GENOMICS AND EFFECTOROMICS OF XANTHOMONADS

Transcription

1 GENOMICS AND EFFECTOROMICS OF XANTHOMONADS By NEHA POTNIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

2 2011 Neha Potnis 2

3 To my husband, Deepak, and my parents for their unconditional love and support 3

4 ACKNOWLEDGMENTS I would like to express my gratitude to Dr. Jeffrey B. Jones, my committee chair for his constant support and encouragement. I am thankful to him for sharing his expertise and his ideas at every step during this project. I would also like to thank my co-chair, Dr. David Norman for his guidance and financial support during my graduate studies. I would also like to extend my gratitude to my committee members, Dr. Boris Vinatzer, Dr. Jim Preston, and Dr. Jeffrey Rollins for their valuable suggestions in my project and support. I really appreciate valuable guidance from Dr. Robert Stall. I would like to thank Jerry Minsavage for technical help during the experiments, helpful suggestions and constructive criticism. Virginia Chow contributed to the identification of genes encoding glycohydrolases involved in cell wall deconstruction and their respective genome organizations. During research work, I collaborated with Dr. Frank White, Dr. Ralf Koebnik, Dr. Brian Staskawicz, and Dr. Joao Setubal to write research articles and reviews. I would like to thank them all for giving me the opportunity. I thank my labmates Jose Figueiredo, Franklin Behlau, Jason Hong, Mine Hantal, and Hu Yang for co-operation and assistance and for making the lab, a pleasant place, to work. I would also like to thank faculty and staff of the Plant Pathology department. I am grateful to my Indian friends here in Gainesville for their support and lively company during my stay here. I warmly thank my loving husband, Deepak, who has been supportive throughout my PhD, with all his love and encouragement. My heartfelt thanks go to my parents for supporting my decision to fly here away from them, who have been so caring and loving. They helped me to shape my career and always guided me at every step in my life. Thank you all for making this possible. 4

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES... 9 LIST OF FIGURES ABSTRACT CHAPTER 1 XANTHOMONAS-PLANT INTERACTIONS AND GENOMICS Background Type III Secreted Effectors and Their Role in Plant-Pathogen Interactions Avirulence Genes Contributions of Comparative Genomics Era Project Goal and Objectives COMPARATIVE GENOMICS REVEALS DIVERSITY AMONG XANTHOMONADS INFECTING TOMATO AND PEPPER Background Materials and Methods Genome Sequencing Assembly and Annotation Whole Genome Comparisons Phylogenetic Analysis Phylogeny Reconstruction Prediction of Effector Repertoires, Cloning of Candidate Effectors and Confirmation Using AvrBs2 Reporter Gene Assay Cloning of Pepper Specificity Genes in Xp Results Draft Genome Sequences of Xv Strain 1111, Xp Strain and Xg Strain 101 were Obtained by Combining Roche-454 (Pyrosequencing) and Illumina GA2 (Solexa) Sequencing Data Relationships of the Strains to Other Xanthomonads using Whole Genome Comparisons Four Xanthomonads Show Variation in the Organization of the Type III Secretion Gene Clusters A Reporter Gene Assay Confirms Translocation of Novel Type III Effectors Core Effectors among Four Xanthomonads Give Insights into Infection Strategies of the Pathogen

6 Effectors Unique to Xp Might be Responsible for Restricting Growth on Pepper Species-Specific Effectors Few Effectors are Shared among Phylogenetically Related Group Strains Xg Shows Evidence of Effector Acquisition by Horizontal Gene Transfer All Four Xanthomonads Contain Ax21 Coding Gene but Only Xcv Contains a Functional Sulfation Gene Two Type II Secretion Systems are Conserved in All Four Xanthomonas Genomes Xanthomonads Possess Diverse Repertoires of Cell-Wall Degrading Enzymes, which are Present in Diverse Genomic Arrangement Patterns Genes Involved in Several Type IV Secretion Systems are Present in Genomes and Plasmids Type V Secreted Adhesins Function in Synergism During Pathogenesis Type VI Secretion System is Present in Xcv, Xv and Xp LPS Locus Displays Remarkable Variation In Sequence and Number of Coding Genes and Shows Host Specific Variation Analysis of DSF Cell-Cell Signaling System Cyclic Di-GMP Signaling Copper Resistance (cop) Genes are Present in Xv and Copper Homeostasis (coh) Genes are Present in All Strains Genes Unique to Xp as Compared to Pepper Pathogens Give Clues to its Predominance over Xcv in the Field and Host Specificity Pepper Pathogenicity/Aggressiveness Factors Increased In Planta Growth of Xp Genes Specific to Xg as Compared to Other Tomato/Pepper Pathogens may Explain its Aggressive Nature on Tomato and Pepper Genes Common to All Tomato Pathogens but Absent from Other Sequenced Xanthomonads The Evolution of Pathogenicity Clusters Corresponds to the MLST-Based Phylogeny Concluding Remarks AVIRULENCE PROTEINS AVRBS7 FROM XANTHOMONAS GARDNERI AND AVRBS1.1 FROM XANTHOMONAS EUVESICATORIA ELICIT HYPERSENSITIVE RESISTANCE RESPONSE IN PEPPER Background Materials and Methods Plant Material and Plant Inoculations Bacterial Strains, Plasmids and Media Library Preparation and Isolation of Clone with Avirulence Activity Deletion Mutant Construction Bacterial Population Dynamics in Infiltrated Leaf Tissue Determination of Electrolyte Leakage from Infiltrated Leaf Tissue Site Directed Mutagenesis of avrbs Sequence Analysis and Protein Homology Modeling

7 Results Identification of Resistance in Pepper against Bacterial Spot Xanthomonads and Development of Introgression Lines Carrying the Resistance Gene AvrBs7 from Xv444 Elicits HR in Pepper cv. ECW-70R AvrBs1.1 from Xcv str Elicits Delayed HR on ECW-70R Genetic Analysis of Bs7 Resistance in ECW-70R In-Planta Growth Studies and Electrolyte Leakage A Catalytic Tyrosine Phosphatase Domain Might be Responsible for Recognition by the BS7 R Gene Product in ECW-70R There is Difference in the Timings of HR Elicitation by AvrBs7 and AvrBs Avirulence Proteins AvrBs7 and AvrBs1.1 Display Similar Tertiary Protein Structure Host Specificity of Bacterial Spot Strains Avr Genes avrbs7 and avrbs1.1 are Encoded on a Large Transmissible Plasmid Concluding Remarks APPLICATION OF BIOINFORMATICS FOR TYPE III EFFECTOR SIGNAL ANALYSIS AND ITS INTERACTION WITH CHAPERONE Background Materials and Methods Data-Mining Strategy Bacterial Strains, Plasmids and Media Plant Material and Plant Inoculations In Planta Reporter Gene Assay Site-Directed Alanine Mutagenesis Yeast Two-Hybrid Assay In Vitro Pull Down Assay Results General Characteristics of Secretion and Translocation Signals in N Terminal Region of Xanthomonas Type III Effectors Screening Whole Genomes for Candidate Type III Effectors First 70 Amino Acids of XopF1 are Sufficient for Translocation into the Plant Cell Type III Effector XopF1 is Dependent on Global Chaperone HpaB for its Translocation First 40 Amino Acids of XopF1 are not enough for Translocation into Plant Cells Secondary Structure Analysis of XopF1 Effector Alanine Mutagenesis in Alpha Helix Regions Abolished HR of the Effector- Reporter Fusion Complex Yeast Two-Hybrid Assay In Vitro Pull Down Assay Concluding Remarks

8 5 PATHOGENIC STRATEGIES OF XANTHOMONAS GENUS ON PLANTS: LESSONS LEARNT FROM GENOMICS Background Materials and Methods Xanthomonas Genomes and Tools Used for Comparison Database for Xanthomonas Pathogenicity Factors Effectors Database Compilation Effector Analysis of the Test Case of Citrus Pathogens Results Type II Secretion Systems Type III Secretion System Type III-Secreted Effectors A CaseStudy Screening for Candidate Type III Effectors from Draft Genomes and Possible Host Range Determinants The three citrus canker genomes have important differences in regard to their repertoires of type III secreted effectors Effectors XopAI and XopE3 may play a role in citrus canker Additional differences in effector repertoires among CC genomes Adhesins Lipopolysaccharides and Xanthan Toxins Concluding Remarks SUMMARY AND DISCUSSION LIST OF REFERENCES BIOGRAPHICAL SKETCH

9 LIST OF TABLES Table page 2-1 General sequencing and combined (454 and solexa) de novo assembly features of draft genomes of Xv, Xp and Xg Whole genome comparisons using MUMmer dnadiff program Core effectors present in all four tomato and pepper xanthomonads Type III effectors specific to each species Effectors specific to particular groups of species Evidence of horizontal gene transfer using Alien Hunter analysis Repertoire of cell wall degrading enzymes in xanthomonads Type VI secretion clusters in different xanthomonads Genes/contigs representing T6SS in draft genomes as compared to Xcv A comparison of rpf cluster from rpfb to rpfg found across a range of Xanthomonas genomes Genes unique to Xp, grouped in clusters Genes common to all pepper pathogens but absent from Xp Genes present in all four tomato and pepper pathogens but absent from all other sequenced xanthomonads List of bacterial strains and plasmids used in this study List of bacterial strains and plasmids used in this study Xanthomonas species and pathovars within species show host and tissuespecificity Xop nomenclature for xanthomonas effectors Core effector genes from xanthomonads and their role in pathogenicity/ induction of resistance Variable effectors which contribute to the pathogenicity Putative effectors found in the XAC, XauB, and XauC genome sequences

10 LIST OF FIGURES Figure page 2-1 Maximum likelihood tree based on orthologous genes from xanthomonads and Stenotrophomonas Comparison of type III secretion system cluster, its associated type III effector genes and helper genes of three draft genomes with already sequenced xanthomonads AvrBs2-based HR assay confirms translocation of novel effectors Xylanase cluster organization Schematic representation of type IV secretion system cluster common to Xp, Xv and Xg (Plasmid borne) Schematic representation of type IV secretion cluster unique to Xg (plasmid borne) Schematic representation of chromosomal type IV cluster organization in Xcv, Xv, Xp and Xg The Structure and phylogeny of the LPS cluster Pepper specificity genes increasing in planta growth of Xp Correlation between phylogenies based on Multi-Locus Sequence Typing (MLST) core genome and pathogenicity clusters Phenotype observed in leaves of ECW-70R 48 hr after infiltration with bacterial suspesions (adjusted to 10 8 cfu/ml) Phenotype on ECW-70R 24 hr and 48 hr post-infiltration by wild type strains, transconjugants and mutants Time course of bacterial population growth after infiltration of leaves of pepper genotypes ECW and ECW-70R with suspensions of Xg51 transconjugants and mutant strains Electrolyte leakage from pepper genotypes ECW-70R (A and C) and ECW (B and D) after infiltration of leaves with suspensions adjusted to 10 8 cfu/ml of (Xg51) wild type, transconjugants and mutant strains Tyrosine phosphatase domain is essential for HR elicitation on ECW-70R Alignment of avrbs1.1 and avrbs7 amino acid sequences using clustalw

11 3-7 Fusion protein containing N-terminal of avrbs7 and C terminal of avrbs1.1 does not elicit HR on ECW-70R Three dimensional structures of the two avirulence proteins based on homology modeling Phenotype on ECW-20R 24 hr post-infiltration by wild type strains and transconjugants Phenotype on ECW-20R 24 hr post-infiltration by wild type strains, transconjugants, and mutants Phenotype on ECW-20R 24 hr post-infiltration by wild type strains, transconjugants and alanine mutants Secondary structure prediction by PsiPred for first 70 amino acid region of XopF1. Cylinder represents predicted alpha helix Secondary structure prediction by garnier for first 70 amino acid region of XopF1. H indicates alpha helix Yeast two hybrid interaction between alanine mutants of XopF and HpaB chaperone In vitro pull down assay showing binding of HpaB chaperone to XopF1 variants

12 Chair: Jeffrey B. Jones Cochair: David J. Norman Major: Plant Pathology Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GENOMICS AND EFFECTOROMICS OF XANTHOMONADS By Neha Potnis August 2011 Bacterial spot disease is a major concern for tomato and pepper growers. There are four species of xanthomonads associated with this disease classified into four distinct genotypic groups, Xanthomonas euvesicatoria, Xanthomonas vesicatoria, Xanthomonas perforans and Xanthomonas gardneri. Various disease control strategies have been used including application of copper bactericides, antibiotics, biocontrol methods such as phage therapy, and breeding for disease resistance. We are approaching the issue from two perspectives. The first approach includes identifying virulence factors from different species of bacterial spot xanthomonads and studying their role in the disease development. A strain of Xanthomonas euvesicatoria (Xcv str ) has already been sequenced. We have sequenced representatives of the other three species. A comparative genomic analysis has enlightened the commonalities and differences in virulence factors among the four species and has provided possible clues to the understanding of host range specificity and aggressiveness of strains. Important pathogenicity factors of xanthomonads are type III effectors. We have also developed a program to identify these effectors from the draft genomes using computational methods. The regulation of type III effectors during pathogenesis is achieved with the 12

13 help of chaperones. We have focused our studies on effector-chaperone interactions and the role of these interactions during disease development. The knowledge gained from comparative analysis is expected to aid in better understanding of host-pathogen interactions and devising durable control strategies. The second approach consisted of searching for disease resistance genes. We found a new source of resistance in pepper to Xanthomonas gardneri and Xanthomonas euvesicatoria. Genetic segregation analysis indicated the monogenic nature of resistance, confirming a new gene-for-gene interaction in addition to five already characterized interactions in pepper. Characterization of avirulence genes avrbs7 and avrbs1.1 from the two xanthomonads indicated presence of a tyrosine phosphatase domain, which might be important for eliciting the resistance response in pepper. Further efforts will be directed towards understanding the mechanism of the resistance and the importance of domains from avirulence genes in pathogen virulence. 13

14 CHAPTER 1 XANTHOMONAS-PLANT INTERACTIONS AND GENOMICS Background Xanthomonads belong to a genus representing around 27 species infecting more than 400 plant species including dicots and monocots. Many of them exhibit tissue specificity, colonizing either xylem vessels or mesophyll apoplasts of the host. A combination of virulence or pathogenicity factors is used by xanthomonads during infection of plants. Co-ordinated expression of these virulence factors is governed by a set of regulatory genes. Xanthomonads persist as epiphytes before the entry into the plant surfaces via natural openings. Type V-secreted multiple adhesins play an important role during the adhesion, entry and colonization process. Multiple twocomponent signaling cascades are then activated which, in turn, lead to the activation of different secretion systems and the release of virulence factors (Qian et al. 2008). Successful infection, establishment and survival of a pathogen depend on different virulence factors secreted and translocated by different secretion systems, which allow the pathogen to multiply and avoid host defense responses. Type III Secreted Effectors and Their Role in Plant-Pathogen Interactions Among virulence factors, type III secreted effectors are the major contributors to pathogenicity in most Gram-negative pathogens. Type III secretion systems enable pathogens to transport their effector proteins inside the host plant cells upon induction by plant-derived signals (Alfano and Collmer 2004). Type III secreted effector proteins (T3SEs) are involved in virulence by modulating and suppressing host defense responses (White et al. 2009). Each strain possesses its repertoire of T3SEs, which determine the compatibility and subsequent patterns of pathogen growth. The diversity 14

15 in tissue and host specificities of members of the genus is also reflected in the diversity of T3SEs between pathovars and species. Many Xanthomonas T3SEs are known to function to suppress pathogen molecular associated pattern (PAMP) triggered immune (PTI) responses during the early stages of infection (Metz et al. 2005; Hotson et al. 2003; Kim et al. 2008; Kim et al. 2009). XopX from X. euvesicatoria was found to increase the susceptibility of Nicotiana benthamiana to both Xanthomonas and Pseudomonas species (Metz et al. 2005). The T3SEs XopN and XopD have also been shown to reduce PTI and to affect host developmental pathways that may play a role in host defense (Kim et al. 2008; Kim et al. 2009). XopD possesses cysteine protease activity and mimics endogenous plant SUMO isopeptidases. It targets SUMO-conjugated proteins by hydrolyzing them and disrupts the regulation of SUMO mediated pathways (Kim et al. 2008). XopN is another virulence factor on tomato that suppresses defense-related gene expression and callose deposition in tomato. XopN interacts with the cytoplasmic domain of TARK1 and 4 along with tomato isoforms (Kim et al. 2009). Effector XopJ suppresses defenserelated callose deposition and host protein extracellular secretion (Bartetzko et al. 2009). Another XopJ family member, avrrxv, interacts with host protein (Whalen et al. 2008) and is also hypothesized to bind to MAPKs and to interfere with the signaling cascade. Some T3SEs are known to be recognized by host defense surveillance systems. Such effectors elicit a rapid hypersensitive reaction (HR) and effector-triggered immuninty (ETI). T3SEs are also known to suppress R gene mediated defense, which has also been called effector-triggered immunity (ETI) to distinguish ETI from PTI, and 15

16 some evidence suggests that some T3-effectors of Xanthomonas are involved in ETI suppression (Rosebrock et al. 2007). AvrBsT, a member of the XopJ/YopJ family, triggers an HR in a single landrace of Arabidopsis with a recessive mutation in a gene for carboxylesterase, which was named SUPPRESSOR OF AVRBST-ELICITED RESISTANCE1 (SOBER1). A model has been proposed that, in the absence of the esterase activity, AvrBsT can suppress recognition by a host R-gene product (Cunnac et al. 2007). Mutations of individual effector genes do not always result in a change in virulence, which might implicate redundancy of effectors (Castaneda et al. 2005). Some effectors from xanthomonads have been characterized for their contribution towards virulence on their respective hosts. AvrBs2, which is a core type III effector in xanthomonads, has been shown to be a virulence factor only in X. campestris pv. vesicatoria (Kearney and Staskawicz 1990). TAL (Transcription Activator-Like) effectors constitute a major family, members of which are known to impart virulence and are responsible for disease symptoms such as in citrus canker. PthA, of X. citri pv. citri is required for increase in bacterial populations during canker progression and for hypertrophy (Swarup et al. 1991). A similar symptom was associated with the AvrBs3 from Xcv in pepper (Marois et al. 2002). In rice, TAL effectors are responsible for symptoms such as chlorosis and watersoaking (Yang and White 2004). PthA has been shown to interact with the citrus proteins involved in ubiquitination, DNA repair in addition to already characterized interaction with α-importin (Domingues et al. 2010). Another effector belonging to the AvrBs3 family, AvrHah1 from X. gardneri is shown to contribute to increased watersoaking and necrosis on pepper (Schornack et al. 2008). 16

17 In addition to the already mentioned XopN and XopD effectors, other effectors known to contribute to virulence and disease symptoms include XopX (Metz et al. 2005), XopAE (Kim et al. 2003), and XopAH (AvrXccC, Wang et al. 2007). Avirulence Genes Flor s gene-for gene model (Flor 1971) proposes presence of avirulence (avr) genes from pathogens interacting with the corresponding plant resistance (R) genes. However, the nature of the avr gene was not known until the cloning of the first avr gene avra from a soybean pathogen Pseudomonas syringae pv. glycinea race 6 (Staskawicz et al. 1984). This gene, when transferred to other races of P. syringae pv. glycinea, conferred resistance against soybean cultivars containing the Rpg2 resistance gene. Another type of avr gene, called heterologous avr genes was identified after interspecies or inter-pathovar transfer. An example of a heterologous avr gene is avrrxv from X. campestris pv. vesicatoria, which when introduced into pathogenic strains of X. campestris pv. phaseoli elicited resistance on beans (Whalen et al. 1988). Although heterologous avr genes contribute to the host range of pathogens, they can not be said to be host range determinants since the strain of origin of a heterologous avr gene does not become virulent on the host carrying the corresponding R gene if devoid of the avr gene. e.g. an avrrxv deletion mutant of Xcv does not become pathogenic on beans (Whalen et al. 1988). Later, most avr proteins were shown to be secreted by the type III secretion systems, hence are type III effector proteins. Plants have evolved R genes in response to a few effector genes to recognize them and elicit R-gene mediated HR. The race continues and the pathogen then modifies its effector gene set in order to avoid this recognition. Evolution of the pathogen acts as strong selection pressure on the host. 17

18 Since these effector genes come in direct contact with the host environment, they are under strong selective pressure and hence subjected to rapid evolution in an attempt to avoid recognition by host defenses (Stavrinides et al. 2006). Avr genes show signs of horizontal gene transfer. Some avr genes, e.g. avrbs3 family genes, are located on plasmids. A few avr genes, e.g. avrb from P. syringae, belong to a region of low GC content (Tamaki et al. 1988). Few of the avr genes, e.g. avrxv3, are flanked by IS elements (Astua-Monge et al. 2000a). These signs indicate their role in genetic variation contributing to the evolution of the pathogen. Few avirulence genes from xanthomonads have been characterized for the presence of biochemical motifs or functions, which might give clues to their mechanism of eliciting resistance. According to Swords et al. (1996), AvrBs2 might have enzymatic function due to its similarity to agrocinopine synthetase from Agrobacterium tumefaciens. Kearney and Staskawicz (1990) suggested that AvrBs2 had a dual function eliciting resistance in Bs2 carrying pepper plants and promoting virulence on pepper lacking Bs2. AvrBs3 possesses a DNA binding domain, nuclear localization domain and transcriptional activator domain (Van den Ackerveken et al. 1996) and is shown to act as transcriptional activator by binding to the promoter of upa20 (upregulated by AvrBs3), a cell size regulator in susceptible pepper, and in turn induce hypertrophy (Kay et al. 2007). Interestingly there is also a upa box in the promoter of Bs3 resistance gene in resistant peppers (Romer et al. 2007). AvrXv4, AvrRxv, AvrBsT contain acetyl transferase and C55 cysteine ubiquitin-like protease domain (White et al. 2009). AvrRxv has been shown to interact with a regulatory eukaryotic protein called protein and induce cell death response (Whalen et al. 2008). Understanding R-Avr 18

