Genomics and Effectoromics of Xanthomonads

GENOMICS AND EFFECTOROMICS OF XANTHOMONADS

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

2011

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

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.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 10

ABSTRACT ...... 12

CHAPTER

1 XANTHOMONAS-PLANT INTERACTIONS AND GENOMICS ...... 14

Background ...... 14 Type III Secreted Effectors and Their Role in Plant-Pathogen Interactions ...... 14 Avirulence Genes ...... 17 Contributions of Comparative Genomics Era ...... 19 Project Goal and Objectives ...... 20

2 COMPARATIVE GENOMICS REVEALS DIVERSITY AMONG XANTHOMONADS INFECTING TOMATO AND PEPPER ...... 21

Background ...... 21 Materials and Methods ...... 24 Genome Sequencing ...... 24 Assembly and Annotation ...... 25 Whole Genome Comparisons ...... 25 Phylogenetic Analysis ...... 26 Phylogeny Reconstruction ...... 26 Prediction of Effector Repertoires, Cloning of Candidate Effectors and Confirmation Using AvrBs2 Reporter Gene Assay ...... 27 Cloning of Pepper Specificity Genes in Xp...... 28 Results ...... 28 Draft Genome Sequences of Xv Strain 1111, Xp Strain 91-118 and Xg Strain 101 were Obtained by Combining Roche-454 (Pyrosequencing) and Illumina GA2 (Solexa) Sequencing Data...... 28 Relationships of the Strains to Other Xanthomonads using Whole Genome Comparisons ...... 29 Four Xanthomonads Show Variation in the Organization of the Type III Secretion Gene Clusters ...... 30 A Reporter Gene Assay Confirms Translocation of Novel Type III Effectors .... 30 Core Effectors among Four Xanthomonads Give Insights into Infection Strategies of the Pathogen ...... 31

Effectors Unique to Xp Might be Responsible for Restricting Growth on Pepper...... 32 Species-Specific Effectors ...... 33 Few Effectors are Shared among Phylogenetically Related Group Strains ...... 35 Xg Shows Evidence of Effector Acquisition by Horizontal Gene Transfer...... 35 All Four Xanthomonads Contain Ax21 Coding Gene but Only Xcv Contains a Functional Sulfation Gene...... 36 Two Type II Secretion Systems are Conserved in All Four Xanthomonas Genomes...... 37 Xanthomonads Possess Diverse Repertoires of Cell-Wall Degrading Enzymes, which are Present in Diverse Genomic Arrangement Patterns. .... 38 Genes Involved in Several Type IV Secretion Systems are Present in Genomes and Plasmids ...... 40 Type V Secreted Adhesins Function in Synergism During Pathogenesis ...... 41 Type VI Secretion System is Present in Xcv, Xv and Xp ...... 42 LPS Locus Displays Remarkable Variation In Sequence and Number of Coding Genes and Shows Host Specific Variation ...... 43 Analysis of DSF Cell-Cell Signaling System ...... 44 Cyclic Di-GMP Signaling ...... 45 Copper Resistance (cop) Genes are Present in Xv and Copper Homeostasis (coh) Genes are Present in All Strains ...... 46 Genes Unique to Xp as Compared to Pepper Pathogens Give Clues to its Predominance over Xcv in the Field and Host Specificity ...... 47 Pepper Pathogenicity/Aggressiveness Factors Increased In Planta Growth of Xp ...... 48 Genes Specific to Xg as Compared to Other Tomato/Pepper Pathogens may Explain its Aggressive Nature on Tomato and Pepper ...... 48 Genes Common to All Tomato Pathogens but Absent from Other Sequenced Xanthomonads ...... 49 The Evolution of Pathogenicity Clusters Corresponds to the MLST-Based Phylogeny ...... 50 Concluding Remarks ...... 50

3 AVIRULENCE PROTEINS AVRBS7 FROM XANTHOMONAS GARDNERI AND AVRBS1.1 FROM XANTHOMONAS EUVESICATORIA ELICIT HYPERSENSITIVE RESISTANCE RESPONSE IN PEPPER ...... 78

Background ...... 78 Materials and Methods ...... 79 Plant Material and Plant Inoculations ...... 79 Bacterial Strains, Plasmids and Media ...... 80 Library Preparation and Isolation of Clone with Avirulence Activity ...... 80 Deletion Mutant Construction ...... 81 Bacterial Population Dynamics in Infiltrated Leaf Tissue ...... 81 Determination of Electrolyte Leakage from Infiltrated Leaf Tissue ...... 82 Site Directed Mutagenesis of avrBs7 ...... 82 Sequence Analysis and Protein Homology Modeling ...... 83

Results ...... 83 Identification of Resistance in Pepper against Bacterial Spot Xanthomonads and Development of Introgression Lines Carrying the Resistance Gene ...... 83 AvrBs7 from Xv444 Elicits HR in Pepper cv. ECW-70R...... 84 AvrBs1.1 from Xcv str 85-10 Elicits Delayed HR on ECW-70R...... 85 Genetic Analysis of Bs7 Resistance in ECW-70R ...... 85 In-Planta Growth Studies and Electrolyte Leakage ...... 87 A Catalytic Tyrosine Phosphatase Domain Might be Responsible for Recognition by the BS7 R Gene Product in ECW-70R...... 88 There is Difference in the Timings of HR Elicitation by AvrBs7 and AvrBs1.1...... 89 Avirulence Proteins AvrBs7 and AvrBs1.1 Display Similar Tertiary Protein Structure...... 90 Host Specificity of Bacterial Spot Strains...... 90 Avr Genes avrBs7 and avrBs1.1 are Encoded on a Large Transmissible Plasmid...... 91 Concluding Remarks ...... 91

4 APPLICATION OF BIOINFORMATICS FOR TYPE III EFFECTOR SIGNAL ANALYSIS AND ITS INTERACTION WITH CHAPERONE ...... 105

Background ...... 105 Materials and Methods ...... 109 Data-Mining Strategy ...... 109 Bacterial Strains, Plasmids and Media ...... 109 Plant Material and Plant Inoculations ...... 110 In Planta Reporter Gene Assay ...... 110 Site-Directed Alanine Mutagenesis ...... 111 Yeast Two-Hybrid Assay ...... 111 In Vitro Pull Down Assay ...... 112 Results ...... 114 General Characteristics of Secretion and Translocation Signals in N Terminal Region of Xanthomonas Type III Effectors ...... 114 Screening Whole Genomes for Candidate Type III Effectors ...... 115 First 70 Amino Acids of XopF1 are Sufficient for Translocation into the Plant Cell...... 116 Type III Effector XopF1 is Dependent on Global Chaperone HpaB for its Translocation...... 117 First 40 Amino Acids of XopF1 are not enough for Translocation into Plant Cells...... 117 Secondary Structure Analysis of XopF1 Effector...... 117 Alanine Mutagenesis in Alpha Helix Regions Abolished HR of the Effector- Reporter Fusion Complex...... 119 Yeast Two-Hybrid Assay ...... 119 In Vitro Pull Down Assay ...... 120 Concluding Remarks ...... 121

5 PATHOGENIC STRATEGIES OF XANTHOMONAS GENUS ON PLANTS: LESSONS LEARNT FROM GENOMICS ...... 133

Background ...... 133 Materials and Methods ...... 134 Xanthomonas Genomes and Tools Used for Comparison ...... 134 Database for Xanthomonas Pathogenicity Factors ...... 134 Effectors Database Compilation ...... 135 Effector Analysis of the Test Case of Citrus Pathogens ...... 135 Results ...... 135 Type II Secretion Systems ...... 135 Type III Secretion System ...... 137 Type III-Secreted Effectors ...... 137 A CaseStudy – Screening for Candidate Type III Effectors from Draft Genomes and Possible Host Range Determinants...... 139 The three citrus canker genomes have important differences in regard to their repertoires of type III secreted effectors ...... 140 Effectors XopAI and XopE3 may play a role in citrus canker ...... 142 Additional differences in effector repertoires among CC genomes ...... 145 Adhesins ...... 146 Lipopolysaccharides and Xanthan ...... 148 Toxins ...... 148 Concluding Remarks ...... 149

6 SUMMARY AND DISCUSSION ...... 162

LIST OF REFERENCES ...... 169

BIOGRAPHICAL SKETCH ...... 189

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...... 53

2-2 Whole genome comparisons using MUMmer dnadiff program...... 53

2-3 Core effectors present in all four tomato and pepper xanthomonads ...... 54

2-4 Type III effectors specific to each species ...... 55

2-5 Effectors specific to particular groups of species ...... 57

2-6 Evidence of horizontal gene transfer using Alien Hunter analysis ...... 58

2-7 Repertoire of cell wall degrading enzymes in xanthomonads...... 59

2-8 Type VI secretion clusters in different xanthomonads...... 60

2-9 Genes/contigs representing T6SS in draft genomes as compared to Xcv...... 61

2-10 A comparison of rpf cluster from rpfB to rpfG found across a range of Xanthomonas genomes...... 61

2-11 Genes unique to Xp, grouped in clusters...... 62

2-12 Genes common to all pepper pathogens but absent from Xp...... 64

2-13 Genes present in all four tomato and pepper pathogens but absent from all other sequenced xanthomonads...... 67

3-1 List of bacterial strains and plasmids used in this study ...... 96

4-1 List of bacterial strains and plasmids used in this study ...... 125

5-1 Xanthomonas species and pathovars within species show host and tissue- specificity...... 150

5-2 Xop nomenclature for xanthomonas effectors ...... 152

5-3 Core effector genes from xanthomonads and their role in pathogenicity/ induction of resistance ...... 155

5-4 Variable effectors which contribute to the pathogenicity ...... 157

5-5 Putative effectors found in the XAC, XauB, and XauC genome sequences. .... 159

LIST OF FIGURES

Figure page

2-1 Maximum likelihood tree based on orthologous genes from xanthomonads and Stenotrophomonas...... 68

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...... 69

2-3 AvrBs2-based HR assay confirms translocation of novel effectors...... 70

2-4 Xylanase cluster organization...... 71

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

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

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

2-8 The Structure and phylogeny of the LPS cluster ...... 75

2-9 Pepper specificity genes increasing in planta growth of Xp...... 76

2-10 Correlation between phylogenies based on Multi-Locus Sequence Typing (MLST) core genome and pathogenicity clusters...... 77

3-1 Phenotype observed in leaves of ECW-70R 48 hr after infiltration with bacterial suspesions (adjusted to 108 cfu/ml) ...... 97

3-2 Phenotype on ECW-70R 24 hr and 48 hr post-infiltration by wild type strains, transconjugants and mutants...... 98

3-3 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...... 99

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 108 cfu/ml of (Xg51) wild type, transconjugants and mutant strains...... 100

3-5 Tyrosine phosphatase domain is essential for HR elicitation on ECW-70R...... 101

3-6 Alignment of avrBs1.1 and avrBs7 amino acid sequences using clustalw ...... 102

3-7 Fusion protein containing N-terminal of avrBs7 and C terminal of avrBs1.1 does not elicit HR on ECW-70R...... 103

3-8 Three dimensional structures of the two avirulence proteins based on homology modeling...... 104

4-1 Phenotype on ECW-20R 24 hr post-infiltration by wild type strains and transconjugants...... 126

4-2 Phenotype on ECW-20R 24 hr post-infiltration by wild type strains, transconjugants, and mutants...... 127

4-3 Phenotype on ECW-20R 24 hr post-infiltration by wild type strains, transconjugants and alanine mutants...... 128

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

4-5 Secondary structure prediction by garnier for first 70 amino acid region of XopF1. H indicates alpha helix...... 130

4-6 Yeast two hybrid interaction between alanine mutants of XopF11-70 and HpaB chaperone...... 131

4-7 In vitro pull down assay showing binding of HpaB chaperone to XopF1 variants...... 132

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

Neha Potnis

August 2011

Chair: Jeffrey B. Jones Cochair: David J. Norman Major: Plant Pathology

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.

85-10) 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

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.

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 two- component 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

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 14-3-3 isoforms (Kim et al. 2009). Effector XopJ suppresses defense-

related callose deposition and host protein extracellular secretion (Bartetzko et al.

2009). Another XopJ family member, avrRxv, interacts with host 14-3-3 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

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).

