Journal of Biotechnology 106 (2003) 203–214

Genomic approaches in Xanthomonas campestris pv. vesicatoria allow fishing for virulence genes Daniela Büttner, Laurent Noël1 , Frank Thieme, Ulla Bonas∗ Institut für Genetik, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle (Saale), Germany Received 28 March 2003; received in revised form 18 June 2003; accepted 16 July 2003

Abstract Xanthomonas campestris pv. vesicatoria is an economically important pathogen of pepper and tomato and has been established as a model organism to study bacterial infection strategies. In the last two decades, intensive genetic and molecular analyses led to the isolation of many genes that play a role in the intimate molecular relationship with the host plant. Essential for pathogenicity is a type III protein secretion system, which delivers bacterial effector proteins into the host cell. Currently, the genome of X. campestris pv. vesicatoria is being sequenced. The availability of genomic sequence information will pave the way for the identification of new bacterial virulence factors by bioinformatic approaches. In this article, we will present preliminary data from the genomic sequence analysis and describe recent and novel studies to identify bacterial type III effector genes. © 2003 Elsevier B.V. All rights reserved. Keywords: Avirulence proteins; hrp Genes; Pathogenicity island; Plant-inducible promoter box (PIP box); Translocation

1. Introduction Bacteria belonging to the genus Xanthomonas are one of the most omnipresent groups of Gram-negative plant pathogenic bacteria and cause a variety of diseases in multiple plants (Leyns et al., 1984). Xanthomonas campestris pathovar (pv.) vesicatoria (also designated X. axonopodis pv. vesicatoria; Vauterin et al., 2000) is the causal agent of bacterial spot disease in pepper (Capsicum subspecies (spp.)) and tomato (Lycopersicon spp.). This disease affects all above∗ Corresponding author. Tel.: +49-345-5526290; fax: +49-345-5527277. E-mail address: [email protected] (U. Bonas). 1 Present address: Max-Planck Institut für Züchtungsforschung, Carl-von-Linne-Weg 10, D-50829 Köln, Germany.

ground plant parts and results in considerable yield losses due to defoliation and severely spotted fruits that are not salable. Bacterial spot occurs worldwide but is most pernicious in regions with a warm and humid climate. During natural infections, X. campestris pv. vesicatoria enters the host tissue via openings in the plant surface, such as stomata or wounds, and multiplies in the intercellular spaces. Colonization of the apoplast is locally restricted and induces the macroscopically visible appearance of disease symptoms, so-called watersoaked lesions, that later on become necrotic (Stall, 1995). Pathogenicity of X. campestris pv. vesicatoria depends on a type III secretion (TTS) system, which is highly conserved in plant and animal pathogenic bacteria and mediates the transport of bacterial effector proteins into the host cell cytosol (Hueck, 1998;

0168-1656/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2003.07.012

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Fig. 1. Model for the interaction of X. campestris pv. vesicatoria with susceptible and resistant host plants. The bacterial TTS system spans both bacterial membranes and is associated with an extracellular pilus that presumably crosses the plant cell wall (200 nm thick, not drawn to scale). Upon contact with the host cell, effector proteins are delivered into the host cell cytosol with the help of the predicted TTS translocon, a bacterial protein complex that contains HrpF and inserts into the plant cell membrane. In susceptible plant cells, effectors interfere with host cellular pathways, ultimately leading to the formation of disease symptoms. In resistant plants, however, effector proteins are recognized by matching plant resistance (R) proteins and induce the HR. Photographs of plant phenotypes were taken 3 days after inoculation of a high-density bacterial culture into leaves of susceptible and resistant pepper plants. PM, plasma membrane; TTS, type III secretion.