19 gene interactions and the role of avr genes in virulence and resistance can assist in screening for broad and durable resistance. Contributions of Comparative Genomics Era To date nearly 10 complete genome sequences of xanthomonads are available, 9 draft genome sequences have also been published. The complete genomes were sequenced using the Sanger sequencing method. These genomes belong to different species of xanthomonads and provide reference sequences for the new draft genome sequences. With the advent of next generation sequencing methods such as 454 pyrosequencing, solexa/illumina, and SOLiD sequencing, draft genome sequencing has become cost effective and time saving. This next generation or second-generation sequencing has resulted in a quantum leap in the availability of raw genomic data (Fuller et al. 2009). Yet genomic regions that are exceptionally rich in G+C are still obstacles for second-generation sequencing and may lead to gaps when the results of individual sequencing reactions are assembled to reconstruct the complete genomic sequence. Another hazard complicating the assembly process is repetitive sequences, such as insertion sequence (IS) elements, which seem to be present in all xanthomonads, and presence of TAL effectors, which contain repetitive elements, which are not easy to assemble. As a result, next generation sequencing in xanthomonads results in draft genomes with the assembled sequence contigs disrupted by gaps. To complete the draft genomes to finished genomes further requires Sanger sequencing for gap closure and sequence polishing reactions, such as cloning constructs like BACs or fosmids or PCR products. The increasing scale of genomics provides rapid means for identifying virulence/pathogenicity factors and to generate of new hypotheses to explain the 19

20 complexities of host-pathogen interactions. Comparative genomics has raised a number of questions as to how diverse species evolved diverse host range and tissue specificities, as to the role of type III effectors, regulation of pathogenicity factors, and molecular and evolutionary mechanisms driving evolution of genomes. Comparative genomics has now given rise to the omics field, focusing on functional aspects of the genes involved in plant-pathogen interactions. Project Goal and Objectives The aim of this project was to study diversity among xanthomonads with respect to their pathogenicity factors with a special focus on type III effectors using comparative genomics tools. Comparative genomics has provided different hypotheses regarding the role of certain pathogenicity factors during infection, in the host specificity. We have experimentally verified the role of several genes. This study will give insight into the pathogenicity and virulence strategies used by pathogen during infection, which should help design new control strategies. In this study, the four objectives were: I) Comparative genomics of xanthomonads infecting tomato and pepper; II) Isolation and characterization of an avirulence gene corresponding to the R gene from a pepper genotype; III) Application of bioinformatics tools to the identification of type III effectors; and identification and characterization of chaperone HpaB-binding site in type III effectors of xanthomonads; and IV) Comparative genomics and study of pathogenicity factors from all xanthomonads. 20

21 CHAPTER 2 COMPARATIVE GENOMICS REVEALS DIVERSITY AMONG XANTHOMONADS INFECTING TOMATO AND PEPPER 1 Background Bacterial spot disease of tomato and pepper presents a serious agricultural problem worldwide, leading to significant crop losses especially in regions with a warm and humid climate. The disease is characterized by necrotic lesions on leaves, sepals and fruits, reducing yield and fruit quality (Pohronezny and Volin 1983). The disease is caused by a relatively diverse set of bacterial strains within the genus Xanthomonas; strain nomenclature and classification for the strains that infect pepper and tomato have gone through considerable taxonomic revision in recent years. Currently, the pathogens are classified into four distinct pathogen groups (A, B, C, and D) within the genus Xanthomonas. Strains belonging to groups A, B and D infect both tomato and pepper. Group C strains are pathogenic only on tomato (Jones et al. 1998b; Jones et al. 2000). These phenotypically and genotypically distinct strains have different geographic distributions. Strains of group A and B are found worldwide. C strains have been increasingly found in the U.S., Mexico, Brazil, Korea and regions bordering the Indian Ocean, and D group strains are found in the former Yugoslavia, Canada, Costa Rica, U.S., Brazil and regions of the Indian Ocean (Bouzar et al. 1996; Bouzar et al. 1999; Kim et al. 2010; Hamza et al. 2010; Myung et al. 2009). Three of the four groups except for D were originally described as a single pathovar within Xanthomonas campestris and referred to as X. campestris pv. vesicatoria. The D group consisted of a strain isolated from tomato that had been designated Pseudomonas gardneri for many years 1 Reprinted with permission from Potnis et al

22 (Sutic 1957) although De Ley provided evidence for placement in the genus Xanthomonas (De Ley 1978). Subsequently all four groups were classified as separate species on the basis of physiological and molecular characteristics as follows: Xanthomonas euvesicatoria (group A), Xanthomonas vesicatoria (group B), Xanthomonas perforans (group C), and Xanthomonas gardneri (group D) (Jones et al. 2004). Based on 16S rrna analysis, X. euvesicatoria str (A group) and X. perforans (C group) together form a monophyletic group, whereas X. vesicatoria (B group) and X. gardneri (D group) cluster together with X. campestris pv. campestris (Xcc) Xcc str (Jones et al. 2004). Recently, a phylogenetic tree was constructed based on MLST (multi-locus sequence typing) data for A, B, C and D group strains and other xanthomonads (Almeida et al. 2010). The MLST approach revealed that X. euvesicatoria and X. perforans form a group along with X. citri str X. gardneri is most closely related to X. campestris pv. campestris strains while X. vesicatoria forms a distinct clade (Almeida et al. 2010). This diversity among the four groups makes the Xanthomonas-tomato/pepper system an excellent example to study pathogen coevolution, as distinct species have converged on a common host. While integrated management approaches for control of bacterial spot disease are available, the development of host resistance is more economical and environmentally benign for the control of the disease (Obradovic et al. 2004; Louws et al. 2001). Host resistance may also be required to replace the loss of some integrated management tools. Use of copper and streptomycin sprays over the years, for example, has led to the development of resistant strains (Bouzar et al. 1999). At the same time, genetic 22

23 resistance has been lost due to race shifts in pathogen populations (Kearney et al. 1990; Gassmann et al. 2000; Stall et al. 2009). Designing new and possibly durable resistance requires knowledge of pathogenicity factors possessed by the four groups. Many candidate pathogenicity factors have been identified in strains of Xanthomonas. A number of virulence factors are employed by xanthomonads to gain entry into leaf or fruit tissue, and gain access to nutrients, while simultaneously overcoming or suppressing plant defenses. Different secretion systems and their effectors have been shown to contribute to the virulence of plant pathogens. The type III secretion system (T3SS) encoded by the hrp (Hypersensitive Response and Pathogenicity) gene cluster (Bonas et al. 1991; Kim et al. 2003) and type III secreted effectors have been widely studied for their role in hypersensitivity and pathogenicity. Effectors common between strains are believed to be responsible for conserved virulence function and avoidance of host defense. Differences in effector suites have evolved in closely related strains of plant pathogens and strain-specific effectors may help to escape recognition by host-specific defenses (Nimura et al. 2005; Grant et al. 2006; Sarkar et al. 2006; Rohmer et al. 2004; White et al. 2009; Moreira et al. 2010). Important insights into pathogenicity mechanisms of X. euvesicatoria str (hereafter, Xcv) have been obtained with its genome sequence (Thieme et al. 2005). Here we report draft genome sequences of type strains of the other three bacterial spot pathogen species: X. vesicatoria strain 1111 (Xv 1111) (ATCC 35937), X. perforans strain (Xp ), and X. gardneri strain 101 (Xg 101) (ATCC 19865). We have annotated and analyzed predicted pathogenicity factors in the draft genomes. 23

24 Additionally, we have investigated differentiation between xanthomonads that might explain differences in disease phenotypes and in host range. Materials and Methods Genome Sequencing Xv, Xp and Xg were sequenced by 454-pyrosequencing (Margulies et al. 2005) at core DNA sequencing facility, ICBR, University of Florida. Xanthomonas isolates were grown overnight in nutrient broth. Genomic DNA was isolated using CTAB-NaCl extraction method (Ausubel et al. 1994) and resuspended in TE buffer (10 mm Tris ph 8, 1 mm EDTA ph 8). Libraries of fragmented genomics DNA were sequenced on 454- Genome Sequencer, FLX instrument at Interdisciplinary Center for Biotechnology Research (ICBR) at UF. De novo assemblies were constructed using 454 Newbler Assembler (Margulies et al. 2005). The three draft genomes were obtained with around 10X coverage. For Illumina sequencing, the Xanthomonas strains were purified from singlecolony and grown overnight in liquid cultures. Genomic DNA was isolated by phenol extraction and precipitated twice with isopropanol, then dissolved in TE buffer. DNA was then purified by cesium chloride density gradient centrifugation and precipitated with 95% ethanol, then dissolved in TE buffer. Libraries of fragmented genomic DNA with adapters for paired-end sequencing were prepared according to the protocol provided by Illumina, Inc. with minor modifications. The libraries were sequenced on the 2G Genome Analyzer at Center of Genome Research & Biocomputing at Oregon State University and post-processed using a standard Illumina pipeline (Bentley 2006). We obtained approximately 8-10 million 60-bp reads for each genome, providing roughly 95X predicted coverage. 24

25 Assembly and Annotation De novo assembly was generated on Newbler assembler using 454-sequencing reads for each genome. CLC workbench (CLC Genomics Workbench 2010) was used in the next step for combining 454-based contigs with illumina reads, wherein, 454 based contigs were used as long reads to fill in gaps generated during combined de novo assembly. These combined assemblies of each genome were uploaded on IMG- JGI (Joint Genome Institute, Walnut Creek, California) server for gene calling. The gene prediction was carried out using GeneMark. Pfam, InterPro, COGs assignments were carried out for identified genes. Pathogenicity clusters described in the paper were manually annotated. Whole Genome Comparisons We aligned draft genomes against reference Xanthomonas genomes using nucmer (Kurtz et al. 2004) of MUMmer program (version 3.20) and dnadiff was used to calculate percentage of aligned sequences. We have also compared genomes using the MUM index (Delonger et al. 2009) to measure distances between two genomes. The maximal unique exact matches index (MUMi) distance calculation was performed using the Mummer program (version 3.20). Mummer was run on concatenated contigs or replicons (achieved by inserting a string of 20 symbols N between contig or replicon sequences) of each genome. The distance calculations performed using the MUMi script are based on the number of maximal unique matches of a given minimal length shared by two genomes being compared. MUMi values vary from 0 for identical genomes to 1 for very distant genomes (Delonger et al. 2009). 25

26 Phylogenetic Analysis MLST sequences (fusa, gapa, glta, gyrb, lacf, lepa) for all the genomes were obtained in concatenated form from PAMDB website ( Genes and their corresponding amino acid sequences spanning gum, hrp cluster were downloaded from NCBI genbank sequences of sequenced genomes. Amino acid sequences of proteins of these clusters for Xcv and Xcc were used as query to search for homology against draft genomes of Xp, Xv and Xg. The amino acid sequences were then concatenated for each pathogenicity cluster and then aligned using CLUSTALW ignoring gaps. Neighbor-joining trees were constructed with bootstrap value for 1000 replicates using MEGA4 (Tamura et al. 2007). Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 2723 positions in the final dataset. Phylogeny Reconstruction We used a supermatrix approach as in previous work (Moreira et.al. 2010). Protein sequences of six Xanthomonas genomes (ingroups) and the S. maltophilia R551-3 genome (outgroup) were clustered in 5,096 families using OrthoMCL (Li et al. 2003). We then selected families with one and only one representative from each of the ingroup genomes and at most one outgroup protein, resulting in 2,282 families. Their sequences were aligned using MUSCLE (Edgar 2004) and the resulting alignments were concatenated. Non-informative columns were removed using Gblocks (Castresana 2000), resulting in 792,079 positions. RAxML (Stamatakis 2006) with the PROTGAMMAWAGF model was used to build the final tree. 26

27 Prediction of Effector Repertoires, Cloning of Candidate Effectors and Confirmation Using AvrBs2 Reporter Gene Assay A database was created collecting all the known plant and animal pathogen effectors. Using all these known effectors as query, tblastn analysis was performed against all contigs of the draft genomes of Xv, Xg and Xp with e-value of (Altschul et al. 1997). Pfam domains were searched for possible domains found in known effectors in predicted set of ORFs of draft genome sequences. Candidate effectors were classified according to the nomenclature and classification scheme for effectors in xanthomonads according to currently accepted nomenclature (White et al. 2009). Candidate effectors showing <45% identity at amino acid level to the known effectors were confirmed for their translocation using avrbs2 reporter gene assay. N-terminal 100 amino acid region along with upstream 500 bps sequence of candidate genes were PCR amplified using primers with BglII restriction sites at the 5 ends. Following digestion with BglII, PCR amplicons were ligated with BglII-digested pbs(bglii::avrbs ::HA) (courtesy of Dr. Mary Beth Mudgett, Stanford university), and later transformed into E. coli DH5α. In-frame fusions were confirmed by DNA sequencing using F20 and R24 primers. BamHI-KpnI fragments containing the candidate gene fused to avrbs2 was then cloned into pufr034. Resulting plasmids were then introduced into Xcv pepper race 6 (TED3 containing mutation in avrbs2) by tri-parental mating. The resulting Xcv strains were inoculated on Bs2 pepper cv. ECW 20R and kept at 28 o C in growth room. After 24 hours, strong HR was indicating successful translocation of candidate effector fusions. 27

28 Cloning of Pepper Specificity Genes in Xp. The three genes mentioned above were cloned individually and in combination in plafr3 vector and conjugated in Xp avrxv3 mutant PM1. The PM1 transconjugants with the three individual genes and combined ones along with virulent Xcv pepper race 6 strain were infiltrated at 10 5 CFU/ml concentration in pepper cv. ECW and leaves were sampled at every 48 hours after inoculation. The samples were plated on nutrient agar, incubated at 27 o C and CFU/ml counts were enumerated. Experiment was carried out in triplicate and repeated three times. Results Draft Genome Sequences of Xv Strain 1111, Xp Strain and Xg Strain 101 were Obtained by Combining Roche-454 (Pyrosequencing) and Illumina GA2 (Solexa) Sequencing Data. Initially, we sequenced Xv strain 1111 (ATCC 35937) (hereafter Xv), Xp strain (hereafter Xp) and Xg strain 101 (ATCC 19865) (hereafter Xg) by 454 pyrosequencing (Margulies et al. 2005). De novo assembly using Newbler assembler resulted in 4181, 2360 and 4540 contigs, respectively, for Xv, Xp and Xg, with approximately 10-fold coverage for each strain. Many pathogenicity genes, including type III effectors, existed in the form of fragments given the relatively low coverage of the 454-based assembly. More complete assemblies were obtained using Illumina sequencing (Bentley 2006). De novo assemblies of around 100-fold coverage were constructed from the Illumina data alone or combined with pre-assembled 454 long reads using CLC Genomic Workbench (CLC Genomics Workbench 2010). Combined 454 and Illumina sequencing produced a much better assembly than either technology alone (Table 2-1). Therefore, combined assemblies were chosen for all subsequent analyses. The average contig size in the combined 454 and Illumina assemblies was 28

29 around 18 kb for Xv and Xp, and 10 kb for Xg. The N50 (minimum number of contigs needed to cover 50% of the assembly) values were 37 and 40 for Xv and Xp, respectively, and 83 for Xg indicating that final assemblies consist of a few large contigs allowing reasonably accurate whole genome comparisons. The three strains were deduced to contain plasmids as evidenced by the presence of genes that are known to be involved in plasmid maintenance (e.g. parb/f genes). We have used adjacency to such genes to infer occurrence of certain other genes on plasmids. Relationships of the Strains to Other Xanthomonads using Whole Genome Comparisons 16S rrna analysis and MLST-based phylogenetic analysis showed the diversity among the four bacterial spot species. We carried out phylogenetic analysis based on orthologous protein-coding genes from draft genomes and reference xanthomonads (Figure 2-1). Whole genome comparisons were performed using the MUMi index (Delonger et al. 2009) to assess pairwise distance between the draft genomes and available reference Xanthomonas genomes as shown in the phylogenetic tree and the distance matrix. Another program, dnadiff, based on nucmer (Kurtz et al. 2004) showed the extent of homologies among the shared regions of the genomes by pairwise comparisons (Table 2-2). All of the methods yielded consistent results: we were able to ascertain that among the three newly sequenced strains in relationship to the previously sequenced strains, Xp and Xcv form the closest pair, which is in turn closest to X. citri pv. citri (Xac). Next, Xg is closest to Xcc, with Xv forming a clade with Xg and the Xcc species group (Figure 2-1). 29

30 Four Xanthomonads Show Variation in the Organization of the Type III Secretion Gene Clusters Annotation of the respective type III secretion gene clusters, or hrp genes showed that Xp has an almost identical and syntenic hrp cluster to that of Xcv (Figure 2-2). The most notable difference is that hpag and hpaf encode the fusion protein XopAE in Xp, while they are present as separate genes in Xcv. Adjacent hypothetical protein XCV0410 (126 amino acid protein) is absent from Xp. Xv and Xg show greater similarity to the core hrp cluster genes of Xcc than to that of Xcv. Xv and Xg contain hrpw associated with the hrp cluster as in Xcc. Additionally, xopd in Xv and Xg is not associated with the hrp cluster as in Xcc (referred to as psv in Xcc). PsvA shows 74% and 84% sequence identity to the respective homologs from Xv and Xg. XopA (hpa1) from Xcv seems to be absent from Xv and Xg. Interestingly, we found a novel candidate effector gene (named xopz2) upstream of hrpw in Xv and Xg (See below). Finally, the hrp-associated effector xopf1 is conserved and intact in all four tomato and pepper pathogens. A Reporter Gene Assay Confirms Translocation of Novel Type III Effectors We identified and annotated T3SS effectors from the three newly sequenced xanthomonads (See Methods). Several candidate effectors, which had not yet been experimentally confirmed in xanthomonads, and candidate effectors with plausible translocation motifs were identified. Corroborative evidence for T3SS-mediated translocation of the candidate effectors was assessed by constructing fusion genes with the C-terminal end of AvrBs2 coding sequence (avrbs aa ) in a race 6 strain of X. euvesicatoria. Translocation was measured in pepper cv. ECW 20R, containing the resistance gene Bs2. Genes xopao, xopg, xopam, and XGA_0724 (belonging to the 30

31 avrbs1 class of effectors), of which homologs were previously found in Pseudomonas species, were demonstrated to direct AvrBs2-specific hypersensitive reactions in ECW 20R (Table 2-4, Table 2-5; Figure 2-3). Another candidate effector gene xopz2, associated with the hrp clusters in Xv and Xg (Figure 2-2), was also functional in the AvrBs2-based assay. Thus, we identified five effectors that have not been previously recognized in Xanthomonas and showed their functionality. Core Effectors among Four Xanthomonads Give Insights into Infection Strategies of the Pathogen Comparing the draft genome sequences of the three xanthomonads with that of Xcv allowed us to identify the core effectors conserved in all four strains as well as strain-specific effectors (Tables 2-3, 2-4 and 2-5). At least 11 effector genes form a core set of common effectors for xanthomonads infecting tomato and pepper (Table 2-3). Of these 11, eight effector genes (avrbs2, xopk, xopl, xopn, xopq, xopr, xopx and xopz) were found to be conserved in all sequenced xanthomonads including the three draft genomes presented here with the exceptions of X. albilineans and X. campestris pv. armoraciae. These genes might be necessary for maintaining pathogenicity of these xanthomonads in a wide range of host plants. XopN has been reported to suppress PAMP-triggered immunity by interacting with tomato TARK1 and TFT1 (Kim et al. 2009). XopF1 is conserved in tomato and pepper xanthomonads. Although a homolog of xopf1 is found in Xcc, the respective gene is truncated (Silva et al. 2002). Hence, xopf1 is a potential pathogenicity determinant in tomato. A xopf1 deletion mutant of Xcv did not show any difference in virulence when compared to wild type Xcv on the susceptible cultivar of pepper cv. ECW, suggesting XopF1 is not the lone factor for pathogenicity of Xcv on pepper 31

32 (Buttner et al. 2007). Another effector gene, xopd, is associated with the hrp gene cluster in Xcv and Xp. However, xopd appears to have translocated to another location in the genome in case of Xg, Xv and Xcc strains. XopD is annotated as Psv virulence protein in Xcc genome (Silva et al. 2002) and has been shown to be a chimeric protein sharing a C terminus with XopD from Xcv (Stavrinides et al. 2006). Although xopd homologs from Xv and Xg are syntenic with the psv gene in Xcc, Xv and Xg have intact full-length copies of xopd as in Xcv, indicating that the xopd could be another effector exclusive to the tomato pathogens and a possible pathogenicity determinant in tomato. XopD has been shown to enhance pathogen survival in tomato leaves by delaying symptom development (Kim et al. 2008). Two tandem copies of xopx are found in Xg. However, one gene in Xg appears to be inactive due to a frameshift mutation. In Xp, the two copies of xopx are found in different locations in the genome with neighboring genes, including chaperone gene groel, which is also duplicated. Orthologs of xopz are also found in all four xanthomonads, with 82% identity for Xcv and Xp and 35% identity for Xg and Xv. Apart from low sequence identity in Xv and Xg, gene-specific rearrangements appear to have occurred within each ortholog. We propose that the overall low amino acid relatedness of this effector in Xv and Xg warrants assigning the proteins to a new family within the xopz class, named xopz2, while the orthologs from Xcv and Xp belong to family of xopz1 as originally described in Xoo (see above, Figure 2-2, Table 2-5). Effectors Unique to Xp Might be Responsible for Restricting Growth on Pepper. Xp is pathogenic only on tomato. The avirulence gene, avrxv3, present in Xp, was previously shown to elicit an HR in pepper cv. ECW (Astua-Monge et al. 2000a). An avrxv3 knockout mutant of Xp is not virulent in pepper cv. ECW indicating that other 32