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 inter-

species 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.

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 14-

3-3 protein and induce cell death response (Whalen et al. 2008). Understanding R-Avr

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

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.

CHAPTER 2 COMPARATIVE GENOMICS REVEALS DIVERSITY AMONG XANTHOMONADS INFECTING TOMATO AND PEPPER1

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. 2011.

(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. 85-10 (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. 33913 (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. 306. 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 co-

evolution, 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

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. 85-10

(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 91-118 (Xp 91-118), and X. gardneri strain 101 (Xg 101) (ATCC 19865). We have

annotated and analyzed predicted pathogenicity factors in the draft genomes.

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 single-

colony 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.

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).

Phylogenetic Analysis

MLST sequences (fusA, gapA, gltA, gyrB, lacF, lepA) for all the genomes were

obtained in concatenated form from PAMDB website (http://pamdb.org). 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.

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 0.00001

(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::avrBs262-574::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 28oC in growth room. After 24 hours, strong HR was indicating

successful translocation of candidate effector fusions.

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 91-118 ∆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 105 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 27oC 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 91-118 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 91-

118 (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

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).

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 (avrBs262-574aa) 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

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

(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

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 Aw, 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

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%),

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

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 (axYS22) 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

Xcv (93% identity with Xoo PXO99 protein), Xp (94%), Xv (91%), and Xg (88%). The axYS22 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 axYS22 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.

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-O-

methylglucuronate 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

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-O- methylglucuronate 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.

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.

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

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).

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

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

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.

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

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).

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 (91-118∆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

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

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

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

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.

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 296 291 552 N50* 37 40 83 Mean contig length 18,686 18,082 10,014

Longest contig 153,834 133,836 88,536

Total length of 5,531,090 5,262,127 5,528,125 contigs

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 % of average genome aligned identity for the aligned sequences Xp Xcv 85.57 98.1 Xac 85.91 93.8 Xcc 74.23 87.36 Xoo MAFF 77.32 90.5 Xg Xcv 78.44 88.57 Xac 79.71 88.05 Xcc 83.33 88.83 Xoo MAFF 72.83 87.9 Xv Xcv 83.11 87.86 Xcc 76.35 87.37 Xac 80.07 87.90 Xoo MAFF 69.26 87.68

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 Kearney and diester Staskawicz, phosphodiesteras 1990. e XopD XCV0437 XVE_2372 XPE_2945 XGA_3151 C48-family SUMO Roden et al., cysteine protease 2004 (Ulp1 protease family); EAR motif XopF1 XCV0414 XVE_3220 XPE_2922 XGA_2763 - Roden et al., 2004 XopK XCV3215 XVE_0354 XPE_1077 XGA_3563 - 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 XCV4438 - XPE_0810 XGA_0949 Inosine uridine Roden et nucleoside N- al.,2004 ribohydrolase XopR XCV0285 XVE_0593 XPE_1215, XGA_1761 - Furutani et XPE_3295 al., 2009 XopX XCV0572 XVE_ 3610 XPE_1488 XGA_3272 - Metz et al., XVE_3609 XPE_1553 (second 2005 (partial) copy with frameshift) XopZ1 XCV2059 + (*) XPE_2869 +(*) - Furutani et al., 2009 XopAD XCV4315 XVE_4177 XPE_4269 XGA_0755 SKWP repeat Guidot et al., /4314/431 protein 2007 3 *Xv and Xg contain effector xopZ2 belonging to the same family xopZ.

Table 2-4. Type III effectors specific to each species Effector Locus tags Effector Pfam domains/ Comments/Reference homolog biochemical motifs

Effectors specific to Xv XopJ2 XVE_4840 AvrBsT C55-family Minsavage et al., 1990 (partial); cysteine protease

XVE_3769 or Ser/Thr (partial) acetyltransferase XopAG XVE_2415 AvrGf1 - Rybak et al., 2009 XopAI XVE_4756 XAC3230 - Moreira et al., 2010

Effectors specific to Xg class avrBs1 XGA_0724 AvrA - This study (84% identity) AvrHah1 (Fragmented XGA_4845/ AvrBs3 Transcriptional AvrBs3 present in few in assembly) XGA_3187 activator, nuclear euvesicatoria strains. localization Schornack et al., 2008 XopAO XGA_1250 AvrRpm1 - This study (61% identity) 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 White et al., 2009 dehalogenase-like hydrolase XopJ4 XPE_1427 AvrXv4 SUMO protease Astua-Monge et al., (experimental); 2000b; Roden et al., YopJ-like serine 2004. threonine acetyl transferase domain (predicted) XopAF XPE_4185 AvrXv3 Transcriptional Astua-Monge et al., activator domain 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 Roden et al., 2004 transferase domain and haloacid dehalogenase-like hydrolase

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

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 XCV2280, XVE_1190, Putative Thieme et al., 2007 XGA_2887 transglutaminase XopG XCV1298, XVE_4501, M27 family peptidase This study XGA_4777 clostridium toxin Effectors common to Xv, Xg but absent from Xp and Xcv XopAM XVE_4676, XGA_3942 - 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 May not be T3SE; Xu et hexapeptide repeats al., 2006 (X-[STAV]-X-[LIV]- [GAED]-X)- acyltransferase enzyme activity 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, XCV0581 - 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, XCV2942 - 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, XCV0657 - Furutani et al., 2009 XopAK XPE_4569, XCV3786 - 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_2975 - Mukaihara et al., 2010

Table 2-6. Evidence of horizontal gene transfer using Alien Hunter analysis Gene/ Locus tag Score for % tRNA/transposase/mo Evidenc Gene Alien GC bile genetic elements e of cluster Hunter in the vicinity HGT (Threshold = 12.496) avrBs1 XGA_0724 32.031 45 Transposase Good

xopAO XGA_1250 13.735 48 Predicted to be Good located on plasmid

xopAS XGA_0764/XGA_0 19.844 59 Transposase Good 765

xopG XGA_4501 21.272 50 ISxac2 transposase in Good XVE_4777 Xcv XCV1298

xopAQ XGA_2091 Does not 51 Could not be Weak belong to predicted anomalous region xopZ2 XGA_2762 Does not 69 IS30 transposase XVE_3221 belong to 3000 bps apart Weak anomalous region

LPS XPE_3787 to Belongs to 50 No Good cluster XPE_3795 anomalous region

Bacterioci XPE_0786 to Belongs to 50 tranposase Good n cluster XPE_0790 anomalous region

Table 2-7. Repertoire of cell wall degrading enzymes in xanthomonads. Gene Family Enzymatic Xp Xac Xcv Xv Xg Xcc str. Xoo name function 33913 str.

KACC

Xylanases xyn10A GH10 Endo-β-1,4- 2014 4254 4360 2337 1172 4118 4429 xylanase xyn10B 2016 4252 4358 - - - 4428 EC:3.2.1.8 xyn10C 2020 4249 4355 2333 0341 4115 -

aguA GH67 α- 4318 4227 4333 4712 2473 4102 4419 glucuronidase

EC:3.2.1.139

xyn51A GH51 β-D-Arabino- 0180 1286 1335 1029/1030 2303 1191 1317 furanosidase

EC:3.2.1.55

xyn5A GH5 Endo-β-1,4- 4682 0933/34 0965 - - 0857 3618 xylanase partial

EC:3.2.1.8

Glucanases cel8A GH8 Endo-1,4-β-D 1965 3516 3641 0432 - - - glucanase

cel9A GH9 2345 2522 2704 1327 0588 2387 -

Pectate lyases pel1A PL1 Pectate lyase 3841 3562 3687 1933 4024 0645 0821

pel1B EC:4.2.2.2 1563 2986 3132 3512 0893 2815 -

pel1C - 2373 2569 - - - -

pel3A PL3 Pectate lyase - 2922 - 3222 2761 1219 -

EC:4.2.2.2

pel4A PL4 Rhamno- 1975 3505 3632 2592 4531 3377/78/79 1078 galacturonan lyase

EC:4.2.2.-

pel9A PL9 Pectate lyase - - 2278 1927 1853 - 2265

EC:4.2.2.2

Table 2-7. continued Gene Family Enzymatic Xp Xac Xcv Xv Xg Xcc str. Xoo name function 33913 str.

KACC pel10A PL10 Pectate lyase - - - 4069 5124 0122 -

EC:4.2.2.2

Table 2-8. Type VI secretion clusters in different xanthomonads. Strain T6SS #1 T6SS #2 T6SS #3 Phosphorylation- Kinase / Phosphatase / - Kinase / Phosphatase / type regulators: Forkhead Forkhead AraC-type - - AraC regulators: Xvm NCPPB702 YES / / Xvm NCPPB4381 YES / / Xaub / / YES Xauc / / YES Xac / / XAC4116 - XAC4148 Xv / / YES Xp YES / YES Xcv XCV2120 - XCV2143 / XCV4206 - XCV4244 Xoo KACC10331 XOO3034 - XOO3052 XOO3466 - / XOO3517 Xoo MAFF 311018 XOO2886 - XOO2906 XOO3286 - / XOO3319 Xoo PXO99A XOO0245 - XOO0270 XOO2029 - / XOO2060 Xoc BLS256 XOC2523 - XOC2545 XOC1309 - / XOC1370 Xg / / / Xcc ATCC33913 / / / Xcc 8004 / / / Xca 756C / / / Xalb / / /

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 XCV2120- Contig XCV4244- Contig XCV4244- XCV2127(N) 120 XCV4236(N) 233 XCV4216 Contig XCV2127(i) Contig XCV4236(i) 287 287 Contig XCV2127(i) Contig XCV4236(i) 288 288 Contig XCV2127(i) Contig XCV4236(i) 291 291 Contig XCV2127(i) Contig XCV4236(i) 238 238 Contig XCV2127(i) Contig XCV4236(i) 254 254 Contig 90 XCV2127(C)- Contig 44 XCV4236(C)- XCV2137(N) XCV4216 Contig XCV2137(C)- no XCV4215 no XCV4215 240 XCV2144 homolog homolog Contig XCV4214- Contig XCV4214- 116 XCV4209(N) 183 XCV4212(N) Contig XCV4209(C)- Contig XCV4213(N)- 133 XCV4206(N) 148 XCV4206(N) Contig XCV4206(i) 233 Contig XCV4206(C) Contig XCV4206(C) 195 175

Table 2-10. 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 XCV1921 2934 0530 2948 rpfF XC_2332 XOO2869 XCV1920 2932 0528 2950 rpfC XC_2333 XOO2870 XCV1919 2930 0526 2952 rpfH XC_2334 Absent XCV1918 2928/2926* 0524 2954 rpfG XC_2335 XOO2871 XCV1917 2924 0522 2956

Table 2-11. 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 Predicted transcription regulator XPE_2401 containing HTH domain Uncharacterized protein conserved XPE_2402 in bacteria XPE_2403 present in Xv XPE_2404 plasmid mobilization system XPE_3894 relaxase XPE_3895 XCV1122 52% 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

Table 2-11. 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 XPE_3601 xanthomonads 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_37550 95% hypothetical protein XPE_3367 XCV0352 XPE_3368 XCV0353 no hit to any plant XPE_3369 pathogen hypothetical protein no hit to any plant XPE_3370 pathogen hypothetical protein no hit to any plant XPE_3371 pathogen hypothetical protein no hit to any plant XPE_3372 pathogen hypothetical protein no hit to any plant Activator of Hsp90 ATPase XPE_3373 pathogen homolog 1-like protein. 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 XPE_1379 plant pathogen XPE_1380 EF-hand calcium binding protein Cluster 11- Upstream, downstream flanking genes also present in Xcv XPE_0135 XAC3183 Hypothetical protein XPE_0136 XAC3182 Hypothetical protein Cluster 12 signal peptide, transmemb XPE_0734 helices XPE_0735 hypothetical protein Xccb100_0356, vasculorum and XPE_0736 musacearum hypothetical protein

Table 2-11. continued Distribution of flanking Locus tag in Xp/Gene OID genes Function Cluster 13 Type I site-specific restriction- modification 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 restriction- modification system, R (restriction) subunit and related XPE_2194 Xoo helicases XPE_2195 Xoo, Xoc hypothetical protein

