 µl samples were spotted in nitrocellulose membranes. The amount of radiolabeled ligand bound to the protein was determined using a previously described method (Roelofs et al., 2011) using Image J software (Schneider et al., 2012b). Effectiveness of the competitor was calculated by dividing the fraction bound in the presence of competitor by the fraction bound when no competitor was added to the mix.  Protein alignments and tertiary structure modeling Alignment of the amino acid sequences of NtrC (E. coli), FleQ (Pseudomonas aeruginosa), FlrA (Vibrio cholerae) and HrpS (E. amylovora) was performed using ClustalW. Prediction of the tertiary structure of HrpS was carried out using the protein structure homology-modelling server SWISS-MODEL (Biasini et al., 2014), which identified and used the crystal structure of NtrC1 in homodimeric state from Aquifex aeolicus (4l4u.1) as the best template for HrpS 3D structure modeling based on the amino acid sequence of HrpS. The predicted model was visualized using the Swiss PDB viewer (Guex & Peitsch, 1997).   100 IV. Results C-di-GMP synthesized by four DGCs in E. amylovora negatively regulates the expression of hrp genes at the transcriptional level Our previous studies demonstrated that elevated c-di-GMP levels via DGC gene (edcC and edcE) overexpression resulted in a significant repression in the expression of a transcriptional fusion of the hrpA promoter region and GFP; however, no significant changes in gene expression were detected for a transcriptional fusion of the regulatory gene hrpS promoter at 7 h after induction  (Edmunds et al., 2013). These findings suggested that the c-di-GMP-dependent regulation of Hrp-T3SS takes place downstream of the regulatory cascade involved in the activation of transcription of the alternative sigma factor gene hrpL. In addition, our results indicated that c-di-GMP produced by more than one DGC is involved in the regulation of hrp-T3SS gene expression in E. amylovora.   In order to determine the role of the additional DGCs encoded in the genome of this pathogen (edcA, edcB and edcD) in the regulation of virulence through the transcriptional regulation of hrp genes, expression levels of hrpL were evaluated in the WT strain Ea1189 and strains overexpressing individuals DGCs.  Overexpression of all of the DGC genes except edcA, resulted in a significant reduction in the transcript levels of hrpL at 6 and 18 h after induction on IPTG-amended hrpMM (Fig 4.1). Moreover a noticeably stronger repression of hrpL was evidenced upon overexpression of edcD at the two studied time points compared with the other DGC genes, suggesting that c-di-GMP produced by this DGC has a stronger effect on the regulation of virulence by repression of hrp genes. Therefore, we selected this gene for subsequent analyses on hrp-T3SS-related gene expression.  101 Figure 4.1. Expression of hrpL in E. amylovora Ea1189 overexpressing individual DGCs.  Quantitation in gene expression after DGC overexpression at 6 h and 18 h of induction in HrpMM was determined using RT-qPCR analyses. Expression data were normalized to the recA endogenous gene control. Results represent three different experiments and error bars denote standard deviation of the mean. Symbols above the bars indicate statistically significant t test (P < 0.05)    Insight into HrpS tertiary structure hrpS 54-dependent EBP family of transcriptional activators (Wei et al., 2000). Interestingly, it lacks the cis-acting regulatory domain at the N-terminus of the protein, typically found in other members of this protein family (Fig 4.2A). Analysis of the protein sequence and an amino acid alignment with other 54-dependent EBPs revealed the HrpS harbors canonical Walker A and Walker B motifs, involved in the ATP binding and hydrolysis, respectively (Bose et al., 2008, Bush & Dixon, 2012), along with the 54-interacting GAFTGA motif in the AAA+ central domain (Fig 4.2B). In addition, one of the arginine residues involved in c-di-GMP binding in the c-di-GMP receptors FlrA and FleQ (Srivastava et al., 2013, Matsuyama et al., 2016), is also conserved in HrpS. In order to determine other candidate c-di-GMP binding sites or residues in HrpS, the 111.190.910.80*0.66*0.77*0.61*0.42*0.11*0.77*0.35*00.20.40.60.811.21.41.66h18hhrpL relative gene expression (fold)Ea1189Ea1189 + edcA OEEa1189 + edcB OEEa1189 + edcC OEEa1189 + edcD OEEa1189 + edcE OE 102 tertiary structure of the protein was modeled based on the three-dimensional structure of the founder protein NtrC from A. eaolicus (Fig 4.2C). Based on evidence of the arginine residue preference for c-di-GMP binding in other receptor proteins and our modeling analysis, R63 in HrpS was selected as a candidate c-di-GMP binding residue, since this residue aligns with binding residues R137 in FlrA and R185 in FleQ, is similarly located downstream of the Walker A motif, and flanks a pocket located in the interphase of two HrpS monomers that can act as a di-nucleotide binding site.   103 Figure 4.2. Structure of HrpS.  (A) Schematic representation of HrpS protein domains. (B) ClustalW alignment of AAA+ domain amino acid sequences of members of the 54-dependent EBP family NtrC (E. coli), HrpS (E. amylovora), FleQ (P. aeruginosa) and FlrA (V. chloreae). Red boxes indicate important motifs for AAA+ domain activity. Red arrow indicates the conserved arginine implicated in c-di-GMP binding. (C) Homology-based model of HrpS. Two monomers (light and dark green) are depicted in a dimeric state based on the crystal structure of NtrC from A. aeolicus. Candidate c-di-GMP binding residue R63 is highlighted in red. A.    B.   C.          104 C-di-GMP binds to HrpS In collaboration with Dr. Chris Waters and Geoffrey Severin (Microbiology and Molecular Genetics department, MSU), the binding of c-di-GMP to HrpS was studied using the DRaCALA binding technique (Roelofs et al., 2011). This method takes advantage of the property of nitrocellulose membranes to retain protein and protein-ligand complexes and separate them from free ligands through solvent capillary force. Cell lysates of E. coli BL21 DE3 expressing HrpS-6xHis or a HrpSR63H-6xHis tagged version of the protein were assayed for c-di-GMP binding by DRaCALA using 32P-c-di-GMP as ligand. The same strain expressing the c-di-GMP receptor VpsR from V. cholerae was used as positive control. Positive signals were obtained for both VpsR and HrpS (Fig 4.3A). In addition, a weaker signal was obtained for cell lysates of the HrpSR63H-6xHis expressing strain, supporting the hypothesis that R63 might play an important role in binding to c-di-GMP. In addition to studies with cell lysates, purified HrpS-6xHis protein was assayed -fluo-aminohexylcarbamoyl-c-di--AHC-cdiGMP), a fluorescent analog of the secondary messenger by DRaCALA. Although binding to purified -AHC-cdiGMP-DRaCALA assay (Fig 4.3B). Taken together these results strongly support the hypothesis of HrpS acting as a receptor molecule of c-di-GMP in E. amylovora.   105 Figure 4.3. C-di-GMP-HrpS binding assays.  (A) DRaCALA binding assay using 32P-c-di-GMP and cell lysates of E. coli BL21 DE3 overexpressing HrpS-6xHis and HrpSR63H-6xHis. E. coli expressing the empty vector was used as negative control. VpsR-6xHis was used as a positive control. (B) DRaCALA binding assay -AHC-cdiGMP (4 nM). (C) Evaluation of c-di-GMP-HrpS binding specificity using purified HrpS and 32P-c-di-GMP in the presence of 100 µM GTP or c-di-GMP (unlabeled). (D) Competition effectiveness was calculated for unlabeled c-di-GMP, GTP, GMP and pGpG at concentrations ranging from 160 nM to 100 µM. Error bars denote standard deviation of the mean. A.  B.      C.        106  D.    A purified preparation of HrpS, provided by our collaborators at Rutgers University (Dr. Matthew Neiditch and Atul Khataokar), interested in solving the X-ray crystal structure of HrpS and the HrpS-c-di-GMP protein complex, was similarly examined for binding to 32P-c-di-GMP. In addition, the ability of unlabeled c-di-GMP and GTP to outcompete 32P-c-di-GMP, was evaluated to determine binding specificity. A strong binding signal was obtained after addition of 32P-c-di-GMP. Interestingly, both unlabeled c-di-GMP and GTP provided at an excess concentration of 100 µM were able to outcompete 32P-c-di-GMP, suggesting that HrpS might also bind to other nucleotide messenger molecules (Fig 4.3C). To further investigate this possibility, additional competition experiments were performed using other nucleotide signaling  107 molecules, and the effectiveness of competitor was determined by quantifying the 32P-c-di-GMP fraction bound in the presence of competitor, normalized by maximum potential 32P-c-di-GMP fraction bound in the absence of competitor (referred as Percentage of no competed fraction). While unlabeled c-di-GMP, GTP and GMP equally outcompeted 32P-c-di-GMP, unlabeled pGpG displaced the radiolabeled c-di-GMP even at the lowest assayed concentration (160 nM; Fig 4.3D). These results suggest that pGpG, the degradation product of c-di-GMP by EAL domain-containing PDEs, binds with high affinity to HrpS.  pGpG is involved in the regulation of hrp genes  In order to investigate if pGpG exhibits regulatory activity over hrp genes, the expression levels of both hrp-T3SS structural and regulatory genes were evaluated in the WT strain Ea1189 overexpressing the oligoribonuclease (orn) gene, which encodes the primary PDE responsible for the final step of pGpG degradation in the c-di-GMP degradation pathway (Cohen et al., 2015, Orr et al., 2015). Ea1189 overexpressing edcD was used as control. At both 6 and 18 h after induction a significant reduction in transcript levels of hrpL and the effector genes dspE and hrpN was evidenced for both overexpressing strains (Fig 4.4). Interestingly, at the two evaluated time points we detected a significantly lower transcripts levels of the regulatory genes hrpS, hrpX, and hrpY when orn was overexpressed compared to the WT control, with a stronger repression pattern evidenced at 18 h after induction. Similarly, overexpression of the DGC gene edcD induced a significant reduction in the expression levels of the all genes involved in hrpL 54 factor gene rpoN at 18h. These results indicated that in addition to binding to HrpS, c-di-GMP and its degradation product pGpG exert an antagonistic regulatory control of gene expression upstream of hrpL in the pathway for hrp-T3SS expression. 108   Figure 4.4. Expression of hrpL, dspE, hrpN, hrpS, hrpX, hrpY and rpoN in WT strain Ea1189 overexpression the oligoribonuclease (orn) gene and edcD at 6 and 18 h of induction in HrpMM.  Expression data were normalized to the recA endogenous gene control. Results represent four different experiments and error bars denote standard deviation of the mean. Symbols above the bars indicate statistically significant differences from the WT t test (P < 0.05)  109 V. Discussion  The Hrp-T3SS represents one of the main pathogenicity factors of E. amylovora, as mutant strains lacking the hrp/dsp gene cluster are characterized for their inability to infect and multiply within host cells and cause disease symptoms (Steinberger & Beer, 1988, Oh & Beer, 2005a). This system is responsible for the translocation of at least five effector proteins from the bacterial cell to the host plant cytoplasm (Oh & Beer, 2005a), where, similar to other pathogen effector proteins, they are thought to suppress the plant immunity signaling triggered by the recognition of pathogen-associated molecular patterns (PAMPs) (Jones & Dangl, 2006). Expression of the hrp-T3SS and its effector proteins has been determined to occur within the first 48 h after inoculation of flowers stigmas, which in natural infections, is the main entry point of the pathogen to the host (Pester et al., 2012). Once E. amylovora reaches the vascular tissue, the establishment of cellular aggregates and biofilm formation in the xylem promote the systemic spread of the pathogen throughout the plant (Koczan et al., 2009, Koczan et al., 2011a). Thus, a fine-tuned regulation of the expression of hrp-T3SS at the temporal level (i.e. high levels at early infection stages vs. decreased expression at late infection stages) is critical for successful host colonization and disease development.   Two independent pathways regulating the expression of the alternative sigma factor gene hrpL, the master regulator of structural hrp-T3SS gene expression, have been described to be a54-54), its modulator protein YhbH and the IHF (Fig 4.5) (Wei et al., 2000, Ancona et al., 2014, Lee & Zhao, 2015). In addition, our previous studies demonstrated that the secondary messenger molecule c-di-GMP to inversely regulate virulence  110 through the repression of hrp-T3SS structural genes, the activation of EPS biosynthesis and biofilm formation (Edmunds et al., 2013). This observations led us to hypothesize that the regulatory c-di-GMP signaling system is responsible for the tight and specific temporal switch in gene expression during the course of host infection. The results presented in this study indicated that c-di-GMP represses the expression of hrp genes by two different mechanisms: i) by binding 54-dependent EBP activator HrpS, abrogating the transcription of hrpL by RpoN, and ii) by negatively regulating the expression of other genes encoding regulatory proteins implicated in the activation of hrpL transcription.  54-dependent EBP family members possess a cis acting regulatory domain at the N terminal region of the protein that recognizes environmental signals and consequently regulates the activity of the AAA+ central domain (Schumacher et al., 2006).  HrpS from E. amylovora lacks the N terminal regulatory portion, similar to HrpS and HrpR from the plant pathogen Pseudomonas syringae (Hutcheson et al., 2001). However in the case of these two EBPs that bind to form a heterohexamer to control the expression of hrpL, their activity is regulated by protein-protein interactions with control proteins HrpV and HrpG (Jovanovic et al., 2011). On the other hand, regulation of HrpS at the transcriptional level by the TCST system HrpX/HrpY is exhibited in the closely related plant pathogens Pantoea stewartii and Dickeya dadantii (Merighi et al., 2003, Yap et al., 2005). However, regulation of HrpS from E. amylovora has been demonstrated to be independent of HrpX/HrpY (Wei et al., 2000, Zhao et al., 2009c), indicating the existence of an unknown molecular mechanism for the regulation of this protein activity. The data obtained in this study suggests that c-di-GMP binds downstream of the Walker A motif via the R63 residue in the AAA+ central domain of HrpS from E. amylovora  111 to exert regulatory control over its transcriptional target hrpL. C-di-GMP binding to HrpS could sterically hinder ATP binding and hydrolysis by obstructing the active sites, destabilizing the HrpS homohexamer as observed for FleQ from P. aeruginosa (Matsuyama et al., 2016) and thus preventing its binding to the UAS upstream of the hrpL promoter, as has been demonstrated for FlrA and its target promoter flrBC (Srivastava et al., 2013). However, the specific mechanisms of c-di-GMP dependent control of hrp genes in E. amylovora deserve further investigation.  