33 factors are associated with host specificity. Comparing effector repertoires of the pepper pathogens Xg, Xcv, and Xv with Xp may provide clues to the factors that are responsible for reduced virulence (Table 2-5). Besides avrxv3, the only effectors present in Xp and absent or inactive in Xg, Xv and Xcv are xopc2, xopae and xopj4 (avrxv4) (Table 2-4). The gene avrxv4 is absent from other sequenced xanthomonads and shows gene-for-gene interaction with the Xv4 resistance gene from the wild tomato relative Solanum pennellii but does not contribute to restricted growth of Xp on pepper (Astua-Monge et al. 2000b). The effector xopc2 is a homolog of the effector rsp1239 from Ralstonia solanacearum GMI1000 and xopae encodes an LRR protein with homology to the R. solanacearum effector PopC. Both genes, xopc2 and xopae, are truncated in Xcv. Therefore, these two effectors may trigger immunity in pepper. Interestingly, Xp contains a paralog of xopp. The two copies are found next to each other in the genome and share 75% identity at the amino acid level. The second copy is next to the candidate effector xopc2, which is unique to Xp among tomato and pepper pathogens. Effectors xopc2 and xopp may both act to restrict growth in pepper. Moreover, there are at least two effectors, xope2 and xopg, present in the pepper pathogens Xcv, Xv and Xg but absent from Xp. These effectors may be essential pathogenicity factors in pepper. Species-Specific Effectors Xv possesses two unique effector genes, xopag (avrgf1) and xopai (Table 2-4). A phylogenetic analysis of xopag showed that xopag from Xv is closely related to xopag from X. citri A w, which has been shown to be responsible for causing an HR on grapefruit (Rybak et al. 2009). XopAI is a chimeric protein, which contains a conserved myristoylation motif at its N terminus, like XopJ1. This effector class also includes the 33

34 homolog XAC3230 from Xac as well as XAUB_26830 and XAUC_23780 from X. fuscans subsp. aurantifolii strains B and C, respectively (Moreira et al. 2010). The presence of transposons and phage elements in close proximity helps to explain the evolution of this novel effector in Xac by terminal reassortment (Stavrinides et al. 2006). Xv also contains effector gene avrbst, which is responsible for the hypersensitive response on pepper. Loss of the plasmid containing avrbst in Xcv strain 75-3 allows the strain to cause disease on pepper (Minsavage et al. 1990). Xg contains at least two effectors, avrhah1 (an avrbs3-like effector gene) and xopb as does Xcv, and share sequence identity of 82% and 86% respectively to the corresponding effectors of Xcv. However, avrhah1 appears to specify a different phenotype when compared to avrbs3 from Xcv. AvrHah1 was shown to be responsible for increased watersoaking on pepper cv. ECW-50R and 60R, whereas Xcv strains carrying avrbs3 show a phenotype that consists of small raised fleck lesions on pepper (Schornack et al. 2008). Another effector gene, xopb, has a PIP box at the 5 end in Xcv, whereas the homolog in Xg does not contain a PIP box. Neighboring genes to xopb in the respective strains are completely different between genomes, suggesting lack of synteny between the two species in this region (Table 2-5). XopB from Xg is 92% identical at the amino acid level to the homolog in Xcv. Deletion mutants of xopb from Xcv did not show any difference in virulence, indicating it does not contribute significantly to virulence (Noel et al. 2001). However, xopb may contribute to virulence in Xg. We also identified eight effector genes that are unique to Xcv (Table 2-4). With the exception of xopaa (early chlorosis factor), all of these genes belong to regions of low GC content compared to average genome GC content (64.75%): avrbs1 (42%), 34

35 xopc1 (48%), xopj1 (xopj) (57%), xopj3 (avrrxv) (52%), xopo (52%), xopaj (avrrxo1) (51%). Few Effectors are Shared among Phylogenetically Related Group Strains Although Xp and Xcv, and Xv and Xg form distinct phylogenetic groups (Figure 2-1), relatively few effectors are shared between these closely related strains. For Xp and Xcv, they share at least six effectors xope1, xopf2, xopp, xopv, xopak, xopap, which are absent from the other two genomes (Table 2-5). Xv and Xg appear to be most closely related to strains of X. campestris pv. campestris, and this relationship is reflected in the suite of effector genes. In fact, Xg and Xv share four effector genes with Xcc, namely, xopam, avrxcca1, hrpw and xopz2, with the caveat that hrpw and avrxcca1 may not function as intracellular effectors (Table 2-5). Furthermore, the genomic regions containing these genes are syntenic in Xg, Xv and Xcc. Xg Shows Evidence of Effector Acquisition by Horizontal Gene Transfer. Effector homologs of avra, hopas1 and avrrpm1 from P. syringae pv. tomato T1 and P. syringae pv. syringae B728a are found in Xg with 79%, 41% and 61% identity at the amino acid level, respectively (Table 2-4). Other X. gardneri strains also contain these effectors based on PCR screening (data not shown). These three effectors, XGA_0724 (belonging to avrbs1 class), XGA_0764/XGA_0765 (xopas) and XGA_1250 (xopao), are unique to X. gardneri. The C terminal region of XGA_0724 shows 53% identity to avrbs1 from Xcv. Hence according to the Xanthomonas effector nomenclature (White et al. 2009), XGA_0724 from Xg was placed under the class avrbs1. XGA_0764/XGA_0765 and XGA_1250 have not yet been reported to be found in xanthomonads and were assigned to new classes xopas and xopao. X. gardneri strains have been found to be associated with tomato and have a lower optimum 35

36 temperature for disease development similar to that of pathovars of Pseudomonas syringae (Araujo et.al. 2010). A high score by Alien_hunter analysis (Vernikos and Parkhill 2006), along with very low GC content (45% for XGA_0724 and 48% for XGA_01250, 59% for XGA_0764/XGA_0765) and the proximity of mobile genetic elements provides evidence for horizontal gene transfer (Table 2-6). Effector xopas appears to be separated into two ORFs XGA_0764 and XGA_0765 by internal stop codon. The functionality of effector xopas needs to be confirmed by in planta reporter gene assay. AvrA of P. syringae pv. tomato PT23 was shown to contribute to virulence on tomato plants (Lorang et al. 1994). Acquisition of XGA_0724 by Xg might have conferred increased virulence on tomato. AvrRpm1 from P. syringae pv. syringae possesses a myristoylation motif, which is absent from homologs in Xg. This modification in Xg might have been acquired to escape host recognition. Another candidate effector gene, xopaq, in Xg is found 68 bps downstream of a perfect PIP box. The gene shows 65% identity at the amino acid level to rip6/11, a novel effector from R. solanacearum RS1000 (Mukaihara et al. 2010). All Four Xanthomonads Contain Ax21 Coding Gene but Only Xcv Contains a Functional Sulfation Gene. The ax21 (activator of XA21-mediated immunity) gene is conserved among Xanthomonas species and is predicted to encode a type I-secreted protein that may serve as a quorum sensing signaling molecule (Lee et al. 2008). A 17-amino acid sulfated peptide from the N-terminal region of Xanthomonas oryzae pv. oryzae (Xoo) Ax21 (axy S 22) was shown to bind and activate the XA21 receptor kinase from rice, demonstrating that Ax21 is a conserved pathogen-associated molecular pattern (PAMP) that can activate plant immune signaling (Lee et al. 2009). The ax21 gene is present in 36

37 Xcv (93% identity with Xoo PXO99 protein), Xp (94%), Xv (91%), and Xg (88%). The axy S 22 peptide is 100% conserved in Xcv, Xp and Xv, while in Xg there is a change from leucine to isoleucine at residue 20; this is unlikely to alter the activity of the peptide, since changing this residue to alanine had no effect on recognition by XA21 (Lee et al. 2008). Recognition of axy S 22 by the XA21 receptor requires sulfation of tyrosine 22, which requires the putative sulfotransferase RaxST. In contrast to ax21, the raxst gene is more variable in these genomes, which is consistent with a report of sequence differences in this gene among Xoo strains (da Silva et al. 2004). Furthermore, in Xp, there is a single-nucleotide insertion at position 65, causing a frameshift mutation. The Xv and Xg genomes do not contain raxst; therefore, the ax21 gene products may be nonfunctional in these strains. These findings have implications for the further study of the role of Ax21 in quorum sensing and virulence, as well as for the usefulness of the XA21 receptor to confer resistance to xanthomonads in crop plants. Two Type II Secretion Systems are Conserved in All Four Xanthomonas Genomes. Most cell-wall degrading enzymes, such as cellulases, polygalacturonases, xylanases, and proteases, are secreted by a type II secretion system (T2SS). The Xps T2SS, present in all xanthomonads, has been studied for its contribution to virulence in Xcc and Xoo (Jha et al. 2005; Wang et al. 2008). Another T2SS cluster, known as the Xcs system, is found only in certain species of Xanthomonas, e.g. Xcc, Xac, and Xcv. The Xps system secretes xylanases and proteases and is under control of hrpg and hrpx (Szczesny et al. 2010), indicating differential regulation. Both Xps and Xcs systems are present in all three draft genomes. 37

38 Xanthomonads Possess Diverse Repertoires of Cell-Wall Degrading Enzymes, which are Present in Diverse Genomic Arrangement Patterns. Each species of Xanthomonas has its own collection of genes encoding endoxylanases, endoglucanases, and pectate lyases, which contribute to cell wall deconstruction during pathogenesis. We have compared these repertoires from the three draft genomes and other xanthomonads as detailed in Table 2-7. The genes are designated for different families of glycosyl hydrolases (GH) and polysaccharide lyases (PL) that include the enzymes that cleave glycosidic bonds in the structural polysaccharides of plant cell walls. Genes encoding secreted endoxylanases regulated by the xps genes have been described for their contributions to virulence, including XCV0965 (Szczesny et al. 2010) encoding a GH30 endoxyalanase. The GH30 family catalyses the cleavage of methylglucuronoxylans in the cell walls of monocots and dicots at a β-1,4-xylosidic bond penultimate to one linking the xylose residue that is substituted by an α-1,2-linked 4-Omethylglucuronate residue (Hulbert and Preston 2001; St. John et al. 2006). Such an enzyme secreted by Erwinia chrysanthemi generates oligosaccharides that are not assimilated for growth, suggesting a function in which it contributes to cell wall deconstruction for access to pectates for growth substrate. It is interesting to note the orthologous genes encoding GH30 enzymes are absent in Xg and Xv, with a truncated xyn30 gene in Xac. On the basis of sequence homology, xyn30 genes may also contribute to virulence in Xoo, Xcc and Xp. The more common GH10 endoxylanases, which occur in several bacterial and fungal phyla, have been implicated in the virulence of plant pathogenic bacteria and fungi (Sun et al. 2005; Goesaert et al. 2003). In Xoo, deletion of the gene encoding a 38

39 GH10 xyn10b resulted in diminished virulence (Rajeshwari et al. 2005). All sequenced Xanthomonas genomes contain either two or three copies of xyn10 genes, all of which are within a gene cluster that may comprise a single operon (Figure 2-4). The GH10 endoxylanases are the best studied of all of the xylanases, and structure/function relationships may be inferred on the basis of gene sequence. The action of these enzymes on glucuronoxylans generates xylotriose, xylobiose, and small amounts of xylose that generally serve as substrates for growth. Also generated is methylglucuronoxylotriose, that is formed to the extent that xylose residues in the β-1,4 xylan backbone are substituted with α-1,2-linked 4-O-methylglucuronate residues (Biely et al. 1997). An adjacent gene cluster in an opposite orientation contains an agu67 gene encoding a GH67 α-glucuronidase that serves to catayze the removal of 4-Omethylglucuronate from the reducing terminus of methylglucuronoxylotriose. This activity provides a synergistic function to the overall xylanolytic process to generate xylotriose, which is converted to xylose by xylanases and xylosidases for complete metabolism (Preston et al. 2003). The coregulation of operons encoding XynB and Agu67 enzymes occurs as a logical condition to coordinate expression of genes that encode these and additional enzymes that collectively process glucuronxylans and glucuronoarabinoxylans for complete metabolism. The accessory enzymes and transporters necessary for the function of these enzymes are embedded within these operons in Gram positive bacteria (Shulami et al. 1999; Shulami et al. 2007; Chow et al. 2007) and share similarities noted here with Xanthomonas spp. These include the genes encoding two glycohydrolases, a β-xylosidase and an α-l-arabinofuranosidase. 39

40 Also included in this cluster are genes encoding enzymes for intracellular metabolism of glucuronate and xylose, including glucuronate isomerase; xylulose isomerase; D- mannonate dehydratase; and D-mannonate oxidoreductase. Genes encoding mannitol dehydrogenase and the hexuronate transporter, as well as the TonB-dependent receptor and LacI transcriptional regulator, flank these two operons. The arrangement and content of xylanolytic enzymes differentiate Xanthomonas species into three groups (Figure 2-4). Here, we propose a common nomenclature for xylanases, the genes for which have been annotated in the sequenced genomes. Members of the first group are Xac, Xcv and Xp in which all three genes encoding GH10 endoxylanases (xyn10a, xyn10b and xyn10c) are present, and with additional genes further downstream in this cluster. Members of the second group are Xcc, Xv and Xg in which genes encoding two of the three endoxylanases are present (xyn10a and xyn10c) and where one or more of the the downstream genes are absent. Xoo strains represent a third group in which a different set of two endoxylanase encoding genes are present (xyn10a and xyn10b) and where the β-galactosidase and gluconolactonase genes flanking xyn10c are absent. It is noteworthy that the organization of genes in the cluster encoding the α-glucuronidase is conserved across Xanthomonas species. Genes Involved in Several Type IV Secretion Systems are Present in Genomes and Plasmids Like Xcv, the tomato pathogens, Xg, Xv and Xp, also appear to contain more than one copy of a type IV secretion system (T4SS) cluster (Figure 2-5, 2-6). Two T4SS clusters (Vir and Dot/Icm type) are present in Xcv, and genes belonging to both of these systems are found on plasmids (Thieme et al. 2005). The Dot/Icm type system is absent from Xv, Xp and Xg. 40

41 In Xv and Xp, genes for one T4SS are on a plasmid and the second one on the chromosome while in Xg, two T4SS gene clusters are on a plasmid and one is on the chromosome. The two T4SS clusters on plasmids of Xg do not show any similarity to the genes for T4SS in Xac, Xcv, Xcc and Xoo. Of the two T4SS clusters in Xg, one is also found in Xv and Xp. This cluster appears to be exclusive to these three tomato pathogens (Figure 2-5). The genes belonging to this cluster show low (30-45%) identity to the T4SS clusters from Ralstonia, Burkholderia, Bradyrhizobium, and Stenotrophomonas maltophilia. The other cluster from Xg, which is absent from Xv and Xp, shows very high identity (98%) and synteny to the T4SS cluster of Burkholderia multivorans and around 89% identity to a T4SS cluster of Acidovorax avenae subsp. citrulli (Figure 2-6). Apart from the plasmid borne T4SS genes, Xcv also contains a portion of a type IV system cluster on the chromosome and consists of VirB6, VirB8, VirB9, VirD4 genes. This chromosomal cluster is flanked by a transposon element (IS1477) that might indicate its horizontal gene transfer. Xp, Xg and Xv genomes contain a complete chromosomal T4SS cluster showing high identity to the T4SS chromosomal clusters from Xcc (Figure 2-7). Type V Secreted Adhesins Function in Synergism During Pathogenesis Different adhesins have been shown to function at different stages of the infection process starting with attachment, entry, later survival inside host tissue and colonization by promoting virulence (El Tahir et al. 2000; Das et al. 2009). FhaB hemagglutinin, important for leaf attachment, survival inside plant tissue and biofilm formation, is present in all four tomato pathogens. In Xcv, fhab is divided into two separate open reading frames, XCV1860 and XCV1861, with the two-partner secretion domains being 41

42 present in XCV1860. Sequence alignment indicates that fhab is possibly inactivated in Xcv by the internal stop codon that separates XCV1860 from XCV1861. In the case of Xoo PXO99A, the Xanthomonas adhesin-like proteins XadA and XadB promote virulence by enhancing colonization of the leaf surface and leaf entry through hydathode (Das et al. 2009). As in Xcv and Xac, Xp encodes two copies of xada, while Xv and Xg possess a single ortholog of xada as does Xcc. YapH and the type IV pilus protein PilQ were shown to be involved in virulence in Xoo during later stages of growth and migration in xylem vessels. In Xcv, Xc, and XooKACC, two copies of yaph are present. There are two pilq orthologs in Xcv and only one in other sequenced xanthomonads. Next to the fhab and fhac adhesin genes, hms operon is present in the genomes of xanthomonads, the homologs of which are pga operon genes in E. coli involved in biofilm formation (Wang et al. 2004). Type VI Secretion System is Present in Xcv, Xv and Xp Type VI secretion system (T6SS) has been shown recently to contribute to host pathogen interactions during pathogenesis in Vibrio cholerae, Burkholderia pseudomallei and Pseudomonas aeruginosa. Hcp (Haemolysin-coregulated protein) and Vgr (valine-glycine repeats) proteins are exported by the T6SS (Boyer et al. 2009). T6SS clusters can be assigned to three different types in xanthomonads (Table 2-8). Xcv and Xp possess two types of T6SSs (type 1 and 3); whereas Xv contains only a single type of T6SS, type 3 (Table 2-9). As in Xcc, there is no T6SS cluster in Xg (Table 2-8). 42

43 LPS Locus Displays Remarkable Variation In Sequence and Number of Coding Genes and Shows Host Specific Variation The lipopolysaccharide (LPS) biosynthesis cluster has been studied in detail in Xcc (Vorholter et al. 2001), which comprises three regions; region 1 from wxca to wxce involved in biosynthesis of water soluble LPS antigen; region 2 (gmd, rmd) coding for LPS core genes; and region 3 from wxck to wxco coding for enzymes for modification of nucleotide sugars and sugar translocation systems. This LPS biosynthesis locus is positioned between highly conserved housekeeping genes, namely cystathionine gamma lyase (metb) and electron transport flavoprotein (etfa), as reported in other xanthomonads (Patil et al. 2007). Comparison of this cluster from draft genomes to the already sequenced xanthomonads revealed high variability in the number of genes and their sequences. Xv and Xg have an identical type of LPS gene cluster of 17.7 kb encoding 14 open reading frames (Figure 2-8) which is similar in organization and sequence identity to the LPS locus from Xcc strains. Interestingly, Xg and Xv also contain two glycosyl transferases involved in synthesis of xylosylated polyrhamnan as seen in Xcc (Molinaro et al. 2003), in contrast to glycosyl transferases (wbda1, wbda2) involved in synthesis of polymannan in Xcv (Thieme et al. 2005). This suggests that basic structure of O-antigen in Xg and Xv is similar to Xcc. The three tomato/pepper pathogens Xcv, Xv and Xg have retained an ancestral type of LPS gene cluster (Figure 2-8). On the other hand, Xp has acquired a novel LPS gene cluster during the course of evolution and is completely different in sequence and number of genes that are encoded. In Xp, this LPS locus is 17.3 kb long and encodes 12 ORFs, all of which are absent in the corresponding genomic region of Xcv, Xv or Xg. Also the first five ORFs flanking the metb side of the LPS locus in Xp (Figure 2-8, ORFs colored in red) showed 43

44 very low or no identity to region 1 of the LPS locus in the other xanthomonads. However, these ORFs still belong to the same Pfam families (Finn et al. 2010) that are usually present in this region, for example, ABC transporters and glycosyl transferases. The second half of the LPS cluster flanking etfa side encodes six ORFs, which are homologs of the LPS cluster genes from Xac, Xcm and Xoo. Phylogenetic insight based on conserved metb and etfa genes that flank the LPS locus suggest that the ancestor of all the Xanthomonas pathogens of pepper and tomato studied in this paper had the same LPS gene cluster, however putative horizontal gene transfer events at this locus have led to the acquisition of a novel LPS gene cluster in Xp. Alien_hunter analysis also supports this acquisition with a high score showing this region to belong to an anomalous region (Table 2-6). This event might have played a major role in changing the specificity of Xp towards tomato and its dominance over its relative(s) as reported previously (Jones et al. 1998a), similar to variant epidemic strain of Vibrio cholerae, reported to be a major reason for its emergence and cholera outbreak during the 1990 s in the Indian subcontinent (Mooi and Bik 1997). Identity in terms of sequences and gene organization among pepper pathogens and absence of those genes from X. perforans and a novel LPS cluster in the tomato pathogen X. perforans suggest a role of this cluster in host specific variation. Analysis of DSF Cell-Cell Signaling System RpfC/RpfG are two-component signaling factors and are involved in DSF (diffusible signal factor) cell-cell signaling (Slater et al. 2000; He and Zhang 2008; Dow 2008, Ryan et al. 2010), known to co-ordinate virulence and biofilm gene expression. The genomes of Xv, Xp, and Xg carry an rpf (regulation of pathogenicity factors) gene cluster (Table 2-10) that is found in all xanthomonads and which encodes components 44

45 governing the synthesis and perception of the signal molecule DSF (He and Zhang 2008; Dow 2008). The Rpf of the DSF system regulates the synthesis of virulence factors and biofilm formation and is required for the full virulence of Xcc, Xac, Xoc, and Xoo (Barber et al. 1997; Dow et al. 2003; Chatterjee and Sonti 2002; Siciliano et al. 2006; Wang et al. 2007a). RpfF is responsible for the synthesis of DSF, whereas, RpfC and RpfG are implicated in DSF perception and signal transduction (Slater et al. 2000; He and Zhang 2008; Dow 2008; Ryan et al. 2010). RpfC is a complex sensor kinase, whereas RpfG is a response regulator with a CheY-like receiver domain that is attached to an HD-GYP domain. HD-GYP domains act in degradation of the second messenger cyclic di-gmp (Ryan et al. 2006). In addition to genes encoding these products, Xg and Xp have rpfh, which encodes a membrane protein related to the sensory input domain RpfC but whose function is unknown. Xv contains rpfh but with an internal stop codon. rpfh is present in Xcv and Xcc, and absent in Xac and Xoo. Cyclic Di-GMP Signaling Cyclic di-gmp is a second messenger known to regulate a range of functions in diverse bacteria, including the virulence of animal and plant pathogens (Romling et al. 2005; Jenal and Malone 2006; Hengge 2009). The cellular level of cyclic di-gmp is controlled by a balance between synthesis by GGDEF domain diguanylate cyclases and degradation by HD-GYP or EAL domain phosphodiesterases. GGDEF, EAL and HD- GYP domains are largely found in combination with other signaling domains, suggesting that their activities in cyclic di-gmp turnover can be modulated by environmental cues. A number of proteins involved in cyclic di-gmp signaling have been implicated in virulence of Xcc (Ryan et al. 2007; He et al. 2009). The genome of Xcv encodes 3 proteins with an HD-GYP domain and 33 proteins with GGDEF and /or EAL domains. 45

