Comparative proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the susceptible and the resistant cultivars of Brassica oleracea

Black rot of cruciferous plants, caused by Xanthomonas campestris pv. campestris , causes severe losses in agriculture around the world. This disease affects several cultures, including cabbage and broccoli, among others. Proteome studies of this bacterium have been reported; however, most of them were performed using the bacterium grown under culture media conditions. Recently, we have analyzed the proteome of X. campestris pv. campestris during the interaction with the susceptible cultivar of Brassica oleracea and several proteins were identified. The objective of the present study was to analyze the expressed proteins of X. campestris pv. campestris during the interaction with the resistant cultivar of B. oleracea . The bacterium was infiltrated in the leaves of the resistant plant and recovered for protein extraction and two-dimensional electrophoresis. The protein profile was compared with that of the bacterium isolated from the susceptible host and the results obtained revealed a group of proteins exclusive to the resistant interaction. Among the proteins identified in this study were plant and bacterium proteins, some of which were exclusively expressed during the resistant interaction.     

Identification of genes in Xanthomonas campestris pv. vesicatoria induced during its interaction with tomato

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease of tomato and pepper. The disease process is interactive and very intricate and involves a plethora of genes in the pathogen and in the host. In the pathogen, different genes are activated in response to the changing environment to enable it to survive, adapt, evade host defenses, propagate, and damage the host. To understand the disease process, it is imperative to broaden our understanding of the gene machinery that participates in it, and the most reliable way is to identify these genes in vivo. Here, we have adapted a recombinase-based in vivo expression technology (RIVET) to study the genes activated in X. campestris pv. vesicatoria during its interaction with one of its hosts, tomato. This is the first study that demonstrates the feasibility of this approach for identifying in vivo induced genes in a plant pathogen. RIVET revealed 61 unique X. campestris pv. vesicatoria genes or operons that delineate a picture of the different processes involved in the pathogen-host interaction. To further explore the role of some of these genes, we generated knockout mutants for 13 genes and characterized their ability to grow in planta and to cause disease symptoms. This analysis revealed several genes that may be important for the interaction of the pathogen with its host, including a citH homologue gene, encoding a citrate transporter, which was shown to be required for wild-type levels of virulence.     

Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidylprolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the amino-acid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Escherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors of X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.     

Genetic organization of the lexA,recA and recX genes in Xanthomonas campestris

A gene cluster containing lexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported lexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different X. campestris pathovar. The abundance of RecA protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas lexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.     

The A protein of the filamentous bacteriophage Cf of Xanthomonas campestris pv. citri.

Filamentous bacteriophages have very strict host specificities. Experiments were performed to investigate whether the A protein of the filamentous phage Cf, which infects Xanthomonas campestris pv. citri but not X. campestris pv. oryzae, is involved in determining Cf's host specificity. The gene encoding the A protein of Cf was cloned and expressed in X. campestris pv. citri. The genomic DNA of another filamentous bacteriophage, Xf, which infects X. campestris pv. oryzae but not X. campestris pv. citri, was then introduced by electroporation into X. campestris pv. citri that had expressed the A protein of Cf. The progeny phages thus produced were able to infect both X. campestris pv. oryzae and X. campestris pv. citri, indicating that the A protein of Cf was incorporated into the viral particles of Xf and conferred upon Xf the ability to infect the host of Cf. Inactivation of the A protein gene abolished the infectivity of Cf. The results of this study indicate that the A protein of Cf is responsible for controlling the host specificity of Cf.     

Genomic approaches in Xanthomonas campestris pv. vesicatoria allow fishing for virulence genes

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.     

Expression of a subcloned alpha-amylase gene under the control of a Xanthomonas campestris promoter

Abstract Using the promoter probe pKK232-8 a 0.6-kb fragment containing an active promoter sequence from Xanthomonas campestris pv campestris was cloned. Two new plasmids were constructed: (a) pAP2, which contains the amy gene from Bacillus subtilis cloned between the Eco RI and Hin dIII sites in the pMFY40 plasmid, and (b) pAP2X, obtained after introduction of the cloned X. campestris promoter upstream from the amy gene. These plasmids were introduced into amylolytic and non-amylolytic strains of X. campestris pv campestris and pv manihotis , respectively. Quantification of alpha-amylase specific activity in liquid culture showed that the introduction of a Xanthomonas promoter doubled the expression of amy gene when the host strain was the pathovar campestris but had little effect on the strain from pathovar manihotis . This difference in the promoter activity might indicate that the cloned promoter is specific and could be involved in pathovar differentiation or plant-pathogen interaction.     