One of the most interesting results arising from our studies is the possible role of the c-di-GMP intermediate degradation product pGpG as a signaling molecule per se, in the regulation of hrp-T3SS expression. Stelitano and collaborators (2013) determined that pGpG binds with higher affinity than c-di-GMP to PA4781, a HD-GYP domain-containing PDE from P. aeruginosa, suggesting that in that case pGpG could act a signaling molecule alternative to c-di-GMP. Our binding assays indicate that pGpG readily displaces c-di-GMP bound to HrpS which led us to hypothesize an actual role as a signal for this linear molecule in the activation of hrp-T3SS expression. After infection of flowers or leaves, and migration to the host vasculature, E. amylovora cells aggregate, forming biofilms inside the xylem vessel. Moreover, it has been demonstrated that lux-expressing E. amylovora cells forming biofilms inside the xylem burst out to the surrounding parenchymal tissue (Bogs et al., 1998) where expression of the hrp-T3SS is again needed in order to colonize these living cells and spread within the host plant. Based on our results, we can propose a model where high c-di-GMP intracellular concentration in E. amylovora cells established in biofilms inside the host vascular tissue reaches a threshold which leads to the accumulation of pGpG. This molecule in turn displaces c-di-GMP from its binding site in HrpS, allowing the proper formation and stabilization of the HrpS homohexamer and  112 binding to the hrpL promoter. In addition pGpG appears to also have a role in the transcriptional activation of hrp regulatory genes, including hrpS and hrpX/hrpY suggesting that high intracellular levels of this molecule may be one of the main signals for hrp-T3SS expression (Fig 4.5). Figure 4.5. Working model for c-di-GMP-dependent regulation of hrp-T3SS gene expression. C-di-GMP synthesized by GGDEF containing DGC, binds to HrpS abrogating the binding of HrpS to hrpL promoter sequences and subsequent transcription. C-di-GMP is degraded by EAL-domain containing PDEs thereby generating the intermediate molecule pGpG. When pGpG levels reach a threshold, it displaces c-di-GMP bound to HrpS, and activates the expression of hrp regulatory genes (hrpXY, hrpS and rpoN) to activate the transcription of hrp-T3SS structural genes through activation of hrpL transcription.      113 This study presents evidence for the first time of nucleotide-signaling regulation of virulence through transcriptional activation and repression of hrpL by HrpS, and consequently repression of hrp-T3SS structural genes. Our results suggest that orchestration of the expression of pathogenicity factors in E. amylovora requires the action of at least two nucleotide signaling molecules, c-di-GMP and pGpG, and involves a multi-tiered regulatory system that tightly and specifically controls the transcription of multiple targets required for the assembly of the Hrp-T3SS and the translocation of effector proteins.   114  Chapter 5: Conclusions and future directions     115 I. Summary of work C-di-GMP regulation is a powerful and efficient mechanism that provides pathogenic bacteria with the ability to rapidly reprogram the expression of diverse phenotypic traits necessary to adapt to the different environmental conditions or pathogenicity status requirements during the course of host invasion, population establishment, and posterior dissemination to new hosts. The efforts of almost 30 years to decipher the complexity of regulatory systems involving nucleotide signaling molecules has provided the scientific community with a comprehensive overview of the generalities and specificities of these signaling systems. C-di-GMP dependent internal or external signal and the subsequent activation of the enzymatic action of DGCs and PDEs, the binding of c-di-GMP to the appropriate receptor molecule (protein or RNA), and the specific regulation of the target component at transcriptional, post-transcriptional, or post-translation levels.   My doctoral research has focused on the study of c-di-GMP-dependent regulation in the plant pathogen Erwinia amylovora, the causal agent of the economically important fire blight disease of rosaceous plants. Previous research in this pathogen has focused on understanding the molecular biology of the bacterium, mainly focused on the identification of pathogenicity and virulence factors encoded in the genome of E. amylovora that are critical for symptom production and disease development. These include, the Hrp-T3SS, the suite of effector proteins translocated to the host cytoplasm and their cognate chaperone partners, the exopolysaccharides amylovoran and levan, and the formation of biofilms inside the vasculature of the host. My dissertation research investigates for the first time the role of a nucleotide secondary messenger  116 in the timely regulation of these virulence and pathogenicity factors. The initial goal for this research was to explore the genome of E. amylovora for genes encoding proteins associated with c-di-GMP metabolism and to determine the importance of this signaling molecule on genetic regulation of pathogenicity and virulence factors. Based on previous research in other related enterobacteria, I hypothesized that c-di-GMP was actively produced by E. amylovora, and was employed by this pathogen to regulate the expression of the different pathogenicity and virulence factors, already described. My research provides a first overview of c-di-GMP-dependent regulation in E. amylovora and uncovers some of the critical components of this regulatory system for the control of biofilm formation and virulence via hrp-T3SS gene expression.   Chapter 2 describes the existence of eight genes encoding proteins harboring c-di-GMP-metabolizing domains: three phosphodiesterases (PDE) with an EAL domain (epdA, epdB and epdC), four diguanylate cyclases with GGDEF domains (edcB, edcC, edcD and edcE), and one protein containing both GGDEF and EAL domains (edcA). For further experiments in this chapter and in my dissertation in general, I focused on the characterization of the GGDEF containing enzymes as a first approach to study the c-di-GMP-dependent regulatory system in E. amylovora. Although basal levels of c-di-GMP in the wild type strain Ea1189 are very low, overexpression studies revealed that all of the GGDEF-domain harboring enzymes are able to actively synthesize c-di-GMP. This indication prompted me to study four different phenotypes considered to be crucial for E. amylovora pathogenesis: flagellar motility, virulence in the infection models studied in our laboratory (immature pear fruits and apple shoots), biosynthesis of amylovoran and formation on biofilms. The results strongly suggested that the regulation of the transition between motility and sessility in E. amylovora fits the inverse regulatory pattern  117 previously described for many bacterial species: high c-di-GMP intracellular levels activates in vitro biofilm formation and amylovoran biosynthesis while repressing swarming motility and vice versa. Moreover, virulence was determined to be repressed as well, by elevated c-di-GMP conditions. This result was surprising, as previous research conducted in our lab demonstrated that strains lacking the ability to form biofilms (i.e. ams operon mutant strain) are usually avirulent due to the inability to move systemically through the vascular tissue. However, our results were also the first indication of c-di-GMP as a master regulator of differential temporal expression of pathogenicity and virulence factors. The results presented in chapter 2 also provided the first clues on regulation at the transcriptional level for genes related to hrp-T3SS expression and amylovoran biosynthesis.  After studying the enzymatic activity of c-di-GMP catabolic enzymes and the effect of this secondary molecule on different phenotypic outputs, I decided to explore the existence and function of c-di-GMP receptor proteins potentially exerting the regulatory roles over said outputs after binding to the secondary messenger. In chapter 3, I addressed the role of the cellulose catalytic subunit BcsA in the regulation of biofilm formation through c-di-GMP-dependent cellulose biosynthesis. This protein is a homolog of BcsA from Komagataeibacter xylinum, and it was earlier described as specifically binding to c-di-GMP through its PilZ domain. This domain can be easily predicted by bioinformatics analyses; as a result, proteins containing a PilZ domain are the best described type of c-di-GMP binding receptors.  BcsA is part of a multi-protein complex required for the biosynthesis and export of the exopolysaccharide cellulose in bacterial systems. As demonstrated in other bacterial species, c-di-GMP binds allosterically to the PilZ domain in BcsA which promotes a conformational change in the protein, resulting in the post-translational activation of cellulose biosynthesis.  118 Moreover, cellulose has been described as a critical component of biofilms produced by several bacterial species. Since neither the role of cellulose in the pathogenesis of E. amylovora, nor the effect of c-di-GMP on the regulation of this cellular process have been studied before, I decided to investigate, if similar to other bacterial systems, c-di-GMP exerted post-transcriptional regulation of cellulose biosynthesis in E. amylovora, and how this process is involved in the successful formation of biofilms inside the vascular system of the host. Analyses using a gene knockout mutant of bcsA revealed that cellulose is produced by E. amylovora, and is a critical component of biofilms assembled by this phytopathogen in vitro and inside the xylem of apple leaves. In planta studies of biofilms formed by strains lacking the ability to produce cellulose indicate that this EPS is required for the appropriate assembly of three-dimensional structures to establish a stable biofilm inside the xylem that allows bacteria to persist and migrate down the host vasculature. Moreover, my results suggest that the defect on the biofilm architecture represents a disadvantage for the pathogen, as reduced virulence was detected in bcsA mutant strain. In addition, studies using DGC-overexpression strains and point mutations of the critical residues for c-di-GMP binding in the PilZ domain of BcsA strongly suggest that in this pathogen, c-di-GMP also activates cellulose biosynthesis at a post transcriptional level.   Given the evidence of c-di-GMP-dependent transcriptional regulation of virulence through transcriptional repression of hrp genes presented in chapter 2, I decided to investigate the molecular mechanism for such regulation by identifying potential c-di-GMP receptors involved in the transcriptional activation (or repression) of hrpL, which encodes the master regulator of hrp-54-dependent enhancer binding protein (EBP), required for the activation of hrpL transcription, appeared to be a good candidate since several members of this family of proteins have been described as c-di-GMP  119 receptors. Initial results from experiments addressing c-di-GMP binding to HrpS suggest that this EBP actually binds to c-di-GMP. Intriguingly, assays aimed to determine the specificity of this binding, using other signaling nucleotides as competitors indicated the possibility of HrpS binding to other guanosine-containing molecules. Interestingly, the linear nucleotide pGpG appears to displace c-di-GMP bound to HrpS, even at concentrations as low as 160 nM, suggesting that pGpG might actually be a ligand for HrpS. Although this hypothesis requires further exploration, the possible appearance of pGpG in the picture of virulence regulation, opens new avenues for obtaining a comprehensive mechanistic insight of the control of virulence at a temporal level. In fact, results presented in Chapter 4, indicate that c-di-GMP and pGpG have antagonistic roles in the transcriptional activation of not only structural hrp-T3SS genes through hrpL but also through hrpS itself and other regulatory genes acting upstream of hrpL. The results presented in this doctoral dissertation provide evidence of the significance and importance of c-di-GMP for the global regulation of key pathogenicity-related phenotypes, as well as the implications of different components of this signaling network in the orchestration of pathogenicity, survival within the host and ultimate spread of the pathogen to other host plants.    120 II. Future directions  The work presented in this dissertation identified several key components of the c-di-GMP regulatory network that governs the expression of virulence and pathogenicity factors in E. amylovora. However, many questions that deserve further exploration arose from these studies. In Chapter 2, five proteins containing a GGDEF domain were characterized to be enzymatically active in the biosynthesis of c-di-GMP; however one of them, EdcA, which also harbors an EAL domain was not implicated in the regulation on any of the phenotypic outputs evaluated under the studied conditions, including cellulose biosynthesis in Chapter 3. As mentioned in Chapter 2 this could be due to an antagonistic PDE activity of the EAL domain that masks the DGC activity. Alternatively, c-di-GMP synthesized by EdcA can be involved in the regulation of cellular processes that were not studied in this dissertation research, but are yet important for the survival and/or pathogenicity of E. amylovora. To elucidate these questions, further investigation on the PDE activity of the EAL domain of EdcA needs to be carried out as well as the identification of genes associated target processes using an edcA mutant strain for high throughput transcriptional profiling.  