46 As in other Xanthomonas spp, the HD-GYP domain proteins are completely conserved in Xcv, Xv, Xg and Xp. There is also almost complete conservation of GGDEF/EAL domain proteins between Xcv and three draft genomes, although Xv has no ortholog of XCV1982. In addition, the EAL domain protein (XCVd0150) encoded on a plasmid in Xcv is absent in the other strains. Copper Resistance (cop) Genes are Present in Xv and Copper Homeostasis (coh) Genes are Present in All Strains Among the Xcv, Xv, Xp and Xg strains sequenced, Xv is the only one resistant to copper and the only strain harboring a set of plasmid borne genes, namely copl, copa, copb, copm, copg, copc, copd, and copf that are also present in copper resistant strains of Xac (unpublished data/ Behlau, F. personal communication) and S. maltophilia (Crossmann et al. 2008). Genes copa and copb have been previously annotated as copper resistance related genes for many different xanthomonad genomes including Xoo, Xoc, Xcv, Xac and Xcc. Homologs of these genes are also present in Xv, Xg and Xp and are located on the chromosome. Additionally, upstream of copa on the chromosome of all strains, there is an ORF that shares homology with plasmid copl. In contrast to what has been published, chromosomal copa and copb are not responsible for copper resistance but likely for copper homeostasis and/or tolerance. While strains harboring the plasmid-borne cop genes, like in Xv, are resistant to copper and can grow on MGY agar (manitol-glutamate yeast agar) amended with up to 400 mg L -1 of copper sulfate pentahydrate, strains that have only the chromosomal cop genes as for Xcv, Xp and Xg, are sensitive to copper and can only grow on media amended up to 75 mg L -1 of copper. Nucleotide sequence of plasmid cop genes in Xv are 98% similar to the ones found in Xac and Stenotrophomonas, whereas 46

47 chromosomal coplab from Xv is 83% identical to homolog ORFs in Xcv, Xg and Xp. When copl, copa and copb genes from Xv located on the plasmid are compared to the homologs on the chromosome of the same strain, the identity of nucleotide sequences is 27, 73, and 65%, respectively. To avoid further confusion or misinterpretation, we suggest that the nomenclature of the chromosomal copl, copa and copb genes in xanthomonads should be changed to cohl, coha and cohb, respectively, referring to copper homeostasis genes. New nomenclature has been adopted in the annotation of the draft genomes. Genes Unique to Xp as Compared to Pepper Pathogens Give Clues to its Predominance over Xcv in the Field and Host Specificity Thirteen gene clusters were found to be specific to the tomato pathogen Xp when compared to the other three strains (Table 2-11). A part of the clusters are syntenic to the genomic regions specific to the three pepper pathogens, suggesting the replacement of these genomic regions from pepper pathogens in correspond to these region in Xp. These replaced regions in Xp might provide potential candidates for host range determinants. Most notable among these regions was the LPS cluster genes (See above). Other such regions include the avirulence genes avrxv3 and avrxv4, a TIR-like domain containing protein, oxidoreductases, and bacteriocin-like proteins that were not found in any other sequenced xanthomonads. Importance of bacteriocin-like genes in Xp has already been studied for its predominance in the field over T1 strains (Hert et al. 2005; Tudor-Nelson 2003). Alien_hunter analysis showed that the bacteriocin BCN-A region belongs to an anomalous region indicating possible horizontal gene transfer of this region (Table 2-6). 47

48 Pepper Pathogenicity/Aggressiveness Factors Increased In Planta Growth of Xp Comparison of proteomes of Xv, Xg, Xcv against Xp showed 68 genes exclusive to pepper pathogens which might be candidate virulence factors on pepper (Table 2-12). These include 16 genes with known function, 35 coding for mobile genetic elements, and 17 genes with unknown function/hypothetical proteins. Out of the 16 genes with known function, xopg was confirmed to be a type III effector using the avrbs2 reporter gene assay and 6 genes belong to the LPS biosynthesis gene cluster. These 16 genes were searched against already sequenced genomes of Xac, Xcc and Xoo. The wxco gene, which codes for O-antigen, has been identified to be a virulence factor in the X. fuscans bean pathosystem by subtractive hybridization (Alavi et al. 2008). Three genes, XCV1298, XCV1839 and wxco, were initially selected for the verification of their contribution to virulence in pepper. Individual genes along with their promoter regions were cloned into plafr3 and conjugated individually and in combination into X. perforans ME24 ( avrxv3), which no longer elicits an HR in pepper. However, in planta growth of ME24 is more similar to that of an avirulent strain than the virulent pepper strain TED3 race 6. ME24 transconjugants carrying wxco and XCV1839 in combination showed increased in planta growth and also comparatively increased number of lesions on pepper cv. ECW when compared to ME24 revealing that these two genes play in fact a role in pepper pathogenicity (Figure 2-9). Genes Specific to Xg as Compared to Other Tomato/Pepper Pathogens may Explain its Aggressive Nature on Tomato and Pepper Comparison of genes from Xg against Xcv, Xp and Xv genes showed the presence of 625 genes specific to Xg. These include four type III effectors (avrbs1 member, xopao, avrhah1, xopaq), twenty-one genes belonging to the unique type IV 48

49 secretion system cluster and associated genes. These genes can be speculated to contribute to the aggressive nature of Xg strains on tomato and pepper. Xg also contains a unique beta xylosidase not present in any other xanthomonads. Moreover, Xg contains XGA_3730 coding for a hemolysin-type calcium-binding repeat containing protein, a homolog of which is found in Xylella strains with 55% sequence identity. In Xylella, this gene is annotated as a member of a family of pore forming toxins/rtx toxins. Its homolog is also found in other plant pathogens (i.e. P. syringae pv. syringae B728a and R. solanacearum GMI1000). This protein has been described as a type I effector in X. fastidiosa str. temecula (PD1506) (Reddy et al. 2007). RTX toxin family members, especially of the hemolysin type, have been shown to be virulence factors in a variety of cell types in eukaryotes (Lally et al. 1999; Linhartova et al. 2010). Finally, a gene XGA_0603 coding for lanthionine synthetase (lantibiotic biosynthesis) is found among these Xg specific genes, a homolog of which is found in Xvm NCPPB702. LanL enzymes in pathogenic bacteria contribute to virulence by modifying the host signaling pathways, in most cases by inactivating MAPKs (Goto et al. 2010). Genes Common to All Tomato Pathogens but Absent from Other Sequenced Xanthomonads In order to see what defines the tomato pathogens, we compared the four sequenced genomes (Xv, Xp, Xg and Xcv) to other sequenced xanthomonads. We found seven genes that were conserved in all four tomato pathogens and absent from most of other sequenced xanthomonads with the exception of Xcm, Xvm, Xau, which possess homologs for six out of these seven genes (Table 2-13). Only the hypothetical protein XCV2641 seems to be specific to the four tomato pathogens. This gene shows only 35% sequence identity to a gene from Xvm and Xcm. A homolog of the 49

50 hypothetical protein, XCV4416 was found in Xau, but is absent from all other sequenced xanthomonads. Genes homologous in Xcm and Xvm include two transposase genes both belonging to the transposase 17 superfamily (XCV0615, XCV0623), XCV0041 (putative penicillin amidase fragment), XCV0111 (lignostilbene-alpha, beta dioxygenase), XCV0112 (uncharacterized protein conserved in bacteria) (Table 2-13). Interestingly, XCV0111 encodes a protein known to be involved in phenylpropanoid degradation. Phenylpropanoids are well known plant secondary metabolites induced during defense response upon pathogen attack (Dixon et al. 2002). It appears that the four tomato pathogens along with Xvm and Xcm have acquired this function to disarm the basal plant defense. The Evolution of Pathogenicity Clusters Corresponds to the MLST-Based Phylogeny The correlation between tree topology using MLST and phylogeny based on the sequences of pathogenicity clusters and the avrbs2 effector gene, which is found in all xanthomonads, was tested. Based on MLST, Xp and Xcv group together along with Xac while Xg is more closely related to Xcc. Xv forms a different clade and is more closely related to the Xcc group. As can be seen in Figure 2-10, phylogeny based on MLST is congruent with phylogeny based on the pathogenicity clusters (gum, hrp cluster) and based on the avrbs2 effector, suggesting that overall these clusters were vertically inherited from the most recent common ancestor of these strains. Concluding Remarks The interaction of Xanthomonas strains with tomato and pepper represents a model system for studying plant-pathogen co-evolution because of the diversity present among the strains causing bacterial spot. Although the four Xanthomonas species infect 50

51 the same host, tomato, and cause very similar disease, they are genetically diverse pathogens. The comparative genomic analysis has provided insights into the evolution of these strains. Whole genome comparisons revealed that Xg and Xv are more closely related to Xcc than Xcv and Xp. A few pathogenicity clusters, such as hrp, xcs and xps of Xg and Xv, were similar in terms of genetic organization and sequence identity to Xcc. However, a few pathogenicity clusters of the four strains belonging to four phylogenetic groups showed different evolutionary origins. While the pepper pathogens Xcv, Xv and Xg possess similar LPS biosynthesis cluster, part of the LPS cluster from Xp is similar to the one from Xac. Xv contains few effectors, including xopag (avrgf1) and xopai the latter of which was previously found to be unique to citrus pathogens Xac, Xaub and Xauc (Moreira et al. 2010). Xg has a number of effectors homologous to P. syringae type III effectors suggesting probable horizontal transfer of these effectors. Xg contains a unique T4SS along with the one that is exclusive to Xp, Xv and Xg. Xp has two T6SSs, as found in Xcv. Xv has only one T6SS, which is similar to that of Xac. Xg has no T6SS as seen for Xcc. While Xg and Xv show close relationship to Xcc based on whole genome comparisons, few pathogenicity clusters mentioned above seem to be conserved among tomato/pepper Xanthomonads. Type III effectors have been investigated for their contribution to pathogenicity and host-range specificity. In addition to homologs of the known effectors, we identified novel effectors in the draft genomes. By comparing effector repertoires of tomato pathogens, two possible candidate pathogenicity determinants, xopf1 and xopd, were identified, of which xopd is responsible for delaying symptom development, and in turn, is important for pathogen survival. Unique genes present in Xg include the novel 51

52 effectors xopao, xopaq, xopas and an avrbs1 member as well as a few other virulence factors, which have been characterized in other plant pathogens and which could explain the aggressive nature of Xg on pepper. Each species contains at least three unique type III effectors, which could explain host preferences among the strains and their aggressiveness on tomato/pepper. Comparison of the LPS clusters between the four species revealed significant variation. Xp has acquired a novel LPS cluster during evolution, which might be responsible for its predominance and its limited host range. As seen from the in planta growth assay of Xp avrxv3 mutant carrying the LPS O-antigen from Xcv, the LPS cluster from pepper pathogens can be a contributor to the increased in planta growth of Xp avrxv3 mutant on pepper, but is not the absolute virulence determinant. Use of the XA21 receptor similar to the Xoo-rice system in Xcv tomato/pepper could be one of the ways to confer resistance to xanthomonads due to presence of a similar AX21 peptide and a functional rax system in Xcv. Common and unique genes encoding enzymes involved in cell wall deconstruction are candidates for further study to define host preference and virulence. In conclusion, comparison of draft genomes obtained by next generation sequencing has allowed an in-depth study of diverse groups of bacterial spot pathogens at the genomic level. This analysis will serve as a basis to infer evolution of new virulent strains and overcoming existing host resistance. The knowledge of potential virulence or pathogenicity factors is expected to aid in devising effective control strategies and breeding for durable resistance in tomato and pepper cultivars. 52

53 Table 2-1. General sequencing and combined (454 and solexa) de novo assembly features of draft genomes of Xv, Xp and Xg. Xv Xp Xg Number of contigs N50* Mean contig length 18,686 18,082 10,014 Longest contig 153, ,836 88,536 Total length of contigs 5,531,090 5,262,127 5,528,125 Table 2-2. Whole genome comparisons using MUMmer dnadiff program. % coverage of the aligned contigs and % identities of the respective contigs against reference genomes has been shown for each draft genome. Genome comparison % of contigs of draft genome aligned % of average identity for the aligned sequences Xp Xcv Xac Xcc Xoo MAFF Xg Xcv Xac Xcc Xoo MAFF Xv Xcv Xcc Xac Xoo MAFF

54 Table 2-3. Core effectors present in all four tomato and pepper xanthomonads Effector Xcv Xv Xp Xg Pfam domains Ref class AvrBs2 XCV0052 XVE_4395 XPE_2126 XGA_3805 Glycerophosphoryl diester phosphodiesteras e Kearney and Staskawicz, XopD XCV0437 XVE_2372 XPE_2945 XGA_3151 C48-family SUMO cysteine protease (Ulp1 protease family); EAR motif Roden et al., 2004 XopF1 XCV0414 XVE_3220 XPE_2922 XGA_ Roden et al., 2004 XopK XCV3215 XVE_0354 XPE_1077 XGA_ Furutani et al., 2009 XopL XCV3220 XVE_0359 XPE_1073 XGA_0320 LRR protein Jiang et al., 2009 XopN XCV2944 XVE_0564 XPE_1640 XGA_0350 ARM/HEAT repeat Kim et al., 2009 XopQ XCV XPE_0810 XGA_0949 Inosine uridine nucleoside N- ribohydrolase XopR XCV0285 XVE_0593 XPE_1215, XPE_3295 XopX XCV0572 XVE_ 3610 XVE_3609 (partial) XPE_1488 XPE_1553 Roden et al.,2004 XGA_ Furutani et al., 2009 XGA_3272 (second copy with frameshift) - Metz et al., 2005 XopZ1 XCV (*) XPE_2869 +(*) - Furutani et al., 2009 XopAD XCV4315 /4314/431 3 XVE_4177 XPE_4269 XGA_0755 SKWP repeat protein *Xv and Xg contain effector xopz2 belonging to the same family xopz. Guidot et al.,

55 Table 2-4. Type III effectors specific to each species Effector Locus tags Effector homolog Pfam domains/ biochemical motifs Comments/Reference Effectors specific to Xv XopJ2 XVE_4840 (partial); XVE_3769 (partial) AvrBsT C55-family cysteine protease or Ser/Thr acetyltransferase Minsavage et al., 1990 XopAG XVE_2415 AvrGf1 - Rybak et al., 2009 XopAI XVE_4756 XAC Moreira et al., 2010 Effectors specific to Xg class avrbs1 XGA_0724 AvrA (84% identity) AvrHah1 (Fragmented in assembly) XGA_4845/ XGA_3187 AvrBs3 XopAO XGA_1250 AvrRpm1 (61% identity) - This study Transcriptional activator, nuclear localization AvrBs3 present in few euvesicatoria strains. Schornack et al., This study XopAQ XGA_2091 Rip6/rip11 No known domains Mukaihara et al., 2010 XopAS XGA_0764/0765 HopAS1 No known domains This study Effectors specific to Xp XopC2 XPE_3585 Rsp1239 Haloacid dehalogenase-like hydrolase XopJ4 XPE_1427 AvrXv4 SUMO protease (experimental); YopJ-like serine threonine acetyl transferase domain (predicted) XopAF XPE_4185 AvrXv3 Transcriptional activator domain White et al., 2009 Astua-Monge et al., 2000b; Roden et al., Astua-Monge et al., 2000a XopAE XPE_2919 HpaF/G LRR protein White et al., 2009 Effectors specific to Xcv AvrBs1 XCVd0104 AvrBs1 - Thieme et al., 2005 XopC1 XCV2435 XopC Phosphoribosyl transferase domain and haloacid dehalogenase-like hydrolase Roden et al.,

56 Table 2-4. continued Effector Locus tags Effector homolog Pfam domains/ biochemical motifs XopJ1 XCV2156 XopJ C55-family cysteine protease or Ser/Thr acetyltransferase XopJ3 XCV0471 AvrRxv C55-family cysteine protease or Ser/Thr acetyltransferase Comments/Reference Roden et al., 2004 Thieme et al., 2005 XopO XCV1055 Unknown Thieme et al., 2005 XopAA XCV3785 ECF Early chlorosis factor, proteasome/cyclosome repeat Thieme et al., 2005 XopAI XCV4428 AvrRxo1 - Thieme et al.,

57 Table 2-5. Effectors specific to particular groups of species Effector class Locus tags Pfam domains Comments/ References Effectors common to all pepper pathogens Xv, Xcv and Xg XopE2 XopG XCV2280, XVE_1190, XGA_2887 XCV1298, XVE_4501, XGA_4777 Putative transglutaminase M27 family peptidase clostridium toxin Thieme et al., 2007 This study Effectors common to Xv, Xg but absent from Xp and Xcv XopAM XVE_4676, XGA_ This study HrpW XVE_3222, XGA_2761 Pectate lyase HrpW associated with hrp cluster, May not be T3SE; Park et al., 2006 AvrXccA1 XVE_5046, XGA_0679 LbH domain containing hexapeptide repeats (X-[STAV]-X-[LIV]- [GAED]-X)- acyltransferase enzyme activity May not be T3SE; Xu et al., 2006 XopZ2 XGA_2762, XVE_3221 Not known This study; Associated with hrp cluster. Effectors common to Xg and Xcv but absent from Xp and Xv XopB XGA_4392, XCV Noel et al., 2001 Effectors common to Xp and Xcv but absent from Xg and Xv XopE1 XPE_1224, XCV0294 Putative Thieme et al., 2007 transglutaminase XopF2 XPE_1639, XCV Roden et al., 2004 XopI XPE_3711, XCV0806 F-box domain Thieme, 2008 XopP XPE_3586, Roden et al., 2004 XPE_4695(Partial), XCV1236 XopV XPE_4158, XCV Furutani et al., 2009 XopAK XPE_4569, XCV Not confirmed to be effector in Xanthomonas; Homolog of effector in Pseudomonas. XopAP XPE_1567, XCV3138 Lipase class III 45% identity to homolog in Xp; Homolog of rip38 from R. solanacearum RS1000; Mukaihara et al., 2010 Effectors present in Xv and Xp but absent from Xg and Xcv XopAR XVE_3216, XPE_ Mukaihara et al.,

58 Table 2-6. Evidence of horizontal gene transfer using Alien Hunter analysis Gene/ Gene cluster Locus tag Score for Alien Hunter (Threshold = ) % GC trna/transposase/mo bile genetic elements in the vicinity Evidenc e of HGT avrbs1 XGA_ Transposase Good xopao XGA_ Predicted to be located on plasmid Good xopas XGA_0764/XGA_ Transposase Good xopg XGA_4501 XVE_4777 XCV ISxac2 transposase in Xcv Good xopaq XGA_2091 Does not belong to anomalous region xopz2 XGA_2762 XVE_3221 Does not belong to anomalous region 51 Could not be predicted Weak 69 IS30 transposase 3000 bps apart Weak LPS cluster XPE_3787 to XPE_3795 Belongs to anomalous region 50 No Good Bacterioci n cluster XPE_0786 to XPE_0790 Belongs to anomalous region 50 tranposase Good 58

59 Table 2-7. Repertoire of cell wall degrading enzymes in xanthomonads. Gene name Family Enzymatic function Xp Xac Xcv Xv Xg Xcc str Xoo str. KACC Xylanases xyn10a GH10 Endo-β-1, xyn10b xylanase EC: xyn10c agua GH67 α- glucuronidase EC: xyn51a GH51 β-d-arabinofuranosidase / EC: xyn5a GH5 Endo-β-1,4- xylanase EC: /34 partial cel8a GH8 Endo-1,4-β-D glucanase Glucanases cel9a GH pel1a PL1 Pectate lyase Pectate lyases pel1b EC: pel1c pel3a PL3 Pectate lyase EC: pel4a PL4 Rhamnogalacturonan lyase /78/ EC: pel9a PL9 Pectate lyase EC:

60 Table 2-7. continued Gene name Family Enzymatic function Xp Xac Xcv Xv Xg Xcc str Xoo str. KACC pel10a PL10 Pectate lyase EC: Table 2-8. Type VI secretion clusters in different xanthomonads. Strain T6SS #1 T6SS #2 T6SS #3 Phosphorylationtype regulators: AraC-type regulators: Kinase / Phosphatase / Forkhead - Kinase / Phosphatase / Forkhead - - AraC Xvm NCPPB702 YES / / Xvm NCPPB4381 YES / / Xaub / / YES Xauc / / YES Xac / / XAC XAC4148 Xv / / YES Xp YES / YES Xcv XCV XCV2143 / XCV XCV4244 Xoo KACC10331 XOO XOO3052 XOO / XOO3517 Xoo MAFF XOO XOO2906 XOO / XOO3319 Xoo PXO99A XOO XOO0270 XOO / XOO2060 Xoc BLS256 XOC XOC2545 XOC / XOC1370 Xg / / / Xcc ATCC33913 / / / Xcc 8004 / / / Xca 756C / / / Xalb / / / 60