Table 2-12. Genes common to all pepper pathogens but absent from Xp. Locus tag in Xcv85- Gene symbol Product name Evidence to be 10 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 Alavi, SM et. al., translocase 2008 in X. fuscans – bean pathosystem and this study. XCV3718 gmd GDP-mannose 4,6- dehydratase (EC: 4.2.1.47) XCV3720 wxcB Putative protein kinase XCV3722 wzm O-antigen ABC transporter permease XCV4257 rpmB LSU ribosomal protein L28P

Table 2-12. continued Locus tag in Xcv85- Gene symbol Product name Evidence to be 10 involved in pathogenicity/ virulence XCV1298 Type III effector (homolog of hopH1 from Pseudomonas syringae)

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

Table 2-12. continued Locus tag in Xcv85- Gene symbol Product name Evidence to be 10 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 XCV4421

Table 2-13. 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 Xanthomonas campestris pv. –COG belonging to musacearum NCPPB4381 transposase, inactive derivatives XCV2641 Hypothetical protein X. c. musacearum and X. c. 0.65 vasculorum (identity 37, 31% respectively) XCV4416 Hypothetical protein Pectobacterium carotovorum 0.57 X. fuscans pv. aurantifolii XCV0615 Transposase 17 Acidovorax, X. c. musacearum and X. 0.62 superfamily Hypothetical c. vasculorum protein COG1943 (transposase, inactivated derivates) XCV0112 COG4704 Stenotrophomonas, X. c. musacearum 0.65 uncharacterized protein and X. c. vasculorum conserved in bacteria XCV0111 putative lignostilbene- Stenotrophomonas, Ralstonia, X. c. 0.66 alpha,beta-dioxygenase- musacearum and X. c. vasculorum phenylpropanoid compound degradation

XCV0041 putative penicillin Ralstonia, X. c. musacearum and X. c. 0.64 amidase (fragment) vasculorum

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.

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.

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

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.

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

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

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

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.

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.

Figure 2-10. 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.

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.

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 26oC during 12

hr light and 15oC, 12 hr dark cycle. Plants were observed for 48 hr following inoculations

for development of HR/ watersoaking symptoms.

A four to five cm2 area of fully expanded leaves was infiltrated using a hypodermic

needle and syringe with bacterial suspension adjusted to 108 CFU/ml (Hibberd et al.

1987). All experiments were carried out in three replicates.

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 28oC 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 37oC 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 28oC

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 -80oC.

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, pXv444-352, containing a 20 kb insert

gave an HR on ECW-70R. This clone was selected for further subcloning. A 5kb

BamHI- BamHI DNA fragment subclone from the pXv444-352 clone was transferred into pBlueScript (Stratagene, CA). This 5 kb subclone pXv444-352Bam8 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 pXv444-352Bam8 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 ORF-

deleted 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 105 CFU/ml using sterile tapwater and then

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 15oC-26oC (12 hr dark/light period) for 8 days. Leaves were sampled every 48 hr. An area of 1cm2 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 28oC for three days. Colonies were then counted and the bacterial

populations were calculated as cfu/cm2 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 108 CFU/ml (O.D at 600nm = 0.3).

Inoculated plants were kept in the growth room at 26oC during 12 hr light period and

15oC 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 28oC.

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

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 bell-

shaped, 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 F2 population were obtained after self- pollinating a F1 plant. Segregation of resistance in the F2 population seemed to occur,

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 bcF1 generation were screened for resistance to Xv444, which provided a strong HR in resistant plants. In a third backcross, F1 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 7th backcross was obtained that did not appear to have the

fertility problems present in the early population of the interspecific cross and was

th uniform in resistance. A population in the 7 bcF4 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

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 39.734 kDa. It shows 67% sequence identity at the amino acid level to avrBs1.1 (XCVd0105) from Xcv

str. 85-10, whose genome was sequenced previously. The possibility that avrBs1.1 from

Xcv str.85-10 was responsible for HR when infiltrated into leaves of ECW-70R was

pursued experimentally.

AvrBs1.1 from Xcv str 85-10 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 F2 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 F2 population.

A total of 166 F2 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 (χ2 =

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 F2 population. Out of a total 59 F2 plants tested, 24 plants (40%)

were susceptible, showing χ2 = 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 F2 population exhibited 71 resistant (66%) plants and 36 (34%)

susceptible plants, giving an χ2 value of 4.264 with the p value slightly lower than 0.05.

The same avr gene when introduced into Xcv TED3 race 6 and inoculated in the leaves

of 59 F2 plants yielded 40 resistant (68%) plants and 19 susceptible (32%) plants, with

the χ2 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.

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/cm2 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).

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 (Cys265

→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-

specificity phosphatase activity. Xanthomonas campestris pv. campestris str. 33913

along with Xcc str. 8004 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

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 14-3-3 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).

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

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 7th

backcross was referred to as ECW-70R, which was used to determine genetic

inheritance of resistance. Segregation analysis of the F2 population identified a gene-

for-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. 85-10 elicits delayed HR on ECW-70R. The segregation of resistance

genes in an F2 population was identical after inoculation with Xv444 and Xcv85-10. The corresponding avr genes in Xv444 and Xcv str. 85-10 have been isolated and characterized. The avr gene from Xv444 has been referred to as avrBs7, while Xcv str.

85-10 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

around catalytic residues in avrBs7 as well as Cys265 →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 p130Cas 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).

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. 85-10 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.

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.

Table 3-1. List of bacterial strains and plasmids used in this study Strain designation Relevant characteristics Source or reference

Xanthomonas euvesicatoria 85-10 Pepper race 2, tomato race 1, Rifr Minsavage et.al. 1990 TED3 Pepper race 6 E3 CuR, HR(+) ECW 70R, avrBs1.1(+) R. E. Stall (Florida, 1960) Xv718 CuR, HR(+) ECW 70R, avrBs1.1(+) .Jones (Puerto Rico 1991) Xv881 CuS, HR(-) ECW 70R, avrBs1.1(-) Jones (Mexico, 1992) Xv669 CuS, HR(-) ECW 70R, avrBs1.1(-) Jones Xv1025 CuR, HR(-) ECW 70R, avrBs1.1(-) Jones (Mexico, 1992) Xv818 CuS, 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 Tcrrlx+RK2 replicon, Tcr Staskawicz et.al., 1987 pBlueScript II KS +/- Phagemid sequencing vector, Apr Stratagene, La Jolla, CA pRK2073 Spr Tra+, helper plasmid Figurski and Helinski 1979

A B

Figure 3-1. Phenotype observed in leaves of ECW-70R 48 hr after infiltration with bacterial suspesions (adjusted to 108 cfu/ml) A) Xv444, and B)Xg51

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 108 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.

9 ECW-70R 9 ECW 8 8 ) ) 2 7 2 7

6 6 (CFU/cm (CFU/cm 10 10 5 5 Log Log 4 4

3 3 0 2 4 6 8 0 2 4 6 8 A Days after infiltration Days after infiltration B

10 ECW-70R 10 ECW 9 9 ) ) 2 8 2 8 7 7 (CFU/cm 6 (CFU/cm 6 10 10 5 5 Log Log 4 4 3 3 0 2 4 6 8 0 2 4 6 8 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 105 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.

300 ECW-70R 300 ECW 250 250 200 200 150 150 Mhos Mhos μ 100 μ 100 50 50 0 0 0 12 24 36 48 60 0 12 24 36 48 60 A Hours after infiltration Hours after infiltration B

300 ECW-70R 250 ECW 250 200 200 150 150 Mhos Mhos μ μ 100 100

50 50

0 0 0 12 24 36 48 60 0 12 24 36 48 60 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 108 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

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 108 cfu/ml.

101

avrBs1.1 MPNKISGSIAPSASSDAMKSADCAENIKEEVVSKHVHQAVPAELADLPSRQPPRSKTA-L avrBs7 MPNPVSRSSTSSVSGKGSDDADVVADIKQEAVVEPGNQSTPHGLEGLA----PRSKTARD *** :* * :.*.*... ..** . :**:*.* : :*:.* * .*. ****** avrBs1.1 YQVIQKFRDPLPLPPPPTSHPVLAYDRDLGSS-DNFRSSDEFDLPESLNPTGWKNLHVSG avrBs7 LSLIKKFSNPLPLPQRPTEIPVLQYDRSPRSSSDNFRSSDDFDLPESCNPTGWKDLHVSG .:*:** :***** **. *** ***. ** *******:****** ******:***** avrBs1.1 SGSIASIGQITRLRPSKERPVVVLDAREESHAIVGGYPGTWRTPNNWGNAGKSRDEALAD avrBs7 SGSIASISQITRLNPSRDRPVIVLDVREESHAIVGGYPATWRAPNNWANVGKSREEVLAD *******.*****.**::***:***.************.***:****.*.****:*.*** avrBs1.1 EQQRIQALKSQETVHIFHRKDVKSEARNPRGATLSKPLIFSEEELVRAAGAKYVRLTVTD avrBs7 EHEKIRAIKSQETVQILHRKDVKNGFPNPRSVKLSNPSIFSEEELVRNAGAEYLRLTVTD *:::*:*:******:*:******. ***...**:* ********* ***:*:****** avrBs1.1 HLSPRADDIDAFIAMEREMAHDERLHVHCGMGLGRTTIFIVMHDILRNAAMLSFDDIIER avrBs7 HLGPRADDIDAFVRMERNMAPHERLHVHCGVGQGRTGIFIAMHDILRNAHIISFEDIIKR **.*********: ***:** .********:* *** ***.******** ::**:***:* avrBs1.1 QRKFNPGRSLDNNKDVSDKGRSEFRNERSEFLPLFYEYAKQNPKGQPLLWSEWLDHNA-- avrBs7 QLAFNPGRALDFNKDVSHEGRSDFRNDRLELISLFYEYAKSNPNGQPSLWSEWLRAANKT * *****:** *****.:***:***:* *::.*******.**:*** ******

Figure 3-6. Alignment of avrBs1.1 and avrBs7 amino acid sequences using clustalw

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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 108 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 85-10

103

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.

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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 50-100 most N-terminal amino acids of some

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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

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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

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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 effector-

chaperone 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

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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 28oC on nutrient

agar (NA) plates. For plant inoculations, the cultures were suspended in sterile tap

water and adjusted to A600= 0.3 with a spectrophotometer (Spectronic 20, Spectronic

UNICAM, U.S.). E. coli strains or transformants were grown at 37oC on Luria-Bertani

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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 28oC 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 -80oC. Yeast strain CG1945 was grown on YPD agar (Peptone

2%, Yeast 1%, glucose 2%, pH 5.8) or YPD broth at 30oC overnight. For prepration of yeast competent cells, the culture was grown in YPD broth until mid-log phase

(OD600=0.8-1.0).

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 25oC to

35oC (day/night). Plant leaves were infiltrated with 108 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

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BglII, PCR amplicons were ligated with BglII-digested pBS(BglII::avrBs262-574::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 28oC 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(XopF11-70::avrBs262-

574::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, XopF11-70, 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

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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. 630417). Transformants grown on this

medium were transferred to minimal SD agar amended with –Trp-Leu-His supplement

(Clonetech cat. No. 630419) and checked for presence of growth after 72 hr of

incubation at 30oC (Nodzon and Song 2004).

In Vitro Pull Down Assay

XopF1, XopF11-70, 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 37oC. A 500

μl aliquot of the overnight cultures was transferred into LB broth containing 50 μl Amp,

o 200μl 50% glucose and continued growth at 37 C until OD600=0.6. Protein expression

was induced by adding 25 μl of 1M IPTG and growth was continued for 3 more hr at

37oC. The cells were pelleted by centrifuging at 6000 rpm for 10min at 4oC. Supernatant

was discarded and cell pellets were weighed and stored at -20oC until further

purification steps. The above cell pellet was thawed and resuspended in 5 ml of 1X

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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 4oC. 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 4oC 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 -20oC 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 FLAG-

tagged protein and MBP-tagged protein were rocked at 4oC overnight. Resin-bound

protein samples were washed with 1X MBP buffer five times by centrifuging at 1000 rpm

for 1min at 4oC. 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 17.53 g, pH 7.5, bring volume to 2 L)] for 10 min at room

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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 10000-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

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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

85-10 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

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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 50-100 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 avrBs262-574 in pBlueScript using a BglII restriction enzyme site.