A central theme that emerged from the work presented in this dissertation is the redundancy in regulatory activity of c-di-GMP synthesized by different DGCs. With the exception of EdcA, the c-di-GMP-synthesizing activity of all other DCGs encoded in the genome of E. amylovora appears to similarly regulate all the phenotypic outputs studied in this project, with some degree of variation. This finding suggests that E. amylovora produces a general c-di-GMP pool to ensure the appropriate expression of pathogenicity factors during the course of infection and highlights the importance of these phenotypes for survival and pathogenesis. However, it cannot be ruled out that artificial levels of c-di-GMP biosynthesis induced by DGC  121 overexpression is masking c-di-GMP specificities for selected phenotypes. It is also possible that the EAL-containing enzymes encoded in the genome of E. amylovora target the global c-di-GMP pool to exert regulation on specific phenotypic outputs. Studies with gene knockout mutants of edc and epd genes, and complemented strains could help to differentiate between globally and specifically regulated processes. Cellulose biosynthesis and its regulation by c-di-GMP in E. amylovora is a newly studied area that provides new avenues for investigating the colonization, persistence, and systemic movement of the bacterial pathogen inside the vascular tissue of host plants. An important question arising from the results presented in Chapter 3 is how E. amylovora orchestrates the expression of at least three EPSs (amylovoran, levan and cellulose), which are required for the formation of a stable biofilm inside the host. Evaluation of a transcriptional fusion of amsG with lux determined that c-di-GMP positively regulates the biosynthesis of amylovoran at the transcriptional level. However, the specific mechanisms and receptor molecules involved in such regulation remain elusive. In addition, it is still unknown if c-di-GMP-dependent signaling also regulates the production of levan. It would be also interesting to study the individual contribution of each one of the EPSs for biofilm formation and if there is a temporal requirement for their expression. In the last chapter of my dissertation, I focused on studying the regulation of virulence through Hrp-T3SS regulation. The data presented in this chapter indicate that repression and activation of hrp gene expression is a tightly and specifically controlled process that requires the activation of a complex global regulatory system involving the action of many key components. Although c-di-GMP appears to bind to HrpS in the DRaCALA assays, determination of actual binding with purified HrpS and other binding assays would confirm that this nucleotide molecule  122 is a HrpS ligand. Moreover, the apparent displacement of c-di-GMP by other guanosine-containing nucleotides, especially the c-di-GMP degradation product pGpG, opens the avenue for a whole new set of questions about the efficient reprogramming of HrpS activity and subsequent expression of hrp genes. Much more work needs to be done to determine if and how the signaling nucleotides interacting with HrpS affect its conformation and binding to the upstream regulatory sequence in the hrpL promoter. Concentration quantification of the signaling molecules in the wild type strain would provide a first indication of the basal levels of these molecules. In addition, genetic analysis (gene expression) and phenotypic studies (pathogenicity in infection models) need to be performed in order to determine the implications of these signaling molecules in the regulation of virulence.    123           APPENDIX  124 Regulation of effector translocation by type III secretion chaperone proteins and HrpN in Erwinia amylovora I. Abstract Translocation of effector proteins into the host cytoplasm through the type III secretion system (T3SS) is a critical pathogenicity determinant of Gram-negative bacterial pathogens. Moreover, the successful delivery of many effector proteins depends on the association with type III secretion (T3S) chaperones and other regulatory proteins in the bacterial cytoplasm prior to their export to the plant cell in order to establish a secretion hierarchy and enhance the translocation process. Erwinia amylovora, secretes at least four effector proteins: DspE, Eop1, Eop3 and Eop4. DspE specifically interacts with the T3S chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the Hrp-T3SS. In addition, the harpin protein HrpN has been demonstrated to be required for efficient translocation of DspE. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the T3S chaperones DspF, Esc1 and Esc3 and the harpin protein HrpN in yeast. Using site-directed mutagenesis, secretion and translocation assays, we demonstrated that T3S chaperones have additive roles for the translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Additionally, we demonstrated that HrpN is required for the efficient translocation of effector proteins other than DspE suggesting the possibility that HrpN might work as a translocator protein. Collectively, these results indicate that T3S chaperone proteins and HrpN exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora. 125 II. Introduction The delivery of effector proteins via the type III secretion system (T3SS) is a critical process for host-pathogen interactions in Gram-negative bacterial pathogens, since it enables the manipulation of the host cellular machinery and activities that benefit pathogen virulence (Büttner & He, 2009). Frequently, the translocation of these proteins depends on a secretion signal localized in the N-the effector mRNA (Miao & Miller, 2000, Mudgett et al., 2000, Blaylock et al., 2008). Some conserved patterns in the signal region, such as amino acid frequency, G+C content and secondary structure are often used in computational approaches for in silico prediction and identification of type III secreted substrates (Arnold et al., 2009, Samudrala et al., 2009, Schechter et al., 2012). The translocation efficiency of many effector proteins also depends on a physical association with cytoplasmic type III secretion (T3S) chaperone proteins (Luo et al., 2001, Matsumoto & Young, 2009, Triplett et al., 2009a). T3S chaperones are low molecular weight acidic proteins (pI 4-5) which form dimeric structures that bind to one or multiple effectors. Despite exhibiting low overall amino acid residue similarity, T3S chaperones share a conserved three-dimensional structure and similar modes of interaction with their effector partners (Ghosh, 2004, Cornelis, 2006). Genes of T3S chaperones are often localized adjacent to their associated effector, suggesting a co-regulation at the transcriptional and translational level. For example, translational coupled expression of the effector protein SptP and its chaperone SicP has been demonstrated in Salmonella Typhimurium (Button & Galán, 2011), indicating that bacteria finely regulate the expression of chaperone/effector complexes at both transcriptional and translational levels to ensure high levels of effector translocation when the T3SS is assembled.  126 T3S chaperones may also interact with non-secreted proteins, such as transcription factors, in order to upregulate the expression of effector genes and facilitate the global regulation of T3S (Darwin & Miller, 2001). In addition, T3S chaperones have been demonstrated to interact with ATPases associated with the T3SS. This interaction induces the docking, unfolding and release of the effector protein to the secretion system (Akeda & Galán, 2005, Cooper et al., 2010). In plant pathogenic bacteria, only few T3S chaperone proteins have been characterized, and these proteins share a highly conserved tertiary structure with characterized chaperones from human pathogenic bacteria. These similarities have been used to model the three-dimensional structure of chaperone proteins from plant pathogens, as well as to identify critical residues in the interaction with their cognate effectors (Triplett et al., 2010b). In addition to enhancing the translocation of effector proteins through the T3SS, chaperones from plant pathogens have been demonstrated to exhibit regulatory functions. In Pseudomonas syringae, several T3S chaperone proteins function in protecting effectors from degradation by the Lon protease in permissive conditions for T3S (Losada & Hutcheson, 2005). Furthermore, the T3S chaperone HpaB from Xanthomonas campestris has been shown to be involved in the establishment of a secretion hierarchy that allows the secretion of T3SS components prior to the mobilization of effector proteins through this system (Lorenz et al., 2008). Additionally, HpaB interacts with the T3S ATPase HrcN which promotes the dissociation of the chaperone/effector complex and posterior translocation of the effector protein (Lorenz & Buttner, 2009). Erwinia amylovora, the causal agent of fire blight disease of rosaceous plants, secretes at least four effector proteins in vitro under Hrp-T3SS-inducing conditions: DspA/E (DspE henceforth), Eop1, AvrRpt2Ea/Eop4 (Eop4 henceforth) and Eop3 (Nissinen et al., 2007a). Among these, only DspE is required for pathogenicity, multiplication in planta, and for disease  127 promotion by the alteration of host defenses, as it induces cell death in both host and non-host plants (Gaudriault et al., 1997a, Boureau et al., 2006a). DspE is a large protein (198 kDa) that interacts with at least four proteins from apple (Meng et al., 2006a); however, the functional role of DspE in bacterial pathogenesis remains unknown. DspE specifically interacts with the T3S chaperone protein DspF, which stabilizes the effector protein in the cytoplasm preventing intracellular degradation, and promotes its efficient translocation through the Hrp-T3SS (Gaudriault et al., 2002a). However, a dspF mutant does not lack pathogenic ability, but exhibits reduced virulence and is still able to translocate the N terminal region of DspE (Triplett et al., 2009a, Oh et al., 2010), suggesting that other proteins may be involved in the secretion of this effector protein in the absence of or in addition to its cognate T3S chaperone. Indeed, recent studies indicate that the harpin protein HrpN is also required for the efficient translocation of DspE into plant cells (Bocsanczy et al., 2008c). HrpN was first characterized as a cell-free elicitor of the hypersensitive response (HR) in plants when inoculated in the intracellular spaces of tobacco leaves (Wei et al., 1992) suggesting that it might have a translocator role in T3S. The effector protein Eop1, a member of the YopJ family of proteins, is also translocated by the Hrp-T3SS. Likewise, the eop1 gene is located adjacent to a T3S chaperone gene, named orfA (Oh & Beer, 2005b). It has been demonstrated that this T3S chaperone interacts not only with Eop1 but also with DspE in yeast (Asselin et al., 2006a), suggesting that T3S chaperones in E. amylovora may be involved in the translocation of non-partner effectors. However, there are no reports concerning the roles of proteins other than DspF and HrpN in the regulation of effector translocation in this plant pathogen. We hypothesized that DspF and HrpN act cooperatively to modulate the translocation of DspE and possibly other effector proteins in E. amylovora. We also hypothesized that putative  128 chaperones of Eop1 and Eop3 were involved in mediating the translocation of their cognate effectors and DspE. In the present study, we identified functional interactions between effector proteins DspE, Eop1, and Eop3 with the T3S chaperones DspF, Esc1 and Esc3 and the harpin protein HrpN. Additionally, using site-directed mutagenesis and translocation assays, we determine how these proteins play a functional role in regulating effector translocation dynamics.  III. Materials and Methods Bacterial strains, plasmids, growth conditions, and genetic techniques.  The bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were growth at 28°C in Luria-Bertani (LB) broth and agar unless otherwise noticed. Media were amended with ampicillin (Amp; 50 mg L-1), chloramphenicol (Cm; 10 mg L-1), gentamicin (Gm; 10 mg L-1) or kanamycin (Km; 25 mg L-1) as necessary. PCR, restriction digestions, gene cloning and gel electrophoresis were performed according to standard methods (Sambrook et al., 2001).  Mutant construction.  Chromosomal mutants were constructed using an adaptatipreviously described (Datsenko & Wanner, 2000, Zhao et al., 2005a, Triplett et al., 2009a). Briefly, Cm and Km resistance cassettes were amplified from template plasmids pKD3 and pKD4, using primers with 50 bp overhangs, homologous with the gene of interest. PCR products were purified and electroporated into E. amylovora strain Ea1189 expressing the genes of redexo recombinases from the pKD46 plasmid. Resultant colonies were screened for antibiotic resistance, and gene disruption was verified by PCR and sequencing.  In order to create dspF/esc3 and  129 dspF/esc1/esc3 strains by repetitive growth cycles without antibiotic selection and including heat shock at 37°C. Cured strains were transformed with pCP20 in order to resolve antibiotic resistances by the thermo-inducible resolvase encoded in this plasmid. Transformants were tested for Amp, Cm and Km sensitivity prior to initiating the next round of mutagenesis.  Table A.1. Bacterial strains and plasmids used in Appendix A. Strain or plasmid Characteristics a Source or reference Escherichia coli strain    F-80dlacZ M15 (lacZYA-argF)U169 endA1 recA1 hsdR17 (rk-mk+)deoR thi-1 supE44 gyrA96 relA1 - Invitrogen, Carlsbad, CA, USA    Erwinia amylovora strains   Ea1189 Wild type (Burse et al., 2004b) dspF dspF deletion mutant, KmR (Triplett et al., 2009a) Ea1189esc1  esc1 deletion mutant, CmR This study Ea1189esc3 esc3 deletion mutant, CmR This study Ea1189hrpN hrpN deletion mutant, CmR This study Ea1189dspF/esc1 dspF/esc1 deletion mutant, KmR CmR This study Ea1189dspF/esc3 dspF/esc3 deletion mutant, KmR CmR This study Ea1189dspF/hrpN dspF/hrpN deletion mutant, KmR CmR This study Ea1189dspF/esc1/esc3 dspF/esc1/esc3 deletion mutant, CmR This study Ea1189dspF/esc1/esc3/hrpN dspF/esc1/esc3/hrpN deletion mutant, CmR This study    Plasmids   pMJH20 pWSK29 containing codons 2 to 406 of CyaA, AmpR (Miao & Miller, 2000) pLRT198 pBRR1MCS-5 expressing DspF, GmR (Triplett et al., 2009a) pLRT8 pMHJ20 expressing DspE(1-15)-CyaA (Triplett et al., 2009a) pLRT201 pMHJ20 expressing DspE(1-737)-CyaA (Triplett et al., 2009a) pLRT177 pMHJ20 expressing Eop1-CyaA This study pLRT209 pMHJ20 expressing Eop3-CyaA This study pLRT210 pMHJ20 expressing Eop4-CyaA This study pGilda HIS3 LexA BD bait vector, AmpR Clontech, Mountain View, CA, USA pB42AD TRP1 B42 AD bait vector, AmpR Clontech pB42-HA-T Positive control prey vector, AmpR Clontech pLexA-53 Positive control bait vector, AmpR Clontech pLRT192 LexA-DspE(1-800) (Triplett et al., 2009a) pLRT13 LexA-DspE(1-150) (Triplett et al., 2009a) pLRT111 LexA-Eop1 This study  130       pLRT169 LexA-Eop1(1-200) This study pLRT231 LexA-Eop1(135-263) This study pLRT232 LexA-Eop1(264-402) This study pLRT167 LexA-Eop3 This study pLRT168 LexA-Eop4 This study pLFC7 LexA-HrpN This study pLFC3 B42-HA-HrpN This study pLRT215 B42-HA-DspF (Triplett et al., 2009a) pLRT226 B42-HA-Esc1 This study pLRT227 B42-HA-Esc3 This study pKD46 Redexo recombinases, AmpR (Datsenko & Wanner, 2000) pKD3 Mutagenesis cassette template, CmR (Datsenko & Wanner, 2000) pKD4 Mutagenesis cassette template, KmR (Datsenko & Wanner, 2000) pCP20 Temperature sensitive replication and induction of FLP synthesis, AmpR (Cherepanov & Wackernagel, 1995) (a) KmR, CmR, GmR and AmpR indicate resistance to kanamycin, chloramphenicol, gentamicin and ampicillin respectively.    Virulence assays.  Strain virulence was evaluated using immature pear fruit and apple shoot assays as previously described (Zhao et al., 2005a, Koczan et al., 2011b). Briefly, for immature pear assays, bacterial 4 CFU mL-1 in 0.5x sterile phosphate-buffered saline (PBS). Three microliters of the bacterial suspension were inoculated on previously stab-wounded surface-sterilized immature pears and incubated at 28°C. ImageJ software (National Institutes of Health; Bethesda, MD, U.S.A.) was used to quantify lesion area at 4 days post-inoculation (dpi). Pear assays were done in triplicate and each experiment was repeated at least three times.    131 Yeast two hybrid assays.  