Cornelis and Van Gijsegem, 2000) (Fig. 1). The TTS system spans both bacterial membranes and is associated with a pilus structure, which presumably spans the plant cell wall and serves as a conduit for secreted proteins as has recently been demonstrated for the plant pathogens Pseudomonas syringae and Erwinia amylovora (Brown et al., 2001; Jin and He, 2001; Jin et al., 2001; Li et al., 2002). In X. campestris pv. vesicatoria, intensive genetic and biochemical studies have led to the identification of a number of proteins that are involved in the type III-mediated host–pathogen interaction (for review see Büttner and Bonas, 2002). Among those are components of the TTS system as well as type III-secreted effector proteins that are delivered into the host cell where they presumably interfere with cellular processes and suppress unspecific plant defense (Brown et al., 1995; Vivian and Arnold, 2000; White et al., 2000). However, the full repertoire of effector proteins has yet to be deciphered. The availability of genomic sequence information from X. campestris pv. vesicatoria will provide a powerful tool to uncover effector genes

by homology searches and bioinformatic approaches. To date, the genomes of two members from the genus Xanthomonas have already been sequenced. The genome analysis of Xanthomonas axonopodis pv. citri, the causal agent of citrus canker, and X. campestris pv. campestris, a vascular pathogen that causes black rot in crucifers, revealed a number of common and specific virulence gene candidates that might determine the interaction with the respective host plants (Da Silva et al., 2002). In this article, we will highlight our current knowledge on pathogenicity strategies of X. campestris pv. vesicatoria and describe molecular and bioinformatic approaches to identify bacterial virulence genes.

2. The hrp pathogenicity island encodes the TTS system and secreted proteins In X. campestris pv. vesicatoria, the TTS system is encoded by the 23 kb chromosomal hrp

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205

hrpA

hrpB

hrpC

hrpD hrpE

A

xopD

hrpF

N

xopA

tR

OR F4

OR F1

X. campestris pv. vesicatoria

IS C

H

T

hrpA

N

J

hrpB

U V

C

hrpC

Q RS A

B

F G

hrpD hrpE

hrpF

tR N A

hrc hpa

hrc hpa 2 1

C

T

hrpA

N

J

hrpB

U V

P

hrpC

Q RS A

B

hr pW

X. axonopodis pv. citri

hrpD hrpE

F hrpF

X. campestris pv. campestris hrc hpa 2 1

C

T

N

J

U V

P

Q RS A

B 2 kb

Fig. 2. hrp Regions in different xanthomonads. Schematic overview of the sequenced hrp regions from X. campestris pv. vesicatoria strain 85-10 (accession numbers U33548, AF056246), X. axonopodis pv. citri strain 306 (accession numbers AE011665, AE011666, AE011667) and X. campestris pv. campestris strain ATCC33913 (accession numbers AE012221, AE012222, AE012223). Grey areas correspond to homologous sequences, hatched boxes to xop genes with low G + C content; hrc and hpa genes are represented in dark and light grey, respectively. Arrows indicate the direction of transcription, black dots and squares refer to PIP and hrp boxes, respectively. hrp, Hypersensitive response and pathogenicity; hpa, hrp associated; hrc, hrp conserved; xop, Xanthomonas outer protein.

(hypersensitive response and pathogenicity) gene cluster which has originally been identified genetically as a key pathogenicity determinant (Bonas et al., 1991) and comprises six transcriptional units designated from hrpA to hrpF (Fenselau et al., 1992; Fenselau and Bonas, 1995; Wengelnik et al., 1996a; Huguet and Bonas, 1997; Huguet et al., 1998; Rossier et al., 2000) (Fig. 2). Nine hrp genes (termed hrc for hrp conserved) are conserved in plant pathogenic bacteria and encode TTS components that are also present in animal pathogens (Bogdanove et al., 1996; He, 1998). Hrc proteins presumably constitute the core components of the secretion apparatus in the inner and outer membrane. By contrast, the role of non-conserved Hrp proteins is less clear. Exceptions are HrpF, a putative component of the type III translocon in the plant plasma membrane (Büttner et al., 2002) (Fig. 1), and HrpE1, which is probably the major subunit of the Hrp pilus (T. Ojanen-Reuhs, E. Weber, R. Koebnik and U. Bonas, unpublished data). Interestingly, the left hrp region of X. campestris pv. vesicatoria contains an insertion sequence (IS)-like element as well as putative effector genes with low G+C content compared to the genomic average (64% over 100 kb; Noël et al., 2002). Mobile genetic elements as