The portion of avrBs2 effector, avrBs262-574, lacks its own secretion and translocation signal. Therefore, avrBs262-574 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 xopF11-70: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.

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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 (xopF11-

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

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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; 52nd, 57th and 59th residues) and another one as QAEDVAA (three β-motif

residues underlined in the sequence; 25th, 29th and 31st residues) within the 25 through

33 helix region.

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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

3 33 and 47 through 59 to alanine using 10 diluted pBS(xopF11-70 : 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 xopF11-70 fragment from the avrBs2

fragment. The mutated xopF11-70 fragment was then again fused in frame to pBS

(avrBs262-574) and transferred to pLAFR3 to get pLAFR3 [xopF1(1-70; 27-33= 4A, 1C, 1S, 1 deletion): avrBs262-574] and pLAFR3 [xopF1(1-70; 47-59= 9A, 1S): avrBs262-574] 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, XopF11-70, 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 XopF1-70, both

alanine mutants, and HpaB (Figure 4-6).

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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 XopF11-70 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.

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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

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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 50-100 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 avrBs262-574 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.

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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 50-77 residues. It was shown that residues 50-77 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 30th amino acid and one around 50th

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

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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 in- planta 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.

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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 Tcrrlx+RK2 replicon, Tcr Staskawicz et al. 1987 pBlueScript II KS +/- Phagemid sequencing vector, Stratagene, La Jolla, CA Apr pRK2073 Spr Tra+, helper plasmid Figurski and Helinski 1979 pBS(BglII::avrBs262-574::HA) Phagemid sequencing vector, Mary Beth Mudgett, Apr Stanford university pDBLeu Kmr Song, University of Florida pPC86 Apr Song, University of Florida pMAL86 MBP binding domain, Apr Song, University of Florida pFLAG-CTC Apr,contains FLAG tag on C Jerry Minsavage terminus r + r pLAFR3[xopF1(1-70; 27-33= 4A, 1C, Tc rlx RK2 replicon, Tc This study 1S, 1 deletion): avrBs262-574] containing XopF1 alanine mutant r + r pLAFR3[xopF1(1-70; 47-59= 9A, 1S): Tc rlx RK2 replicon, Tc This study avrBs262-574] containing XopF1 alanine mutant r pDBLeu(xopF1(1-70; 27-33= 4A, 1C, Km , XopF1 variant fused in This study 1S, 1 deletion)) frame with LexA binding domain r pDBLeu[xopF1(1-70; 47-59= 9A, 1S)] Km , XopF1 variant fused in This study frame with LexA binding domain pPC86 (hpaB) Apr This study r pMAL86(xopF1(1-70; 27-33= 4A, 1C, Ap , XopF1 variant fused in This study 1S, 1 deletion)) frame with MBP binding domain r pMAL86[xopF1(1-70; 47-59= 9A, 1S)] Ap , XopF1 variant fused in This study frame with MBP binding domain pFLAG-CTC (hpaB) HpaB fused In frame with C This study terminal FLAG tag

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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(xopF11-70:avrBs2) clone; TED3 race 6 transconjugants carrying pLAFR3(xopF11-40:avrBs2) clone ; TED3 race 6.

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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(xopF11-70:avrBs2) clone; TED3 race 6 ∆hpaB mutant transconjugants carrying pLAFR3(xopF11-70:avrBs2) clone.

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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 (xopF11-70: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

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

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. 10 . 20 . 30 . 40 . 50 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 . 60 . 70 . 80 . 90 . 100 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.

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Figure 4-6. Yeast two hybrid interaction between alanine mutants of XopF11-70 and HpaB chaperone. Empty vector control contains pDBLeu and pPC86 empty vectors.

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Figure 4-7. In vitro pull down assay showing binding of HpaB chaperone to XopF1 variants. Lane 1: Kaleidoscope prestained standard, 2: XopF11-70 27 through 33 alanine mutant fused to MBP tag; 3: XopF11-70 47 through 59 alanine mutant fused to MBP tag; 4. XopF11-70 fused to MBP tag; 5. Empty; 6: total protein containing FLAG protein; 7: MBP protein; all pulled down using HpaB- FLAG-CTC tag.

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CHAPTER 5 PATHOGENIC STRATEGIES OF XANTHOMONAS GENUS ON PLANTS: LESSONS LEARNT FROM GENOMICS1

Background

Xanthomonas, the genus belonging to the gamma subdivision of the

proteobacteria, comprises of 27 species. These diverse species are known to cause

diseases on nearly 400 plant hosts, including both eudicots and monocots (Buttner and

Bonas 2010). Xanthomonas species cause serious diseases of a wide variety of

economically important crops including rice, citrus, banana, cabbage, tomato, pepper

and bean (Chan and Goodwin 1999). Pathogenic species and pathovars within species

show a high degree of host plant specificity and many exhibit tissue-specificity, invading

either the xylem elements of the vascular system or the intercellular spaces of the

mesophyll tissue of the host. For instance, Xanthomonas campestris includes pathovars

that (collectively) infect different brassicaceous, and other plant species, and

Xanthomonas oryzae, a species specific to rice and some wild relatives, comprises

pathovars that either invade through the vascular system (X. oryzae pv. oryzae) or colonize the intercellular spaces of the parenchyma tissue (X. oryzae pv. oryzicola). As with X. oryzae, the X. campestris group also includes vascular and non-vascular colonizers, exemplified by X. campestris pv. campestris and X. campestris pv. armoraciae respectively. Bacteria enter the host either through stomates to colonize the mesophyll parenchyma, or through hydathodes or wounds to spread systemically through the vascular system.

1 Reprinted with permission from White et al. 2009; Moreira et al. 2010; and Ryan et al. 2011.

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In order to study plant-pathogen interactions at the molecular level, Xanthomonas- plant system can be used as a model due to diversity among xanthomonads with respect to hosts and tissue types. At the time of writing, the complete genome sequences of 10 Xanthomonas species have been determined and draft genomes of a further 9 species are available, in total comprising seven species and eight pathovars

(Table 5-1). Sequencing projects currently in train will provide data on further species and pathovars (Ryan et al. 2009). In addition, complete and draft genome sequences are available for the related bacteria, Xylella fastidiosa and Stenotrophomonas species including S. maltophilia (Ryan et al. 2009). In order to get insights into the diversity of xanthomonads, their pathogenic adaptation colonizing wide variety of plants, and evolutionary mechanisms, we compared pathogenicity clusters, and individual pathogenicity/ virulence factors, especially type III effectors from all sequenced xanthomonads. The results mentioned in this Chapter have been published in three research articles – White et al. 2009, Moreira et al. 2010, and Ryan et al.2011.

Materials and Methods

Xanthomonas Genomes and Tools Used for Comparison

As listed in table 5-1, available Xanthomonas genome sequences were downloaded from NCBI Genbank. IMG-JGI website (http://img.jgi.doe.gov/er/) was used as interface for comparative genomics. Few recently published Xanthomonas genomes are not yet available on this website, in such a case, comparisons are made manually using blast (Altschul et al. 1997).

Database for Xanthomonas Pathogenicity Factors

Protein sequences of pathogenicity factors including all type secretion systems, other virulence factors such as LPS, EPS of known plant pathogens, especially

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characterized in terms of function in xanthomonads were downloaded from NCBI. The known pathogenicity factors were searched in IMG interface and searched for homologs within all xanthomonads.

Effectors Database Compilation

New xop nomenclature was introduced for type III effectors of xanthomonads. All known plant and animal pathogen effectors including known xanthomonas effectors were collected as a database and were used as query with an e-value threshold ≤ 10-5 for tblastn analysis. Pfam domains were searched for possible domains in the identified set. Nomenclature and classification scheme is available on www.xanthomonas.org

Effector Analysis of the Test Case of Citrus Pathogens

The candidate T3SS effectors in the XauB and XauC genomes were identified using tBLASTn (Altschul et al. 1997) analysis and Pfam domain (Finn et al. 2010) searches. For tBLASTn analysis, all known plant and animal pathogen effectors were used as query with an e-value threshold ≤ 10-5. Pfam domains were searched for possible domains found in known effectors in the predicted set of ORFs of draft genome sequences. Candidate effectors were classified according to the nomenclature and classification scheme for effectors in xanthomonads recently described by White et al.

(2009).

Results

Type II Secretion Systems

Plant cell wall-degrading enzymes such as cellulase, polygalacturonase, xylanase, and protease are secreted by type II secretion systems (T2SS). All Xanthomonas spp. and the phylogenetically related Xylella fastidiosa and Stenotrophomonas spp. possess a T2SS system called Xps. A second T2SS system known as the Xcs system is found

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in certain species that include X. campestris pv. campestris, X. armoraciae pv. citri and

X. campestris pv. vesicatoria. The Xps T2SS has been shown to contribute to virulence in X. campestris pv. campestris and Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola and X. campestris pv. vesicatoria (Dow et al. 1987; Jha et al. 2007;

Wang et al. 2008; Szczesny et al. 2010). In contrast the Xcs T2SS does not appear to have a function in virulence (Szczesny et al. 2010).

Interestingly, homologs of T2SS substrates from other Xanthomonas species are not secreted by the T2SS of X. campestris pv. vesicatoria. This finding indicates that the substrate specificity of T2SS secretion systems differs significantly among

Xanthomonas spp. (Szczesny et al. 2010). Comparative analysis of the XpsD protein, which forms the secretion channel in the Xps system, reveals amino acid differences at three positions (residues 494, 696, 698) that correlated with vascular or mesophyll tissue specificity among the strains analysed (Lu et al. 2008). However, subsequent analysis of the mesophyllic (bacterial spot) pathogens X. gardneri and X. vesicatoria indicated that this correlation no longer held for position 494 (unpublished data). It is not known whether these differences in XpsD relate to the substrate specificity of the various Xps T2SS. All Xanthomonas genomes have an extensive number of genes for cell wall degrading enzymes. Whereas some genes such as cbhA encoding a cellobiosidase are found only in the xylem-invading Xanthomonas spp. and in Xylella fastidiosa, others are conserved in all species. Differences in the complement of genes for cell wall degrading enzymes between species may reflect the differences in symptoms produced, rather than host or tissue preference (Table 2-7).

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Type III Secretion System

The type III secretion system (T3SS) is encoded by the hrp gene cluster (for

hypersensitive response and pathogenicity). This system “injects” effector proteins into

the cytoplasm of the host cell that interfere in diverse host processes in order to

promote disease (Nimura et al. 2005; Grant et al. 2006; Sarkar et al. 2006; Rohmer et al. 2004; White et al. 2009). X. albilineans does not possess the typical hrp gene cluster found in all other Xanthomonas species, but encodes a unique T3SS similar to the SPI-

1 (Salmonella Pathogenicity Island-1) found in Erwinia spp. (Pieretti et al. 2009). The

function of this SPI-1 T3SS is unknown, although its occurrence in insect bacterial

pathogens and symbionts has led to the suggestion that X. albilineans, which is not

known to be insect-transmitted, may have an insect-associated lifestyle. However

Xylella fastidiosa, which is insect vectored, possesses neither an Hrp-like nor a SPI-1-

like T3SS.

Type III-Secreted Effectors

Genome-enabled bioinformatic analyses have provided insights into the diverse

repertoires of type III-secreted effectors possessed by Xanthomonas with clues to their

possible contribution towards host specificity (White et al. 2009). So far, a total of 52

effector families have been identified along with three harpin proteins; helper or

accessory proteins that assist in the translocation of the effectors (Table 5-2). The

majority of sequenced Xanthomonas genomes contain a core set of nine effector genes

(xopR, avrBs2, xopK, xopL, xopN, xopP, xopQ, xopX, xopZ). The exceptions are X.

campestris pv. armoraciae, which only has six known effectors, and X. albilineans,

which has none. A study of 132 strains comprising eighteen pathovars of X. axonopodis

identified additional core effector genes (pthA, xopF1, xopE2 and avrXacE3) for this

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species (Hajri et al. 2009). Mutation of genes encoding some core effectors leads to reduced virulence and fitness of the pathogen (Table 5-3) (Kim et al. 2009; Kearney and

Staskawicz 1990; Metz et al. 2005; Duan et al. 1999; Al-saadi et al. 2007; Song and

Yang 2010; Gassmann et al. 2000), although this is not always the case (Roden et al.

