The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G؉C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gramnegative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides. Xanthomonas campestris pv. vesicatoria (also designatedXanthomonas axonopodis pv. vesicatoria [101] or Xanthomonas euvesicatoria [46]) is a gram-negative, rod-shaped ␥-proteobacterium with a high genomic GϩC content. Members of the genus Xanthomonas represent an omnipresent group of plantpathogenic bacteria which infect most economically important crop plants and cause a broad variety of diseases (54). X. campestris pv. vesicatoria, the causative agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.) plants, enters the plant tissue through stomata and wounds. Bacterial colonization of plant intercellular spaces is locally restricted and induces macroscopically visible disease symptoms, so-called water-soaked lesions that later become necrotic (91). The disease results in defoliation and severely spotted fruits, both of which cause massive yield losses. Bacterial spot disease occurs worldwide but is most pernicious in regions with a warm and humid climate.Pathogenicity of X. campestris pv. vesicatoria depends on a type III protein secretion system (TTSS) (11, 17), which is highly conserved among plant and animal pathogenic bacteria (24, 97). In X. campestris pv. vesicatoria, the TTSS is encoded by the chromosomal hrp gene cluster (hypersensitive response and pathogenicity) (11) and translocates effector proteins into the plant cell (96). Once inside the plant cytoplasm, the effectors modulate host cell processes, such as suppression of the plant basal defense mechanisms, for the benefit of the pathog...

The Hrp type III protein secretion system (TTSS) is essential for pathogenicity of gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. cDNA-amplified fragment length polymorphism and reverse transcription-PCR analyses identified new genes, regulated by key hrp regulator HrpG, in the regions flanking the hrp gene cluster. Sequence analysis revealed genes encoding HpaG, a predicted leucine-rich repeatcontaining protein, the lysozyme-like HpaH protein, and XopA and XopD, which are similar in sequence to Hpa1 from Xanthomonas oryzae pv. oryzae and PsvA from Pseudomonas syringae, respectively. XopA and XopD (Xanthomonas outer proteins) are secreted by the Xanthomonas Hrp TTSS and thus represent putative effector proteins. Mutations in xopA, but not in xopD, resulted in reduced bacterial growth in planta and delayed plant reactions in susceptible and resistant host plants. Since the xopD promoter contains a putative hrp box, which is characteristic of hrpL-regulated genes in P. syringae and Erwinia spp., the gene was probably acquired by horizontal gene transfer. Interestingly, the regions flanking the hrp gene cluster also contain insertion sequences and genes for a putative transposase and a tRNA Arg . These features suggest that the hrp gene cluster of X. campestris pv. vesicatoria is part of a pathogenicity island.

SummaryThe Hrp type III protein secretion system is essential for pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. Expression of the hrp gene cluster is controlled by HrpG, a two-component response regulator, and HrpX, an AraC-type transcriptional activator. Using the cDNA-AFLP technique, 30 hrpG-induced (hgi ) and five hrpG-repressed (hgr ) cDNA fragments were identified, defining a large hrpG-regulon in X. campestris pv. vesicatoria. Expression of most genes in the hrpG-regulon was dependent on hrpX. Seven cDNA fragments map to the known hrp gene cluster and flanking regions. All other genes appear to be scattered over the chromosome and endogenous plasmids. Sequence analysis identified genes encoding putative extracellular proteases, a putative transcriptional regulator and XopJ and XopB (Xanthomonas outer proteins), homologues of YopJ from Yersinia spp. and the avirulence protein AvrPphD of Pseudomonas syringae respectively. XopB is secreted by the Hrp type III secretion system. Analysis of deletion mutants in several hgi genes revealed a new virulence locus. This study demonstrates that cDNA-AFLP is a powerful tool to study prokaryotic transcriptomes and to identify genes contributing to Xanthomonas virulence and putative effector proteins.

The pathogenicity of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Expression of the hrp operons is strongly induced in planta and in a special minimal medium and depends on two regulatory proteins, HrpG and HrpX. In this study, DNA affinity enrichment was used to demonstrate that the AraC-type transcriptional activator HrpX binds to a conserved cis-regulatory element, the plant-inducible promoter (

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