well as sequences that differ in G + C content from the genomic DNA are typical features of pathogenicity islands (PAIs) that presumably have been acquired during evolution by horizontal gene transfer (Hacker and Kaper, 2000). For the hrp PAI from X. campestris pv. vesicatoria, this hypothesis is supported by the finding that one of the putative effector genes in this region is preceded by a hrp box-like motif, a typical promoter motif of hrp and effector genes from P. syringae (Innes et al., 1993; Xiao et al., 1994; Xiao and Hutcheson, 1994; Fouts et al., 2002) (Fig. 2). Furthermore, horizontal transfer of the hrp region among different strains from X. campestris pv. vesicatoria has indeed been shown (Basim et al., 1999). It is worth noting that sequences homologous to the left hrp region from X. campestris pv. vesicatoria are missing in the genomes of X. axonopodis pv. citri and X. campestris pv. campestris. Comparative sequence analysis of the hrp gene clusters from all three xanthomonads revealed that—despite a high conservation of the core region—the flanking sequences differ in gene content and orientation (Fig. 2). These findings emphasize the importance of genomic sequence analysis of closely related pathogens in order to unravel differences in the virulence equipment that might bear host-specific functions.

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3. hrp Genes are co-regulated with putative virulence factors In X. campestris pv. vesicatoria, hrp genes are not constitutively expressed but are induced in planta and in minimal medium (Wengelnik and Bonas, 1996). Two regulatory proteins, HrpG and HrpX, have been identified that are essential for hrp gene induction. HrpG, a transcriptional activator of the OmpR family of two-component response regulators, is activated by a yet unidentified plant-derived signal and controls the expression of a genome-wide regulon including hrp and effector genes as well as TTS-unrelated genes (Wengelnik et al., 1996b; Astua-Monge et al., 2000a; Noël et al., 2001). Furthermore, HrpG induces the expression of HrpX, an AraC-type transcriptional activator, which in turn is essential for the expression of most members of the HrpG regulon (Wengelnik and Bonas, 1996; Noël et al., 2001). Many hrpG/hrpX-regulated genes contain a plant-inducible promoter box (PIP box; consensus TTCGC-N15 -TTCGC) in their promoter regions which presumably serves as a regulatory element (Büttner and Bonas, 2002). However, the PIP box might not be sufficient to confer HrpX inducibility since it is also present in hrpG/hrpX-independent promoters (Ciesiolka et al., 1999). New genes belonging to the hrpG regulon have recently been identified by cDNA-AFLP (amplified fragment length polymorphism)-based transcript comparisons between a wild-type strain and a strain expressing a constitutively active version of HrpG (HrpG*) (Wengelnik et al., 1999; Noël et al., 2001). Sequence analysis of hrpG-induced cDNA fragments identified several genes with homology to known effectors from other plant pathogenic bacteria. The corresponding gene products—designated Xops (Xanthomonas outer proteins) (Table 1)—are indeed secreted by the TTS system (Noël et al., 2001, 2002). Furthermore, HrpG* regulates the expression of genes encoding predicted proteins with homology to transcriptional regulators, degradative enzymes and XadA, a member of the family of adhesin-like proteins from plant and animal pathogenic bacteria (Noël et al., 2001). The co-expression of TTS-unrelated genes with the hrp gene cluster suggests that the corresponding gene products are involved in the host–pathogen interaction. In many bacteria, degradative enzymes such as proteases, cellulases or pectate lyases, which