2004a). Similarly, mutation of genes encoding some variable effectors influences virulence and disease symptomology (Table 5-4) (Kim et al. 2003; Wang et al. 2007b) whereas mutation of others has no apparent effect (Roden et al. 2004b). Mutation in individual genes may not result in change in virulence phenotype because of redundancy of function between effectors that may or may not share sequence similarity.

A few effector genes have been found to be associated only with certain species or with different species that attack common hosts. For instance xopAL1, xopAC, xopAD, xopAH, and xopAL2 are unique to X. campestris strains pathogenic on cruciferous plants, with the exception of X. campestris pv. armoraciae, which only has xopAL1 and xopAC . The xopE3 and xopAI genes are specific to the citrus pathogens

X. axonopodis pv. citri strain 306, X. fuscans pv. aurantifolii B and X. fuscans pv. aurantifolii C and are localized in a region identified as genomic island (Moreira et al.

2010). XopE4, a member of the XopE family, is an effector found uniquely in X. fuscans subsp. aurantifolii strains B and C but not in any other Xanthomonas spp. (Moreira et al.

2010). The contribution of these genes to the ability to cause disease in specific hosts remains to be tested. Multiple copies of some effectors may also be responsible for host range, an example being the AvrBs3/PthA family of TAL-like effectors of X. oryzae. pv. oryzae, X. oryzae. pv. oryzicola, and X. campestris pv. vesicatoria.

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The role of effectors in restriction of host range through an action as avirulence determinants has also been described. One example is the avrGf1 gene responsible for exclusion of grapefruit from the host range of X. axonopodis pv. citri Aw strain (Rybak et al. 2009). Interestingly, the effector gene xopAG which belongs to the same family as avrGf1 has also been found to limit the host range of X. fuscans pv. aurantifolii strain C to exclude grapefruit. The X. fuscans pv. aurantifolii B strain, which causes disease in grapefruit, has an almost identical xopAG gene that is inactivated by transposon insertion (Moreira et al. 2010). An avirulence gene, avrXv3, restricts the host range of X. perforans to tomato, giving HR on pepper (Astua-Monge et al. 2000a). A microarray based on the complete genome sequence of X. campestris pv. campestris strain 8004 was used investigated the genetic diversity and host specificity of this pathovar by array-based comparative genome hybridization analyses of 18 virulent strains. This analysis led to the identification of avrXccC and avrXccE1 as determinants of host specificity of X. campestris pv. campestris strain 8004 on mustard and Chinese cabbage respectively and avrBs1 as determinant of non-host resistance on pepper

ECW10R (He et al. 2007).

A CaseStudy – Screening for Candidate Type III Effectors from Draft Genomes and Possible Host Range Determinants.

Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002.

Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. We have sequenced the genomes

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of strains B and C to draft status. We have compared their genomic content to X. citri

subsp. citri and to other Xanthomonas genomes, with special emphasis on type III

secreted effector repertoires.

The three citrus canker genomes have important differences in regard to their repertoires of type III secreted effectors

The hrp/hrc genes encoding the T3SS are basically the same and found in the same order in all three Citrus Canker (CC) genomes. However, there are notable differences in the three putative T3SS-secreted effector repertoires.

A list of twenty-seven T3SS effector genes predicted in the genomes of the CC strains is shown in Table 5-5. Effectors are important determinants of virulence and host

range in many plant pathogenic bacteria, in particular in Xanthomonas sp. and

Pseudomonas syringae (Alfano and Collmer 2004). Comparison of effector repertoires

between the three CC genomes and all other Xanthomonas genomes can thus give us

important clues. The effector genes avrBs2, xopL, xopQ, and xopX are present in all

three CC genomes, in all sequenced genomes of other Xanthomonas species, and in all

X. citri and most Xanthomonas strains that were surveyed by PCR and hybridization for

these genes by Hajri et al. (2009). These effectors thus belong to the Xanthomonas

core set of effectors possibly important for pathogenicity on all plants. The putative

effector genes xopK, xopR, and xopZ also belong to this group since they can be found

in all sequenced Xanthomonas genomes. However, no data exist for these effectors in

regard to other Xanthomonas strains (Hajri et al. 2009). The effector genes xopI, xopV,

xopAD, and xopAK are present in all three CC genomes and in several, but not all,

sequenced Xanthomonas genomes. These effectors, therefore, might contribute to

disease in some plant species while they might trigger immunity in others.

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As already mentioned, PthA is well known to be an important X. citri effector that plays an essential role in citrus canker, while limiting the host range of CC strains to citrus because it triggers immunity in all other tested plant species (see references above). The pthA gene is a member of the avrBS3 family of effector genes, members of which are present in most Xanthomonas genomes and in some R. solanacearum genomes (Heuer et al. 2007). However, only PthA is known to induce citrus canker.

Besides pthA (XACb0065), three paralogs of pthA are also present in the Xac genome

(XACa0022, XACa0039, and XACb0015). All four copies are found on plasmids. The three paralogs do not seem to play an important role in citrus canker (Swarup et al.

1991). We found two pthA homologs in the XauB genome (XAUB_40130 and

XAUB_28490) and two in the XauC genome (XAUC_22430 and

XAUC_24060/XAUC_09900 [the latter is a single gene with halves in different contigs]).

Not all of these genes have been completely assembled due to the repetitive regions found in avrBS3 family members. However, El Yacoubi et al. (2007) previously assembled a pthA homolog (pthB [GenBank: 2657482]) from the pXcB plasmid

[GenBank: NC_005240] of a XauB strain with the same repeat copy number (i.e. 17.5) as pthA, and Al-Saadi et al. (2007) sequenced and assembled another homolog (pthC

[GenBank: EF473088]) from a XauC strain. These genes functionally complemented a pthA deletion in Xac without affecting host range (Al-Saadi et al. 2007). The

XAUC_22430 gene has 99% nucleotide identity to pthC and thus probably corresponds to pthC and would be the functional pthA homolog of XauC. We do not have enough data to confidently report on the repeat copy number of the other three Xau pth

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homologs, but a phylogenetic analysis (see below) suggests that XAUB_28490 is the

functional pthA homolog of XauB.

Effectors XopAI and XopE3 may play a role in citrus canker

A comparison of effectors present in all three CC strains with those present in fully

sequenced Xanthomonas species, and data from the study by Hajri et al. (2009),

suggest that two additional putative effectors may play a special role in citrus canker.

These are XopAI and XopE3. Both are present in all three CC genomes.

The putative effector xopAI is not found in any other sequenced Xanthomonas species and it was not included in the Hajri et al. (2009) analysis. We do have evidence that it is present in Xanthomonas vesicatoria str. 1111 (Potnis et al. 2011). Interestingly, the C-terminal region of XopAI has similarity to predicted ADP-ribosyl transferase domains of the effector HopO1-1 of Pseudomonas syringae and of hypothetical proteins in Acidovorax citrulli, Ralstonia solanacearum, and other bacteria. The N-terminus has high similarity to the N-terminus of the effector XopE2 of X. campestris pv. vesicatoria

85-10 as well the N-termini of a number of other Xanthomonas and Pseudomonas

syringae effectors (more on the N-terminal region of xopAI below).

XopE3 belongs to the HopX/AvrPphE family of effectors. Effectors belonging to

this family have been found in diverse phytopathogenic bacteria including Ralstonia,

Pseudomonas, Acidovorax, and Xanthomonas, suggesting their conserved role in

virulence on a wide range of hosts. Sequences from this family have similarity to the

transglutaminase superfamily of enzymes, which are responsible for modification of host

proteins (Nimchuk et al. 2007). The HopX/AvrPphE effector from Pseudomonas

syringae has been shown to be involved in host protein proteolysis, thereby suppressing

host defenses (Nimchuk et al. 2007; Schechter et al. 2004). In xanthomonads, multiple

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effectors belonging to this group have been found, such as xopE1, xopE2, xopE3, xopE4. XopE1 and xopE2 have been found in most of the xanthomonads. XopE3 effector gene homologs have been found by PCR and dot-blot hybridization methods in some Xanthomonas axonopodis strains belonging to the alfalfae, anacardii, glycines, phaseoli, malvacearum, fuscans, mangiferae, indicae, and citrumelo pathovars (Hajri et al. 2009). However, sequences of xopE3 from these strains could not be compared against homologs from CC strains since sequence data from the X. axonopodis strains mentioned are not currently available. Phylogenetic analysis of hopX orthologs shows that the xopE3 effector genes found in the CC strains group together with hopX1 effector genes from pseudomonads (data not shown).

Although all hopX orthologs show conservation of the catalytic triad (Cys, His, Asp residues) as well as the conserved domain “GRGN” N-terminal to the triad, the region

C-terminal to the triad shows high degree of variability. This variable region has been hypothesized to be responsible for targeting different host proteins (Nimchuk et al.

2007). In fact, while some AvrPphE (hopX) alleles from P. syringae pv. phaseolicola strains trigger gene for gene disease resistance in some bean cultivars, other alleles were shown to be virulent on these same cultivars. Amino acid differences in the C- terminal region of AvrPphE were identified between alleles (Stevens et al. 1998).

Similarly, comparing XopE3 homologs from different strains at the amino acid level and their corresponding reactions on different hosts might give clues regarding the variable

C-terminal domains of XopE3 family members and might determine whether this variability is responsible for targeting different proteins in different host species.

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Both xopE3 and xopAI belong to an interesting Xac chromosomal region of approximately 15 kbp in size that has been hypothesized to be a genomic island

(Moreira et al. 2005). An alignment of the Xac chromosome sequence with the chromosome sequences of X. campestris pv. campestris str. ATCC33913 and X. oryzae pv. oryzae str. PX099A strongly suggests that this region is an insertion (data not shown). The presence of three transposase genes and two phage-related genes in the region provides additional evidence for this hypothesis. The central part of this region (7 kbp) duplicates a region found in Xac plasmid pXAC64, suggesting a chromosome- plasmid DNA exchange. In the plasmid we find the effector gene xopE2 (XACb0011), which - as described above - shares its N-terminal region with xopAI (XAC3230).

Transposons and phage elements in this region might thus have been responsible for a shuffling process, described as terminal reassortment (Stavrinides et al. 2006), resulting in the novel effector gene xopAI. Although we can characterize this region completely only in Xac, XauB and XauC contigs contain the most important elements of this region. Next to xopE3 (XAC3224) we find gene XAC3225, whose product is annotated as tranglycosylase mltB. This gene has strong similarity (e-value 10-133,

100% coverage) to hopAJ1 from P. syringae pv. tomato strain DC3000, where it is annotated as a T3SS helper protein. Although the hopAJ1 gene is not itself a T3SS substrate, it contributes to effector translocation (Oh et al. 2007). A mutant with a deletion of XAC3225 has reduced ability to cause canker (mutant phenotypes include a reduction in water soaking, hyperplasia, and necrosis compared to wild type) (Laia et al.

2009). We thus conclude that the effector and effector-related genes in this region probably play an important role in citrus canker.

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Additional differences in effector repertoires among CC genomes

In addition to the pth differences noted above, other effectors that distinguish the

Xac genome from the two Xau genomes are XopB, XopE4, XopJ (AvrXccB), XopAF

(avrXv3), and XopAG, which are all present in both Xau genomes but absent from Xac strain 306. (AvrXccB homologs were found in two Xac strains by Hajri et al. (2009). The absence of these effectors from Xac strain 306 raises the possibility that these effectors might be responsible for limiting the host range of both B and C strains. Interestingly,

XauB and XauC strains both contain xopAG, an effector gene belonging to the same effector family as avrGf1 from X. citri Aw, which has been shown to be responsible for triggering a hypersensitive defense response in C. paradisi (grapefruit) (Rybak et al.