Identification and expression profiling of tomato genes differentially regulated during a resistance response to Xanthomonas campestris pv. vesicatoria

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. Plants of the tomato line Hawaii 7981 are resistant to race T3 of X. campestris pv. vesicatoria expressing the type III effector protein AvrXv3 and develop a typical hypersensitive response upon bacterial challenge. A combination of suppression subtractive hybridization and microarray analysis identified a large set of cDNAs that are induced or repressed during the resistance response of Hawaii 7981 plants to X. campestris pv. vesicatoria T3 bacteria. Sequence analysis of the isolated cDNAs revealed that they correspond to 426 nonredundant genes, which were designated as XRE (Xanthomonas-regulated) genes and were classified into more than 20 functional classes. The largest functional groups contain genes involved in defense, stress responses, protein synthesis, signaling, and photosynthesis. Analysis of XRE expression kinetics during the tomato resistance response to X. campestris pv. vesicatoria T3 revealed six clusters of genes with coordinate expression. In addition, by using isogenic X. campestris pv. vesicatoria T2 strains differing only by the avrXv3 avirulence gene, we found that 77% of the identified XRE genes were directly modulated by expression of the AvrXv3 effector protein. Interestingly, 64% of the XRE genes were also induced in tomato during an incompatible interaction with an avirulent strain of Pseudomonas syringae pv. tomato. The identification and expression analysis of X. campestris pv. vesicatoria T3-modulated genes, which may be involved in the control or in the execution of plant defense responses, set the stage for the dissection of signaling and cellular responses activated in tomato plants during the onset of spot disease resistance.     

Characterization of Xanthomonas campestris pv. pruni strains from different hosts by pathogenicity tests and analysis of whole-cell fatty acids and whole-cell proteins

Strains of Xanthomonas campestris pv. pruni obtained from Prunus armeniaca. P. domestica, P. persica and P. salicina in different geographical areas were compared for pathogenicity, fatty acid and wholecell protein analysis. Four strains, one per each host plant, were inoculated at the same time, on the foliage of P. armeniaca, P. avium, P. persica and P. salicina cultivars . Mean content of fatty acids of X.c. pv. pruni strains were also compared with those of many strains of X.c. pv. campestris , pv. graminis , pv. hyacinthii , pv. pelargonii and pv. vasculorum. Strains showed a remarkable homogeneity in fatty acids content and whole-cell protein profiles and principal component and cluster analysis did not reveal any grouping according to original host or geographical origin. However, X.c . pv. pruni strains can be grouped apart from the other X. campestris pathovars. There appears to be no pathogenic specialization among the strains tested, however, they varied in aggressiveness to host plants and host plant in susceptibility. The most of the strains were able to cross-infect species other that from where they were originally isolated, although, P. avium did not show any symptom of disease. P. persica cv. Sentry and P. salicina cv. Globe Sun, recently licensed as resistant to X.c. pv. pruni. were infected, although to a lesser extent, by some strains.     

The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

Deoxyribonucleic acid relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae

Twenty Xanthomonas campestris pathotype strains, three non-pathotype strains, and one strain of X. fragariae were studied by S1 DNA:DNA hybridization tests. The results of these tests do not support the retention of X. campestris as a single species. DNA reassociation values among many of the strains were low. Three clusters of closely related strains were observed, but nine strains did not cluster. Xanthomonas campestris pv. secalis was more closely related to X. fragariae than to any other X. campestris pathovar. Mapping the host family upon a three-dimensional genomic distance matrix of the xanthomonads suggested that strains attacking the same plant family usually show some relationship, but only a distant one. Thus, pathogenicity toward members of the same host family is not a measure of the genomic relationships of xanthomonads.     

DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.     

Xanthomonas campestris pv. campestris possesses a single gluconeogenic pathway that is required for virulence

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.