are often secreted into the extracellular medium by the type II secretion system, have indeed been shown to contribute to virulence (Alfano and Collmer, 1996; Sandkvist, 2001). Furthermore, adhesins presumably mediate bacterial attachment to the host cell (Soto and Hultgren, 1999; Rojas et al., 2002). In X. campestris pv. vesicatoria, xadA is down-regulated by HrpG*, suggesting that the corresponding gene product is only required in the initial phase of infection during which the TTS system might not play an essential role. Taken together, cDNA-AFLP-based transcriptome analyses in X. campestris pv. vesicatoria have underlined the suitability of expression studies to unravel putative bacterial virulence genes (see also Fig. 3). However, due to technical limitations not all hrpGregulated genes could be identified so far. For future research, the availability of genomic sequence information will provide an important tool for the quantitative and saturated analysis of the hrpG regulon by microarray experiments.

4. The molecular host–pathogen crosstalk is mediated by type III effector proteins Once the bacteria are in the vicinity of the host cell, they start to deliver effector proteins across the plant plasma membrane. Due to the low in vitro secretion efficiency of many plant pathogens including X. campestris pv. vesicatoria, it has been difficult to unravel the full spectrum of type III-secreted proteins. Thus, many effectors have been identified genetically as the products of avirulence (avr) genes that betray the pathogen to the surveillance system of resistant plants. Recognition of Avr proteins by corresponding plant resistance (R) gene products leads to the induction of specific defense responses that often culminate in the hypersensitive response (HR), a rapid local cell death at the infection site, which can easily be scored (Klement, 1982; Staskawicz et al., 2001) (Fig. 1). In X. campestris pv. vesicatoria, eight Avr proteins, which are all most likely translocated into the plant cell, as well as six candidate effectors have been uncovered so far (Table 1). Type III-dependent translocation of several effector proteins from X. campestris pv. vesicatoria has recently been demonstrated by the use of reporters such as the adenylate cyclase and

Table 1 Known and putative effector genes from X. campestris pv. vesicatoria G + C content (%)

Expressionc

Present in Xac/Xccd

Homolog of gene product (organism; accession number)/ effector protein familye

Reference

P

42

Constitutive

−/+

AvrA (P. syringae pv. glycinea; AAA25725)

C

64

nd

+/+

Agrocinopine synthase (A. tumefaciens; AAO15364)

avrBs3

P

67

Constitutivef

+/−

AvrBs3 family

Ronald and Staskawicz, 1988; Swanson et al., 1988; Escolar et al., 2001 Kearney and Staskawicz, 1990; Swords et al., 1996; Mudgett et al., 2000 Bonas et al., 1989; Van den Ackerveken et al., 1996; Szurek et al., 2001; Marois et al., 2002

avrBs4

P

67

Constitutivef

+/−

AvrBs3 family

avrBsT

P

42

Constitutive

−/+

YopJ/AvrRxv family

avrRxv

C

51

Constitutive; PIP box

−/+

YopJ/AvrRxv family

avrXv3 avrXv4

nd nd

54 47

Induced; PIP box nd; PIP box

−/− −/+

YopJ/AvrRxv family

65

Induced; PIP box

+/+

C (hrp region)

48

Induced; PIP box

+/+

xopB

nd

55

Induced

−/−

xopC xopD

C C (hrp region)

47 54

Induced Induced; hrp box

−/− −/+

Effector genes avrBs1 √

avrBs2

Effector gene candidates hpaA C (hrp region) √

xopA

HpaA (X. oryzae pv. oryzae; BAB07864) Hpa1 (X. oryzae pv. oryzae; AAC95121) AvrPphD (P. syringae pv. phaseolicola; CAC16699) PsvA (P. syringae pv. eriobotryae; BAA87062) YopJ/AvrRxv family