61 Table 2-9. Genes/contigs representing T6SS in draft genomes as compared to Xcv. T6SS subtype #1 T6SS subtype #3 Xp XCV homologs Xp XCV homologs Xv XCV homologs Contig 33 Contig 287 Contig 288 Contig 291 Contig 238 Contig 254 Contig 90 Contig 240 XCV2127(i) XCV2127(i) XCV2127(i) XCV2127(i) Contig 120 Contig 287 Contig 288 Contig 291 Contig 238 Contig 254 Contig 44 no homolog Contig 116 Contig 133 Contig 233 Contig 195 XCV4236(i) XCV4236(i) XCV4236(i) XCV4236(i) XCV2127(C)- XCV2137(N) XCV2137(C)- XCV2144 XCV4236(C)- XCV4216 XCV4215 XCV4206(C) Contig 233 no homolog Contig 183 Contig 148 Contig 175 XCV2120- XCV2127(N) XCV2127(i) XCV4244- XCV4236(N) XCV4236(i) XCV4244- XCV4216 XCV4215 XCV4214- XCV4209(N) XCV4209(C)- XCV4206(N) XCV4206(i) XCV4214- XCV4212(N) XCV4213(N)- XCV4206(N) XCV4206(C) Table A comparison of rpf cluster from rpfb to rpfg found across a range of Xanthomonas genomes. Gene Xcc8004 Xoo Xcv Xv Xp Xg Name rpfb XC_2331 XOO2868 XCV rpff XC_2332 XOO2869 XCV rpfc XC_2333 XOO2870 XCV rpfh XC_2334 Absent XCV /2926* rpfg XC_2335 XOO2871 XCV

62 Table Genes unique to Xp, grouped in clusters. Distribution of flanking Locus tag in Xp/ Gene OID genes Function Cluster 1- LPS cluster genes XPE_3787 to XPE_3795 Present in Xcv Lipopolysaccharide biosynthesis cluster Cluster 2- Chemotaxis protein histidine kinase inactivated by transposase carrying 3 genes (in yellow) along with it in Xp. XPE_4460 In all 4 chemotaxis protein histidine kinase XPE_4461 In all 4 transposase XPE_4462 Fe-S oxidoreductase XPE_4463 XPE_4464 XPE_4465 In all 4 chemotaxis protein histidine kinase Cluster 3- Carrying unique genes in Xp not present in any plant pathogens XPE_1809 Transposase TIR-like domain, cyclic nucleic acid XPE_1810 binding domain XPE_1811 Hypothetical protein XPE_1812 Hypothetical protein XPE_1813 Hypothetical protein Cluster 4- avrxv4 and phage genes in the neighborhood Cluster 5- XopC from Xcv is replaced by other unique genes in Xp XPE_3067 present in 306 hypothetical protein XPE_3068 present in 306 hypothetical protein XPE_3069 XAC2120 XPE_3070 mdmc from Xac306 Predicted O-methyltransferase XPE_3071 not called in gene calling Hypothetical protein in 306 Cluster 6- Carrying bacteriocin genes XPE_0786 to XPE_0790 Cluster 7- flanked by phage integrase XPE_2401 Predicted transcription regulator containing HTH domain XPE_2402 Uncharacterized protein conserved in bacteria XPE_2403 present in Xv XPE_2404 XPE_3894 plasmid mobilization system relaxase XPE_3895 XCV % hypothetical protein [Legionella pneumophila str. Corby] XPE_3896 predicted ATPase XPE_3897 hypo protein from Legionella XPE_3898 present in Xv, Xg XPE_3899 in Xv, XCV1116 XPE_3900 XccB100_3109 exonuclease VII 62

63 Table continued Locus tag in Xp/Gene OID Distribution of flanking genes Function Cluster 8- Upstream flanking genes are conserved in order in all xanthomonads; while downstream are transposase genes in Xcv. present in all xanthomonads XPE_3601 XPE_3602 XPE_3603 XPE_3604 XPE_3605 XPE_3606 XPE_3607 XPE_3608 XPE_3609 cluster 9- Flanking genes conserved in Xcv XPE_3366 XAUB_ % hypothetical protein XPE_3367 XCV0352 XPE_3368 XCV0353 XPE_3369 no hit to any plant pathogen hypothetical protein XPE_3370 no hit to any plant pathogen hypothetical protein XPE_3371 no hit to any plant pathogen hypothetical protein XPE_3372 no hit to any plant pathogen hypothetical protein XPE_3373 no hit to any plant pathogen no hit to any other XPE_3374 plant pathogen hypothetical protein Cluster 10- Upstream and downstream flanking genes present in Xcv no hit to any other XPE_1376 plant pathogen No hit to any other XPE_1377 plant pathogen no hit to any other XPE_1378 plant pathogen no hit to any other plant pathogen Activator of Hsp90 ATPase homolog 1-like protein. XPE_1379 XPE_1380 Cluster 11- Upstream, downstream flanking genes also present in Xcv XPE_0135 XAC3183 Hypothetical protein XPE_0136 XAC3182 Hypothetical protein Cluster 12 XPE_0734 XPE_0735 XPE_0736 Xccb100_0356, vasculorum and musacearum EF-hand calcium binding protein signal peptide, transmemb helices hypothetical protein hypothetical protein 63

64 Table continued Locus tag in Xp/Gene OID Cluster 13 Distribution of flanking genes Function Type I site-specific restrictionmodification system, R (restriction) subunit and related helicases XPE_2183 In Xoo Uncharacterized conserved XPE_2187 Xoo protein Uncharacterized conserved XPE_2190 Xoo protein Type I restriction-modification system methyltransferase XPE_2192 Xoo subunit Type I site-specific restrictionmodification system, R (restriction) subunit and related XPE_2194 Xoo helicases XPE_2195 Xoo, Xoc hypothetical protein Table Genes common to all pepper pathogens but absent from Xp. Locus tag in Xcv85-10 Gene symbol Product name Evidence to be involved in pathogenicity/ virulence Genes with the known functions XCV2278 Pectate lyase precursor XCV3713 wxcl Glycosyltransferase XCV3715 wxcn Putative membrane protein involved in synthesis of cell surface polysaccharide XCV3716 wxco Putative carbohydrate translocase XCV3718 gmd GDP-mannose 4,6- dehydratase (EC: ) XCV3720 wxcb Putative protein kinase XCV3722 wzm O-antigen ABC transporter permease XCV4257 rpmb LSU ribosomal protein L28P Alavi, SM et. al., 2008 in X. fuscans bean pathosystem and this study. 64

65 Table continued Locus tag in Xcv85-10 Gene symbol Product name Evidence to be involved in pathogenicity/ XCV1298 Type III effector (homolog of hoph1 from Pseudomonas syringae) virulence XCV1839 Hypothetical protein This study XCVc0007 kfra KfrA protein XCV0510 hsds1 Type I site-specific deoxyribonuclease (specificity subunit) XCV0513 hsdm1 Type I site-specific deoxyribonuclease (modification subunit) XCV2820 Putative type IV pilus assembly protein PilV XCV3312 Transcriptional regulator, AraC family XCV2191 Putative DoxD-like family membrane protein Genes coding for mobile genetic elements XCVb0012 Putative ISxac3 transposase (fragment) XCVb0018 tnpr Tn5045 resolvase XCVc0040 Site-specific recombinase/resolvase family protein XCVd0025 ISxac3 transposase (fragment) XCVd0071 Phage integrase family protein XCVd0097 tnpa Tn5044 transposase XCVd0109 tnpr Tn5045 resolvase XCVd0115 Tn5044 traposase XCV0355 ISxac3 transposase XCV0619 Transposase XCV0706 ISxac3 transposase XCV1118 ISxac3 transposase XCV1553 Phage-related integrase XCV1698 ISxac3 transposase XCV1843 ISxac3 transposase XCV1848 Putative integrase/recombinase XCV2158 ISxac3 transposase XCV2217 Phage-related integrase 65

66 Table continued Locus tag in Xcv85-10 Gene symbol Product name Evidence to be involved in pathogenicity/ virulence XCV2261 Phage-related integrase XCV2263 ISxac3 transposase (fragment) XCV2273 Tn5044 transposase XCV2295 Putative ISxac3 transposase (fragment) XCV2439 Tn5044 trasposase XCV2453 Filamentous phage Cf1c protein XCV2461 Filamentous phage philf related protein XCV2474 Filamentous phage Cf1c protein XCV2477 ISXac3 transposase XCV2484 Phage-related integrase XCV2615 Integrase XCV2690 ISxac3 transposase XCV2712 Putative transposase (fragment) XCV2867 ISxac3 trasposase XCV3384 ISxac3 trasposase XCV3397 ISxac3 trasposase XCV3410 ISxac3 trasposase Genes with function unknown XCVd0055 XCV0648 XCV1188 XCV1189 XCV1187 XCV1303 XCV1596 XCV1937 XCV2455 XCV2857 XCV2958 XCV3162 XCV3326 XCV3986 XCV4135 XCV4262 XCV

67 Table Genes present in all four tomato and pepper pathogens but absent from all other sequenced xanthomonads. Locus tag for Possible function Homolog present in any other genera GC content Xcv85-10 XCV0623 Transposase 17 In Stenotrophomonas, Acidovorax 0.59 superfamily Hypo protein COG belonging to transposase, inactive derivatives Xanthomonas campestris pv. musacearum NCPPB4381 XCV2641 Hypothetical protein X. c. musacearum and X. c vasculorum (identity 37, 31% respectively) XCV4416 Hypothetical protein Pectobacterium carotovorum X. fuscans pv. aurantifolii 0.57 XCV0615 Transposase 17 superfamily Hypothetical protein COG1943 (transposase, inactivated derivates) XCV0112 COG4704 uncharacterized protein conserved in bacteria XCV0111 putative lignostilbenealpha,beta-dioxygenasephenylpropanoid compound degradation Acidovorax, X. c. musacearum and X. c. vasculorum Stenotrophomonas, X. c. musacearum and X. c. vasculorum Stenotrophomonas, Ralstonia, X. c. musacearum and X. c. vasculorum XCV0041 putative penicillin amidase (fragment) Ralstonia, X. c. musacearum and X. c. vasculorum

68 Figure 2-1. Maximum likelihood tree based on orthologous genes from xanthomonads and Stenotrophomonas. Concatenated amino acid sequences of the orthologous genes from four bacterial spot pathogen strains along with other sequenced xanthomonads were considered in the analysis. Stenotrophomonas maltophilia was used as an outgroup. The evolutionary history was inferred using the Maximum likelihood method. The tree is drawn to scale, with branch lengths corresponding to the evolutionary distances. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. 68

69 Figure 2-2. Comparison of type III secretion system cluster, its associated type III effector genes and helper genes of three draft genomes with already sequenced xanthomonads. Type III secretion gene clusters in five strains are shown. Boxes of the same color indicate orthologous genes. Genes of special interest discussed in the paper are labeled. Xp has near identical hrp cluster as Xcv; Xv and Xg contain mosaic hrp cluster with organization and gene content similar to Xcc, but associated effectors are similar to Xcv along with novel effector gene associated with the cluster. 69

70 Figure 2-3. AvrBs2-based HR assay confirms translocation of novel effectors. Hypersensitive response reaction indicating presence of translocation signal was recorded 24 hr after inoculation on pepper cv. ECW20R with candidate effectors xopz2 (a), avrbs1 (b), xopg (d), xopam (e), xopao (f) conjugated in race 6 strain along with control race 6 strain (c). All the strains showed watersoaking on pepper cv. ECW after 48 hr after inoculation 70

71 Figure 2-4. Xylanase cluster organization. Three types of cluster organizations can be found within xanthomonads. A) Found in Xac, Xcv and Xp containing three endoxylanase genes xyn10a, xyn10b and xyn10c; B) Found in Xcc, Xv and Xg containing two endoxylanases xyn10a and xyn10c; and C) Found in Xoo containing xyn10a and xyn10b within endoxylanase operon. 71

72 Figure 2-5. Schematic representation of type IV secretion system cluster common to Xp, Xv and Xg (Plasmid borne). 72

73 Figure 2-6. Schematic representation of type IV secretion cluster unique to Xg (plasmid borne). 73

74 Figure 2-7. Schematic representation of chromosomal type IV cluster organization in Xcv, Xv, Xp and Xg. 74

75 Figure 2-8. The Structure and phylogeny of the LPS cluster. Schematic comparison of LPS gene clusters described in the present study. Genes conserved in different strains are given identical color. Genes specific to individual strains are given unique color. Hpo pro indicates an ORF encoding for a hypothetical protein. The red color-coded genes in Xp genes are absent in any of the sequenced xanthomonads. 75

76 Figure 2-9. Pepper specificity genes increasing in planta growth of Xp. In planta growth of PM1 transconjugants (combined 2 [XCV1839+wxcO]; combined 3 [XCV1839+wxcO+xopG]); PM1 and pepper virulent strain pepper race 6 represented in log (CFU/cm2 of leaf tissue) at 0, 2, 4, and 6 days post inoculation. 76

77 Figure Correlation between phylogenies based on Multi-Locus Sequence Typing (MLST) core genome and pathogenicity clusters: Concatenated amino acid sequences of the six genes fusa, gapa, glta, gyrb, lacf, lepa from four bacterial spot pathogen strains along with other sequenced xanthomonads are considered in the analysis. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA4. 77

78 CHAPTER 3 AVIRULENCE PROTEINS AVRBS7 FROM XANTHOMONAS GARDNERI AND AVRBS1.1 FROM XANTHOMONAS EUVESICATORIA ELICIT HYPERSENSITIVE RESISTANCE RESPONSE IN PEPPER Background Bacterial spot of tomato and pepper is a disease leading to significant yield loss (Pohronezny and Volin 1983). Based on the current classification, there are four genetically distinct groups of xanthomonads infecting tomato and pepper. These have been named as Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X. gardneri (Jones et al. 2000, 2004). These groups are sufficiently different to be assigned to different species (Jones et al. 1998, Bouzar et al. 1999). Chemical control strategies such as sprays of copper/ streptomycin have not significantly helped control of the disease (Bouzar et al. 1999). Efforts have been focused on breeding for resistance in tomato and pepper cultivars. Five resistance genes namely, Bs1, Bs2, Bs3, Bs4 and BsT have been identified so far in pepper giving a hypersensitive type of resistance with the corresponding avr genes from the pathogen being characterized (Stall et al. 2009). Two recessive resistance genes, bs5 and bs6, leading to non-hypersensitive type of resistance confer quantitative, or multigenic resistance that has been shown to be more durable (Stall et al. 2009). Screening for novel resistance genes continues to be important since the pathogen evolves to selective pressure. Plant pathogenic xanthomonads possess a type III secretion system (T3SS), encoded by a hypersensitive response and pathogenicity (hrp) cluster (Bonas et al.1991). Effector proteins are secreted through the T3SS. They interfere with host immunity and manipulate host cellular processes (Buttner and He 2009, Grant et al. 78

79 2006). Avirulence proteins are type III effectors that are recognized in particular host genotypes by plant R gene products. Many avirulence proteins have been characterized and in some cases, the mechanism of R-gene interaction has been studied. In the absence of the corresponding R gene, avr proteins/ effectors act as virulence factors contributing to susceptibility (Kjemtrup et al. 2000). Avirulence genes have also been studied for their contribution to pathogen evolution. A classic example is AvrBs2 eliciting resistance in pepper containing Bs2. AvrBs2 is known to have an enzymatic function, required for virulence activity. Pathogens have evolved in such a way that mutations in avrbs2 retain enzyme activity but lose recognition by Bs2. In particular, mutants with single base changes at nucleotide site 1386 resulted in loss of recognition by Bs2 without a loss in aggressiveness (Gassmann et al. 2000). Materials and Methods Plant Material and Plant Inoculations Several Capsicum genotypes were collected by Rosana Rodrigues in Brazil and screened for resistance against races of the bacterial spot xanthomonads. Pepper (Capsicum annuum L.) cv. Early Calwonder (ECW), its near-isogenic line ECW-70R, F1 cross (ECW-70R ECW), two hundred and twenty-five F2 plants, and backcrosses [(ECW-70R ECW) ECW-70R; (ECW-70R ECW) ECW] were grown in the greenhouse. Inoculated plants were kept in a greenhouse under a 26 o C during 12 hr light and 15 o C, 12 hr dark cycle. Plants were observed for 48 hr following inoculations for development of HR/ watersoaking symptoms. A four to five cm 2 area of fully expanded leaves was infiltrated using a hypodermic needle and syringe with bacterial suspension adjusted to 10 8 CFU/ml (Hibberd et al. 1987). All experiments were carried out in three replicates. 79

80 Bacterial Strains, Plasmids and Media Bacterial strains and plasmids used and developed in this study are listed in Table 3-1. All Xanthomonas strains were grown at 28 o C on nutrient agar (NA) plates. The cultures were suspended in sterile tapwater and the optical density (O.D.) at 600 nm was adjusted to 0.3 with a spectrophotometer (Spectronic 20, Spectronic UNICAM, U.S.) for plant inoculations. E. coli strains or transformants were grown at 37 o C on Luria-Bertani agar/broth (Maniatis et al. 1982) amended with appropriate antibiotics. Protocols for ligation of plasmids and transformation into E. coli DH5α were according to Maniatis et al. (1982). These plasmids were mobilized from E. coli into Xanthomonas recipient strains by triparental mating with the aid of prk2073 helper plasmid (Ditta et al. 1980; Figurski and Helinski 1979). After incubating the matings on NYG agar at 28 o C overnight (Daniels et al. 1984), the growth was suspended in 2 ml sterile tapwater and plated on NA plates containing appropriate antibiotics. The resulting transconjugants were then grown to obtain pure cultures. Antibiotics used in following final concentrations: ampicillin 125 μg/ml; kanamycin 50 μg/ml; rifamycin 100 μg/ml; spectinomycin 50 μg/ml; and tetracycline 12.5 μg/ml. All strains and constructs were stored in 20% glycerol stocks at -80 o C. Library Preparation and Isolation of Clone with Avirulence Activity A genomic library of X. gardneri Xv444 was constructed in E. coli using the cosmid vector plafr3 following the protocol as described earlier (Staskawicz et al. 1987; Minsavage et al. 1990). Individual clones were mobilized by triparental mating into recipient X. gardneri Xg51, a strain virulent on pepper cv. ECW-70R. Transconjugants were inoculated into the leaves of pepper cv. ECW-70R to screen for the clone eliciting a hypersensitive response (HR). A cosmid clone, pxv , containing a 20 kb insert 80

81 gave an HR on ECW-70R. This clone was selected for further subcloning. A 5kb BamHI- BamHI DNA fragment subclone from the pxv clone was transferred into pbluescript (Stratagene, CA). This 5 kb subclone pxv bam8 was sequenced at the ICBR (Interdisciplinary Center for Biotechnology Research) sequencing facility (University of Florida, Gainesville, FL) with the Applied Biosystems (Foster city, CA) model 373 system. Further subcloning of the pxv bam8 clone was achieved using restriction enzymes BamHI, EcoRI and PstI. The digestion products were tested for HR in leaves of pepper cv. ECW-70R by cloning them into plafr3 and by mobilization into recipient strain Xg51. Deletion Mutant Construction An in-frame deletion of the coding region of avrbs7 in the vector pgemt-easy was carried out. PCR primers were designed in the outward directions to create the deletion and to add BamHI restriction enzyme sites. The PCR product was purified and digested with BamHI. After re-ligation and transformation into E.coli DH5α, a clone lacking the complete ORF of avrbs7 and containing only flanking regions of avrbs7 was chosen. The deletion was confirmed by sequencing the insert in pgemt-easy vector. The ORFdeleted fragment of the gene with flanking regions was excised from pgemt-easy and cloned into the suicide vector pok1. This deletion mutant gene was conjugated into XV444 using homologous recombination as previously described and mutants were identified by PCR (Huguet et al. 1998). Bacterial Population Dynamics in Infiltrated Leaf Tissue Internal bacterial populations were determined at selected time intervals after inoculation. Xanthomonas culture suspensions (wild type, mutant, recipient carrying avr clones) were diluted to a concentration of 10 5 CFU/ml using sterile tapwater and then 81

82 infiltrated into the leaves of the parental lines of pepper cvs. ECW-70R and ECW. The inoculated plants were incubated in the growth room maintained at 15 o C-26 o C (12 hr dark/light period) for 8 days. Leaves were sampled every 48 hr. An area of 1cm 2 of leaf tissue was cut from the infiltrated area using a sterile cork-borer. Using sterile forceps the leaf disk was then placed into a sterile tube containing 1ml sterile tapwater and triturated. Standard 10-fold dilution plating onto NA plates was carried out and plates were incubated at 28 o C for three days. Colonies were then counted and the bacterial populations were calculated as cfu/cm 2 of leaf area. Determination of Electrolyte Leakage from Infiltrated Leaf Tissue The amount of tissue damage after inoculation of leaves with the different Xanthomonas strains listed above was estimated to assess necrosis by quantifying electrolyte leakage as described previously (Cook and Stall 1968). Leaves were infiltrated with bacterial suspensions adjusted to 10 8 CFU/ml (O.D at 600nm = 0.3). Inoculated plants were kept in the growth room at 26 o C during 12 hr light period and 15 o C during 12 hr dark period. Electrolyte leakage was expressed as the increase in conductivity [calculated by difference in the two readings (measured in μmhos)] per hour at 28 o C. Site Directed Mutagenesis of avrbs7 Catalytic site residues of avrbs7 were mutated to alanine by a PCR-mutagenesis approach using primers designed as follows: Avr1.1AlaF 5 GCGCTAGCGGCCGCCGCAGCCACATGCAGCCT 3 and Avr1.1AlaR 5 GCGCTAGCGCAGCTGCAGCCATCTTCATTGCT 3. Both primers had NheI overhangs at the 5 end. pgemt-easy:avrbs7 was used as template for mutagenesis PCR. The amplified PCR product was purified using the Qiagen spin kit and subsequently 82