hrpN, eop3, eop4 and eop1 (full-gene and fragments) were cloned in fusion with the LexA binding domain into the bait vector pGilda (Clontech; Mountain View, CA, USA) using BamHI and XhoI restriction sites. esc1, esc3 and hrpN were digested with BamHI and EcoRI and cloned into the prey vector pB42AD. Prey and bait constructs were co-transformed into Saccharomyces cerevisiae EGY48 (pLacZ) using the Frozen- Corporation; Irvine, CA, USA). Transformants were selected on minimal synthetic dropout (SD)-galactose/raffinose medium amended with -Ura/-His/-Trp dropout. Positive interaction was screened on SD-galactose medium amended with -Ura/-His/-Trp/-Leu dropout and 5-bromo-4-chloro-3-indolyl--galactopyranoside (X-gal; 80 mg L-1). A yeast strain co-transformed with pLexA-53, containing p53, and pB42AD-T, containing SV40 large T-antigen, was used as a positive control for protein interaction.  CyaA translocation assay.  eop1, eop3 and eop4 genes were amplified by PCR and ligated into the XbaI and SmaI restriction sites of pMJH20 in fusion with the catalytic domain of CyaA from Bordetella pertussis (Miao & Miller, 2000). Bacterial strains were transformed with the CyaA fusion constructs and stable expression was confirmed by immunoblot using anti-CyaA antibody (Santa 8 CFU mL-1 in PBS and were syringe-infiltrated into fully expanded leaves of 8 wk-old Nicotiana tabacum cv. Samsun plants. Nine hours after inoculation, leaf discs of 1 cm in diameter were collected from the infiltrated areas, and were immediately frozen in liquid nitrogen. Samples  132 were processed for cAMP quantification as previously described (Schechter et al., 2004). were centrifuged 5 min at 3000 g and cAMP levels in supernatants were measured using the cAMP EIA Kit (Cayman chemical; instructions. Protein concentration in samples was determined using the BCA Protein Assay Kit. Statistical analyses were done using a one-way analysis of variance, and mean separation was accomplished using the Tukey-Kramer HDS test.   Protein secretion assay.  Liquid cultures of the wild type strain Ea1189 and mutant strains expressing the DspE(1-737)-Cya, Eop1-CyaA, and Eop3-CyaA fusions were grown overnight in 50 mL LB medium at 28°C. Cell pellets were resuspended in 40 mL of Hrp-inducing minimal medium, pH 5.7 (Huynh et al., 1989) and induced for 24 h at 24°C. Induced cultures were pelleted and supernatants treated with 0.5 mM phenyl methylsulfonyl fluoride (PMSF) and concentrated (300x) using the Amicon Ultra-15 Centrifugal Filter Unit (30 kDa molecular cut-off; Millipore; Billerica, MA, USA). Protein concentrations were measured using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific; Rockfort, IL, USA). Secretion of DspE(1-737)-CyaA was detected by immunoblot using anti-CyaA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-DnaK antibody (LifeSpan BioSciences Inc;Seattle, WA, USA) was used as a lysis control.    133 IV. Results DspF is partially required for DspE translocation into host cells but is not required for DspE secretion in vitro.  We have previously demonstrated that the N terminus of DspE fused with an adenylate cyclase reporter (CyaA) is stably expressed and translocated into tobacco cells by the wild-type strain E. amylovora Ea1189 (Triplett et al., 2009a). To assess the importance of DspF for the successful delivery of DspE into host cells, we compared the expression, secretion and translocation levels of the DspE(1-737)-CyaA fusion from Ea1189 andspF mutant, as described in the methods section. cAMP accumulation due to DspE(1-737)-CyaA translocation was significantly dspF when compared with the wild type and dspF-complemented strains (Fig A.1.A). However, no clear differences in DspE(1-737)-CyaA expression and secretion in vitro dspF strains (Fig A.1.B). Together, these results suggest that DspE(1-737) is expressed and secreted in the absence of DspF and that this chaperone protein is only partially required for the efficient translocation of DspE(1-737) into the host cytoplasm.   134 Figure A.1. Impact of DspF on DspE(1-737)-CyaA translocation.  (A) cAMP accumulation in N. tabacum dspF and dspF/dspF expressing DspE(1-737)-CyaA.  Ea1189 expressing DspE(1-15)-CyaA was used as negative control. Results are the means and error bars represent the SED. Letters above bars denote statistically significant different groups (Tukey-Kramer HDS test, P< 0.05). cAMP accumulation was quantified for duplicate for each strain with similar results. (B) Immunoblot analysis of DspE(1-737)-CyaA expression and secretion dspF. Immmunoblot with DnaK antisera was performed as cell lysis control. The assay was repeated twice with similar results.  T3S chaperones and HrpN interact with multiple effector proteins in yeast.  T3S chaperone genes have often been found as short open reading frames (ORFs) located adjacent to the genes encoding their cognate effector proteins (Cornelis, 2006). Kim and Beer  135 reported the presence of two putative T3S chaperone genes named orfA and orfC located adjacent to eop1 and the harpin gene hrpW, respectively (Kim & Beer, 1998b). The recent release of the E. amylovora strain ATCC49946 genome sequence (Sebaihia et al., 2010a) allowed the identification of a third putative chaperone gene located upstream of the putative effector gene eop3 (Fig A.2A). Prediction of the secondary structure for this 137-amino acid chaperone protein using 3D-Jury (Ginalski et al., 2003) and the Phyre server (Kelley & Sternberg, 2009) -helical motifsand an acidic pI, both characteristic of T3S chaperones. Conversely, the secondary structure predicted for the protein encoded by orfC did not match the structural characteristics of T3S chaperones. Additionally, annotation of sequences surrounding the secreted effector Eop4 and the putative effector gene hopPtoCEa  (Zhao et al., 2005a) did not reveal the presence of any ORF with the characteristics of a T3S chaperone gene. These results indicate that in addition to DspE, two additional effector proteins in E. amylovora are encoded adjacent to confirmed or putative chaperone genes. Because these effector proteins are named Eop1 and Eop3, we propose the putative genes encoding chaperone proteins be named esc1 and esc3 for Erwinia secretion chaperones 1 and 3, respectively.   136 Figure A.2. Interactions of T3S chaperones, effector proteins and HrpN in E. amylovora.  (A) Schematic diagram of gene organization of effector genes eop1, eop3 and eop4 and their putative chaperone partners. Depiction of these regions based on analysis of the E. amylovora strain ATCC49946 genome (accession number FN666575). White-labeled ORFs are predicted to encode putative T3S chaperone proteins. Unlabeled ORFs are predicted to encode hypothetical proteins of functions unrelated to the T3S. (B) Yeast two hybrid interactions between prey fusions of DspF, the putative chaperones Esc1 and Esc3, and HrpN in the pB42AD vector, and bait fusions of the effector proteins Eop1, Eop3, Eop4, N- terminal portions of DspE and HrpN in the pGilda vector. Positive interactions were identified when a yeast colony harboring a particular prey/bait fusion pair turned blue. ++, strong interaction; +, weak interaction; - no interaction.   137 Similar to other T3S chaperone proteins, DspF has been shown to interact with more than one effector protein in yeast two hybrid experiments (Asselin et al., 2006a). In order to assess whether DspF, Esc1, and Esc3 interact with multiple T3S effector proteins in E. amylovora, a yeast two hybrid assay was performed. All of the chaperone proteins fused with a B42-hemagglutinin (HA) tag interacted with fusions of DspE(1-800), Eop1, and Eop3 with the LexA binding domain, but did not interact with Eop4-LexA (Fig A.2B). In contrast with DspF, that interacts with residues 51- 100 of DspE (Triplett et al., 2009a), B42-HA-Esc1 and B42-HA-Esc3 did not interact with the DspF-binding domain in the N terminal region of DspE-LexA (Figure A.2B), indicating that the interaction domain for these chaperones is located elsewhere in the effector. In addition, the chaperone binding domains of Eop1 were mapped with further yeast studies. While the N terminal 200 residues of Eop1 interacted strongly with its partner chaperone B42-HA-Esc1, a weaker interaction with B42-HA-DspF was observed. The interaction of residues 135  402 in the C terminus with B42-HA-DspF and B42-HA-Esc3 was stronger than that with B42-HA-Esc1 (Fig A.2B). Interaction of HrpN with effector and chaperone proteins was also evaluated in yeast. B42-HA-HrpN interacted with DspE(1-800)-LexA and Eop1-LexA, but not with Eop3-LexA (Fig A.2B). In addition, only the constructs expressing B42-HA-DspF and B42-HA-Esc1 interacted with HrpN-LexA, suggesting that HrpN does not interact with the effector protein Eop3 or its partner chaperone Esc3. All of the effector interactions observed with HrpN were weaker than the chaperone-effector interactions.   138 Simultaneous expression of dspF, esc1, esc3 and hrpN genes is required for full virulence of E. amylovora and for secretion and translocation of DspE(1-737)-CyaA.  Virulence and translocation levels of the N terminal region of DspE into host cells are dramatically reduced in either dspF (Figure A.1A) (Gaudriault et al., 2002; Triplett et al., 2009) or hrpN mutants (Bocsanczy et al., 2008c). However, a small amount of DspE is translocated in the absence of these proteins, and both deletion mutants are still pathogenic. To determine whether the additional T3S chaperone proteins Esc1 and Esc3 have an additive effect for the efficient translocation of DspE, a series of mutant strains was constructed and evaluated for virulence in immature pears and for DspE(1-737)-CyaA secretion and translocation. While single deletions of esc1 and esc3 did not have a significant effect on virulence, double deletion mutants dspF/esc1 dspF/esc3 showed a reduction of virulence that was statistically equivalent with the reduction in virulence in the dspF and hrpN mutant backgrounds (Fig A.3A and A.dspF/hrpN strain showed a complete loss of pathogenicity, indicating that the T3S chaperone DspF and HrpN are simultaneously required for E. amylovora to effectively colonize its host and cause disease. Interestingly, a mutant strain lacking the three T3S chaperone genes still caused disease at the same level as double deletion mutants, suggesting that the roles played by Esc1 and Esc3 are minor for the virulence of E. amylovora.   139 Figure A.3. Impact of DspF, Esc1, Esc3 and HrpN for the virulence of E. amylovora.  (A) Lesion size on dspF, esc1, esc3, hrpN, dspF/esc1, dspF/esc3, dspF/hrpN, dspF/esc1/esc3, dspF/esc1/esc3/hrpN. T3SS strain was used as negative control. Results are the means and error bars represent the SED. Different letters above bars denote statistically significant differences (Tukey-Kramer HDS test, P< 0.05) (B)  Symptom development on stab-wounded immature pears at 4 dpi with Ea1189 and mutant strains. (C) Immunoblot analysis of DspE(1-737)-CyaA expression and secretion from the Ea1189 and mutant strains in the extracellular media. Supernatant (SN) and pellet (P) fractions were immunoblotted with CyaA antisera. (D) cAMP accumulation in tobacco leaves inoculated with Ea1189, and mutant strains expressing DspE(1-737)-CyaA. Ea1189 expressing DspE(1-15)-CyaA was used as negative control.  Results are the means and error bars represent the SED. Different letters above bars denote statistically significant differences (Tukey-Kramer HDS test, P< 0.05).       140   Secretion and translocation of DspE(1-737) was evaluated in mutant strains expressing DspE(1-737)-CyaA under hrp-inducing conditions. While DspE(1-737)-CyaA was expressed by all of the strains, secretion of DspE(1-737)-CyaA to the medium was not detected in the triple mutant dspF/esc1/esc3 dspF/esc1/esc3/hrpN backgrounds (Fig A.3C). Furthermore, while cAMP accumulation due to translocation of DspE(1-737)-esc1 esc3 single mutants was not significantly different from the Ea1189 wild type, reduced levels of cAMP were dspF and for all double and triple mutants deleted in dspF and esc1 and/or esc3 (Fig A.3D). cAMP accumulation levels monitoring translocation from  141 hrpN and dspF/hrpN mutant backgrounds were further reduced and statistically equivalent. The lowest level of translocation observed was for the dspF/esc1/esc3/hrpN quadruple mutant which was not significantly different from the DspE(1-15)-CyaA fusion, previously reported to be a non-translocated fragment of DspE (Triplett et al., 2009a). Our results indicate that T3S chaperones in E. amylovora act in conjunction with the harpin protein HrpN in order to effectively deliver DspE into the host cytoplasm through the Hrp-T3SS. DspF negatively affects the translocation of Eop1-CyaA and Eop3-CyaA, but not Eop4-CyaA.  To determine whether the translocation of the effector proteins Eop1, Eop3 and Eop4 was also facilitated by DspF, accumulation of cAMP in tobacco leaves fusions was evaluated after infiltration of Eop1-CyaA, Eop3-CyaA and Eop4-CyaA from a dspF mutant background. Translocation of Eop1-CyaA and Eop3-CyaA from dspF elicited a greater accumulation of cAMP compared to dspF/dspF complemented strain (Fig A.4A and A.4B). Conversely, cAMP accumulation due to Eop4-CyaA translocation was not affected by the deletion of dspF (Fig A.4C). These observations suggest that DspF may play an inhibitory role in the translocation of Eop1 and Eop3.   142 Figure A.4. cAMP accumulation of tobacco leaves mediated by E. amylovora strains expressing fusion proteins.  cAMP accumulation was measured on tobacco leaves infiltrated dspF and dspF/dspF, expressing Eop1-CyaA (A), Eop3-CyaA (B) and Eop4-CyaA (C). Ea1189 expressing DspE(1-15)-CyaA was used as negative control. Results represent the means and error bars represent the SED. Different letters above bars denote statistically significant differences (Tukey-Kramer HDS test, P< 0.05).    143 HrpN is required for efficient translocation of Eop1-CyaA and Eop3-CyaA into plant cells.  In order to determine whether HrpN is also involved in facilitating the translocation of the Eop1 and Eop3 effector proteins, secretion and translocation of CyaA fusions was evaluated in hrpN, Ea1189dspF/hrpN, dspF/esc1/esc3, and dspF/esc1/esc3/hrpN strains. Whereas Eop1-CyaA and Eop3-CyaA were expressed and secreted by all of the mutant strains (Fig A.5C and A.5D), cAMP accumulation levels due to Eop1-CyaA and Eop3-CyaA translocation were significantly reduced in the hrpN mutant strain as compared with the Ea1189 wild type, and at a level that was statistically equivalent to that of the dspF/esc1/esc3/hrpN quadruple mutant (Fig A.5A and A.5B). In contrast, cAMP accumulation elicited by Eop1-CyaA was not significantly affected by simultaneous deletion of dspF and hrpN or the dspF, esc1 and esc3 T3S chaperone genes. These findings indicate that, similar to DspE, HrpN is also required for full levels of Eop1 and Eop3 translocation into plant cells. However, the absence of DspF enhances the translocation levels of these two effector proteins, even in the absence of HrpN. Moreover, the chaperone proteins Esc1 and Esc3 also play a role in promoting the translocation of Eop1-CyaA and Eop3-CyaA, as the levels of cAMP accumulation from the dspF/esc1/esc3 triple mutant was significantly reduced when compared with those from the dspF mutant background.   144 Figure A.5. Secretion and translocation of Eop1 and Eop3 into tobacco plants.  (A) cAMP levels in tobacco after infiltration with Eal189dspF, hrpN, Ea1dspF/hrpN, dspF/esc1/esc3, dspF/esc1/esc3/hrpN expressing Eopl-CyaA. (B).cAMP levels in tobacco after infiltration with the same parent strains expressing Eop3-CyaA. Ea1189 expressing DspE(1-15)-CyaA was used as negative control. Results represent the means and error bars represent the SED. Different letters above bars denote statistically significant differences (Tukey-Kramer HDS test, P< 0.05). (C) Immunoblot analysis of DspE(1-737)-CyaA expression and secretion from the Ea1189 and mutant strains in the extracellular media. Supernatant (SN) and pellet (P) fractions were immunoblotted with CyaA antisera.  V. Discussion As a complex process, T3S exhibits various mechanisms of regulation at all of the different stages in the assembly and translocation processes (Cornelis, 2006). In fact, a hierarchical organization in effector protein delivery through the Hrp-T3SS has been demonstrated for several animal pathogens (Mills et al., 2008, Lara-Tejero et al., 2011).  145 However, mechanisms regulating Hrp-T3SS assembly and the translocation of pre-formed proteins in plant pathogenic bacteria are still poorly understood. In this study, we examined the roles of DspF, other T3S chaperones, and HrpN in effector secretion and translocation efficiency as well as their effect on bacterial virulence. The capacity of the N-terminal region of DspE for DspF-independent translocation observed previously (Triplett et al., 2009) and the interaction of LexA-DspE(1-800) with B42-HA-Esc1and B42-HA-Esc3 observed in this study, led us to hypothesize that T3S chaperone proteins other than DspF might be also involved in the efficient translocation of DspE into the host cell. While mutations of esc1 or esc3 do not have a significant effect on virulence, our secretion and translocation assays indicated that the activity of the T3S chaperones on DspE secretion and translocation is additive, as secretion of DspE(1-737)-CyaA could not be detected from dspF/esc1/esc3 mutant, and this strain induces less cAMP accumulation than its wild type, single, or double mutant counterparts. Although most of the T3S chaperones interact with a single effector protein, T3S chaperones that bind to multiple target effectors (multiple cargo) have been described in several animal pathogenic bacteria, such as SrcA and InvB from Salmonella enterica serovar Typhimurium and CesT from EPEC (Creasey et al., 2003, Ehrbar et al., 2004, Cooper et al., 2010), as well as in plant pathogens such as HpaB from Xanthomonas campestris pv. vesicatoria and ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato (Büttner et al., 2004, Kabisch et al., 2005). Our yeast two-hybrid studies indicated that DspF, Esc1 and Esc3 bind not only to their cognate effector partner, but also to other effector proteins, and in the case of DspE, these T3S chaperones function cooperatively in DspE translocation into the plant cell. This finding is consistent with previous studies in Chlamydia pneumoniae where the T3S chaperones Ssc1 and Ssc4 bind forming a  146 complex that interacts with the N terminal region of the effector protein CopN and promotes its secretion through the T3SS (Silva-Herzog et al., 2011). HrpN  is involved in the translocation of DspE into the plant cell as well as the elicitation of callose deposition in apple leaves (Bocsanczy et al., 2008c, Boureau et al., 2011b). However, hrpN mutant background expressing DspE(1-737)-CyaA is significantly higher than the accumulation induced by DspE(1-15)-CyaA, a non-translocated portion of this effector, suggesting that HrpN is not completely required for DspE translocation. This result led us to hypothesize that HrpN and T3S chaperones might have additive and redundant functions in the translocation of DspE and that this cooperative behavior assures maximal levels of DspE translocation into the host cell. Despite the lack of virulence, our translocation studies demonstrated that deletion of hrpN in a dspF mutant background did not reduce the levels of DspE(1-737)-CyaA translocation, suggesting that both Esc1 and Esc3 play a minor but important role in effector translocation. Indeed, the translocation of DspE(1-737)-CyaA from the dspF/esc1/esc3/hrpN quadruple mutant was completely eliminated. These results suggest that at least four proteins, three T3S chaperones and HrpN, are required for maximal levels of translocation of DspE into plant cells and for DspE secretion to the extracellular medium. Our yeast two-hybrid experiments also allowed us to detect a strong interaction between LexA-HrpN and B42-HA-DspF and a weak interaction between HrpN and the N terminus of DspE, suggesting that HrpN and DspF might associate to promote the recruitment of DspE for the T3S. Cooperation between functional semi-redundant proteins for translocation of T3SS substrates is consistent with previous reports in P. syringae pv. tomato where four harpin proteins form a consortium that serves as a translocator complex required for extracellular secretion and translocation of effector proteins (Kvitko et al., 2007). In E. amylovora, two  147 harpins, HrpN and HrpW, have been postulated to be candidate translocator proteins due to their capacity to elicit HR responses when inoculated on tobacco leaves and to the putative pectate lyase domain in the C terminus of HrpW (Wei et al., 1992, Gaudriault et al., 1998). It is possible that these harpin proteins associate in a pore-forming complex after T3S in order to ensure the translocation of effectors into the plant cell.  In a previous report, we have identified the chaperone binding domain (CBD) of DspF to be within residues 51- 100 in the N terminal region of DspE (Triplett et al., 2009a). Interestingly, yeast two-hybrid results suggest that the CBDs for Esc1 and Esc3 are located in the C-terminal portion of the effector protein, ruling out the possibility of heterodimerization with DspF. The presence of CBDs in regions other than the N terminus of effector proteins has been reported previously in P. syringae pv. tomato for the T3S chaperones ShcO1, ShcS1 and ShcS2, which bind to the middle third portion of HopO1-1 (Guo et al., 2005). The same pattern of CBD distribution was observed for Eop1;  the CBD for the cognate T3S chaperone Esc1 is located between residues 1- 100, while DspF and Esc3 CBDs are located within the last 200 residues of the effector protein.  In contrast to DspE and Eop4, our experiments indicated that translocation of Eop1 and Eop3 is negatively affected by DspF since cAMP accumulation from a dspF strain expressing Eop1-CyaA or Eop3-CyaA is significantly greater than the accumulation induced by the Ea1189 wild type strain, suggesting that DspF plays an antagonistic role, delaying the translocation of effectors other than DspE. Recent studies have postulated Eop1 and Eop3 as effector proteins exhibiting avirulence functions (Asselin et al., 2011, Bocsanczy et al., 2012) which might explain the antagonistic role of DspF on these effector proteins. On the other hand, we observed  148 that, similar to DspE, translocation levels of Eop1 and Eop3 were significantly impaired in the  We observed that The dspF/hrpN double mutant exhibited a complete lack of pathogenicity, but DspE(1-737)-CyaA translocation levels were statistically equivalent to the hrpN strain and significantly higher than those of the dspF/esc1/esc3 triple mutant. This discrepancy (i.e., DspE translocation was insufficient for virulence) led us to hypothesize that in addition to  translocator activity, HrpN might itself be translocated and act as a virulence protein within the plant cell, along with DspE and other effector proteins. A dual role in regulation of effector secretion as well as virulence activity inside the plant cell has been recently reported for HrpJ in Pseudomonas syringae; whereas inside the bacterial cell HrpJ is required for the secretion of translocator proteins, it also contributes to suppression of early immune responses inside the host cell (Crabill et al., 2012a, Wei & Collmer, 2012b). In addition, T3SS-dependent translocation into plant cells has been previously reported for several translocator proteins from plant pathogens such as HrpF from P. syringae pv. syringae and HrpK from P. syringae pv. tomato (Petnicki-Ocwieja et al., 2005b, Ramos et al., 2007). Indeed, Boczanczy et al. (2008c) reported that the first 323 amino acids of HrpN fused with the CyaA reporter were translocated into N. tabacum cells at levels significantly higher than a Hrp-T3SS mutant expressing the same fusion protein.   These data lead us to propose a model for effector translocation in which after Hrp-T3SS assembly, translocator proteins such as HrpN are recruited and secreted by the Hrp-T3SS in order to act as a pore-forming complex in the host cell wall (Fig A.6). DspF might be involved in the recruitment of HrpN before effector translocation. After the establishment of the translocation machinery, HrpN is translocated into the plant cell. Concurrently, DspF binds to the  149 N terminal region of DspE, while Esc1 and Esc3 bind to the C terminus, inhibiting its folding, stabilizing the effector protein and preventing premature interactions with Hrp-T3SS components as well as promoting the efficient recruitment of the effector protein for T3S. DspF also binds to Eop1 and Eop3 reducing their translocation (Figure A.6). In summary, we determined cooperation between several regulating proteins in order to efficiently deliver a T3S effector into plant cells. Further studies are needed to determine if E. amylovora orchestrates a hierarchical translocation of effectors to colonize its host and cause disease. 150 Figure A.6. Model for translocation of TTS effectors in E. amylovora.  (A). After T3SS assembly, HrpN interacts with DspF which promotes its recruitment for TTS. (B) HrpN and other proteins act as translocators, forming the pore in the plant cell wall for effector translocation and HrpN is translocated into the plant cell. (C) After the establishment of the translocation machinery, DspF promotes the recruitment of DspE to the T3SS binding to the N terminus of the effector, while Esc1 and Esc3 bind to the C terminal region inhibiting mis-folding and stabilizing the protein in the bacterial cytoplasm before TTS. While DspE is translocated, DspF also bind to Eop1 and Eop3 reducing their translocation and promoting maximal levels of translocation of DspE. (D) After DspE reaches maximal levels of translocation into the plant cells, DspF releases Eop1 and Eop3 for their posterior translocation.  151               LITERATURE CITED              152 LITERATURE CITED  Abel, S., Chien, P., Wassmann, P., Schirmer, T., Kaever, V., Laub, Michael T., et al. (2011) Regulatory cohesion of cell cycle and cell differentiation through interlinked phosphorylation and second messenger networks. Mol. Cell, 43, 550-560. Ahmad, I., Lamprokostopoulou, A., Le Guyon, S., Streck, E., Barthel, M., Peters, V., et al. (2011) Complex c-di-GMP signaling networks mediate transition between virulence properties and biofilm formation in Salmonella enterica Serovar Typhimurium. PLoS ONE, 6, e28351. Akeda, Y. and Galán, J. E. (2005) Chaperone release and unfolding of substrates in type III secretion. Nature, 437, 911-915. Al-Karablieh, N., Weingart, H. and Ullrich, M. S. (2009) The outer membrane protein TolC is required for phytoalexin resistance and virulence of the fire blight pathogen Erwinia amylovora. Microbial Biotechnology, 2, 465-475. Aldridge, P., Metzger, M. and Geider, K. (1997) Genetics of sorbitol metabolism in Erwinia amylovora and its influence on bacterial virulence. Mol. Gen. Genet., 256, 611-619. Ancona, V., Li, W. and Zhao, Y. (2014) Alternative sigma factor RpoN and its modulation protein YhbH are indispensable for Erwinia amylovora virulence. Mol. Plant Pathol., 15, 58-66. Arnold, R., Brandmaier, S., Kleine, F., Tischler, P., Heinz, E., Behrens, S., et al. (2009) Sequence-based prediction of type III secreted proteins. PLoS Pathog, 5, e1000376. Asselin, J., Oh, C., Nissinen, R. and Beer, S. (2006a) The secretion of EopB from Erwinia amylovora. (Bazzi, C. and Mazzucchi, U., eds.). Acta Horticulturae, pp. 409-416. Asselin, J., Oh, C., Nissinen, R. and Beer, S. (2006b) The secretion of EopB from Erwinia amylovora. Acta Horticulturae 704, 409-416. Asselin, J. E., Bonasera, J. M., Kim, J. F., Oh, C. S. and Beer, S. V. (2011) Eop1 from a Rubus strain of Erwinia amylovora functions as a host-range limiting factor. Phytopathology, 101, 935-944. Ayers, A. R., Ayers, S. B. and Goodman, R. N. (1979) Extracellular polysaccharide of Erwinia amylovora: a correlation with virulence. Appl. Environ. Microbiol., 38, 659-666. Bailey, T. L. and Gribskov, M. (1998) Combining evidence using p-values: application to sequence homology searches. Bioinformatics, 14, 48-54.  153 Barends, T. R. M., Hartmann, E., Griese, J. J., Beitlich, T., Kirienko, N. V., Ryjenkov, D. A., et al. (2009) Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase. Nature, 459, 1015-1018. Barny, M.-A. (1995) Erwinia amylovora hrpN mutants, blocked in harpin synthesis, express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco. Eur. J. Plant Pathol., 101, 333-340. Bayot, R. and Ries, S. (1986) Role of motility in apple blossom infection by Erwinia amylovora and studies of fire blight control with attractant and repellent compounds. Phytopathology, 76, 441-445. Bellemann, P., Bereswill, S., Berger, S. and Geider, K. (1994) Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis. Int. J. Biol. Macromol., 16, 290-296. Bellemann, P. and Geider, K. (1992) Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization. Journal of General Microbiology, 138, 931-940. Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., et al. (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res., 42, W252-W258. Blaylock, B., Sorg, J. A. and Schneewind, O. (2008) Yersinia enterocolitica type III secretion of YopR requires a structure in its mRNA. Mol. Microbiol., 70, 1210-1222. Bobrov, A. G., Kirillina, O., Ryjenkov, D. A., Waters, C. M., Price, P. A., Fetherston, J. D., et al. (2011) Systematic analysis of cyclic di-GMP signalling enzymes and their role in biofilm formation and virulence in Yersinia pestis. Mol. Microbiol., 79, 533-551. Bocsanczy, A., Beer, S., Perna, N., Biehl, B., Glasner, J., Cartinhour, S., et al. (2008a) Contributions of the genome sequence of Erwinia amylovora to the fire blight community. Acta Horticulturae, 793, 163-170. Bocsanczy, A. M., Nissinen, R. M., Oh, C.-S. and Beer, S. V. (2008b) HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells. Mol. Plant Pathol., 9, 425-434. Bocsanczy, A. M., Nissinen, R. M., Oh, C.-S. and Beer, S. V. (2008c) HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells. Mol Plant Pathol, 9, 425-434. Bocsanczy, A. M., Schneider, D. J., DeClerck, G. A., Cartinhour, S. and Beer, S. V. (2012) HopX1 in Erwinia amylovora Functions as an avirulence protein in apple and is regulated by HrpL. J. Bacteriol., 194, 553-560.  154 Bogdanove, A. J., Bauer, D. W. and Beer, S. V. (1998) Erwinia amylovora secretes DspE, a pathogenicity factor and functional AvrE homolog, through the Hrp (type III secretion) pathway. J. Bacteriol., 180, 2244-2247. Bogs, J., Bruchmüller, I., Erbar, C. and Geider, K. (1998) Colonization of host plants by the fire blight pathogen Erwinia amylovora marked with genes for bioluminescence and fluorescence. Phytopathology, 88, 416-421. Bonn, W. G. and Van der Zwet, T. (2000) Distribution and economic importance of fire blight. In: Fire blight: the disease and its causative agent, Erwinia amylovora. (Vanneste, J., ed.). New York: CABI Publishing, pp. 37-53. Bordeleau, E., Fortier, L.