2009). The xopAG gene from the B and C genomes shows 44% identity to avrGf1 at the amino acid level. The XauB and and XauC genes are almost identical to each other, with one important difference: in XauB xopAG is interrupted by a transposon. Therefore, the incompatibility between XauC and grapefruit and the ability of XauB to cause disease in grapefruit could be explained by this single gene difference. The xopE4 DNA sequence is identical in the two Xau genomes and has similarity to avrXacE3 but only with 31% identity at the amino acid level; this is why we named this gene xopE4 instead of xopE2. Unlike other XopE family members, XopE4 does not have a predicted myristoylation site, suggesting that it may not be targeted to the cell membrane as the other XopE family members.

Presence of an additional effector gene, the avirulence gene avrXccA2, has been shown in some X. aurantifolii B (CFBP3528, CFBP3530) and X. aurantifolii C

(CFBP2866) strains by hybridization and PCR analysis (Hajri et al. 2009). However, this avirulence gene was not found in the two sequenced Xau genomes. A homolog of the

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effector xopF1 (XAUC_20070) was found only in the XauC strain. It is located in a 5-kbp region that lies between the T3SS genes hrpW (XAUC_20020) and hpa3

(XAUC_20080). The same two genes are adjacent in XauB. Two transposases are present in this region, and the sequence of xopF1 has a frameshift, suggesting that this gene is likely the result of a recent insertion and is not active.

There are four effector genes present in the Xac and XauB genomes that have not been found in the genome of XauC: xopE2, xopN, xopP, and xopAE. These effectors could explain the wider host range of Xac and XauB compared to XauC, assuming a virulence activity of these effectors on citrus species. XopN has been shown to interact with the plant protein TARK1 and to interfere with immunity triggered by pathogen- associated molecular patterns (PAMP-triggered immunity) (Kim et al. 2009). Further experiments are required to determine the possible role of XopN in extending host range to lemon, grapefruit and sweet orange. Another effector that could have a similar role is XopAE (a hpaF/PopC homolog) (Noel et al. 2002; Sugio et al. 2005).

The harpin-like protein HrpW with a pectate lyase domain is present in all CC strains. In the sequenced Xac genome, it is not associated with the T3SS gene cluster, whereas in the genomes of XauB and XauC it is. The role of harpin-like proteins like

HrpW as virulence factors or T3SS accessory proteins has not yet been determined in the Xanthomonas genus.

Experiments will need to be performed to confirm translocation of the above putative effectors and their putative function as virulence or avirulence genes.

Adhesins

As with many bacteria, Xanthomonas spp. synthesise adhesins of both the fimbrial and non-fimbrial classes that are involved in bacterial attachment to surfaces and

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contribute to virulence (Ray et al. 2002; Das et al. 2009; Gottig et al. 2009). The non- fimbrial adhesins include filamentous haemagglutinin-like proteins such as FhaB, homologues of the autotransporter adhesin YapH from Yersinia spp. and XadA and

XadB, which are both related to YadA from Yersinia spp. Fimbrial adhesins include type

IV pili and related proteins such the the type IV pilus secretin PilQ (Ray et al. 2002; Das et al. 2009; Gottig et al. 2009). It has been shown for Xanthomonas oryzae pv. oryzae that different adhesins are preferentially involved in the different stages of infection that comprise attachment to leaf surfaces, entry, colonization and later survival inside plant tissue (Das et al. 2009). For example, XadA and XadB affect leaf attachment and entry into the host, but do not affect virulence after wound inoculation whereas PilQ appears to have no role on leaf attachment or entry but has a role when bacteria are within plants. In contrast, FhaB is involved in virulence both in epiphytic and wound inoculations of Xanthomonas axonopodis pv. citri (Gottig et al. 2009). Comparative genome analysis indicates that FhaB is absent in some strains of Xanthomonas campestris pv. campestris and Xanthomonas oryzae pv. oryzae and in Xanthomonas campestris pv. vesicatoria there are two related open reading frames, FhaB1 and

FhaB2. Two copies of xadA and yapH are found in Xanthomonas campestris pv. campestris strain 306 and in Xanthomonas campestris pv. vesicatoria 85-10; two copies two copies of yapH are also present in present In Xanthomonas oryzae pv. oryzae

KACC, and there are two pilQ orthologs in Xanthomonas campestris pv. vesicatoria 85-

10 (Xcv). Although different combinations of adhesins in each species might indicate their specificity towards host tissues as has been speculated for Ralstonia solanacearum (Guidot et al. 2007), no clear relationship is evident.

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Lipopolysaccharides and Xanthan

Both lipopolysaccharides (LPS) and the extracellular polysaccharide xanthan

contribute to the ability of Xanthomonas spp. to cause disease (Qian et al. 2005; Chou

et al. 1997; Rajeshwari and Sonti 2000; Kingsley et al. 1993). A lipopolysaccharide

biosynthesis gene cluster defined by a 14-26 kb region flanked by etfA and metB is

highly variable in terms of identity and size across sequenced xanthomonads. This

variability is seen not only at the species and pathovar level but also between strains,

suggesting very recent horizontal gene transfer (Patil et al. 2007). However, a comparative study for this cluster among sequenced xanthomonads indicated that variation in the LPS biosynthesis cluster was not associated with host or tissue specificity (Lu et al. 2008). The gum gene cluster comprises 12 genes, gumB to gumM,

that encode proteins involved in the synthesis and secretion of xanthan, a

polysaccharide characteristic of the genus Xanthomonas which is important for

virulence (Qian et al. 2005 Chou et al. 1997; Rajeshwari and Sonti 2000). The gum

cluster is absent from Xanthomonas albilineans (Pieretti et al. 2009) whereas Xylella

fastidiosa lacks some of the gum genes but is capable of synthesis of a related

polysaccharide that lacks the terminal mannose residues and is not pyruvylated.

Toxins

The production of toxins in Xanthomonas species is currently thought to be

restricted to Xanthomonas albilineans, which produces albicidin, an important virulence

factor (Pieretti et al. 2009). Albicidin is synthesised by a non-ribosomal pathway via a

hybrid modular nonribosomal peptide synthase- polyketide synthase (NRPS-PKS).

NRPSs related to SyrE, which is responsible for the synthesis of the phytotoxin

syringomycin in Pseudomonas syringae, are encoded by the genomes of Xanthomonas

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axonopodis pv. citri (Van Sluys et al. 2002) and Xanthomonas oryzae pv. oryzicola

(unpublished data). Whether these direct synthesis of toxins is however not known. A

second NRPS found in several Xanthomonas species is related to EntF, which is

involved in the synthesis of the siderophore enterobactin in Escherichia coli.

Concluding Remarks

We have discussed the insights afforded by comparative genomics into the pathogenesis, adaptation to host plants and evolutionary trends of Xanthomonas, the impact that knowledge of genome sequences has made to functional genomics and the new tools that have been developed to derive further benefits from the wealth of information available.

The analysis of genomic data from phytopathogenic bacteria has remarkably expanded the repertoire of genes thought to contribute to virulence in plants. These genes encode proteins involved in a range of cellular functions including adhesion, phytotoxin production, resistance to oxidative stress, degradation of plant cell walls, production of plant hormones, production and injection of effector proteins into host cells and interference with host defences. Comparative genomic analysis between

Xanthomonas spp. has revealed both the conservation of certain genes and gene clusters associated with virulence as well as differences in genetic content that may be related to host and/or tissue specificity. It is evident that differentiation with respect to host- and tissue-specificity does not involve major modifications or wholesale exchange of pathogenicity gene clusters, but is associated instead with subtle changes in a small number of genes within these clusters, and/or differences outside the clusters, potentially among secretory substrates or regulatory targets.

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Table 5-1. Xanthomonas species and pathovars within species show host and tissue-specificity. Species Pathovar Strain Strai Host Disease Geno Plasmid Status Reference n me s abbr size eviati (Mb) on X. campestris ATCC 33913 Xcc Crucifier Black 5.08 n.a. Complete da Silva et campestris s rot al. 2002 X. campestris 8004 Xcc Crucifier Black 5.15 n.a. Complete Qian et al. campestris s rot 2005 X. campestris B100 Xcc Crucifier Black 5.1 n.a. Complete Vorholter campestris s rot et al. 2008 X. armoraciae 756C Xca Crucifier Leaf 4.94 n.a. Complete Unpublish campestris s spot ed X. musacearum NCPPB4381 Xvm Banana Enset 4.7 Complete Unpublish campestris wilt ed X. vasculorum NCPPB702 Xvv Sugarca Gummi 5.4 n.a. Complete Studholm campestris ne ng e et al. (vasicola) disease 2010 X. 85-10 Xcv Tomato/ Leaf 5.42 pXCV3 Complete Thieme et euvesicatoria pepper spot 8; al. 2005 pXVC2; pXCV1 9; pXCV1 83 X. oryzae oryzae KACC10331 Xoo Rice Bacteri 4.94 n.a. Complete Lee et al. al blight 2005 X. oryzae oryzae PXO99A Xoo Rice Bacteri 5.2 n.a. Complete Salzberg al blight et al. 2008 X. oryzae oryzae MAFF 311018 Xoo Rice Bacteri 4.94 n.a. Complete Ochiai et al blight al. 2005 X. oryzae oryzae AX01947 Xoo Rice Bacteri 5.1 Draft Unpublish al blight ed

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Table 5-1. continued Species Pathovar Strain Strai Host Disease Geno Plasmid Status Reference n me s abbr size eviati (Mb) on X. citri 306 Xac Citrus Citrus 5.27 pXAC6 Complete da Silva et axonopodis canker 4;PXCC al.2002 33

X. phaseoli Xap Beans Bacteri n. a Draft Unpublish axonopodis al blight ed X. manihotis Xam Cassava Bacteri n. a. Draft Unpublish axonopodis al blight ed X. fuscans aurantifolii ICPB 11122 Xaub Citrus Citrus 4.7 n.a. Draft Moreira et canker al. 2010 X. fuscans aurantifolii ICPB 10535 Xauc Citrus Citrus 5.0 n.a. Draft Moreira et canker al. 2010 X. oryzae oryzicola BLS256 Xoc Rice Bacteri 4.8 n.a Draft Unpublish al ed streak X. vesicatoria ATCC35937 Xv Pepper Bacteri 5.4 Draft Potnis et and al spot al. 2011 Tomato X. perforans 91-118 Xp Tomato Bacteri 5.1 Draft Potnis et al spot al. 2011 X. gardneri ATCC19865 Xg Pepper Bacteri 5.4 Draft Potnis et and al spot al. 2011 Tomato X. albilineans GPE PC73 Xal Sugarca Leaf 3.77 n.a. Complete Pieretti et ne scald al. 2009)

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Table 5-2. Xop nomenclature for xanthomonas effectors Effector Related Xoo Xac Xcv Xcc Xoo Xcmu Xp class proteins/ MAFF311018 306 85-10 ATCC33913 AXO1947 /Xvm 91- synonyms 118 AvrBs1 AvrA N N XCVd0104 XCC2100 N N N AvrBs2 XOO0148 XAC0076 XCV0052 XCC0052 + + + AvrBs3 Pth, TAL 17 genes 4 genes Y (P14727) N ~8 N N genes XopB N N XCV0581 N N + N (HopD1 ) XopC RSp1239 XOO3221 XAC1210/ XCV2435 Yf + + + XAC1209g XCV1238/ (ACS12858) XCV1237g XopD N N XCV0437 XCC2896 N N + XopEh AvrPphE N XAC0286 XCV0294(E1 XCC1629 N N + (HopX) AvrXccE1 (E1) ) (E2) + HopPmaB XAC3224 XCV2280(E2 (E3) ) XACb0011(E2 ) XopFh Hpa4 XOO0103(F XAC2785(F2) XCV0414 XCC1218 + + + 1) (F1) (F1) + XCV2942 (F2) XopG AvrPtoH XOO4258 N XCV1298 XCC3258 N + N (HopH, HopAP) XopH AvrBs1.1 N N XCVd0105 XCC2099 N N N (HopAO HopPtoD2 ) XopI XOO3626 XAC0754 XCV0806 N + + +

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Table 5-2. continued Effector Related Xoo Xacb Xcvb Xcc Xoo Xcmu Xp classa proteins/ MAFF311018 306 85-10 ATCC33913 AXO1947 /Xvm 91- synonyms 118 XopJ YopJ, N N XCV2156 XCC3731 N + N (HopJ) AvrRxv XCV0471 AvrXv4, Y AvrBsT (AAD39255) Y