Bonas et al., 1993; Ballvora et al., 2001 Minsavage et al., 1990; Escolar et al., 2001 Whalen et al., 1988; Ciesiolka et al., 1999 Astua-Monge et al., 2000a Astua-Monge et al., 2000b Wengelnik and Bonas, 1996; Huguet et al., 1998 Noël et al., 2002 Noël et al., 2001 Noël et al., 2003 Noël et al., 2002

207

xopJ C 57 Induced −/+ Noël et al., 2003 a √, virulence activity demonstrated by knock-out studies. b Location on plasmid (P) or chromosome (C) was determined by Southern blot analysis of chromosomal and plasmid DNA. nd, Not determined. c Expression in planta or under hrp gene-inducing conditions. PIP, plant-inducible promoter. d Presence in the genome of Xac (X. axonopodis pv. citri) and Xcc (X. campestris pv. campestris) was determined by BlastN (http://www.ncbi.nlm.nih.gov/blast/): (+) present; (−) absent. e Homologs have been identified by BlastP (http://www.ncbi.nlm.nih.gov/blast/). The AvrBs3 family (InterPro accession number IPR005042) mainly comprises effector proteins from Xanthomonas spp. that contain 34-amino-acid direct repeats, an acidic activation domain and nuclear localization signals. The YopJ/AvrRxv family (InterPro accession number IPR005083) includes effector proteins of various plant and animal pathogenic bacteria that presumably act as cysteine proteases (Lahaye and Bonas, 2001). f Recent in vitro expression experiments indicate that hrpG* leads to a two to threefold increase in expression (U. Bonas et al., unpublished data).

D. Büttner et al. / Journal of Biotechnology 106 (2003) 203–214

Location on chromosome or plasmidb

Genea

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(A)

promoter elements

secretion signal

eukaryotic features

mobile elements average G+C content

(B)

Molecular approaches Identification by avirulence activity in resistant plants Identification due to association with pathogenicity islands (Alfano et al., 2000; Noël et al., 2002) Amplification by PCR using primers which anneal to conserved sequences associated with avr genes (Arnold et al., 2001) Screen for genes that are induced in planta or co-regulated with hrp genes using cDNA-AFLP, IVET or a reporter transposon (Noël et al., 2001; Boch et al., 2002; Fouts et al., 2002)

Genomic approaches Comparative genomic sequence analyses (Zwiesler-Vollick et al., 2002; PetnickiOcwieja et al., 2002; Salanoubat et al., 2002; da Silva et al., 2002) Identification of export-associated signals based on sequence analysis of novel effector proteins (Guttman et al., 2002; PetnickiOcwieja et al., 2002) Generation of a search matrix for hrp box-associated promoters (Fouts et al., 2002; Zwiesler-Vollick et al., 2002)

In vivo transposon screen using an AvrRpt2 derivative deprived of its secretion signal as a reporter (Guttman et al., 2002)

Fig. 3. Strategies for identification of effector genes in plant pathogenic bacteria. (A) Characteristic features of many effector genes include conserved promoter elements such as PIP or hrp boxes, a different G + C content as well as the vicinity to mobile genetic elements. The predicted proteins often exhibit characteristic N-terminal amino acid compositions that presumably serve as a secretion signal. Several effector proteins contain typical eukaryotic motifs indicating an activity inside the host cell. (B) Summary of molecular and bioinformatic approaches that have been used to identify type III effector genes from plant pathogenic bacteria (Alfano et al., 2000; Arnold et al., 2001; Boch et al., 2002).

an HR-inducing Avr protein deprived of its secretion signal (Mudgett et al., 2000; Guttman and Greenberg, 2001; Casper-Lindley et al., 2002). Furthermore, AvrBs3 from X. campestris pv. vesicatoria was directly localized in nuclei of infected plant cells (Szurek et al., 2002). Nucleocytoplasmic trafficking is presumably mediated by importin ␣ which was identified as a plant interaction partner of AvrBs3 (Szurek et al., 2001). AvrBs3 belongs to a class of effector proteins that have mainly been identified in Xanthomonas spp. (Lahaye and Bonas, 2001). All family members contain nuclear localization signals