83 digested with NheI enzyme. The digested product was re-ligated and transformed into E. coli DH5α. The mutated gene construct was then moved to the Xanthomonas compatible plasmid plafr3. The plafr3 clone was then mobilized into two recipients, Xg51 and X. euvesicatoria TED3 by tri-parental mating. A similar protocol was followed for the single amino acid mutation (Cys to Ser) within the catalytic site of avrbs7, with the following primer set. CysmutF1 5 GCGGTGCACTCAGGGGTCGGCCA 3 and CysmutR1 5 GCGGTGCACATGCAGCCTCTCAT 3. Sequence Analysis and Protein Homology Modeling Sequence analysis was carried out using several programs including blast (Altschul et al. 1997) and pfam (Finn et al. 2010). Results Identification of Resistance in Pepper against Bacterial Spot Xanthomonads and Development of Introgression Lines Carrying the Resistance Gene Several Capsicum genotypes were collected in Brazil and were tested for HR against races of Xanthomonas euvesicatoria. One genotype gave an HR when inoculated with races 1, 2, 3 and 6. This line had been designated as 1556 and was a member of Capsicum chinense. The fruit of this line was of the elongated chili type and pungent. Since this line appeared to have broad resistance to different races of the bacterial spot xanthomonads, crosses were made with Capsicum annuum cultivar Early Calwonder (ECW) for the purpose of transferring the resistance to a plant with bellshaped, nonpungent fruit. Since the first cross was interspecific, only a few seeds were obtained. Some of the seeds germinated and a few plants of a F 2 population were obtained after selfpollinating a F 1 plant. Segregation of resistance in the F 2 population seemed to occur, 83

84 but the inheritance of the resistance could not be obtained because of the low number of plants. It was then decided to transfer the resistance via a backcross program to a recurrent susceptible parent (ECW). Plants in each bcf 1 generation were screened for resistance to Xv444, which provided a strong HR in resistant plants. In a third backcross, F 1 plants were male sterile, but female fertile. Instead of pollination of susceptible parent plants, as was done previously in the backcross procedure, pollination of resistant plants with pollen from a susceptible plant was necessary. In the next generation, a plant that was resistant to Xv444 and male fertile was identified. All future backcrosses were based on this plant and were always male fertile. Eventually a population of plants in the 7 th backcross was obtained that did not appear to have the fertility problems present in the early population of the interspecific cross and was uniform in resistance. A population in the 7 th bcf 4 was used to determine the inheritance of the resistance gene, designated as Bs7, the cultigens referred to here as pepper cv. ECW-70R. AvrBs7 from Xv444 Elicits HR in Pepper cv. ECW-70R. Among the different strains of the bacterial spot pathogens tested on pepper cv. ECW-70R, Xv444 was found to elicit a strong HR. An avirulence gene corresponding to the R gene i. e. Bs7 was isolated by mobilizing clones in plafr3 from a genomic DNA library of Xv444 into Xg51, a strain virulent on ECW-70R, by conjugation. A transconjugant carrying subclone pxg-352bam8 elicited HR in ECW-70R. Further subcloning of this 5 kb insert into plafr3 using BamHI, PstI and EcoRI restriction enzymes resulted into three subclones of 1kb, 1.8kb and 2 kb. None of the subclones elicited an HR on pepper cv. ECW-70R. Sequence analysis of the clone showed that there was one ORF spanning the region cut by the restriction enzymes used to 84

85 generate two subclones. This ORF with 500 bps of upstream sequence was cloned by PCR (Primers: avrbs1.1f : 5 CAAGGTGGTGATGGACATGG 3 and avrbs1.1r: 5 GTTGTCACCGCCGACAAGTT 3 ) individually in the pgemt-easy vector and the insert was confirmed by sequencing. The insert was then transferred to plafr3 and conjugated into Xg51. Transconjugants carrying this insert exhibited a strong HR similar to the wild type Xv444 strain by 24 hr after infiltration (Figure 3-1). This ORF was named avrbs7. It consists of 1071 bps encoding a 356 amino acid protein of kda. It shows 67% sequence identity at the amino acid level to avrbs1.1 (XCVd0105) from Xcv str , whose genome was sequenced previously. The possibility that avrbs1.1 from Xcv str was responsible for HR when infiltrated into leaves of ECW-70R was pursued experimentally. AvrBs1.1 from Xcv str Elicits Delayed HR on ECW-70R. The avrbs1.1 gene with 500 bps of upstream sequence was cloned by PCR (8510-Bs1.1F: 5 CGTTTCTACGACAGCACCAA 3 ; 8510-Bs1.1R: 5 CCTCTTGGGGGTTTGAAAAT 3 ) into plafr3 and conjugated in Xg51. Transconjugants carrying avrbs1.1 produced a weak HR by 32 hr post inoculation and, a strong HR was observed by 48 hr (Figure 3-2). The susceptible reaction due to Xg51 was first observed at 72 hr. Genetic Analysis of Bs7 Resistance in ECW-70R Genetic segregation of resistance was analyzed by inoculating an F 2 population with Xg51 transconjugants carrying the avrbs7 clone and Xg51 transconjugants carrying the avrbs1.1 clone. The two clones were also introduced into the Xcv TED3 race 6 background and tested for their phenotype on the F 2 population. 85

86 A total of 166 F 2 plants were infiltrated with Xg51 transconjugants carrying the avrbs7 clone. They were scored for HR or susceptibility at 48 hr post inoculation. One hundred and seventeen plants (70.5%) developed an HR and 49 (29.5%) exhibited a water-soaking phenotype. Analysis of segregation ratio showed a fit to 3:1 ratio (χ 2 = 1.80; P value between 0.2 and 0.1; p value greater than 0.05), confirming that the resistance trait was inherited as a single dominant resistance gene. However, when the avrbs7 clone was introduced into Xcv TED3, there were more susceptible plants than expected in another F 2 population. Out of a total 59 F 2 plants tested, 24 plants (40%) were susceptible, showing χ 2 = 7.93 and a p value between 0.01 and 0.001, showing the close fit to the only Mendelian segregation ratio of 3:1out of all other segregation ratios. In the case of Xg51 transconjugants carrying the avrbs1.1 clone when infiltrated into ECW-70R, the F 2 population exhibited 71 resistant (66%) plants and 36 (34%) susceptible plants, giving an χ 2 value of with the p value slightly lower than The same avr gene when introduced into Xcv TED3 race 6 and inoculated in the leaves of 59 F 2 plants yielded 40 resistant (68%) plants and 19 susceptible (32%) plants, with the χ 2 value of 1.63 and p value between 0.3 and 0.2. This indicated a good fit to a 3:1 Mendelian segregation ratio. In a backcross of an F1 plant with ECW-70R, both avrbs7 and avrbs1.1 clones inted3 caused an HR. Hence we can speculate that AvrBs1.1 might react with the BS7 R gene itself or possibly with another resistance gene closely linked to Bs7. 86

87 In-Planta Growth Studies and Electrolyte Leakage Growth of wild type and individual avr clone transconjugants along with deletion mutants was examined by measuring CFU/cm 2 of inoculated leaf tissue. By 6 days inoculation, Xg51 carrying the avrbs7 clone increased in population size by approximately 3 log, while Xg51 carrying the avrbs1.1 clone showed 0.5 log more growth than the avrbs7 clone and then growth ceased to increase. The wild type virulent strain Xg51 increased up to 4 log by 8 days post inoculation (Figure 3-3A). Wild type Xv444 showed approximately 2.5 log increase in growth by 6 days post inoculation and stayed at the same level afterwards, whereas, deletion mutant Xv444 avrbs7 grew 2 log more compared to wild type Xv444 by day 6 post-inoculation (Figure 3-3C). In summary, strains carrying avr genes grew significantly less compared to strains lacking the avr genes and Xg51 transconjugants carrying avrbs1.1 exhibited more in planta growth compared to Xg51 transconjugants carrying avrbs7 (Figure 3-3A). The rapidity of tissue damage in the inoculated leaf tissue was measured by electrolyte leakage. There was no significant difference between Xg51 carrying avrbs7 or carrying avrbs1.1 compared to Xg51 wild type at 12 hr after inoculation (Figure 3-4A). Similarly, no difference was observed between Xv444 and Xv444 avrbs7 at 12 hr post-inoculation (Figure 3-4B). However, electrolyte leakage in tissue inoculated with Xg51 carrying avrbs7 increased significantly in the following 36 hr, showing peak at 48 hr and then began decreasing. For Xg51 carrying avrbs1.1, electrolyte leakage started increasing slowly after 12 hr with a peak at 60 hr after inoculation. There was a difference in the extent and speed of tissue damage between avrbs7 and avrbs1.1 clones. Significant differences in tissue damage between the Xg51 virulent strain lacking avr genes and those carrying avr genes were observed (Figure 3-4A). 87

88 Electrolyte leakage of Xv444 and Xv444 avrbs7 did not show difference after the first 12 hr but leakage showed a sudden increase for Xv444 in the next 24 hr, showing a peak at 36 hr after inoculation, while that caused by Xv444 avrbs7 remained almost unchanged at 36 hr post-inoculation. In the last 12 hr, tissue damage caused by Xv444 avrbs7 started to increase and that by Xv444 remained constant or started to drop (Figure 3-4C). A Catalytic Tyrosine Phosphatase Domain Might be Responsible for Recognition by the BS7 R Gene Product in ECW-70R. Sequence analysis of both avr genes showed the presence of a tyrosine phosphatase domain in the C-terminal region. AvrBs7 belongs to classical ptyr-specific protein tyrosine phosphatases (PTPs) (pfam family Y_phosphatase PF00102). AvrBs1.1contains a dual specificity phosphatase domain (pfam family DSPc PF00782), indicating its ability to dephosphorylate Ser/Thr phosphate containing proteins in addition to Tyr phosphate containing proteins. Mutation of the catalytic domain (HCGVGQGRTG) in avrbs7 to Alanine residues abolished HR activity of Xg51 carrying the avrbs7 clone (Figure. 3-5) Mutation of Cys residue to Ser residue in catalytic domain of tyrosine phosphatase is known to abolish the enzyme activity (Espinosa et al. 2003). Xg51 transconjugant carrying avrbs7 (Cys 265 Ser) clone failed to exhibit HR on ECW-70R. This implies that tyrosine phosphatase activity of the AvrBs7 avirulence protein might be contributing towards recognition by Bs7 gene transcripts inside the plant cell. Blastp search using avrbs7 and avrbs1.1 as query showed hits with effectors from other plant pathogens. Some effectors from other xanthomonads and from other genera of plant pathogens also possess tyrosine phosphatase activity, specifically dual- 88

89 specificity phosphatase activity. Xanthomonas campestris pv. campestris str along with Xcc str and Xcc str. B100 encode avrbs1.1 effector. Effector avrbs1.1 has been classified as xoph according to the recent classification of xop effectors (White et al. 2009). Pseudomonas syringae pv. tomato str. DC3000 encodes the HopAO1 effector (HopPtoD2) which has been shown to suppress programmed cell death in Nicotiana bentamiana, suggesting its role as virulence factor interfering with MAPK pathway and downstream defense pathway (Espinosa et al. 2003). Similar to AvrBs7, also effector HopAO1 belongs to Y-phosphatase pfam family. Acidovorax citrulli AAC00-1 contains another homolog, Aave_3502 with a dual-specificity phosphatase domain. There is Difference in the Timings of HR Elicitation by AvrBs7 and AvrBs1.1. Xg51 transconjugants carrying avrbs1.1 show a delayed HR compared to Xg51 transconjugants carrying the avrbs7 clone. This phenotype was also confirmed by higher population growth by day 6 and slower tissue damage in case of Xg51 carrying avrbs1.1 compared to avrbs7. We were interested why avrbs1.1 causes a delayed HR. As mentioned above, avrbs7 encodes a conserved catalytic domain for tyrosine phosphatase (HCGVGQGRTG), whereas, avrbs1.1 is predicted to encode a dual specificity phosphatase (HCGMGLGRTT) based on pfam domains. Our hypothesis was that the difference in the timings of tissue damage and HR is due to differences in the amino acid residues at the catalytic domain. We aligned AvrBs7 and AvrBs1.1 using clustalw (Figure 3-6). We fused the N terminal 262 amino acids of AvrBs7 (just upstream of catalytic domain) to the C terminal of AvrBs1.1 containing catalytic domain of AvrBs1.1, in turn replacing catalytic domain of AvrBs7 with that of AvrBs1.1. Exchanging the catalytic domain of AvrBs7 with the catalytic domain of AvrBs1.1 89

90 abolishes the hypersensitive response of AvrBs7 on ECW-70R (Figure 3-7). According to our hypothesis, if the difference between AvrBs7 and AvrBs1.1 was only at the catalytic domains, replacement of AvrBs7 catalytic domain by that of AvrBs1.1 would have altered hypersensitive response of AvrBs7 by delaying it similar to AvrBs1.1, instead of complete loss of HR. However, several explanations can be given for this result. Either this fusion protein might have become inactive due to modification in tertiary structure or replacement of catalytic residues in AvrBs7 makes it no longer capable of recognizing the R gene. Avirulence Proteins AvrBs7 and AvrBs1.1 Display Similar Tertiary Protein Structure. Although AvrBs7 shows only 67% identity to AvrBs1.1 at the amino acid level, the tertiary structure of the respective Avr proteins were similar to each other when predicted by homology modeling (Figure 3-8). Except for the first 50 amino acids (blue helix fragment), within which a motif for ligands is found, appears to be different between AvrBs7 and AvrBs1.1. That motif along with an APCC-D box motif, are the only differences between the two avr proteins when the motifs are compared. The catalytic domain is highlighted in the 3-D structures by pink dots (Figure 3-8). Host Specificity of Bacterial Spot Strains Different strains belonging to the groups A, B and D of bacterial spot xanthomonads were tested for their phenotype on ECW-70R (Table 3-1). The C group (X. perforans) is not pathogenic on pepper (Astua-Monge et al. 2000a). HR was classified into types- strong HR after 24 hr (as seen for avrbs7) and delayed HR after 48 hr (as seen for avrbs1.1). 90

91 We also developed avrbs7 specific primers and avrbs1.1 specific primers to identify the type of avr gene in the strains. PCR amplification results correlated well with the phenotype observed after infiltration, thus classifying strains into those carrying avrbs7 and those carrying avrbs1.1.the avirulence gene avrbs7 was exclusively limited to X. gardneri strains (D group). Gene avrbs1.1 was found in X. euvesicatoria (A group) and X. vesicatoria (B group) strains. X. vesicatoria strain Xv1111, for which the draft genome has been already sequenced (Potnis et al. 2011), also has avrbs1.1 but with an internal stop codon. Thus, the avr gene in this strain is inactive and the strain escapes detection by R gene in ECW-70R. There were strains in each group that did not contain the avr gene. Avr Genes avrbs7 and avrbs1.1 are Encoded on a Large Transmissible Plasmid. The cosmid clone p352-bam8, from which avrbs7 was isolated, contained marginal regions showing identity to avirulence gene avrhah1 (Schornack et al. 2008). The J1 mutant of Xv444, a strain cured of the plasmid encoding AvrHah1, showed a watersoaking phenotype by 3 days after infiltration of ECW-70R (Figure 3-2). This indicates that the two avirulence genes avrbs7 and avrhah1 are encoded on the same plasmid in Xv444. The gene avrbs1.1 from Xcv85-10 is encoded on plasmid pxcv183, upstream of avirulence gene avrbs1 (Thieme et al. 2005). Some of the X. euvesicatoria strains, tested for phenotype on ECW-70R, are also known to carry copper resistance plasmid. Correlation was found in copper resistance and avrbs1.1 phenotype. Concluding Remarks A broad resistance was initially found in pepper Capsicum chinense against several strains of bacterial spot xanthomonads. Since fruits were elongated and 91

92 pungent, the resistance was transferred to another species Capsicum annuum cv. Early Calwonder (ECW). Such interspecific crosses have been carried out in the past for resistance gene transfer (Cook and Guevara 1984; Astua-Monge et al. 2000a). The resistance gene segregated in several backcrosses and a population in the 7 th backcross was referred to as ECW-70R, which was used to determine genetic inheritance of resistance. Segregation analysis of the F 2 population identified a genefor-gene interaction for avrbs7 avirulence gene from Xv444 and corresponding R gene Bs7 from pepper cv. ECW-70R, similar to gene-for gene model explained by Flor (1971). Xcv str elicits delayed HR on ECW-70R. The segregation of resistance genes in an F 2 population was identical after inoculation with Xv444 and Xcv The corresponding avr genes in Xv444 and Xcv str have been isolated and characterized. The avr gene from Xv444 has been referred to as avrbs7, while Xcv str gene was referred to as avrbs1.1 as previously named. Further experiments such as bacterial population dynamics and electrolyte leakage (Cook and Stall 1968) were carried out to confirm that these avirulence genes elicit HR in ECW-70R. Avirulence genes with enzyme activity have been characterized in xanthomonads (Kearney and Staskawicz 1990, Mudgett et al. 2000). Here we present evidence of another avirulence gene possessing enzyme activity, which is required for elicitation of HR. Although avrbs7 and avrbs1.1 share only 67% identity at amino acid level, they have a common characteristic, both belong to the tyrosine phosphatase superfamily. The carboxy-terminal of both avirulence genes contains a consensus PTP active site domain (HCGVGQGRTG for avrbs7 and HCGMGLGRTT for avrbs1.1) along with possible general acid motif (TVTDH) 24 amino acids upstream. Alanine mutagenesis 92

93 around catalytic residues in avrbs7 as well as Cys 265 Ser mutant within catalytic residues in avrbs7 failed to elicit an HR on ECW-70R, so the catalytic domain appears to be important in recognition of the avr protein by R gene transcripts. Characteristic phosphotyrosine recognition region and the immediately following arginine and aspartic acid residues which make H bonds to an acidic side chain and main chain (Fauman and Saper 1996) were located in the two avr protein sequences. Sequence YDR at position 81, 82 and 83 of AvrBs7 represents a possible recognition loop of PTPs. Tyrosine phosphatases have been found as type III effectors in animal as well as plant pathogens. They have been shown to interfere with the host signal transduction pathways, thus functioning as virulence factors. Yersinia pseudotuberculosis secretes type III effector YopH, classic PTPase family protein. YopH, in activated form, dephosphorylates p130 Cas and FAK substrates and thus resists its upstake by the host mammalian cells (Persson et al. 1997). Similarly, Salmonella type III effector SptP possesses tyrosine phosphatase activity which increases pathogen replication by dephosphorylating host AAA+ ATPase VCPs (Humphreys et al. 2009). Pseudomonas syringae pv. tomato DC3000 contains HopPtoD2 i.e. HopAO1 type III effectors, which is a chimeric protein with N terminal region similar to avrpphd hooked onto tyrosine phosphatase containing C terminal region. AvrPphD from Pseudomonas syringae pv. phaseolicola elicits non-host HR on pea (Arnold et al. 2001). Various hopptod alleles have been found in pseudomonads. In contrast to AvrPphD, HopPtoD2 acts as a virulence factor targeting step downstream or independent of MAPK, suppressing plant innate immunity (Bretz et al. 2003; Underwood et al. 2007). 93

94 The difference in the timing of HR elicitation was observed between the transconjugants carrying the avrbs7 clone and those carrying avrbs1.1 clone. The avrbs1.1 clone, along with wild type Xcv str elicited HR 48 hr after infiltration. To explain this difference between the two clones, we compared amino acid sequences of the two clones. Avirulence gene AvrBs7 shares 67% identity with AvrBs1.1 at amino acid level. There are few amino acid differences within and around the catalytic site. Hence, we hypothesized that these amino acid differences within and around the catalytic site could be the reason for the differences in HR timing due to differential activation of R gene. We constructed a fusion protein containing 262 N terminal amino acids of AvrBs7 fused to 90 C-terminal amino acids of AvrBs1.1, thereby exchanging AvrBs7 catalytic site with AvrBs1.1 catalytic site. Exchanging catalytic domains did not change the timing of HR elicitation. HR activity was completely lost when the fusion construct was infiltrated into ECW-70R leaves. There are two possibilities. Either fusion protein was modified in its three dimensional structure and became inactive, or AvrBs1.1 has different substrate specificity than AvrBs7. If the latter is true, AvrBs1.1 might be interacting with the different proteins within plant cell and might be activating the resistance gene transcripts by a different pathway or might be activating another linked resistance gene transcripts. Comparison of three-dimensional structures of AvrBs7 and AvrBs1.1 did not show significant differences in the two structures. In summary, we have identified a gene-for-gene interaction in the Xanthomonas - pepper system in addition to five already identified gene-for-gene interactions (Stall et al. 2009). Avirulence gene avrbs7 has been found to be restricted to D group X. gardneri strains, while its ortholog avrbs1.1 is distributed among A and B group strains. 94

95 Both avr genes are plasmid-borne. Future research on differences in the activation of resistance by AvrBs1.1 and AvrBs7 will contribute to our understanding of the mechanism of activation of this broad resistance. Studying the possible motifs in avirulence proteins will shed light into their possible role in pathogen virulence. 95

96 Table 3-1. List of bacterial strains and plasmids used in this study Strain designation Relevant characteristics Source or reference Xanthomonas euvesicatoria Pepper race 2, tomato race 1, Rif r Minsavage et.al TED3 Pepper race 6 E3 Cu R, HR(+) ECW 70R, avrbs1.1(+) R. E. Stall (Florida, 1960) Xv718 Cu R, HR(+) ECW 70R, avrbs1.1(+).jones (Puerto Rico 1991) Xv881 Cu S, HR(-) ECW 70R, avrbs1.1(-) Jones (Mexico, 1992) Xv669 Cu S, HR(-) ECW 70R, avrbs1.1(-) Jones Xv1025 Cu R, HR(-) ECW 70R, avrbs1.1(-) Jones (Mexico, 1992) Xv818 Cu S, HR(-) ECW 70R, avrbs1.1(-) Jones Xanthomonas gardneri Xg51 HR(-) ECW 70R, avrbs7(-) Minsavage, unpublished Xv444 HR(+) ECW 70R, avrbs7(+) Jones et.al., 2004 Xv444 avrbs7 Xv444, avrbs7 deletion mutant This study J1 Mutant strain of Xv444 cured of the Schornack, et.al., 2008 plasmid carrying avrhah1 XV1927 (BSX104A) HR(+) ECW 70R, avrbs7(+) D. Cuppels, AAFC London; ON, Canada ENA4035 HR(-) ECW 70R, avrbs7(-) Rosana Rodrigues 01T46A HR(+) ECW 70R, avrbs7(+) Jones 02T1A HR(+) ECW 70R, avrbs7(+) Jones 04T5 HR(+) ECW 70R, avrbs7(+) Jones 98T3A HR(-) ECW 70R, avrbs7(-) Jones 00T12B HR(-) ECW 70R, avrbs7(-) Jones 99T4A HR(-) ECW 70R, avrbs7(-) Jones 1782 HR(+) ECW 70R, avrbs7(+) Brazil 1783 HR(+) ECW 70R, avrbs7(+) Brazil Furman 3 HR(+) ECW 70R, avrbs7(+) Minsavage XV451 HR(+) ECW 70R, avrbs7(+) Jones XV1194 HR(-) ECW 70R, avrbs7(-) Jones Escherichia coli DH5α F - recaφ80dlacz M15 Bethesda Research Laboratories, Gaithersburg,MD λpir Host for pok Huguet et.al., 1998 Plasmids plafr3 Tc r rlx + RK2 replicon, Tc r Staskawicz et.al., 1987 pbluescript II KS +/- Phagemid sequencing vector, Ap r Stratagene, La Jolla, CA prk2073 Sp r Tra +, helper plasmid Figurski and Helinski