-C., Malouin, F. and Burrus, V. (2011) c-di-GMP turn-over in Clostridium difficile is controlled by a plethora of diguanylate cyclases and phosphodiesterases. PLoS Genet, 7, e1002039. Bose, D., Joly, N., Pape, T., Rappas, M., Schumacher, J., Buck, M., et al. (2008) Dissecting the ATP hydrolysis pathway of bacterial enhancer-binding proteins. Biochem. Soc. Trans., 36, 83-88. Boughammoura, A., Expert, D. and Franza, T. (2012) Role of the Dickeya dadantii Dps protein. Biometals, 25, 423-433. Boughammoura, A., Matzanke, B. F., Böttger, L., Reverchon, S., Lesuisse, E., Expert, D., et al. (2008) Differential role of ferritins in iron metabolism and virulence of the plant-pathogenic bacterium Erwinia chrysanthemi 3937. J. Bacteriol., 190, 1518-1530. Boureau, T., ElMaarouf-Bouteau, H., Garnier, A., Brisset, M.-N., Perino, C., Pucheu, I., et al. (2006a) DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants. Mol Plant Microbe Interact, 19, 16-24. Boureau, T., ElMaarouf-Bouteau, H., Garnier, A., Brisset, M.-N., Perino, C., Pucheu, I., et al. (2006b) DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants. Mol. Plant-Microbe Interact., 19, 16-24. Boureau, T., Siamer, S., Perino, C., Gaubert, S., Patrit, O., Degrave, A., et al. (2011a) The HrpN effector of Erwinia amylovora, which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves. Mol. Plant-Microbe Interact., 24, 577-584. Boureau, T., Siamer, S., Perino, C., Gaubert, S., Patrit, O., Degrave, A., et al. (2011b) The HrpN effector of Erwinia amylovora, which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves. Mol Plant Microbe Interact, 24, 577-584.  155 Bubán, T. and Orosz-Kovács, Z. (2016) The nectary as the primary site of infection by Erwinia amylovora (Burr.) Winslow et al.: a mini review. Plant Syst. Evol., 238, 183-194. Bugert, P. and Geider, K. (1995) Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Mol. Microbiol., 15, 917-933. Burse, A., Weingart, H. and Ullrich, M. S. (2004a) NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria. Appl. Environ. Microbiol., 70, 693-703. Burse, A., Weingart, H. and Ullrich, M. S. (2004b) The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora. Mol Plant Microbe Interact, 17, 43-54. Burse, A., Weingart, H. and Ullrich, M. S. (2004c) The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora. Mol. Plant-Microbe Interact., 17, 43-54. Bush, M. and Dixon, R. (2012) The role of bacterial enhancer binding proteins as specialized -dependent transcription. Microbiol. Mol. Biol. Rev., 76, 497-529. Büttner, D., Gürlebeck, D., Noël, L. D. and Bonas, U. (2004) HpaB from Xanthomonas campestris pv. vesicatoria acts as an exit control protein in type III-dependent protein secretion. Mol. Microbiol., 54, 755-768. Büttner, D. and He, S. Y. (2009) Type III protein secretion in plant pathogenic bacteria. Plant Physiol., 150, 1656-1664. Button, J. E. and Galán, J. E. (2011) Regulation of chaperone/effector complex synthesis in a bacterial type III secretion system. Mol. Microbiol., 81, 1474-1483. Castiblanco, L. F. and Sundin, G. W. (2015) New insights on molecular regulation of biofilm formation in plant-associated bacteria. J. Integr. Plant Biol., 58, 362-372. Cesbron, S., Paulin, J.-P., Tharaud, M., Barny, M.-A. and Brisset, M.-factor HrpL negatively modulates the flagellar system in the phytopathogenic bacterium Erwinia amylovora under hrp-inducing conditions. FEMS Microbiol. Lett., 257, 221-227. Chalupowicz, L., Zellermann, E. M., Fluegel, M., Dror, O., Eichenlaub, R., Gartemann, K. H., et al. (2011) Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection. Phytopathology, 102, 23-31. Chatterjee, A., Cui, Y., Chaudhuri, S. and Chatterjee, A. K. (2002) Identification of regulators of hrp/hop genes of Erwinia carotovora ssp. carotovora and characterization of HrpLEcc (SigmaLEcc), an alternative sigma factor. Mol. Plant Pathol., 3, 359-370.  156 Chatterjee, S., Killiny, N., Almeida, R. P. P. and Lindow, S. E. (2010) Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission. Mol. Plant-Microbe Interact., 23, 1356-1363. Cherepanov, P. P. and Wackernagel, W. (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene, 158, 9-14. Chin, K.-H., Lee, Y.-C., Tu, Z.-L., Chen, C.-H., Tseng, Y.-H., Yang, J.-M., et al. (2010) The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cellcell signaling to virulence gene expression in Xanthomonas campestris. J. Mol. Biol., 396, 646-662. Chou, S.-H. and Galperin, M. Y. (2016) Diversity of c-di-GMP-binding proteins and mechanisms. J. Bacteriol., 198, 32-46. Cohen, D., Mechold, U., Nevenzal, H., Yarmiyhu, Y., Randall, T. E., Bay, D. C., et al. (2015) Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A., 112, 11359-11364. Cooper, C. A., Zhang, K., Andres, S. N., Fang, Y., Kaniuk, N. A., Hannemann, M., et al. (2010) Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host. PLoS Pathog, 6, e1000751. Cornelis, G. R. (2006) The type III secretion injectisome. Nat Rev Micro, 4, 811-825. Cotter, P. and Stibitz, S. (2007) c-di-GMP-mediated regulation of virulence and biofilm formation. Curr. Opin. Microbiol., 10, 17-23. Crabill, E., Karpisek, A. and Alfano, J. R. (2012a) The Pseudomonas syringae HrpJ protein controls the secretion of type III translocator proteins and has a virulence role inside plant cells. Mol Microbiol, doi: 10.1111/j.1365-2958.2012.08097.x. Crabill, E., Karpisek, A. and Alfano, J. R. (2012b) The Pseudomonas syringae HrpJ protein controls the secretion of type III translocator proteins and has a virulence role inside plant cells. Mol. Microbiol., 85, 225-238. Creasey, E. A., Delahay, R. M., Bishop, A. A., Shaw, R. K., Kenny, B., Knutton, S., et al. (2003) CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map. Mol. Microbiol., 47, 209-221. Danhorn, T. and Fuqua, C. (2007) Biofilm Formation by Plant-Associated Bacteria. Annu. Rev. Microbiol., 61, 401-422. Darnell, C. L., Hussa, E. A. and Visick, K. L. (2008) The putative hybrid sensor kinase SypF coordinates biofilm formation in Vibrio fischeri by acting upstream of two response regulators, SypG and VpsR. J. Bacteriol., 190, 4941-4950.  157 Darwin, K. H. and Miller, V. L. (2001) Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. EMBO J, 20, 1850-1862. Datsenko, K. A. and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci, 97, 6640-6645. de Lorenzo, V. and Timmis, K. N. (1994) Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. In: Methods Enzymol. (Clark, V. L. and Bavoil, P. M., eds.). Academic Press, pp. 386-405. DebRoy, S., Thilmony, R., Kwack, Y.-B., Nomura, K. and He, S. Y. (2004) A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants. Proc. Natl. Acad. Sci. U. S. A., 101, 9927-9932. Degrave, A., Fagard, M., Perino, C., Brisset, M. N., Gaubert, S., Laroche, S., et al. (2008) Erwinia amylovora type threesecreted proteins trigger cell death and defense responses in Arabidopsis thaliana. Mol. Plant-Microbe Interact., 21, 1076-1086. Dellagi, A., Brisset, M.-N., Paulin, J.-P. and Expert, D. (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity. Mol. Plant-Microbe Interact., 11, 734-742. Deng, Y., Schmid, N., Wang, C., Wang, J., Pessi, G., Wu, D., et al. (2012) Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover. Proc. Natl. Acad. Sci. U. S. A., 109, 15479-15484. Dugé De Bernonville, T., Gaucher, M., Flors, V., Gaillard, S., Paulin, J.-P., Dat, J. F., et al. (2012) T3SS-dependent differential modulations of the jasmonic acid pathway in susceptible and resistant genotypes of Malus spp. challenged with Erwinia amylovora. Plant Science, 188189, 1-9. Dunn, A. K., Millikan, D. S., Adin, D. M., Bose, J. L. and Stabb, E. V. (2006) New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl. Environ. Microbiol., 72, 802-810. Eden-Green, S. J. and Billing, E. (1974) Fireblight. Review of Plant Pathology, 53, 353-365. Edmunds, A. C., Castiblanco, L. F., Sundin, G. W. and Waters, C. M. (2013) Cyclic di-GMP modulates the disease progression of Erwinia amylovora. J. Bacteriol., 195, 2155-2165. Ehrbar, K., Hapfelmeier, S., Stecher, B. and Hardt, W.-D. (2004) InvB Is required for type III-dependent secretion of SopA in Salmonella enterica serovar Typhimurium. J. Bacteriol., 186, 1215-1219. Engelhardt, S., Lee, J., Gäbler, Y., Kemmerling, B., Haapalainen, M.-L., Li, C.-M., et al. (2009) Separable roles of the Pseudomonas syringae pv. phaseolicola accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity. Plant J., 57, 706-717.  158 Engl, C., Waite, C. J., McKenna, J. F., Bennett, M. H., Hamann, T. and Buck, M. (2014) Chp8, a diguanylate cyclase from Pseudomonas syringae pv. tomato DC3000, suppresses the pathogen-associated molecular pattern flagellin, increases extracellular polysaccharides, and promotes plant immune evasion. mBio, 5, e01168-01114. Expert, D., Dellagi, A. and Kachadourian, R. (2000) Iron and fire blight: role in pathogenicity of desferrioxamine E, the main siderophore of Erwinia amylovora. In: Fire blight: the disease and its causative agent, Erwinia amylovora. (Vanneste, J., ed.). New York: CABI publising, pp. 179-195. Fang, X., Ahmad, I., Blanka, A., Schottkowski, M., Cimdins, A., Galperin, M. Y., et al. (2014) GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria. Mol. Microbiol., 93, 439-452. Farkas, Á., Mihalik, E., Dorgai, L. and Bubán, T. (2012) Floral traits affecting fire blight infection and management. Trees - Structure and Function, 26, 47-66. Finn, R. D., Mistry, J., Tate, J., Coggill, P., Heger, A., Pollington, J. E., et al. (2010) The Pfam protein families database. Nucleic Acids Res., 38, D211-222. Flemming, H.-C. and Wingender, J. (2010) The biofilm matrix. Nat Rev Micro, 8, 623-633. Fouts, D. E., Abramovitch, R. B., Alfano, J. R., Baldo, A. M., Buell, C. R., Cartinhour, S., et al. (2002) Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor. Proc. Natl. Acad. Sci. U. S. A., 99, 2275-2280. Fralick, J. A. (1996) Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. J. Bacteriol., 178, 5803-5805. Gaudriault, S., Brisset, M. N. and Barny, M. A. (1998) HrpW of Erwinia amylovora, a new Hrp-secreted protein. FEBS Lett., 428, 224-228. Gaudriault, S., Malandrin, L., Paulin, J. P. and Barny, M. A. (1997a) DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way. Mol Microbiol, 26, 1057-1069. Gaudriault, S., Malandrin, L., Paulin, J. P. and Barny, M. A. (1997b) DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way. Mol. Microbiol., 26, 1057-1069. Gaudriault, S., Paulin, J.-P. and Barny, M.-A. (2002a) The DspB/F protein of Erwinia amylovora is a type III secretion chaperone ensuring efficient intrabacterial production of the Hrp-secreted DspA/E pathogenicity factor. Mol Plant Pathol, 3, 313-320.  159 Gaudriault, S., Paulin, J.-P. and Barny, M.-A. (2002b) The DspB/F protein of Erwinia amylovora is a type III secretion chaperone ensuring efficient intrabacterial production of the Hrp-secreted DspA/E pathogenicity factor. Mol. Plant Pathol., 3, 313-320. Ghosh, P. (2004) Process of protein transport by the type III secretion system. Microbiol. Mol. Biol. Rev., 68, 771-795. Ginalski, K., Elofsson, A., Fischer, D. and Rychlewski, L. (2003) 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics, 19, 1015-1018. Goodman, R. N., Huang, J. S. and Huang, P.-Y. (1974) Host-specific phytotoxic polysaccharide from apple tissue infected by Erwinia amylovora. Science, 183, 1081-1082. Gross, M., Geier, G., Rudolph, K. and Geider, K. (1992) Levan and levansucrase synthesized by the fireblight pathogen Erwinia amylovora. Physiol. Mol. Plant Pathol., 40, 371-381. Guex, N. and Peitsch, M. C. (1997) SWISSMODEL and the SwissPdb Viewer: an environment for comparative protein modeling. Electrophoresis, 18, 2714-2723. Guo, M., Chancey, S. T., Tian, F., Ge, Z., Jamir, Y. and Alfano, J. R. (2005) Pseudomonas syringae type III chaperones ShcO1, ShcS1, and ShcS2 facilitate translocation of their cognate effectors and can substitute for each other in the secretion of HopO1-1. J. Bacteriol., 187, 4257-4269. Gürlebeck, D., Thieme, F. and Bonas, U. (2006) Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant. J. Plant Physiol., 163, 233-255. Ham, J. H., Majerczak, D. R., Nomura, K., Mecey, C., Uribe, F., He, S.-Y., et al. (2009) Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs. Mol. Plant-Microbe Interact., 22, 703-712. Hauben, L., Moore, E. R. B., Vauterin, L., Steenackers, M., Mergaert, J., Verdonck, L., et al. (1998) Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst. Appl. Microbiol., 21, 384-397. Hengge, R. (2009) Principles of c-di-GMP signalling in bacteria. Nat Rev Micro, 7, 263-273. Hengge, R., Galperin, M. Y., Ghigo, J.-M., Gomelsky, M., Green, J., Hughes, K. T., et al. (2016) Systematic nomenclature for GGDEF and EAL domain-containing cyclic di-GMP turnover proteins of Escherichia coli. J. Bacteriol., 198, 7-11. Hickman, J. W. and Harwood, C. S. (2008) Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol. Microbiol., 69, 376-389. Higuchi, R., Krummel, B. and Saiki, R. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res., 16, 7351-7367.  160 Hung, C., Zhou, Y., Pinkner, J. S., Dodson, K. W., Crowley, J. R., Heuser, J., et al. (2013) Escherichia coli biofilms have an organized and complex extracellular matrix structure. mBio, 4. Hutcheson, S. W., Bretz, J., Sussan, T., Jin, S. and Pak, K. (2001) Enhancer-binding proteins HrpR and HrpS interact to regulate hrp encoded type III protein secretion in Pseudomonas syringae strains. J. Bacteriol., 183, 5589-5598. Huynh, T., Dahlbeck, D. and Staskawicz, B. (1989) Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. Science, 245, 1374-1377. Jahn, C. E., Selimi, D. A., Barak, J. D. and Charkowski, A. O. (2011) The Dickeya dadantii biofilm matrix consists of cellulose nanofibres, and is an emergent property dependent upon the type III secretion system and the cellulose synthesis operon. Microbiology, 157, 2733-2744. Jenal, U. and Malone, J. (2006) Mechanisms of cyclic-di-GMP signaling in bacteria. Annu. Rev. Genet., 40, 385-407. Jin, Q., Hu, W., Brown, I., McGhee, G., Hart, P., Jones, A. L., et al. (2001) Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae. Mol. Microbiol., 40, 1129-1139. Jones, J. D. G. and Dangl, J. L. (2006) The plant immune system. Nature, 444, 323-329. Jovanovic, M., James, E. H., Burrows, P. C., Rego, F. G. M., Buck, M. and Schumacher, J. (2011) Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity. Nat Commun, 2, 177. Jumel, K., Geider, K. and Harding, S. E. (1997) The solution molecular weight and shape of the bacterial exopolysaccharides amylovoran and stewartan. Int. J. Biol. Macromol., 20, 251-258. Kabisch, U., Landgraf, A., Krause, J., Bonas, U. and Boch, J. (2005) Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector. Microbiology, 151, 269-280. Kader, A., Simm, R., Gerstel, U., Morr, M. and Römling, U. (2006) Hierarchical involvement of various GGDEF domain proteins in rdar morphotype development of Salmonella enterica serovar Typhimurium. Mol. Microbiol., 60, 602-616. Karatan, E. and Watnick, P. (2009) Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol. Mol. Biol. Rev., 73, 310-347. Kelley, L. A. and Sternberg, M. J. E. (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc, 4, 363-371.  