(NLSs) and an acidic activation domain (AAD), typical features of eukaryotic transcription factors indicative of an activity in the host cell nucleus. Indeed, modulation of host gene expression has recently been demonstrated for AvrBs3 (Marois et al., 2002). AvrBs3 is one of the best studied effector proteins from plant pathogenic bacteria. For many effectors, it has been difficult to assign precise molecular functions since they often do not possess conserved sequence motifs or show sequence homologies to proteins with known functions. Furthermore, in many cases the analysis of individual mutant strains revealed little

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phenotypic effects on the interaction with the host plant, indicating that many effector proteins are functionally redundant (Vivian and Arnold, 2000; White et al., 2000) (see also Table 1). Comparative sequence analysis uncovered homologies between several effectors and Xops from X. campestris pv. vesicatoria and known effector proteins from other plant and animal pathogens (Büttner and Bonas, 2003). For instance, at least four members of the YopJ/AvrRxv family of effector proteins have been identified so far in X. campestris pv. vesicatoria (Lahaye and Bonas, 2001). The broad conservation of these effector proteins suggests that they target similar pathways in plant and animal host cells. YopJ from Yersinia spp. was demonstrated to interfere with immune responses of animal cells and presumably acts as a cysteine protease (Orth, 2002). However, whether YopJ homologs exhibit similar functions remains to be investigated. The cocktail of effector proteins that is delivered by X. campestris pv. vesicatoria into the plant cell also comprises pathovar-specific proteins. Several effectors do not possess homologs in X. axonopodis pv. citri and X. campestris pv. campestris (Table 1) and vice versa, suggesting that they have evolved to target specific pathways of the respective host plants. Thus, the functional characterization of non-conserved effector proteins might help to understand the molecular processes that determine host–pathogen interactions and bacterial host range.

5. Genomic approaches to identify type III effectors The identification of type III effectors from plant pathogenic bacteria has recently been fueled by genomic approaches. In X. axonopodis pv. citri, X. campestris pv. campestris and Ralstonia solanacearum, candidate genes have been uncovered due to homologies to known effector genes from other pathogens as well as to the presence of typical eukaryotic features in the predicted gene products, which indicate a function inside the host cell (Da Silva et al., 2002; Salanoubat et al., 2002) (Fig. 3A). Furthermore, in P. syringae, prediction algorithms have been developed that include conserved sequence motifs such as hrp boxes in the promoter regions and characteristic

209

N-terminal amino acid compositions, which presumably serve as export signals of the predicted proteins, as search criteria (Fouts et al., 2002; Guttman et al., 2002; Petnicki-Ocwieja et al., 2002; Zwiesler-Vollick et al., 2002) (Fig. 3). These bioinformatic approaches led to the identification of 38 candidate effectors, many of which were indeed shown to be secreted and translocated (Collmer et al., 2002; Greenberg and Vinatzer, 2003). 5.1. The fishing ground of X. campestris pv. vesicatoria: the genomic sequence of strain 85-10 Currently, the genome of X. campestris pv. vesicatoria strain 85-10 (approximately 5.5 Mb as has been determined by pulse-field gel electrophoresis; R. Stall and E. Hacioglu, personal communication) is being sequenced (http://www.genetik.uni-bielefeld.de/ GenoMik/partner/halle.html). Strain 85-10 was chosen since it has already been used as genetic background in the cDNA-AFLP screen (Noël et al., 2001). In addition, many hrp and effector mutants have been generated in this strain (e.g. Bonas et al., 1991; Rossier et al., 2000; Noël et al., 2002). Strain 85-10 contains 1–2 small plasmids of unknown function as well as a 200 kb plasmid carrying copper resistance genes and the avrBs1 gene (Canteros, 1990). For the sequencing project, three genomic libraries with inserts from one to eight kb in size were generated. The analysis of sequencing data will be achieved using the genome annotation system GenDB, which has been developed at the university of Bielefeld (http://gendb.genetik.unibielefeld.de/). 5.2. Identification of virulence genes from X. campestris pv. vesicatoria by genomic approaches Preliminary sequence analysis of X. campestris pv. vesicatoria strain 85-10 already revealed the complete sequence of hrpG-regulated genes that have previously been identified by cDNA-AFLP (see above). Comparisons of regions containing hrpG-regulated genes to the genomic sequences of X. axonopodis pv. citri and X. campestris pv. campestris uncovered interesting differences in gene content. Several hrpG-induced genes that are present in X. campestris pv. vesicatoria are absent in X. axonopodis pv. citri and/or X. campestris pv. campestris. This is for instance the