97 A B Figure 3-1. Phenotype observed in leaves of ECW-70R 48 hr after infiltration with bacterial suspesions (adjusted to 10 8 cfu/ml) A) Xv444, and B)Xg51 97

98 Figure 3-2. Phenotype on ECW-70R 24 hr and 48 hr post-infiltration by wild type strains, transconjugants and mutants infiltrated with suspension adjusted to 10 8 cfu/ml. Order of inoculation as follows (counterclockwise from top left): 1. Xg51 (plafr3: avrbs7); 2 Xg51 (plafr3: avrbs1.1); 3 Xv444 avrbs7 mutant; 4.Xg51. 98

99 9 ECW-70R 9 ECW 8 8 Log 10 (CFU/cm 2 ) Log 10 (CFU/cm 2 ) A Days after infiltration Days after infiltration B Log 10 (CFU/cm 2 ) ECW-70R Log 10 (CFU/cm 2 ) ECW C Days after infiltration Days after infiltration D Figure 3-3. Time course of bacterial population growth after infiltration of leaves of pepper genotypes ECW and ECW-70R with suspensions (adjusted to 10 5 cfu/ml) of Xg51 transconjugants and mutant strains. A) and C) In planta growth in ECW-70R and B) and D) In planta growth on ECW; Transconjugants used in A) and B) are diamond shape-xg51 transconjugants carrying plafr3 clone; square shaped- Xg51 carrying avrbs7 clone; triangle Xg51 carrying avrbs1.1 clone. Wild type and mutants used in C) and D) are- square shaped: Xv444 wild type; cross : Xv444 avrbs7 mutant. 99

100 μmhos ECW-70R A Hours after infiltration Hours after infiltration B μmhos ECW ECW-70R ECW μmhos μmhos C Hours after infiltration Hours after infiltration D Figure 3-4. Electrolyte leakage from pepper genotypes ECW-70R (A and C) and ECW (B and D) after infiltration of leaves with suspensions adjusted to 10 8 cfu/ml of (Xg51) wild type, transconjugants and mutant strains. Transconjugants used in A) and B) are diamond shape-xg51 transconjugants carrying plafr3 clone; square shaped- Xg51 carrying avrbs7 clone; triangle Xg51 carrying avrbs1.1 clone. Wild type and mutants used in C) and D) are- diamond shaped: Xv444 wild type; square : Xv444 avrbs7 mutant. 100

101 Figure 3-5. Tyrosine phosphatase domain is essential for HR elicitation on ECW-70R. Phenotype on ECW-70R 48 hr post-infiltration by wild type strains, transconjugants and site-directed mutants infiltrated with suspension adjusted to 10 8 cfu/ml. 101

102 avrbs1.1 avrbs7 avrbs1.1 avrbs7 avrbs1.1 avrbs7 avrbs1.1 avrbs7 avrbs1.1 avrbs7 avrbs1.1 avrbs7 MPNKISGSIAPSASSDAMKSADCAENIKEEVVSKHVHQAVPAELADLPSRQPPRSKTA-L MPNPVSRSSTSSVSGKGSDDADVVADIKQEAVVEPGNQSTPHGLEGLA----PRSKTARD *** :* * :.*.*.....**. :**:*.* : :*:.* *.*. ****** YQVIQKFRDPLPLPPPPTSHPVLAYDRDLGSS-DNFRSSDEFDLPESLNPTGWKNLHVSG LSLIKKFSNPLPLPQRPTEIPVLQYDRSPRSSSDNFRSSDDFDLPESCNPTGWKDLHVSG.:*:** :***** **. *** ***. ** *******:****** ******:***** SGSIASIGQITRLRPSKERPVVVLDAREESHAIVGGYPGTWRTPNNWGNAGKSRDEALAD SGSIASISQITRLNPSRDRPVIVLDVREESHAIVGGYPATWRAPNNWANVGKSREEVLAD *******.*****.**::***:***.************.***:****.*.****:*.*** EQQRIQALKSQETVHIFHRKDVKSEARNPRGATLSKPLIFSEEELVRAAGAKYVRLTVTD EHEKIRAIKSQETVQILHRKDVKNGFPNPRSVKLSNPSIFSEEELVRNAGAEYLRLTVTD *:::*:*:******:*:******. ***...**:* ********* ***:*:****** HLSPRADDIDAFIAMEREMAHDERLHVHCGMGLGRTTIFIVMHDILRNAAMLSFDDIIER HLGPRADDIDAFVRMERNMAPHERLHVHCGVGQGRTGIFIAMHDILRNAHIISFEDIIKR **.*********: ***:**.********:* *** ***.******** ::**:***:* QRKFNPGRSLDNNKDVSDKGRSEFRNERSEFLPLFYEYAKQNPKGQPLLWSEWLDHNA-- QLAFNPGRALDFNKDVSHEGRSDFRNDRLELISLFYEYAKSNPNGQPSLWSEWLRAANKT * *****:** *****.:***:***:* *::.*******.**:*** ****** Figure 3-6. Alignment of avrbs1.1 and avrbs7 amino acid sequences using clustalw 102

103 Figure 3-7. Fusion protein containing N-terminal of avrbs7 and C terminal of avrbs1.1 does not elicit HR on ECW-70R. Picture was taken 48 hrs after infiltration with suspensions adjusted to 10 8 cfu/ml. Order of inoculation anticlockwise from top left: Xg51, Xg51 transconjugant carrying fusion clone, Xv444 wild type, Xg51 transconjugant carrying avrbs7 clone, Xg51 transconjugant carrying fusion clone, Xg51 transconjugant carrying fusion clone, Xg51 transconjugant carrying avrbs1.1 clone, Xcv

104 A B Figure 3-8. Three dimensional structures of the two avirulence proteins based on homology modeling. A) Avirulence protein AvrBs7; B) Avirulence protein AvrBs1.1. Catalytic site is present in the groove in both structures. Amino acid residues for catalytic domain tyrosine phosphatase are highlighted as pink dots in both the structures. 104

105 CHAPTER 4 APPLICATION OF BIOINFORMATICS FOR TYPE III EFFECTOR SIGNAL ANALYSIS AND ITS INTERACTION WITH CHAPERONE Background Most Gram-negative plant and animal pathogens possess a highly specialized type III secretion system (T3SS) that injects effector proteins inside host cells, interferes with the host cellular machinery and paralyzes host defense responses (Buttner and He 2009; White et al. 2009). Type III effector proteins contain secretion and translocation signals that recruit the effectors to the T3SS (Schesser et al. 1996; Mudgett et al. 2000; Sory et al. 1995). The mechanism for recruitment and regulation of secretion of the type III effectors is not yet clear. Several models have been proposed to explain nature and location of the secretion signals in type III effectors (Buttner and Bonas 2006). The first 15 amino acid residues contain a signal for secretion through the T3SS and the signal is not conserved at the amino acid level (Boyd et al. 2000; Lloyd et al. 2001; Schechter et al. 2004, Buttner et al. 2006; Triplett et al. 2009). In a few cases, the 5 end of the mrna is said to contain a secretion signal, suggesting co-translational secretion of effectors (Anderson et al. 1999). The first 28 amino acids of AvrBs2 from Xcv is reported to contain a functional secretion signal (Mudgett et al. 2000). Plant and animal pathogen effectors do not share any sequence similarities or conservation in the N terminal region. In pseudomonads, the secretion signal features have been described based on amino acid composition of the N-terminus (Petnicki-Ocwieja et al. 2002). Apart from the secretion signal, effectors also contain an N-terminal translocation signal, which is required to target the protein across the plant plasma membrane. This translocation signal was determined to lie within the most N-terminal amino acids of some 105

106 effectors (Buttner and Bonas 2006; Mudgett et al. 2000, Schechter et al. 2004; Schesser et al. 1996). Although the location of secretion and translocation signals have been predicted, the exact sequence and nature of signals have not yet been completely understood. Different computational programs have been recently developed to explain nature of secretion and translocation signals and to identify the draft genome sequences for putative type III effectors based on characteristic amino acid residues. The SVM-based Identification and Evaluation of Virulence Effectors (SIEVE) algorithm program, by Samudrala et al. (2009), combines different DNA sequence features such as G+C content, with amino acid composition of the 30 most N terminal residues of the protein sequence. The training set used for machine learning support vector algorithm (SVM) consisted of known effectors from Pseudomonas syringae and Salmonella enterica serovar Typhimurium. This program can identify secreted effectors from evolutionarily distant bacteria. Since the training set included the first 30 amino acids of effectors, the putative effectors found with this program can include secreted proteins, which might not necessarily be translocated but just secreted. As with the other methods, false positives such as tra conjugal transfer proteins, LPS antigen proteins, and type IV secreted proteins are obtained. This method also identifies some sequence biases found in the N terminal region of type III secreted effectors, which can t be identified using BLAST. An effectivet3 program proposed by Arnold et al. (2009) is based on N-terminal sequence features such as frequencies of amino acids, residues with particular physico-chemical properties, and considers the first 25 amino-acid residues. The database for machine learning input included known effectors from family Chlamydiae as well as the genera Escherichia, Yersinia and Pseudomonas. Again, this 106

107 method can identify potential secreted proteins, but whether they are translocated into the host cell or not is not known. Modlab by Lower and Schneider (2009), based on neural networks, included all known effectors from genomes of gram- negative bacteria. All of these methods were generalized for identification of type III effectors using models developed on effectors from different genera, indicating that the type III signal is universally conserved. Another machine learning program developed by Yang et al. (2010) used features such as amino acid composition, hydrophobicity, and secondary structure properties of the N terminal residues of known P. syringae effectors and applied the model to predict effectors in rhizobia. Apart from secretion and translocation signals, some of the effectors are also known to contain a chaperone-binding site in the N terminal region. Chaperones are believed to keep the type III effectors in the cytoplasm in a secretion competent state, stabilized and separated from other interaction partners before they are secreted by the T3SS. Chaperones have also been shown to maintain hierarchy of the secreted substrates in case of animal pathogen effectors (Parsot et al. 2003). T3SS chaperones don t share any sequence similarities, but have common characteristic features such as small size, acidic pi, and amphiphilic α-helix in C terminal regions (Feldman and Cornelis 2003). T3SS chaperones have been classified as class I, II and III. Class I chaperones contain approximately 130 amino acids. They are divided into two groups: class IA associate with only one particular effector; while class IB associate with several effectors, exhibiting broad range specificity. Class II chaperones contain 160 amino acids and associate with two translocators. Class IA and II chaperones are located in the neighborhood of their substrates within the genome, while class IB chaperones are 107

108 encoded within type III secretion system components operon. Class III chaperones contain chaperones of the flagellar export system (Parsot et al. 2003; Feldman and Cornelis 2003). HpaB is the only known type III effector chaperone from X. campestris pv. vesicatoria. HpaB can be classified as a class IB chaperone, since it controls the secretion of effectors that do not exhibit any sequence similarities among each other and it is encoded within the hrp operon. HpaB is a major pathogenicity factor essential for translocation of some of the effectors (Buttner et al. 2004). Effectors, which are not translocated in the absence of HpaB, are classified as class A effectors and include XopJ and XopF1 from Xcv. Class B effectors do not require HpaB for translocation; they are translocated in the absence of HpaB but in reduced amounts (Buttner et al. 2006). Examples of class B effectors are xopc and avrbs3 (Buttner et al. 2004; Buttner and Bonas 2006). Chaperone-dependent effectors get privilege in translocation and are translocated early during the infection process (Feldman and Cornelis 2003). Co-crystallization studies have been carried out for some of the effectorchaperone complexes. A crystal structure model for the SipA-InvB complex from Salmonella shows the interaction in which the chaperone-binding domain of the effector wraps around the chaperone dimer and interacts with a helix-binding groove and hydrophobic regions of the chaperone (Stebbins and Galan 2001; Lilic et al. 2006). Recognition of the effector-chaperone complex by the T3SS apparatus is proposed to impose priority for effectors associated with chaperones in getting through the T3SS (Feldman and Cornelis 2003). Various models have been developed in animal pathogen effectors to describe the interaction of a chaperone with an effector, its role in controlling 108

109 hierarchy of the effector translocation, and switch in substrate specificity (Stebbins and Galan 2001; Parsot et al. 2003; Lilic et al. 2006). In this chapter, we have developed another type III effector identification program based on a machine learning algorithm using known type III effectors from xanthomonads. However, unlike other methods of effector identification, we have considered the first 100 amino acid residues of the known effectors with the intention of targeting both secretion as well as translocation signal patterns for effector identification. Along with the knowledge on secretion and translocation signal patterns, we are also interested in studying the role of type III effector chaperones in the regulation of effector translocation. We have selected the known chaperone-dependent effector, XopF1, as a model to describe the nature of its secretion and translocation signal and its HpaB chaperone-binding site. Materials and Methods Data-Mining Strategy Models and scoring matrices for the type III effector motifs were built using the MEME program (Bailey and Elkan 1994) considering the first 100 amino acids of the known type III effectors of Xanthomonas. A motif search program developed in C was implemented to search against the ORFs from the whole genome. Bacterial Strains, Plasmids and Media Bacterial strains and plasmid constructs used and developed in this study are listed in Table 4-1. X. euvesicatoria TED3 race 6 strain was grown at 28 o C on nutrient agar (NA) plates. For plant inoculations, the cultures were suspended in sterile tap water and adjusted to A 600 = 0.3 with a spectrophotometer (Spectronic 20, Spectronic UNICAM, U.S.). E. coli strains or transformants were grown at 37 o C on Luria-Bertani 109

110 agar/broth (Maniatis et al. 1982) amended with appropriate antibiotics. Protocols for ligation of plasmids and transformation into E.coli DH5α strain were according to Maniatis et al. (1982). These plasmids were then mobilized into Xanthomonas recipient strains by conjugation with prk2073 helper plasmid using the triparental mating procedure (Ditta et al. 1980; Figurski and Helinski 1979). After incubating the matings on NYG agar at 28 o C overnight (Daniels et al. 1984), the growth was suspended in 2ml sterile tap water and plated on NA plates with Rifamycin and other appropriate antibiotics. The transconjugants obtained were then grown for pure culture. Antibiotics were used in following final concentrations: Ampicillin 125 μg/ml; kanamycin 50 μg/ml; rifamycin 100 μg/ml; tetracycline 12.5 μg/ml. All strains and constructs were stored in 20% glycerol stocks at -80 o C. Yeast strain CG1945 was grown on YPD agar (Peptone 2%, Yeast 1%, glucose 2%, ph 5.8) or YPD broth at 30 o C overnight. For prepration of yeast competent cells, the culture was grown in YPD broth until mid-log phase (OD 600 = ). Plant Material and Plant Inoculations Pepper (Capsicum annuum L.) cv. Early Calwonder (ECW) and its near-isogenic line, ECW-20R, were grown in the greenhouse set at temperature range of 25 o C to 35 o C (day/night). Plant leaves were infiltrated with 10 8 CFU/ml bacterial suspension using a syringe and hypodermic needle (Hibberd et al. 1987). All experiments were carried out in three replicates. Plants were observed for the next 48 hr for development of HR or watersoaking symptoms. In Planta Reporter Gene Assay The N-terminal region including 500 bps upstream of the genes were PCR amplified using primers with BglII restriction sites at the 5 ends. Following digestion with 110

111 BglII, PCR amplicons were ligated with BglII-digested pbs(bglii::avrbs ::HA) (courtesy of Dr. Mary Beth Mudgett, Stanford university), and later transformed into E. coli DH5α. In-frame fusions were confirmed by DNA sequencing using F20 and R24 primers. BamHI-KpnI fragments containing the candidate gene fused to avrbs2 was then cloned into pufr034/ plafr3. Resulting plasmids were then introduced into Xcv pepper race 6 (TED3 containing mutation in avrbs2) by tri-parental mating. The resulting Xcv strains were inoculated into pepper cv. ECW 20R containing Bs2 and kept at 28 o C in a growth room. After 24 hours, a strong HR indicated successful translocation of candidate effector fusions. Site-Directed Alanine Mutagenesis Amino residues of xopf1 were mutated to alanine by a PCR-mutagenesis approach using primers designed as follows F1-50A - R1: 5 GC GCT AGC GGC CGC CGC AGC CGC TGC GGC CAGGCCCGCAAGCG 3 ; F1-50A F1: 5 GC GCT AGC GCT GCG GCA GCT GCA GCC GGTCGCGCCAGTCCT; F1-30A R1: GC GCT AGC GGC GCA GGC CTGCGTTGG; F1-30A F1: GC GCT AGC GCT GCC GCA GAA CGCGCACCC. All primers had NheI overhangs at 5 end. pbs(xopf ::avrbs ::HA) was used as template for the above mutagenesis PCR to avoid contamination by wild-type plasmid. The amplified PCR product was cleaned and digested with NheI enzyme. The digested product was re-ligated and transformed into E.coli DH5α. Consequently the mutated gene construct was moved to plafr3. It was then mobilized by triparental mating into Xcv TED3 race 6 strain. Yeast Two-Hybrid Assay XopF1, XopF1 1-70, XopF1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion); XopF1 (1-70; 47-59= 9A, 1S) fragments were cloned in fusion with the LexA DNA-binding domain in the SalI and SpeI 111

112 sites of the bait vector pdbleu. HpaB was cloned into the SalI and NotI sites of the prey vector ppc86. Bait and prey constructs were cotransformed into the Saccharomyces cerevisiae CG1945 (placz/his3) using Frozen-EZ Yeast Transformation kit (Promega, U.S.). Transformation mixture was spread on minimal Synthetic dropout (SD) agar amended with Trp-Leu supplement (BD cat no ). Transformants grown on this medium were transferred to minimal SD agar amended with Trp-Leu-His supplement (Clonetech cat. No ) and checked for presence of growth after 72 hr of incubation at 30 o C (Nodzon and Song 2004). In Vitro Pull Down Assay XopF1, XopF1 1-70, XopF1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion); XopF1 (1-70; 47-59= 9A, 1S) fragments were cloned in fusion with maltose-binding domain in SalI and NotI sites of vector pmal86. HpaB was cloned into the NotI and SalI sites with fusion to FLAG-C terminal in pflag-ctc vector. These vectors were then transformed into E.coli DH5α. The transformants carrying inserts were sequenced to confirm in-frame fusion. These vectors were then transferred to expression E.coli strain BL21 (DE3). The cytoplasmic expression of the HpaB-carboxy-terminal FLAG fusion protein and XopF1 variants fused to MBP was carried out by following protocol. Single colony cultures of the above expression E.coli carrying different fusion vectors were grown overnight at 37 o C. A 500 μl aliquot of the overnight cultures was transferred into LB broth containing 50 μl Amp, 200μl 50% glucose and continued growth at 37 o C until OD 600 =0.6. Protein expression was induced by adding 25 μl of 1M IPTG and growth was continued for 3 more hr at 37 o C. The cells were pelleted by centrifuging at 6000 rpm for 10min at 4 o C. Supernatant was discarded and cell pellets were weighed and stored at -20 o C until further purification steps. The above cell pellet was thawed and resuspended in 5 ml of 1X 112

113 MBP buffer (20mM Tris-HCl ph 7.4, 200mM NaCl, 1mM EDTA, 1mM dithiothreitol) per 50 ml culture. The resuspended cells were aliquoted into 1.5 ml eppendorf tubes and sonicated (30s 4 times) (Sonicator setting 9.5, reading =12-15). Fifteen microliters of this sonicated mixture was placed in a separate tube as total protein and stored. For HpaB-FLAG tagged protein and FLAG-CTC protein, samples were stored at this point without further purification. XopF1 variant fusion protein samples were further purified using resin columns. Resin was equilibriated by washing 50 μl of slurry with 500 μl 1X MBP buffer four times at 1000 rpm for 1 min. Sonicated cell suspension was centrifuged at 6500 rpm for 15 min at 4 o C. Fifteen microliters of the supernatant was placed in a separate tube and stored. The remaining supernatant was loaded onto pre-equilibriated resin and shaken gently on a rotary shaker at 4 o C for 1 hr. The resin was then washed five times with 800 μl of 1X MBP buffer. Resin-bound fusion protein samples were stored at -20 o C for later use. Protein concentration in the purified protein samples was checked by running SDS-PAGE gel. Each MBP-tagged XopF1 variant fusion protein sample bound to resin was mixed with FLAG-tagged total protein. The tubes with the combination of mixtures of FLAGtagged protein and MBP-tagged protein were rocked at 4 o C overnight. Resin-bound protein samples were washed with 1X MBP buffer five times by centrifuging at 1000 rpm for 1min at 4 o C. Fifteen microliters of resin-bound protein samples were loaded on 15% SDS-PAGE and were run at 120 V. The proteins separated by SDS-PAGE were transferred to the nitrocellulose membrane by semi-dry blotting for 30 min. The membrane was then incubated in 20 ml TTBS [1ml of tween-20 to 1 L of TBS (10mM Tris 3.12 g; 150 mm NaCl g, ph 7.5, bring volume to 2 L)] for 10 min at room 113