XopK XOO1669 XAC3085 XCV3215 XCC2899 + + + XopL XOO1662 XAC3090 XCV3220 XCC4186 + + +

XopMi +

XopN XOO0315 XAC2786 XCV2944 XCC0231 + + + (HopAU ) XopO AvrRps4 N XAC3666 XCV1055 N + + + (HopK) XCV3786 XopP XOO3222 XAC1208 XCV1236 XCC1247 + + +

XopQ XOO4208 XAC4333 XCV4438 XCC1072 + + + (HopQ) XopR XOO4134 XAC0277 XCV0285 XCC0258 + + +

XopSg +

XopT XOO2210 NA, I N N N N N

XopU XOO2877 N N N + N N

XopV XOO3803 XAC0601 XCV0657 N + + +

XopW XOO0037 N N N + N N

XopX XOO4042 XAC0543 XCV0572 XCC0529 + + + XCC0530 + + XopY XOO1488 N N N + + N

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Table 5-2. continued Effector Related Xoob Xac Xcvb Xcc Xoo Xcmu Xp classa proteins/ MAFF311018 306 85-10 ATCC33913 AXO1947 /Xvm 91- synonyms 118 XopZ AWR XOO2402 XAC2009 XCV2059 XCC1975 + + + (HopAS ) XopAA ECF, XOO2875 N XCV3785 N + + N (HopAE HolPsyAE ) XopAB XOO3150 N N N + + +

XopAC AvrAC N N N XCC2565 N N N

XopAD skwpj XOO4145 XAC4213 XCV4315/ N + + + (BAH47290 XCV4314/ + ) XCV4313g XopAE HpaF XOO0110 XAC0393 XCV0409/ N + + + PopC XCV0408 XopAF AvrXv3 N N N N N + + (HopAF ) XopAG AvrGf1 N Y (ABB84189) N XCC3600 N + N (HopG) XopAH AvrXccC N N N XCC2109 N N N AvrB, AvrC XopAI N XAC3230 N N N N N (HopO1 ) XopAJ AvrRxo1 N N XCV4428 N N N N

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Table 5-3. Core effector genes from xanthomonads and their role in pathogenicity/ induction of resistance Core Effectors Functional domain/ motif Phenotype of mutant Possible role in References pathogenicity/ Induction of resistance

XopR Unknown Similar to wild type Xoo str. Does not contribute Song et al. 2010; PXO99 individually to virulence in Furutani et al. Xoo- rice system 2009

AvrBs2 Sequence related to Mutation in Xcv resulted in Important for pathogen Swords et al. agrocinopine synthase, loss of avirulence, reduced fitness; dual role in 1996; Kearney glycerol virulence on pepper recognition and and Staskawicz phosphodiesterase pathogenicity Bs2 (unusual 1990; Wichman member of NBS-LRR R gene and Bergelson family that does not have 2004. TIR or LZ domain.

XopK Unknown Similar to wild type Xoo Does not contribute Song et al. 2010; PXO99 individually to virulence in Furutani et al. Xoo-rice system 2009

XopL LRR protein Mutant in Xcc less virulent Required for full virulence of Jiang et al. 2009, on Chinese radish; However Xcc on Chinese radish; But Song et al. 2010. In Xoo, similar to wild type Does not contribute individually to virulence in Xoo-rice system

XopN Alpha-helical ARM/HEAT Mutants in Xcv impaired in Reduces PAMP-induced Roden et al. repeats, Irregular alpha- growth and reduced ability gene expression and callose 2004b; Jiang et helical repeats suggesting to elicit disease symptoms deposition in host al. 2008; Kim et multiple protein-protein on susceptible tomato tissue.Interacts with al. 2009. interactions. leaves.Also, virulence factor cytoplasmic domain of in Xcc. TARK1 and 4 tomato 14-3-3 isoforms- TFT1, TFT3, TFT5, TFT6.

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Table 5-3. continued Core Effectors Functional domain/ Phenotype of mutant Possible role in pathogenicity/ References motif Induction of resistance

XopP Unknown No change in phenotype for Xoo Virulence factors in Xcc- Roden et al. PXO99 xopP mutant; No chinese radish system 2004b; Jiang et significant growth defect in Xcv al. 2009; Song et mutant; Required for full al. 2010 virulence and growth of Xcc 8004 in Chinese radish

XopQ Inosine-uridine No significant growth defect in Virulence factors in Xcc- Roden et al. nucleoside N- Xcv mutant; Required for full chinese radish system 2004; Jiang et al. ribohydrolase virulence and growth of Xcc 2009 8004 in Chinese radish

XopX Methionine-rich Reduced in planta growth of Virulence factors; targets Metz et al. 2005 protein Xcv in tomato and pepper basic innate immunity in plants by suppressing host defense function, which in turn allows for greater pathogen growth and an altered visual high titer inoculation response by N. benthamiana; Elicits non-host resistance reaction in N. benthamiana in presence of active T3SS.

XopZ Unknown Mutations in both the copies in Interferes with host innate Song et al. 2010 PXO99 reduced virulence in immunity/ suppresses host terms of lesion length and basal defense responses. bacterial multiplication in rice. Single copy mutation does not show visible change in symptoms.

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Table 5-4. Variable effectors which contribute to the pathogenicity Variable Effector Functional domain/ motif Possible role in pathogenicity/ Distribution References genes Induction of resistance

AvrBsT C55 family cysteine Responsible for cell death in Certain Xcv strains Kim et al. 2010; protease, ser/thr acetyl pepper, but virulence factor in (Plasmid borne) Szczesny et al. (XopJ2) transferase tomato. 2010;

Interacts with SnRK1 and suppresses HR elicited by avrBs1 in resistant peppers.

XopC Phosphoribosyl No significant growth defect in Only in Xcv (xopC); Roden et al. 2004; transferase domain and Xcv mutant on tomato/pepper, Song et al. 2010 haloacid dehalogenase- Only in Xoo strains like hydrolase No change in phenotype in Xoo (xopC2- inactivated PXO99 mutant(xopC2) version in Xac, Xcv)

XopF1 Unknown No significant growth defect in Xcv, Xca, Xoo, Xoc, Roden et al. 2004 Xcv mutant on tomato/pepper Xvv, Xvm.

XopF2 Unknown No significant growth defect in Xcv, Xvm, Xvv Roden et al. 2004 Xcv mutant on tomato/pepper

XopJ C55-family cysteine No significant growth defect in Xcv Roden et al. 2004 protease or Ser/Thr Xcv mutant on tomato/pepper acetyltransferase

XopO Unknown No significant growth defect in Xcv, Xoc Roden et al. 2004 Xcv mutant on tomato/pepper

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Table 5-4. continued Variable Effector Functional domain/ motif Possible role in pathogenicity/ Distribution References genes Induction of resistance

XopAE LRR protein Role in virulence and disease Xag, Xac, Xoo, Xoc, Kim et al. 2003 symptomalogy in X. axonopodis Xvm, Xvv. pv. glycines (Xag) (hpaG/hpaF pseudogene in Xcv)

XopAH Unknown Required for full bacterial Only in Xcc Wang et al. 2007b virulence in the susceptible host (AvrXccC) cabbage (Brassica oleracea) and avirulence in the resistant host mustard (Brassica napiformis L.H. Baily).

XopE2 Putative Required for full virulence and Xcv, Xac and Xcc Jiang et al. 2009 transglutaminase growth of Xcc 8004 in Chinese radish

XopAM Unknown Required for full virulence and Xcc, Xvm, Xvv. Jiang et al. 2009 growth of Xcc 8004 in Chinese radish

PthA/AvrBs3 Transcriptional activator Activate host genes e.g. Xac, Xoo, Xcv, Xg Yang et al. 2004, family hypertrophy related to virulence, 2005; Sugio et al. resulting in pathogen spread and 2007; Schornock et disease al. 2008

XopD C48-family SUMO Virulence factor that mimics plant Xcv, Xcc str. B100. Kim et al. 2008; cysteine protease; EAR SUMO isopeptidase, targets Noel et al. 2002; motif; DNA-binding and SUMO-conjugated protein and (Chimeric version in Hotson et al. 2003; nuclear localization interferes with regulation of host Xcc str. 33913, Chosed et al. 2007 domain proteins, promotes pathogen 8004) multiplication and delays symptom development.

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Table 5-5. Putative effectors found in the XAC, XauB, and XauC genome sequences. Some of the effectors appear to be pseudogenes (indicated by a Ψ). In column ‘effector family’ we provide the standard effector name along with the names of related proteins belonging to the same family; in column ‘effectors’ we provide the reference where the effector indicated was characterized. Effector family XAC XauB XauC Pfam: functional/ References structural domain Candidate effectors common to XAC, XauB, and XAUC

AvrBs2 XAC0076 XAUB_16770 XAUC_23650 Glycerophosphoryl Kearney and diester Staskawicz 1990. phosphodiesterase AvrBs3 XACa0022 XAUB_40130 XAUC_22430 Transcriptional Al-Saadi et al. (pthA1) XAUB_28490 XAUC_24060 activator, nuclear 2007 XACa0039 XAUC_09900 localization (pthA2) XAUC_43080 XACb0015 (pthA3) XACb0065 (pthA4) XopE1 (avrXacE1, XAC0286 XAUB_37010 XAUC_37580 Putative Thieme et al. hopX, avrPphE) transglutaminase 2007 XopE3 (avrXacE2, XAC3224 XAUB_14680 XAUC_00040 Putative Dunger et al. hopX, avrPphE) transglutaminase 2008 XopF2 XAC2785 Ψ XAUB_07540 XAUC_21010 Ψ Gurlebeck et al. /XAUB_07550 Ψ /21000 Ψ 2006 XopI XAC0754 XAUB_39080 XAUC_07100 F-box protein Thieme 2008

XopK XAC3085 XAUB_34090 XAUC_12520 Identified in Xoo by cya assay Furutani et al. 2009 XopL XAC3090 XAUB_34130 contig1165 (725- LRR protein Dunger et al. 3) and 2008 XAUC_02900

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Table 5-5. continued Effector family XAC XauB XauC Pfam: functional/ Effectors structural domain XopQ (hopQ1) XAC4333 XAUB_10220 XAUC_14670 Inosine uridine Roden et al. 2004 nucleoside N- ribohydrolase XopR XAC0277 XAUB_36920 XAUC_37490 Furutani et al. 2009 XopV XAC0601 XAUB_23140 XAUC_21260 Identified in Xoo by cya assay Furutani et al. 2009 XopX (HolPsyAE) XAC0543 XAUB_14760 XAUC_20690 Metz et al. 2005 XopZ (HopAS, AWR) XAC2009 Ctg607 (28419- Ctg1224 (21120- Furutani et al. 29915) and 16957) 2009 XAUB_13710 XopAD (skwp, XAC4213 XAUB_02510 XAUC_34870 SKWP repeat protein Skwp from RSc3401) Ralstonia Guidot et al. 2007 XopAI (HopO1 XAC3230 XAUB_26830 XAUC_23780 ADP-ribosyltransferase Thieme et al. (HopPtoO, HopPtoS), 2007 HopAI1 (HolPtoAI)) XopAK (HopAK1 XAC3666 XAUB_02580 XAUC_32490 Not confirmed to (HopPtoK, be effector in HolPtoAB)C terminal Xanthomonas; domain) homolog of effector in Pseudomonas HrpW (PopW) XAC2922 XAUB_19460 XAUC_20020 Pectate lyase, may not Park et al. 2006 (associated with (associated with be T3SE hrp cluster) hrp cluster) Candidate effectors present in XAC and XauB BUT ABSENT in XauC

XopE2 (avrXacE3, XACb0011 XAUB_31660 - Putative XopE2 found in avrXccE1) transglutaminase another C strain Thieme et al. 2007

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Table 5-5. continued Effector family XAC XauB XauC Pfam: functional/ Effectors structural domain XopN (hopAU1) XAC2786 XAUB_07520 - ARM/HEAT repeat Kim et al. 2009 XopP XAC1208 XAUB_06720 - Roden et al. 2004

XopAE XAC0393 XAUB_19500 - LRR protein Noel et al. 2002 (HpaF/G/PopC)

Candidate effectors present in XauB and XauC BUT ABSENT from XAC XopB (hopD1, - (ctg622 (24545- XAUC_00260 Noel et al. 2001 avrPphD1) 24841) and ctg594 (2-745) XopE4 (HopX) - XAUB_23330 XAUC_31730 New class introduced XopJ (AvrXccB) - XAUB_20830 XAUC_08850 C55-family cysteine Xu et al. 2004 protease or Ser/Thr acetyltransferase XopAF (avrXv3, - XAUB_02310 XAUC_00300 Astua-Monge et HopAF1 (HopPtoJ)) al. 2000a XopAG (AvrGf1, - Ctg529 (1957- XAUC_04910 Rybak et al. 2009 HopG1 (HopPtoG). 2856) HolPtoW)

Candidate effectors present only in XauC XopF1 (Hpa4) - - XAUC_20070 Ψ Roden et al. 2004 /20060 Ψ

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CHAPTER 6 SUMMARY AND DISCUSSION

Bacterial spot disease is a devastating disease on tomato and pepper and has been responsible for significant losses. Prior to 1995, Xanthomonas causing bacterial spot was classified under a single species, Xanthomonas campestris pv. vesicatoria.