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(A)

1

xopJ

type III effector

2

3

4

5

6

regulatory function

7

8

9

10

enzymatic function

1 kb

(B)

ORF

Blast P Predicted function b E-value (InterPro accession number)

Given homology of gene product a %G+C (organism; accession number)

xopJ

56

0.0 XopJ (X. campestris pv. vesicatoria; AAK72486)

YopJ/AvrRxv family protease (IPR005083)

ORF1

62

Two-component system sensor protein (X. axonopodis pv. citri; AAM37020)

Histidine kinase (IPR005467)

ORF2

63

Two-component system regulatory protein 10-49 (X. campestris pv. campestris; AAM41460)

ORF3

61

Two-component system sensor protein (X. axonopodis pv. citri; AAM37020)

10

ORF4

64

Probable nodulation protein (Bradyrhizobium sp. WM9; AAK00179)

5x10-13 Regulatory protein, LuxR family (IPR000792)

ORF5

62

VirH (Agrobacterium tumefaciens; AAL57007)

5x10-87 Cytochrome P 450 enzyme (IPR001128)

ORF6

65

Putative oxidoreductase (Sinorhizobium melilotii; AAK65766)

10-170

ORF7

66

Hypothetical protein Atu 5212 9x10-93 Esterase/ lipase/ thioesterase (Agrobacterium tumefaciens; AAK90583) (IPR000379)

ORF8

65

VirG-like protein (Bradyrhizobium japonicum; AAM12361)

4x10-52 Two-component system response regulator (IPR001789, IPR001867)

ORF9

65

Cytochrome c peroxidase (Bacteroides fragilis; AAL09840)

4x10-39 Cytochrome c peroxidase (IPR004852)

ORF10

66

Sensory box histidine kinase (Chlorobium tepidum TLS; AAM73277)

6x10-28 Two-component system sensor histidine kinase (IPR005467, IPR004358)

10

-148

-122

Two-component system response regulator, LuxR family (IPR001789, IPR000792) Histidine kinase (IPR005467)

Glucose-methanol-choline oxidoreductase (IPR000172, IPR007867)

a

Closest homologs were identified by BlastP (http://www.ncbi.nlm.nih.gov/blast/) based on the matrix BLOSUM 62. b Putative functions were predicted by InterPro (http://www.ebi.ac.uk/interpro/). Fig. 4. Preliminary structure of the region flanking the putative effector gene xopJ from X. campestris pv. vesicatoria strain 85-10. (A) Schematic overview of the xopJ region. Arrows indicate the direction of transcription, different shadings refer to predicted functions of the corresponding gene products as indicated. (B) Homologs and predicted functions of XopJ and the putative gene products encoded in the xopJ-flanking region.

case for xopJ (Noël, 2001; Noël et al., 2001) and several flanking genes (Fig. 4A). Interestingly, xopJ has a low G + C content indicative of horizontal gene transfer and is located adjacent to genes encoding

proteins with homology to transcriptional regulators of two-component systems (Fig. 4B). Whether these proteins are involved in the virulence of X. campestris pv. vesicatoria remains to be investigated.