114 temperature with shaking on rotator mixer. The membrane was then incubated for 1 hr at room temperature in 15ml blocking solution (5 g dry milk to 100ml TTBS) to reduce non-specific binding. Blocking solution was decanted. Fifteen milliliters of primary antibody (anti-flag rabbit antibody) diluted fold dilution in blocking solution was added and then the suspension and membrane were incubted on a rotary shaker for 1 hr. After incubation, the membrane was washed with 15 ml of TTBS, 2 times for a total of 30 min. A 15 ml aliquot of secondary antibody (Anti-rabbit goat IgG antibody conjugated to Alkaline phosphatase) diluted 2500 fold was applied for 1 hr of gentle shaking at room temperature. Secondary antibody was then washed and the membrane was washed with 15 ml TTBS twice, for a total of 30 min. After washing, membrane was transferred to a plastic petri dish. Color development mixture was prepared by dissolving a pill made of alkaline phosphatase buffer (AP), BCIP, NBT in 10 ml of ddh2o. The color development mixture was added to a dish containing the membrane and gently shaken until bands became visible. Development was stopped by discarding the mixture and rinsing with water. Membrane was then allowed to air dry. Results General Characteristics of Secretion and Translocation Signals in N Terminal Region of Xanthomonas Type III Effectors Frequency of each amino acid within the first 50 amino acid region of a set of known secreted and translocated Xanthomonas effectors was determined. Six predictive rules have been developed in case of Pseudomonas effectors (Petnicki- Ocwieja et al. 2002). Pattern and amino acid bias varies to a greater extent in Xanthomonas effectors compared to Pseudomonas effectors. A) First 50 residues are rich in Pro/ Ser/ Ala. In Xcv, Ser content within the first 25 amino acids of known TTS 114

115 substrates varies 8% (HrpB2) and 32% (HrpF). This is higher than Ser content in N termini of non-secreted components of TTSS (between 0% in HrcN and 12% as in HrcT). B) For few effectors like AvrBs1, AvrRxv, AvrBsT, AvrXv3, AvrXv4, XopB, XopF1, XopP, XopN, richness in Ser was observed as was true for Pseudomonas effectors. For effectors belonging to avrbs3 family, the first 50 residues are rich in Pro along with Ala and Arg. C) Similar to Pseudomonas effectors, Ile, Leu, Val can be found in positions 3, 4, 5, not both and preceded by polar amino acids. D) Asp and Glu can occur within first 12 positions, in contrast to Pseudomonas effectors. E) The group of effectors which was mentioned above showing richness in Ser i.e. AvrRxv, AvrXv4, AvrXv3, AvrBsT, XopA, position 5 was found to be occupied by, either, Ile, Leu, Phe, Tyr, or Trp, which are rarely found at position 5 in Pseudomonas effectors. Clearly, due to several differences between the effectors from different species, there is need to develop a separate prosite pattern for xanthomonads for this amino acid bias which can then be employed to screen the draft genomes for candidate effectors. Screening Whole Genomes for Candidate Type III Effectors The first 100 amino acids of known effectors from xanthomonads were used as training set. The MEME program identified a number of motifs, which were assigned based on amino acid frequencies among a group of effectors. Position specific scoring matrices (PSSM) corresponding to each motif were used to create input for the screening program. Model parameters were adjusted by validation against known Xanthomonas effector set and the well characterized Xanthomonas euvesicatoria Xcv genome. The program gave hits for all the known characterized effectors with high scores. However, the major drawback was that it also identified type II and type IV 115

116 substrates; therefore the search strategy is not foolproof. We also searched draft genomes of xanthomonads using the above models. We tested several candidates using the avrbs2 reporter gene assay (Refer Chapter 2) and identified novel effectors from Xv and Xg. First 70 Amino Acids of XopF1 are Sufficient for Translocation into the Plant Cell. The translocation signal of type III effectors in xanthomonads was shown to be located within the most N-terminal amino acid residues (Buttner and Bonas 2006; Mudgett et al. 2000, Schechter et al. 2004; Schesser et al. 1996). Based on the models developed above, candidate translocation signal sites were predicted. The coding sequence corresponding to the first 70 amino acids of XopF1, an effector from Xcv 85-10, along with a 500 bps long upstream region containing the endogenous promoter were fused to avrbs in pbluescript using a BglII restriction enzyme site. The portion of avrbs2 effector, avrbs , lacks its own secretion and translocation signal. Therefore, avrbs cloned alone in plafr3 and conjugated in TED3 race 6 is not translocated into plant cells of pepper cv. ECW-20R. TED3 race 6 was chosen as a recipient since it contains an inactivated version of avrbs2. The xopf :avrbs2 fragment from pbluescript clone was moved to plafr3 resulting in a clone designated as plafr:xopf1-70 and conjugated into Xcv TED3 race 6 by triparental mating. Transconjugants when infiltrated into the leaves of pepper cv. ECW-20R, showed a strong HR by 24 hr post inoculation (Figure 4-1). This shows that the first 70 amino acids contain a translocation signal necessary for delivery of the protein into plant cells. 116

117 Type III Effector XopF1 is Dependent on Global Chaperone HpaB for its Translocation. XopF1 has been shown earlier to be translocated only in the presence of the chaperone, HpaB (Buttner et al. 2006). We were interested in identifying the chaperone binding site(s). plafr:xopf1-70 was conjugated into a TED3 race 6 hpab mutant and infiltrated into leaves of pepper cv. ECW-20R. This fusion construct did not elicit an HR 48 hr after inoculation (Figure 4-2). This indicates that the first 70 amino acids might contain a possible HpaB binding site. First 40 Amino Acids of XopF1 are not enough for Translocation into Plant Cells. Next we decided to narrow down the location of the translocation signal and HpaB binding site within the first 70 amino acids of XopF1. The first 40 amino acids were fused to the reporter gene and transferred to plafr3 resulting in plafr3 (xopf1 1-40:avrBs2) clone, referred to as plafr3:xopf1-40, which was conjugated into TED3 race6. The transconjugant failed to induce an HR on 20R by 48 hr after inoculation (Figure 4-1). One explanation for the absence of HR for above transconjugants is that the translocation signal is missing in the first 40 amino acids. A second possiblity is that the HpaB binding site is missing in the first 40 amino acids and although a translocation signal is present in the first 40 amino acids, the effector will not bind to HpaB and therefore translocation will not occur. A third possibility could be that both translocation signal and HpaB binding site could be overlapping and are missing in first 40 amino acids. Secondary Structure Analysis of XopF1 Effector. Although there is little or no sequence identity among type III effector chaperones, they have similarities in their structures in that they contain acidic dimers with three 117

118 alpha helices and five beta sheets. Specific secondary structures and specific residues are known to be involved in the effector-chaperone binding. These secondary structures include alpha helices and a single three-residue motif, the β-motif. Class I chaperones interact with one or more alpha helices on the effector protein (Lilic et al. 2006). In the case of Erwinia effector DspE and chaperone DspF interaction, DspF was shown to bind in the region rich in alpha helices and the β-motif. Alanine stretch mutagenesis of this region reduced binding of the corresponding chaperone (Triplett et al. 2009). We predicted the secondary structure of the XopF1 effector using secondary structure prediction programs Psipred (Jones 1999) and Garnier (Garnier et al. 1996). Psipred predicted alpha helix rich region from aa 49 through 56 (Figure 4-4), whereas, Garnier predicted alpha helices in two regions, one from 48 through 53 and another from 26 through 35 (Figure 4-5). Mutations or deletions in the β-motif have been shown to lead to loss of effector-chaperone binding (Lilic et al. 2006). Alignment of different type III effector substrates from plant and animal pathogens showed conserved β-motif residues, the first two of which are always hydrophobic and the third one which is mostly hydrophobic (Lilic et al. 2006). We searched for the probable conserved β-motif residues in XopF1 amino acid sequence by alignment. We found β-motifs within α-helix regions (47 through 59) as LRGRRASL (three β-motif residues underlined in the sequence; 52 nd, 57 th and 59 th residues) and another one as QAEDVAA (three β-motif residues underlined in the sequence; 25 th, 29 th and 31 st residues) within the 25 through 33 helix region. 118

119 Alanine Mutagenesis in Alpha Helix Regions Abolished HR of the Effector- Reporter Fusion Complex. We generated alanine mutants of XopF1 mutating amino acid residues 27 through 33 and 47 through 59 to alanine using 10 3 diluted pbs(xopf : avrbs2) as template. The generated PCR product was digested with the enzyme NheI, religated and transformed. The constructs were sequenced to confirm the mutated sequence. Since PCR also introduced random mutations in the avrbs2 gene, we digested the product using the BglII enzyme and separated the mutated xopf fragment from the avrbs2 fragment. The mutated xopf fragment was then again fused in frame to pbs (avrbs ) and transferred to plafr3 to get plafr3 [xopf1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion): avrbs ] and plafr3 [xopf1 (1-70; 47-59= 9A, 1S) : avrbs ] clone. Transconjugants carrying mutations at 27 through 33 and 47 through 59 were inoculated into the leaves of pepper cv. ECW-20R. Neither mutant elicited HR on 20R (Figure 4-3) indicating that either they had lost the HpaB-binding site or they carried a mutation in the translocation signal. Yeast Two-Hybrid Assay XopF1 full-length gene, XopF1 1-70, and both alanine mutants 27 through 33 and 47 through 59 were cloned in frame with the LexA-DNA binding domain in the bait vector pdbleu and HpaB was cloned in the prey vector ppc86. Yeast transformants carrying both these vectors were selected on SD-Leu-Trp-His medium. Y2H assay showed weak interaction of XopF1 full length with HpaB and very weak interaction for XopF 1-70, both alanine mutants, and HpaB (Figure 4-6). 119

120 In Vitro Pull Down Assay To confirm results obtained in Y2H assay, we cloned the above constructs from pdb-leu vector to pmal86 vector using SalI/NotI enzymes. HpaB was cloned into pflag-ctc with in-frame fusion. Fusion proteins containing XopF and the two alanine mutants 27 through 33 and 47 through 59, each fused in-frame with Maltose binding domain were expressed in E.coli BL21 (DE3) and purified using resin. Fusion protein HpaB-FLAG-CTC was expressed and total protein from the cell extracts was stored. This HpaB-FLAG-CTC total protein extract was allowed to bind to resin-bound xopf1 variants individually overnight and then co-immunoprecipitated using anti-flag antibodies. Pull down assay showed that HpaB-FLAG-CTC pulled down all XopF1 variants including two alanine mutants (Figure 4-7). The band observed for the two alanine mutants indicates that HpaB could bind to both alanine mutants. Binding to the two alpha helices might be essential for formation of HpaB-XopF1 complex and hence mutating individual alpha helix still allowed binding of HpaB to the other intact alpha helix of XopF1. This binding might occur to the alanine mutants in vitro but may not be translocated by type III secretion system in planta and hence cannot elicit HR when fused to reporter. In fact, in both alanine mutants, we mutated three β-motif residues as well to alanine, which are part of alpha helix. Mutating β-motif residues to alanine did not change the hydrophobic properties of the residues at that position and could still possibly form the β-motif structure. However, the other interacting regions of alpha helix of XopF1 were mutated in the two mutants. These might not be able to interact with corresponding residues in HpaB and may not form a stable HpaB-XopF1 complex. 120

121 Concluding Remarks Plant and animal pathogens deliver type III effectors into the host cell. Two functional signals are believed to be present in effectors, which direct their journey through the bacterial cytoplasm to the host cell. Usually these signals are present in the N terminal region, with a short N terminal fragment sufficient for secretion into the culture medium and longer one sufficient for translocation in vivo (Mudgett et al. 2000; Sory et al. 1995). Different type III prediction programs have been developed based on the amino acid bias in the N terminal region. But there is no amino acid sequence similarity found in the plant/animal pathogen effectors. In pseudomonads, six predictive rules have been suggested to search for the type III effectors in the draft genome. We compared those six rules to the known Xanthomonas effectors. Richness in Ser content in first 50 amino acids is found in few Xanthomonas effectors, however exception to this would be avrbs3 family effectors. Similar to Pseudomonas effectors, Ile, Leu, Val can be found in positions 3, 4, 5, not both and preceded by polar amino acids. Asp, Glu can occur within first 12 positions, in contrast to pseudomonas effectors. Due to these differences, pattern developed for Pseudomonas effectors cannot be used for searching for the novel effectors in the draft genomes of Xanthomonads. We developed different matrices describing the amino acid biases in the N terminal 100 amino acid residues of known xanthomonas effectors. We used MEME program to develop the matrices and then used C-based program to screen the genomes of xanthomonads for the type III effectors. This program can identity all the known effectors. Apart from these known effectors, it identified other putative candidates. When we used the program to screen our three draft genomes, candidate effectors were assayed using avrbs2 reporter gene assay. We identified 2 novel effectors in the search as mentioned in Chapter 2. The 121

122 drawback of this program is that it also gave type II and type IV secreted proteins as hits suggesting false positive and negatives could not be avoided with this program as well. Here we have reported analysis of N terminal region of type III effector XopF1. We have selected XopF1 as model for the analysis since it is shown to be dependent on HpaB chaperone for its translocation (Buttner et al. 2006). We have demonstrated here that first 70 amino acids of XopF1 effector contain the translocation signal and are sufficient for delivering the fused reporter into the plant cell. Previous work on AvrBs2 has identified the translocation signal present between amino acids (Mudgett et al. 2000). To narrow down the location of translocation signal in XopF1, we cloned coding sequence of first 40 amino acids of XopF1 in frame with reporter gene avrbs and assayed by inoculating the leaves of pepper cv. ECW-20R. The first 40 amino acids failed to deliver the reporter into the plant cell and induce a BS2-based HR. The inability of first 40 amino acids to induce HR could be imparted to absence of translocation signal within first 40 amino acids or absence of chaperone HpaB-binding site within first 40 amino acids. In animal pathogenic type III effectors dependent on chaperone, chaperone-binding site and translocation signal have been shown to be overlapping. This explanation could also be true for XopF1, since XopF1 is not translocated in absence of HpaB. It could be that since a translocation signal is masked by the HpaB binding site and hence only when HpaB binds to it, it is directed for delivery by HpaB to the secretion apparatus. This also could be the distinction between the chaperone dependent and chaperone independent effectors, a separate translocation signal and a separate chaperone-binding site in chaperone independent effectors. 122

123 In Yersinia effector YopE, there is no separate translocation signal, first 2-15 residues are sufficient for delivery into eukaryotic cells along with chaperone SycE binding domain in the region of residues. It was shown that residues are inhibitory to the effector release, however chaperone binding masks that inhibitory effect and allows its translocation (Boyd et al. 2000). Co-crystallization studies have determined effector- chaperone interaction and the residues of effector interacting with the chaperone. According to the model proposed by Lilic et al. (2006), β motif residues are hydrophobic which interact with chaperone hydrophobic residues. Mutation of these single three-residues to glycine resulted in instability of the effector-chaperone complex. We determined secondary structure of XopF1 and found two alpha helices, one around 30 th amino acid and one around 50 th residue. We also found β motif residues in the second helix region. We constructed alanine mutants in the two alpha helices regions. Transconjugants carrying mutations at both sites, 27 through 33 and 47 through 59 were inoculated into the leaves of pepper cv. ECW-20R. Both mutants did not elicit HR on 20R indicating that either they had lost HpaB-binding site or they carried mutation in translocation signal. According to the model proposed based on crystal structures of different effector-chaperone complexes, mutation in β motif residues region makes the complex unstable. Since we mutated β motif residues as well as alpha helices, HpaB could not bind and hence effector XopF1 was not directed towards secretion apparatus by HpaB chaperone. However in vitro experiments such as yeast two hybrid assay and pull-down assay showed that HpaB could bind to both alanine mutants 27 through 33 and 47 through 59, showing possibility that HpaB could have bound to the effector, but the complex might not be active to be 123

124 translocated. HpaB might require both the alpha helices for formation of stable complex. However when one alpha helix was mutated, it might have partially bound to the other alpha helix as seen in the in-vitro experiments. Another possibility is that since we generated alanine mutants, β motif which mainly constitutes hydrophobic residues could have been still active, however the alpha helices were disrupted, chaperone could not have formed stable complex of effector and chaperone and hence not translocated in inplanta experiments. The current analysis of chaperone dependent type III effector of Xanthomonas has opened new areas of study such as regulation of type III effectors by chaperone, role of chaperone dependent effectors. Since HpaB is shown to be essential for pathogenicity, HpaB-dependent effectors might be playing important roles in the establishment of infection (Buttner and Bonas 2006). In animal pathogen effectors, the chaperone is known to be responsible for hierarchy of effectors during translocation (Boyd et al. 2000). Chaperone dependent effectors get privilege in secretion and translocation. If this is true for xanthomonas pathosystem, the chaperone dependent effectors would be important pathogenicity factors in early stages of infection. 124

125 Table 4-1. List of bacterial strains and plasmids used in this study Strain designation Relevant characteristics Source or reference Xanthomonas euvesicatoria TED3 Pepper race 6 Minsavage, Univ of Florida TED3 hpab Race 6 HpaB deletion mutant This study Escherichia coli DH5α F - recaφ80dlacz M15 Bethesda Research Laboratories, Gaithersburg,MD BL21 (DE3) Host for Expression vector Song, University of Florida Plasmids plafr3 Tc r rlx + RK2 replicon, Tc r Staskawicz et al pbluescript II KS +/- Phagemid sequencing vector, Stratagene, La Jolla, CA Ap r prk2073 Sp r Tra +, helper plasmid Figurski and Helinski 1979 pbs(bglii::avrbs ::HA) Phagemid sequencing vector, Ap r Mary Beth Mudgett, Stanford university pdbleu Km r Song, University of Florida ppc86 Ap r Song, University of Florida pmal86 MBP binding domain, Ap r Song, University of Florida pflag-ctc Ap r,contains FLAG tag on C Jerry Minsavage terminus plafr3[xopf1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion): avrbs ] Tc r rlx + RK2 replicon, Tc r containing XopF1 alanine This study plafr3[xopf1 (1-70; 47-59= 9A, 1S) : avrbs ] pdbleu(xopf1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion)) pdbleu[xopf1 (1-70; 47-59= 9A, 1S) ] pmal86(xopf1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion)) mutant Tc r rlx + RK2 replicon, Tc r containing XopF1 alanine mutant Km r, XopF1 variant fused in frame with LexA binding domain Km r, XopF1 variant fused in frame with LexA binding domain This study This study This study ppc86 (hpab) Ap r This study Ap r, XopF1 variant fused in This study frame with MBP binding domain pmal86[xopf1 (1-70; 47-59= 9A, 1S) ] pflag-ctc (hpab) Ap r, XopF1 variant fused in frame with MBP binding domain HpaB fused In frame with C terminal FLAG tag This study This study 125

126 Figure 4-1. Phenotype on ECW-20R 24 hr post-infiltration by wild type strains and transconjugants. First 70 amino acids of xopf1 are sufficient for translocation of the effector into the plant cell, whereas first 40 amino acids are not sufficient for the translocation. The order of inoculation is as follows (anticlockwise, starting with top left): TED3 race 6 transconjugants carrying known Xanthomonas type III effector fused to avrbs2 reporter gene; TED3 race 6 transconjugants carrying plafr3(xopf :avrbs2) clone; TED3 race 6 transconjugants carrying plafr3(xopf :avrbs2) clone ; TED3 race

127 Figure 4-2. Phenotype on ECW-20R 24 hr post-infiltration by wild type strains, transconjugants, and mutants. First 70 amino acids of XopF1 are sufficient for translocation of the effector into the plant cell. However fusion of first 70 amino acids of XopF1 hooked to avrbs2 reporter gene is not translocated in absence of hpab chaperone. Order of inoculation as follows (anticlockwise, starting top left): TED3 race 6; TED3 race 6 transconjugants carrying plafr3(xopf :avrbs2) clone; TED3 race 6 hpab mutant transconjugants carrying plafr3(xopf :avrbs2) clone. 127

128 Figure 4-3. Phenotype on ECW-20R 24 hr post-infiltration by wild type strains, transconjugants and mutants. First 70 amino acids of XopF1 are sufficient for translocation of the effector into the plant cell. Alanine mutants XopF1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion); XopF1 (1-70; 47-59= 9A, 1S) do not show translocation of fusion reporter protein. The order of inoculation is as follows (counterclockwise from top left): TED3 race 6 transconjugants carrying plafr3 (xopf :avrbs2) clone; TED3 race 6; TED3 race 6 transconjugants carrying plafr3 (xopf1 (1-70; 27-33= 4A, 1C, 1S, 1 deletion) : avrbs2) clone.; TED3 race 6 transconjugants carrying plafr3(xopf1 (1-70; 47-59= 9A, 1S) : avrbs2) clone. 128

129 Figure 4-4. Secondary structure prediction by PsiPred for first 70 amino acid region of XopF1. Cylinder represents predicted alpha helix. 129

130 MKLSSDIGTAASRGAASHPPVQPTQAEDVAAPREERAPTGPLAGLASSSA helix HH HHHHHHHHHH H HHH sheet E E EE E EEEE turns T TTT T T T coil CC C CC CCCC CCCC C CCC C ALRGRRASLAGRASPHADEEGAMLGGSHRSDSSQSSQASDATFYTAQVVS helix HHH HHHHHHHHH sheet EEE EEEEEEE turns TT TT TT T coil CC CCCC CCCCC CCCCCCC C CC Figure 4-5. Secondary structure prediction by garnier for first 70 amino acid region of XopF1. H indicates alpha helix. 130

131 Figure 4-6. Yeast two hybrid interaction between alanine mutants of XopF and HpaB chaperone. Empty vector control contains pdbleu and ppc86 empty vectors. 131

132 Figure 4-7. In vitro pull down assay showing binding of HpaB chaperone to XopF1 variants. Lane 1: Kaleidoscope prestained standard, 2: XopF through 33 alanine mutant fused to MBP tag; 3: XopF through 59 alanine mutant fused to MBP tag; 4. XopF fused to MBP tag; 5. Empty; 6: total protein containing FLAG protein; 7: MBP protein; all pulled down using HpaB- FLAG-CTC tag. 132