Jones et al. (2004) carried out multiphasic analysis based on biochemical and molecular analyses and classified bacterial spot xanthomonads into four different genetic groups

A, B, C and D under species namely, Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, X. gardneri. All genetic groups infect tomato. Groups A, B and D infect pepper as well. Avirulence gene avrXv3 responsible for limiting host range of X. perforans on pepper has been identified (Astua-Monge et al. 2000a). However, avrXv3 deletion mutant is not completely virulent on pepper, indicating there are some additional factors responsible for host range specificity. Xcv str.85-10 belonging to A group has already been sequenced (Thieme et al. 2005). In order to get insights into the host range specificity and diversity among the four groups, we sequenced genomes of the strains belonging to the B, C and D groups.

Whole genome comparison using MUMmer and phylogenetic analysis based on orthologous genes showed that Xp is closely related to Xcv; whereas Xg and Xv are more closely related to Xcc. We compared different pathogenicity clusters from draft genomes to the already sequenced xanthomonads. Type II and type III secretion clusters of the two bacterial spot pathogens, Xg and Xv, were similar to Xcc in terms of genetic organization and sequence identity. Type III secreted effectors are important pathogenicity factors and host range determinants. Effectors are translocated into the plant cell and they interfere with the plant immune responses favoring pathogen

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survival, multiplication, and spread. Using a bioinformatics approach and experimental validations, we screened the three bacterial spot draft genomes for effectors and found interesting similarities and differences in the effector repertoires of the bacterial spot xanthomonads. Common effectors like XopF1 and XopD are important pathogenicity candidates on tomato. XopD has been shown to delay symptom development and hence plays a potential role in the development in pathogen survival. Like tomato specific pathogenicity factors, we found effectors common to pepper pathogens and absent from Xp, namely xopE2 and xopG. We identified some unique effectors from Xg.

These include xopAO, xopAQ, xopAS and an avrBs1 member XGA_0724. Homologs of xopAS, xopAO, and XGA_0724 are found in pseudomonads infecting tomato. Based on the evidence of horizontal gene transfer along with the fact that Xg prefers lower optimum temperature for disease development similar to pseudomonads, we can speculate on the acquisition of these effectors in Xg from pseudomonads. Along with the novel effectors, we found a few other virulence factors, homologs of which have been identified and characterized in other plant pathogens. Xg appears to be aggressive on pepper. The unique effectors and other virulence factors could explain the aggressive nature of Xg.

The genomic analysis also provided candidates for pepper pathogenicity factors, one of them being LPS O-antigen. Comparison of the LPS clusters between the four species showed similar organization for all pepper pathogens, while Xp seems to have acquired a novel LPS cluster during evolution. This novel cluster might be responsible for limited host range of Xp. Another possibility is that common LPS antigen of pepper pathogens might be the factor involved in pepper specificity. We tested three candidate

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genes (LPS O-antigen, xopG, and a hypothetical protein XCV1839) experimentally for

their ability to increase in planta growth on pepper by conjugating candidate genes

individually and in combination in Xp ∆avrXv3 mutant. LPS cluster from pepper

pathogens contributes towards increased in planta growth of Xp ∆avrXv3 mutant on

pepper.

The computational predictions based on draft genome sequences of bacterial spot

pathogens have offered a wide scope for the studies involving experimental verification

of potential role of the candidate virulence factors and implementation of the essential

ones in the development of control strategies and breeding for durable resistance.

Screening for durable resistance continues to be a challenge due to plant-

pathogen co-evolution. While screening different Capsicum genotypes, Capsicum

chinense showed a new source of resistance against several strains of bacterial spot

xanthomonads. In an interspecific cross, this new resistance was transferred to

Capsicum annuum cv. Early Calwonder (ECW). The resistance gene was then

segregated in a backcross program and 7th backcross population (ECW-70R) was used

to determine genetic inheritance of the resistance.

X. gardneri strain Xv444 gave a strong HR on ECW-70R. We isolated a clone from

a genomic library of Xv444 showing an avirulence phenotype on ECW-70R by

mobilizing a library of Xv444 into the virulent, recipient strain, Xg51. The ORF showing avirulence activity was referred to as avrBs7. Group A strain Xcv 85-10 shows a delayed HR on the leaves of ECW-70R. Avirulence gene avrBs7 shows 67% identity at amino acid level to avrBs1.1 from Xcv str. 85-10. Inoculation of Xg51 transconjugants carrying avrBs1.1 showed delayed HR on ECW-70R confirming avirulence activity.

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Segregation analysis of F2 population indicated that this resistance trait in ECW-70R is controlled by single dominant resistance gene, which we designated as Bs7.

Comparing in-planta bacterial growth and electrolyte leakage indicating tissue damage showed significant differences between transconjugants carrying avr clones and the virulent wild type strain confirming that AvrBs7 and AvrBs1.1 were responsible for eliciting HR on ECW-70R. Sequence analysis of both avr genes revealed the presence of 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 in the carboxy-terminal regions of both avirulence genes. Alanine mutants at the catalytic site of AvrBs7 did not elicit HR on ECW-70R indicating importance of tyrosine phosphatase domain in recognition of the avr protein by Bs7 gene transcripts. In order to explain the differences in the timing of HR elicitation by the two avr genes, we constructed a fusion protein exchanging AvrBs7 catalytic site with

AvrBs1.1 catalytic site. Exchanging catalytic domains abolished HR activity. AvrBs7 and

AvrBs1.1 possess more or less similar three-dimensional structure.

The newly identified resistance gene Bs7 is effective against few of the tested A, B and D group strains. Avirulence gene avrBs7 has is found only in D group X. gardneri strains, while avrBs1.1 is present among A and B group strains. However, the avr genes appear to be located on the plasmid, making it easier for the pathogen to lose the avr gene and attain virulence. Future studies will be focused on characterization of motifs in the avr genes and their role in pathogenicity.

Type III effectors are secreted and translocated into the host cell via a specialized type III secretion system. The process of translocation of effectors is highly regulated.

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Effectors are believed to contain secretion and translocation signals in the N-terminal region that direct them through the secretion apparatus. In xanthomonads, these two signals are not well characterized. The characteristic features of such signals have been used in the identification of effectors from other plant pathogens e.g. pseudomonads.

We compared the predictive rules for N terminal signal sequences from pseudomonads to that of xanthomonads and we found that although a few Xanthomonas effectors contain Pseudomonas effector-like features, the majority of the amino acid bias features do not strictly follow for Xanthomonas effectors, indicating the need to develop separate predictive rules and programs for screening type III effectors using signal features.

Based on amino acid biases in N-terminal region of Xanthomonas effectors, we searched for motifs and corresponding matrices of each motif were searched within whole genomes using C-based program. Although we could identify all the known effectors, the program also identified type II and type IV secreted proteins with high score hit indicating that false positive and negatives could not be avoided with this program as well. We used the same program to screen our three bacterial spot

Xanthomonas draft genomes. Candidate effectors were tested for their translocation using in planta avrBs2 reporter gene assay. We identified 2 novel effectors, xopZ2, xopAO.

In order to narrow down the location of translocation signal of the Xanthomonas effectors, we selected effector XopF1 as a model. Based on comparative genomic analysis, XopF1 appears to be a tomato-specific effector. It also has been shown to be dependent on chaperone HpaB for its translocation. Without HpaB binding, it is not translocated into the host cell (Buttner et al. 2006). HpaB is an important pathogenicity

166

factor in xanthomonads, hence HpaB dependent effectors also must be playing

important role in the initial steps of pathogen infection. We were interested in identifying

the translocation signal and HpaB binding site in XopF1. The first 70 amino acids of

XopF1 fused to reporter gene avrBs262-574 elicited an HR on ECW-20R in in-planta assay, indicating that first 70 amino acids of XopF1 are sufficient for translocation and hence contain translocation signal as well as HpaB-binding site. However, the first 40 amino acids were not sufficient to deliver the reporter into the plant cell. The first 40 amino acids of XopF1 either lack translocation signal or they lack an HpaB binding site.

Another possibility that the translocation signal and HpaB binding site could be overlapping and absent in first 40 amino acids can’t be disregarded, especially in case of chaperone-dependent effectors. The secondary structure analysis of XopF1 showed presence of two alpha helices, one from amino acid 27 through 33 and another from amino acid 47 through 59. According to the effector-chaperone interaction models proposed by Lilic et al. (2006); alpha helices and β motif residues are involved in the interaction. We found β motif residues in the second helix region. Alanine mutants carrying mutations at both sites, 27 through 33 and 47 through 59 were tested by in- planta assay for their ability to deliver reporter gene into the plant cell and elicit HR on

ECW-20R. Both the mutants failed to elicit HR on 20R indicating that either they had lost HpaB-binding site or translocation signal due to mutations. To confirm the HpaB-

XopF1 protein-protein interaction, we carried out in vitro experiments namely, yeast two- hybrid assay and pull-down assay. In both in-vitro experiments, weak binding of HpaB to XopF1 alanine mutants was observed. Based on in-vitro and in-planta results, we can speculate the model for XopF1 and HpaB interaction. HpaB could be binding to XopF1

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in the alpha helix region and β motif and directing it to the translocation apparatus. In- vitro, HpaB could be binding to the alanine mutants but complex may not be stable and active and hence not translocated in in-planta assay.

Such a model for effector-chaperone interaction for Xanthomonas system will highlight the role of chaperones in regulating the type III effector translocation and in turn regulation of pathogenicity during the infection process.

Apart from studying genomics of bacterial spot xanthomonads, we also analyzed other 15 available Xanthomonas genomes infecting different plant species including dicots and monocots. Diversity among xanthomonads is reflected in their genome organization and the repertoires of pathogenicity genes. We analyzed different pathogenicity clusters among these xanthomonads with respect to their core function in pathogenicity and host as well as tissue specificity. Certain pathogenicity genes, a majority of which are type III effectors, could be associated with their preference on particular hosts. Knowledge based on these analyses could serve as basis to experimentally verify role of such genes in disease progression, host preference, and evolution of xanthomonads and efforts could be directed towards better understanding of plant-pathogen interaction and designing durable disease control strategies.

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BIOGRAPHICAL SKETCH

Neha Potnis was born in Pune, Maharashtra State, India in 1985. She obtained her bachelor’s degree in microbiology in 2005 from University of Pune, India where she specialized in industrial microbiology. During her bachelor’s degree, she was offered a scholarship to conduct research in University of Mysore Indian Academy of Sciences.

During this fellowship, she got opportunity to work under the guidance of Prof. H. S.

Shetty and Dr. Sarosh. The research project was focused on screening for microsatellite markers from Sclerospora graminicola genomic library. This was the first time she worked with a plant pathogen and microbial genetics. In 2005, she was admitted to the

Master of Science program in Microbiology from University of Pune. After completing her master’s studies, she received research assistantship to pursue her Ph.D. studies in

Department of Plant Pathology, University of Florida, under the guidance of Dr. Jeffrey

B. Jones and Dr. David J. Norman. Her research focused on comparative genomics of xanthomonads and study of pathogenicity factors of xanthomonads in disease development.

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