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The identification of virulence genes will be the main emphasis of the X. campestris pv. vesicatoria genome sequencing project. Prediction algorithms will be developed to identify effector genes based on search criteria such as PIP box motifs in promoter regions as well as TTS-associated N-terminal amino acid compositions in the predicted gene products. Furthermore, sequences with low G + C content, association with mobile genetic elements as well as the presence of eukaryotic protein motifs in the predicted gene products will be considered as characteristics of regions encoding putative effector proteins (Fig. 3A). All evaluated candidates will be tested for type III-dependent secretion using an integrative vector that allows translational fusion to a c-myc epitope as C-terminal tag (Noël et al., 2001). Furthermore, protein translocation into the plant cell will be assayed by translational fusions to the adenylate cyclase or an N-terminal deletion derivative of AvrBs3, both of which have recently been established as reporter proteins in our lab (Noël et al., 2003; F. Thieme, S. Köthke and U. Bonas, unpublished data). In addition to bioinformatic studies, we also envisage to upscale in vitro secretion assays in order to dissect the cocktail of secreted proteins by proteome analysis using two-dimensional gel electrophoresis and mass spectrometry. 5.3. Functional characterization of virulence factors The major goal of the post-genomics era will be to assign molecular functions to individual effector proteins. So far, tentative assignments are based on pairwise sequence similarity searches as well as on prediction algorithms that concern protein families and domains (e.g. InterPro; Mulder et al., 2003). In further studies, predicted biochemical activities need to be confirmed by suitable biological approaches. It should be considered that similar proteins can differ in their molecular activities and biological functions. For instance, the finding that genes in the xopJ region encode transcriptional regulators of two-component systems does contribute little to establish their biological role. The contribution of putative virulence factors to the host–pathogen interaction will be assayed by phenotypic analyses of corresponding knock-out strains. For this, infection assays such as spray or dip inoculations have to be used. The standard infection

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procedure (infiltration of high-density bacterial suspensions into the leaf apoplast) is presumably not suitable for the detection of subtle phenotypic variations between wild-type strains and effector mutants. In addition to mutant studies, individual type III effectors will be expressed in planta in order to monitor potential disease-like phenotypes as well as to analyze changes in host gene expression. Indications on potential molecular functions might be provided by the identification of plant virulence targets by protein interactor screens such as yeast two-hybrid studies or co-immunoprecipitation. For these analyses, we will especially focus on effector proteins that are characteristic for X. campestris pv. vesicatoria.

6. Conclusions The genus Xanthomonas comprises a large number of bacterial plant pathogens that infect at least 124 monocotyledonous and 268 dicotyledonous plants (Chan and Goodwin, 1999). So far, the bacterial factors that determine host range and mode of infection in different pathovars are largely unknown. Comparative genomic sequence analyses of X. axonopodis pv. citri, X. campestris pv. campestris and X. campestris pv. vesicatoria have already revealed differences among putative virulence factors that might bear host-specific functions. The obvious differences in the genetic virulence equipment validate genome sequencing of more than one subspecies. Currently, the genomes of X. campestris pv. vesicatoria and X. axonopodis pv. aurantifolii (http://genoma4.iq.usp.br/xanthomonas/) are being sequenced. The successful termination of these sequencing projects will provide the unique opportunity to identify and compare putative virulence factors from four different members of one bacterial genus. In addition, pathovar-specific differences in gene content might give us some clues about targeted approaches for disease control and will last but not least allow the precise PCR-based diagnosis of bacterial diseases.

Acknowledgements We are grateful to J. Boch for critical reading of the manuscript and thank all members of the lab for stimulating discussions. Work on X. campestris pv.

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vesicatoria in our laboratory is supported by grants from the Deutsche Forschungsgemeinschaft and the German ministery of education and research (BMBF) to Ulla Bonas.

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