161 Kim, J. F. and Beer, S. V. (1998a) HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct class. J. Bacteriol., 180, 5203-5210. Kim, J. F. and Beer, S. V. (1998b) HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct class. J Bacteriol, 180, 5203-5210. Koczan, J. M., Lenneman, B. R., McGrath, M. J. and Sundin, G. W. (2011a) Cell surface attachment structures contribute to biofilm formation and xylem colonization by Erwinia amylovora. Appl. Environ. Microbiol., 77, 7031-7039. Koczan, J. M., Lenneman, B. R., McGrath, M. J. and Sundin, G. W. (2011b) Cell surface attachment structures contribute to biofilm formation and xylem colonization by Erwinia amylovora. Appl Environ Microbiol, 77, 7031-7039. Koczan, J. M., McGrath, M. J., Zhao, Y. and Sundin, G. W. (2009) Contribution of Erwinia amylovora exopolysaccharides amylovoran and levan to biofilm formation: Implications in pathogenicity. Phytopathology, 99, 1237-1244. -1,4-endoglucanase secreted by Acetobacter xylinum plays an essential role for the formation of cellulose fiber. Bioscience, Biotechnology, and Biochemistry, 62, 2257-2259. Koutsoudis, M. D., Tsaltas, D., Minogue, T. D. and von Bodman, S. B. (2006) Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii. Proc. Natl. Acad. Sci. U. S. A., 103, 5983-5988. Kovach, M., Phillips, R., Elzer, P., Roop 2nd, R. and Peterson, K. (1994) pBBR1MCS: a broad-host-range cloning vector. BioTechniques, 16, 800. Kovach, M. E., Elzer, P. H., Steven Hill, D., Robertson, G. T., Farris, M. A., Roop Ii, R. M., et al. (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene, 166, 175-176. Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E. L. L. (2001) Predicting transmembrane protein topology with a hidden markov model: application to complete genomes1. J. Mol. Biol., 305, 567-580. Kvitko, B. H., Ramos, A. R., Morello, J. E., Oh, H.-S. and Collmer, A. (2007) Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type III secretion system effectors. J. Bacteriol., 189, 8059-8072. Lamprokostopoulou, A., Monteiro, C., Rhen, M. and Römling, U. (2010) Cyclic di-GMP signalling controls virulence properties of Salmonella enterica serovar Typhimurium at the mucosal lining. Environmental Microbiology, 12, 40-53.  162 Lara-Tejero, M., Kato, J., Wagner, S., Liu, X. and Galán, J. E. (2011) A sorting platform determines the order of protein secretion in bacterial type III systems. Science, 331, 1188-1191. Lee, E. R., Baker, J. L., Weinberg, Z., Sudarsan, N. and Breaker, R. R. (2010) An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science, 329, 845-848. Lee, J. H. and Zhao, Y. (2015) Integration host factor is required for RpoN-dependent hrpL gene expression and controls motility by positively regulating rsmB sRNA in Erwinia amylovora. Phytopathology, 106, 29-36. Lenz, D. H., Mok, K. C., Lilley, B. N., Kulkarni, R. V., Wingreen, N. S. and Bassler, B. L. (2004) The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell, 118, 69-82. Long, T., Tu, K. C., Wang, Y. F., Mehta, P., Ong, N. P., Bassler, B. L., et al. (2009) Quantifying the integration of quorum-sensing signals with single-cell resolution. PLoS Biol., 7, 640-649. Lorenz, C. and Buttner, D. (2009) Functional characterization of the type III secretion ATPase HrcN from the plant pathogen Xanthomonas campestris pv. vesicatoria. J. Bacteriol., 191, 1414-1428. Lorenz, C., Kirchner, O., Egler, M., Stuttmann, J., Bonas, U. and Büttner, D. (2008) HpaA from Xanthomonas is a regulator of type III secretion. Mol. Microbiol., 69, 344-360. Losada, L. C. and Hutcheson, S. W. (2005) Type III secretion chaperones of Pseudomonas syringae protect effectors from Lon-associated degradation. Mol. Microbiol., 55, 941-953. Lu, X.-H., An, S.-Q., Tang, D.-J., McCarthy, Y., Tang, J.-L., Dow, J. M., et al. (2012) RsmA regulates biofilm formation in Xanthomonas campestris through a regulatory network involving cyclic di-GMP and the Clp transcription factor. PLoS ONE, 7, e52646. Luo, Y., Bertero, M. G., Frey, E. A., Pfuetzner, R. A., Wenk, M. R., Creagh, L., et al. (2001) Structural and biochemical characterization of the type III secretion chaperones CesT and SigE. Nat. Struct. Mol. Biol., 8, 1031-1036. Malnoy, M., Martens, S., Norelli, J. L., Barny, M.-A., Sundin, G. W., Smits, T. H. M., et al. (2012) Fire blight: applied genomic insights of the pathogen and host. Annu. Rev. Phytopathol., 50, 475-494. Mann, E. E. and Wozniak, D. J. (2012) Pseudomonas biofilm matrix composition and niche biology. FEMS Microbiol. Rev., 36, 893-916. Martínez-Granero, F., Navazo, A., Barahona, E., Redondo-Nieto, M., González de Heredia, E., Baena, I., et al. (2014) Identification of flgZ as a flagellar gene rncoding a PilZ domain  163 protein that regulates swimming motility and biofilm formation in Pseudomonas. PLoS ONE, 9, e87608. Massie, J. P., Reynolds, E. L., Koestler, B. J., Cong, J.-P., Agostoni, M. and Waters, C. M. (2012) Quantification of high-specificity cyclic diguanylate signaling. Proc. Natl. Acad. Sci. U. S. A., 109, 12746-12751. Matsumoto, H. and Young, G. M. (2009) Essential role of the SycP chaperone in type III secretion of the YspP effector. J. Bacteriol., 191, 1703-1715. Matsuyama, B. Y., Krasteva, P. V., Baraquet, C., Harwood, C. S., Sondermann, H. and Navarro, M. V. A. S. (2016) Mechanistic insights into c-di-GMPdependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A., 113, E209-E218. Mazur, O. and Zimmer, J. (2011) Apo- and cellopentaose-bound structures of the bacterial cellulose synthase subunit BcsZ. J. Biol. Chem., 286, 17601-17606. McCarthy, Y., Ryan, R. P., O'Donovan, K., He, Y.-Q., Jiang, B.-L., Feng, J.-X., et al. (2008) The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris. Mol. Plant Pathol., 9, 819-824. McNally, R. R., Toth, I. K., Cock, P. J. A., Pritchard, L., Hedley, P. E., Morris, J. A., et al. (2012) Genetic characterization of the HrpL regulon of the fire blight pathogen Erwinia amylovora reveals novel virulence factors. Mol. Plant Pathol., 13, 160-173. McNamara, J. T., Morgan, J. L. W. and Zimmer, J. (2015) A Molecular description of cellulose biosynthesis. Annu. Rev. Biochem., 84, 895-921. Meng, X., Bonasera, J. M., Kim, J. F., Nissinen, R. M. and Beer, S. V. (2006a) Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen. Mol Plant Microbe Interact, 19, 53-61. Meng, X., Bonasera, J. M., Kim, J. F., Nissinen, R. M. and Beer, S. V. (2006b) Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen. Mol. Plant-Microbe Interact., 19, 53-61. Merighi, M., Lee, V. T., Hyodo, M., Hayakawa, Y. and Lory, S. (2007) The second messenger bis-(3-5)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa. Mol. Microbiol., 65, 876-895. Merighi, M., Majerczak, D. R., Stover, E. H. and Coplin, D. L. (2003) The HrpX/HrpY two-component aystem activates hrpS expression, the first step in the regulatory cascade controlling the Hrp regulon in Pantoea stewartii subsp. stewartii. Mol. Plant-Microbe Interact., 16, 238-248.  164 Merritt, J. H., Brothers, K. M., Kuchma, S. L. and O'Toole, G. A. (2007) SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function. J. Bacteriol., 189, 8154-8164. Miao, E. A. and Miller, S. I. (2000) A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium. Proc. Natl. Acad. Sci. U. S. A., 97, 7539-7544. Miller, W. G., Leveau, J. H. J. and Lindow, S. E. (2000) Improved gfp and inaZ broad-host-range promoter-probe vectors. Mol. Plant-Microbe Interact., 13, 1243-1250. Mills, E., Baruch, K., Charpentier, X., Kobi, S. and Rosenshine, I. (2008) Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli. Cell Host Microbe, 3, 104-113. Mills, E., Pultz, I. S., Kulasekara, H. D. and Miller, S. I. (2011) The bacterial second messenger c-di-GMP: mechanisms of signalling. Cell. Microbiol., 13, 1122-1129. Morgan, J. L. W., McNamara, J. T. and Zimmer, J. (2014) Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP. Nat. Struct. Mol. Biol., 21, 489-496. Mudgett, M. B., Chesnokova, O., Dahlbeck, D., Clark, E. T., Rossier, O., Bonas, U., et al. (2000) Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants. Proc. Natl. Acad. Sci. U. S. A., 97, 13324-13329. Mukherjee, S., Keitany, G., Li, Y., Wang, Y., Ball, H. L., Goldsmith, E. J., et al. (2006) Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation. Science, 312, 1211-1214. Nakai, T., Sugano, Y., Shoda, M., Sakakibara, H., Oiwa, K., Tuzi, S., et al. (2013) Formation of highly twisted ribbons in a carboxymethylcellulase gene-disrupted strain of a cellulose-producing bacterium. J. Bacteriol., 195, 958-964. Nakka, S., Qi, M. and Zhao, Y. (2010) The Erwinia amylovora PhoPQ system is involved in resistance to antimicrobial peptide and suppresses gene expression of two novel type III secretion systems. Microbiol. Res., 165, 665-673. Newell, P. D., Yoshioka, S., Hvorecny, K. L., Monds, R. D. and O'Toole, G. A. (2011) Systematic analysis of diguanylate cyclases that promote biofilm formation by Pseudomonas fluorescens Pf0-1. J. Bacteriol., 193, 4685-4698. Ng, W.-L. and Bassler, B. L. (2009) Bacterial Quorum-Sensing Network Architectures. Annu. Rev. Genet., 43, 197-222. Nikolskaya, A. N., Mulkidjanian, A. Y., Beech, I. B. and Galperin, M. Y. (2003) MASE1 and MASE2: Two novel integral membrane sensory domains. J. Mol. Microbiol. Biotechnol., 5, 11-16.  165 Nimtz, M., Mort, A., Domke, T., Wray, V., Zhang, Y., Qiu, F., et al. (1996a) Structure of amylovoran, the capsular exopolysaccharide from the fire blight pathogen Erwinia amylovora. Carbohydr. Res., 287, 59-76. Nimtz, M., Mort, A., Domke, T., Wray, V., Zhang, Y. X., Qiu, F., et al. (1996b) Structure of amylovoran, the capsular exopolysaccharide from the fire blight pathogen Erwinia amylovora. Carbohydr. Res., 287, 59-76. Nissan, G., Manulis, S., Weinthal, D. M., Sessa, G. and Barash, I. (2005) Analysis of promoters -factor protein from Pantoea agglomerans pv. gypsophilae. Mol. Plant-Microbe Interact., 18, 634-643. Nissinen, R. M., Ytterberg, A. J., Bogdanove, A. J., Van Wijk, K. J. and Beer, S. V. (2007a) Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels. Mol Plant Pathol, 8, 55-67. Nissinen, R. M., Ytterberg, A. J., Bogdanove, A. J., Van Wijk, K. J. and Beer, S. V. (2007b) Analyses of the secretomes of Erwinia amylovora and selected hrp mutants reveal novel type III secreted proteins and an effect of HrpJ on extracellular harpin levels. Mol. Plant Pathol., 8, 55-67. Norelli, J. L., Jones, A. L. and Aldwinckle, H. S. (2003) Fire blight management in the twenty-first century: using new technologies that enhance host resistance in apple. Plant Dis., 87, 756-765. Oh, C.-S. and Beer, S. V. (2007) AtHIPM, an ortholog of the apple HrpN-interacting protein, is a negative regulator of plant growth and mediates the growth-enhancing effect of HrpN in Arabidopsis. Plant Physiol., 145, 426-436. Oh, C.-S., Martin, G. B. and Beer, S. V. (2007) DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death. Mol. Plant Pathol., 8, 255-265. Oh, C. and Beer, S. V. (2005a) Molecular genetics of Erwinia amylovora involved in the development of fire blight. FEMS Microbiol. Lett., 253, 185-192. Oh, C. and Beer, S. V. (2005b) Molecular genetics of Erwinia amylovora involved in the development of fire blight. FEMS Microbiol Lett, 253, 185-192. Oh, C., Carpenter, S. C. D., Hayes, M. L. and Beer, S. V. (2010) Secretion and translocation signals and DspB/F-binding domains in the type III effector DspA/E of Erwinia amylovora. Microbiology, 156, 1211-1220. Okusu, H., Ma, D. and Nikaido, H. (1996) AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J. Bacteriol., 178, 306-308.  166 Omadjela, O., Narahari, A., Strumillo, J., Mélida, H., Mazur, O., Bulone, V., et al. (2013) BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis. Proc. Natl. Acad. Sci. U. S. A., 110, 17856-17861. Orr, M. W., Donaldson, G. P., Severin, G. B., Wang, J., Sintim, H. O., Waters, C. M., et al. (2015) Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover. Proc. Natl. Acad. Sci. U. S. A., 112, E5048-E5057. Padilla, E., Llobet, E., Doménech-Sánchez, A., Martínez-Martínez, L., Bengoechea, J. A. and Albertí, S. (2010) Klebsiella pneumoniae AcrAB efflux pump contributes to antimicrobial resistance and virulence. Antimicrob. Agents Chemother., 54, 177-183. Pandey, A. and Sonti, R. V. (2010) Role of the FeoB protein and siderophore in promoting virulence of Xanthomonas oryzae pv. oryzae on rice. J. Bacteriol., 192, 3187-3203. Paul, R., Weiser, S., Amiot, N. C., Chan, C., Schirmer, T., Giese, B., et al. (2004) Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev., 18, 715-727. Pérez-Mendoza, D., Coulthurst, S. J., Sanjuán, J. and Salmond, G. P. C. (2011) N-Acetylglucosamine-dependent biofilm formation in Pectobacterium atrosepticum is cryptic and activated by elevated c-di-GMP levels. Microbiology, 157, 3340-3348. Erwinia amylovora expresses fast and simultaneously hrp/dsp virulence genes during flower infection on apple trees. PLoS ONE, 7, e32583. Petnicki-Ocwieja, T., van Dijk, K. and Alfano, J. R. (2005a) The hrpK operon of Pseudomonas syringae pv. tomato DC3000 encodes two proteins secreted by the type III (Hrp) protein secretion system: HopB1 and HrpK, a putative type III translocator. J. Bacteriol., 187, 649-663. Petnicki-Ocwieja, T., van Dijk, K. and Alfano, J. R. (2005b) The hrpK operon of Pseudomonas syringae pv. tomato DC3000 encodes two proteins secreted by the type III (Hrp) protein secretion system: HopB1 and HrpK, a putative type III translocator. J Bacteriol, 187, 649-663. Petrova, O. E. and Sauer, K. (2016) Escaping the biofilm in more than one way: desorption, detachment or dispersion. Curr. Opin. Microbiol., 30, 67-78. Pfaffl, M. W., Horgan, G. W. and Dempfle, L. (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res., 30, e36. Pfeilmeier, S., Saur, I. M.-L., Rathjen, J. P., Zipfel, C. and Malone, J. G. (2016) High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity. Mol. Plant Pathol., 17, 521-531.  167 Plate, L. and Marletta, Michael A. (2012) Nitric oxide modulates bacterial biofilm formation through a multicomponent cyclic-di-GMP signaling network. Mol. Cell, 46, 449-460. Politis, D. J. and Goodman, R. N. (1980) Fine structure of extracellular polysaccharide of Erwinia amylovora. Appl. Environ. Microbiol., 40, 596-607. Pratt, J. T., Tamayo, R., Tischler, A. D. and Camilli, A. (2007) PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae. J. Biol. Chem., 282, 12860-12870. Pusey, P. L. and Curry, E. A. (2004) Temperature and pomaceous flower age related to colonization by Erwinia amylovora and antagonists. Phytopathology, 94, 901-911. Qi, Y., Rao, F., Luo, Z. and Liang, Z.-X. (2009) A flavin cofactor-binding PAS domain regulates c-di-GMP synthesis in AxDGC2 from Acetobacter xylinum. Biochemistry, 48, 10275-10285. Ramos, A. R., Morello, J. E., Ravindran, S., Deng, W.-L., Huang, H.-C. and Collmer, A. (2007) Identification of Pseudomonas syringae pv. syringae 61 type III secretion system Hrp proteins that can travel the type III pathway and contribute to the translocation of effector proteins into plant cells. J. Bacteriol., 189, 5773-5778. Raymundo, A. and Ries, S. (1981) Motility of Erwinia amylovora. Phytopathology, 71, 45-49. Reboutier, D., Frankart, C., Briand, J., Biligui, B., Rona, J.-P., Haapalainen, M., et al. (2007) Antagonistic action of harpin proteins: HrpWea from Erwinia amylovora suppresses HrpNea-induced cell death in Arabidopsis thaliana. J. Cell Sci., 120, 3271-3278. Roelofs, K. G., Wang, J., Sintim, H. O. and Lee, V. T. (2011) Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions. Proc. Natl. Acad. Sci. U. S. A., 108, 15528-15533. Römling, U. (2012) Cyclic di-GMP, an established secondary messenger still speeding up. Environ. Microbiol., 14, 1817-1829. Romling, U. and Amikam, D. (2006) Cyclic di-GMP as a second messenger. Curr. Opin. Microbiol., 9, 218-228. Römling, U. and Galperin, M. Y. (2015) Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions. Trends Microbiol., 23, 545-557. Römling, U., Galperin, M. Y. and Gomelsky, M. (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev., 77, 1-52. Romling, U., Gomelsky, M. and Galperin, M. Y. (2005) C-di-GMP: the dawning of a novel bacterial signalling system. Mol. Microbiol., 57, 629-639.  168 Ross, P., Weinhouse, H., Aloni, Y., Michaeli, D., Weinberger-Ohana, P., Mayer, R., et al. (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature, 325, 279-281. Ryan, R. P. (2013) Cyclic di-GMP signalling and the regulation of bacterial virulence. Microbiology, 159, 1286-1297. Ryan, R. P., Fouhy, Y., Lucey, J. F., Crossman, L. C., Spiro, S., He, Y.-W., et al. (2006a) Cellcell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proceedings of the National Academy of Sciences, 103, 6712-6717. Ryan, R. P., Fouhy, Y., Lucey, J. F., Crossman, L. C., Spiro, S., He, Y. W., et al. (2006b) Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc. Natl. Acad. Sci. U. S. A., 103, 6712-6717. Ryan, R. P., Fouhy, Y., Lucey, J. F., Jiang, B. L., He, Y. Q., Feng, J. X., et al. (2007) Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris. Mol. Microbiol., 63, 429-442. Ryan, R. P., Tolker-Nielsen, Tcyclic di-GMP action in bacteria. Trends Microbiol., 20, 235-242. Ryjenkov, D. A., Simm, R., Römling, U. and Gomelsky, M. (2006) The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria. J. Biol. Chem., 281, 30310-30314. Ryjenkov, D. A., Tarutina, M., Moskvin, O. V. and Gomelsky, M. (2005) Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain. J. Bacteriol., 187, 1792-1798. Sambrook, J., Fritsch, E. F. and Maniatis, T. (2001) Molecular cloning. Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY. Samudrala, R., Heffron, F. and McDermott, J. E. (2009) Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems. PLoS Pathog, 5, e1000375. Saxena, I. M., Kudlicka, K., Okuda, K. and Brown, R. M. (1994) Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization. J. Bacteriol., 176, 5735-5752. Schechter, L. M., Roberts, K. A., Jamir, Y., Alfano, J. R. and Collmer, A. (2004) Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter. J. Bacteriol., 186, 543-555.  169 Schechter, L. M., Valenta, J. C., Schneider, D. J., Collmer, A. and Sakk, E. (2012) Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae. PLoS ONE, 7, e36038. Schmidt, A. J., Ryjenkov, D. A. and Gomelsky, M. (2005) The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains. J. Bacteriol., 187, 4774-4781. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012a) NIH Image to ImageJ: 25 years of image analysis. Nat. Meth., 9, 671-675. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012b) NIH Image to ImageJ: 25 years of image analysis. Nat. Meth., 9, 671-675. Schumacher, J., Joly, N., Rappas, M., Zhang, X. and Buck, M. (2006) Structures and organisation of AAA+ enhancer binding proteins in transcriptional activation. J. Struct. Biol., 156, 190-199. Sebaihia, M., Bocsanczy, A. M., Biehl, B. S., Quail, M. A., Perna, N. T., Glasner, J. D., et al. (2010a) Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946. J Bacteriol, 192, 2020-2021. Sebaihia, M., Bocsanczy, A. M., Biehl, B. S., Quail, M. A., Perna, N. T., Glasner, J. D., et al. (2010b) Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946. J. Bacteriol., 192, 2020-2021. Serra, D. O., Richter, A. M. and Hengge, R. (2013) Cellulose as an architectural element in spatially structured Escherichia coli biofilms. J. Bacteriol., 195, 5540-5554. Silva-Herzog, E., Joseph, S. S., Avery, A. K., Coba, J. A., Wolf, K., Fields, K. A., et al. (2011) Scc1 (CP0432) and Scc4 (CP0033) function as a type III secretion chaperone for CopN of Chlamydia pneumoniae. J. Bacteriol., 193, 3490-3496. Simm, R., Morr, M., Kader, A., Nimtz, M. and Römling, U. (2004) GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol. Microbiol., 53, 1123-1134. Sjulin, T. M. and Beer, S. V. (1978) Mechanism of wilt induction by amylovorin in cotoneaster shoots and its relation to wilting of shoots infected by Erwinia amylovora. Phytopathology, 68, 89-94. Smits, T. and Duffy, B. (2011) Genomics of iron acquisition in the plant pathogen Erwinia amylovora : insights in the biosynthetic pathway of the siderophore desferrioxamine E. Arch. Microbiol., 193, 693-699. Smits, T. H. M., Rezzonico, F., Kamber, T., Blom, J., Goesmann, A., Frey, J. E., et al. (2010) Complete genome sequence of the fire blight pathogen Erwinia amylovora CFBP 1430 and comparison to other Erwinia spp. Mol. Plant-Microbe Interact., 23, 384-393.  170 Solano, C., García, B., Valle, J., Berasain, C., Ghigo, J.-M., Gamazo, C., et al. (2002) Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose. Mol. Microbiol., 43, 793-808. -di-GMP signaling. Curr. Opin. Microbiol., 15, 140-146. Srivastava, D., Harris, R. C. and Waters, C. M. (2011) Integration of cyclic di-GMP and quorum sensing in the control of vpsT and aphA in Vibrio cholerae. J. Bacteriol., 193, 6331-6341. Srivastava, D., Hsieh, M.-L., Khataokar, A., Neiditch, M. B. and Waters, C. M. (2013) Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production. Mol. Microbiol., 90, 1262-1276. Standal, R., Iversen, T. G., Coucheron, D. H., Fjaervik, E., Blatny, J. M. and Valla, S. (1994) A new gene required for cellulose production and a gene encoding cellulolytic activity in Acetobacter xylinum are colocalized with the bcs operon. J. Bacteriol., 176, 665-672. Steinberger, E. M. and Beer, S. V. (1988) Creation and complementation of pathogenicity mutants of Erwinia amylovora. Mol. Plant-Microbe Interact, 1, 135-144. Stelitano, V., Giardina, G., Paiardini, A., Castiglione, N., Cutruzzolà, F. and Rinaldo, S. (2013) C-di-GMP hydrolysis by Pseudomonas aeruginosa HD-GYP phosphodiesterases: analysis of the reaction mechanism and novel roles for pGpG. PLoS ONE, 8, e74920. -dependent transcriptional activators. J. Bacteriol., 185, 1757-1767. Stuurman, N., Bras, C. P., Schlaman, H. R. M., Wijfjes, A. H. M., Bloemberg, G. and Spaink, H. P. (2000) Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants. Mol. Plant-Microbe Interact., 13, 1163-1169. Sudarsan, N., Lee, E. R., Weinberg, Z., Moy, R. H., Kim, J. N., Link, K. H., et al. (2008) Riboswitches in Eubacteria sense the second messenger cyclic di-GMP. Science, 321, 411-413. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. Thomson, S. (1986) The role of the stigma in fire blight infections. Phytopathology, 76, 476-482. Thomson, S. V. (2000) Epidemiology of Fire Blight In: Fire blight: the disease and its causative agent, Erwinia amylovora. (Vanneste, J., ed.). New York: CABI publishing, pp. 9 - 36. Triplett, L. R., Melotto, M. and Sundin, G. W. (2009a) Functional analysis of the N terminus of the Erwinia amylovora secreted effector DspA/E reveals features required for secretion,  171 translocation, and binding to the chaperone DspB/F. Mol Plant Microbe Interact, 22, 1282-1292. Triplett, L. R., Melotto, M. and Sundin, G. W. (2009b) Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F. Mol. Plant-Microbe Interact., 22, 1282-1292. Triplett, L. R., Wedemeyer, W. J. and Sundin, G. W. (2010a) Homology-based modeling of the Erwinia amylovora type III secretion chaperone DspF used to identify amino acids required for virulence and interaction with the effector DspE. Res. Microbiol., 161, 613-618. Triplett, L. R., Wedemeyer, W. J. and Sundin, G. W. (2010b) Homology-based modeling of the Erwinia amylovora type III secretion chaperone DspF used to identify amino acids required for virulence and interaction with the effector DspE. Res Microbiol, 161, 613-618. Tuckerman, J. R., Gonzalez, G. and Gilles-Gonzalez, M.-A. (2011) Cyclic di-GMP activation of polynucleotide phosphorylase signal-dependent RNA processing. J. Mol. Biol., 407, 633-639. Tuckerman, J. R., Gonzalez, G., Sousa, E. H. S., Wan, X., Saito, J. A., Alam, M., et al. (2009) An oxygen-sensing diguanylate cyclase and phosphodiesterase couple for c-di-GMP control. Biochemistry, 48, 9764-9774. Tyson, G. E., Stojanovic, B., Kuklinski, R. F., Divittoria, T. and Sullivan, M. L. (1985) Scanning electron microscopy of Pierce's disease bacterium in petiolar xylem of grape leaves. Phytopathology, 75, 264-269. Van der Zwet, T. (2006) Present worldwide distribution of fire blight and closely related diseases. Acta Horticulturae, 704, 35-36. Vanneste, J. (2000) What is fire blight? Who is Erwinia amylovora? How to control it? In: Fire blight: the disease and its causative agent, Erwinia amylovora. (Vanneste, J., ed.). New York: CABI Publishing, pp. 1-6. Vanneste, J. L. (1995) Erwinia amylovora. In: Pathogenesis and host specificity in plant diseases: histopathological, biochemical, genetic and molecular bases. (Singh, U. S., Singh, R. P. and Kohmoto, K., eds.). Oxford and London: Pergammon Press. Venisse, J.-S., Barny, M.-A., Paulin, J.-P. and Brisset, M.-N. (2003) Involvement of three pathogenicity factors of Erwinia amylovora in the oxidative stress associated with compatible interaction in pear. FEBS Lett., 537, 198-202. Wang, D., Clough, S., Zhao, Y., Korban, S., Sundin, G. and Toth, I. (2011) Regulatory genes and environmental regulation of amylovoran biosynthesis in Erwinia amylovora. Acta Horticulturae, 896, 195-202.  172 Wang, D., Korban, S. S., Pusey, P. L. and Zhao, Y. (2012) AmyR is a novel negative regulator of amylovoran production in Erwinia amylovora. PLoS ONE, 7, e45038. Wang, D., Korban, S. S. and Zhao, Y. (2009) The Rcs phosphorelay system is essential for pathogenicity in Erwinia amylovora. Mol. Plant Pathol., 10, 277-290. Wang, D., Korban, S. S. and Zhao, Y. (2010) Molecular signature of differential virulence in natural isolates of Erwinia amylovora. Phytopathology, 100, 192-198. Waters, C. M., Lu, W., Rabinowitz, J. D. and Bassler, B. L. (2008) Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cyclic di-GMP levels and repression of vpsT. J. Bacteriol., 190, 2527-2536. Wei, H.-L. and Collmer, A. (2012a) Multiple lessons from the multiple functions of a regulator of type III secretion system assembly in the plant pathogen Pseudomonas syringae. Mol. Microbiol., 85, 195-200. Wei, H.-L. and Collmer, A. (2012b) Multiple lessons from the multiple functions of a regulator of type III secretion system assembly in the plant pathogen Pseudomonas syringae. Mol Microbiol, doi: 10.1111/j.1365-2958.2012.08119.x. Wei, Z., Kim, J. F. and Beer, S. V. (2000) Regulation of hrp genes and type III protein secretion in Erwinia amylovora by HrpX/HrpY, a novel two-component system, and HrpS. Mol. Plant-Microbe Interact., 13, 1251-1262. Wei, Z., Laby, R., Zumoff, C., Bauer, D., He, S., Collmer, A., et al. (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science, 257, 85-88. Wei, Z. M. and Beer, S. V. (1995) hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors. J. Bacteriol., 177, 6201-6210. Whitney, J. C. and Howell, P. L. (2013) Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria. Trends Microbiol., 21, 63-72. Wilksch, J. J., Yang, J., Clements, A., Gabbe, J. L., Short, K. R., Cao, H., et al. (2011) MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression. PLoS Pathog, 7, e1002204. Wilson, M., Epton, H. A. S. and Sigee, D. C. (1989) Erwinia amylovora infection of hawthorn blossom. II The stigma. Journal of Phytopathology, 127, 15-28. Wilson, M., Sigee, D. C. and Epton, H. A. S. (1990) Erwinia amylovora Infection of Hawthorn Blossom: III. The Nectary. Journal of Phytopathology, 128, 62-74. Wolfe, A. J. and Visick, K. L. (2008) Get the message out: cyclic di-GMP regulates multiple levels of flagellum-based motility. J. Bacteriol., 190, 463-475.  173 Xu, J., Kim, J., Koestler, B. J., Choi, J.-H., Waters, C. M. and Fuqua, C. (2013) Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile-to-sessile switch. Mol. Microbiol., 89, 929-948. Yang, F., Qian, S., Tian, F., Chen, H., Hutchins, W., Yang, C. H., et al. (2016) The GGDEF-domain protein GdpX1 attenuates motility, exopolysaccharide production and virulence in Xanthomonas oryzae pv. oryzae. J. Appl. Microbiol., DOI: 10.1111/jam.13115. Yang, F., Tian, F., Li, X., Fan, S., Chen, H., Wu, M., et al. (2014) The degenerate EAL-GGDEF domain protein Filp functions as a cyclic di-GMP receptor and specifically interacts with the PilZ-domain protein PXO_02715 to regulate virulence in Xanthomonas oryzae pv. oryzae. Mol. Plant-Microbe Interact., 27, 578-589. Yap, M.-N., Yang, C.-H., Barak, J. D., Jahn, C. E. and Charkowski, A. O. (2005) The Erwinia chrysanthemi type III secretion system Is required for multicellular behavior. J. Bacteriol., 187, 639-648. Yi, X., Yamazaki, A., Biddle, E., Zeng, Q. and Yang, C.-H. (2010) Genetic analysis of two phosphodiesterases reveals cyclic diguanylate regulation of virulence factors in Dickeya dadantii. Mol. Microbiol., 77, 787-800. Zhang, Y. and Geider, K. (1999) Molecular analysis of the rlsA gene regulating levan production by the fireblight pathogen Erwinia amylovora. Physiol. Mol. Plant Pathol., 54, 187-201. Zhao, Y., Blumer, S. E. and Sundin, G. W. (2005a) Identification of Erwinia amylovora genes induced during infection of immature pear tissue. J Bacteriol, 187, 8088-8103. Zhao, Y., Blumer, S. E. and Sundin, G. W. (2005b) Identification of Erwinia amylovora genes induced during infection of immature pear tissue. J. Bacteriol., 187, 8088-8103. Zhao, Y., He, S.-Y. and Sundin, G. W. (2006) The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in Pseudomonas syringae. Mol. Plant-Microbe Interact., 19, 644-654. Zhao, Y., Qi, M. and Wang, D. (2011) Evolution and function of flagellar and non-flagellar type III secretion systems in Erwinia amylovora. Acta Horticulturae, 896, 177-184. Zhao, Y., Sundin, G. W. and Wang, D. (2009a) Construction and analysis of pathogenicity island deletion mutants of Erwinia amylovora. Can. J. Microbiol., 55, 457-464. Zhao, Y., Sundin, G. W. and Wang, D. (2009b) Construction and analysis of pathogenicity island deletion mutants of Erwinia amylovora. Can. J. Microbiol., 55, 457-464. Zhao, Y., Wang, D., Nakka, S., Sundin, G. and Korban, S. (2009c) Systems level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC Genomics, 10, 245.  174 Zogaj, X., Nimtz, M., Rohde, M., Bokranz, W. and Römling, U. (2001) The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix. Mol. Microbiol., 39, 1452-1463. Zorraquino, V., García, B., Latasa, C., Echeverz, M., Toledo-Arana, A., Valle, J., et al. (2013) Coordinated cyclic-di-GMP repression of Salmonella motility through YcgR and cellulose. J. Bacteriol., 195, 417-428.