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.     

Exopolysaccharides of Xanthomonas pathovar strains that infect rice and wheat crops

In order to understand the mode of action of the taxonomically related pathogens Xanthomonas campestris pv. translucens, Xanthomonas oryzae pv. oryzae, and Xanthomonas oryzae pv. oryzicola, which attack wheat and rice crops, we examined the compositional differences of their exopolysaccharides (EPSs). Maximum production of polysaccharide in shake cultures of these pathogens was observed between 24 and 72 h. X. campestris pv. translucens, the leaf streak pathogen of wheat, produced a higher amount of polysaccharide (46.97 microg/ml) at 72 h compared to X. oryzae pv. oryzae (42.02 microg/ml), the bacterial blight pathogen of rice, and X. oryzae pv. oryzicola (41.91 microg/ml), the bacterial leaf streak pathogen of rice. Infrared (FTIR) spectra suggested that the polysaccharides of all three Xanthomonas pathovar strains have an -OH group with intermolecular hydrogen bonding, a C-H group of methyl alkanes, an aldehyde (RCHO) group, a C=C or C=O group, and a C-O group. FTIR spectra also revealed the presence of an acid anhydride group in X. oryzae pv. oryzae, a secondary aromatic or aliphatic amine group in X. campestris pv. translucens, and a primary aromatic or aliphatic amine group in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Nuclear magnetic resonance (NMR) spectra revealed the presence of unsubstituted sugars, an acetyl amine of hexose or pentose, and a beta-anomeric carbon of hexose or pentose in the polysaccharides of all bacteria. NMR spectra also identified the alpha-anomeric carbon of hexose or pentose in all strains, and a branching at the fourth carbon of the sugar only in X. campestris pv. translucens; the presence of an uronic acid molecule (acid anhydride group) in X. oryzae pv. oryzae; and a deoxy sugar, rhamnose, in X. oryzae pv. oryzicola.     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

Culture medium for Xanthomonas campestris pv. oryzae

Y uan , W. 1990. Culture medium for Xanthomonas campestris pv. oryzae. Journal of Applied Bacteriology 69 , 798–805.
Studies on nutrient requirements of four Chinese strains of Xanthomonas campestris pv. oryzae in a modified Watanabe's medium led to the development of a new synthetic medium containing sucrose, sodium glutamate, methionine, KH₂PO₄, NH₄C1 and iron chelated with EDTA. The concentration of each ingredient was optimized based on the number of colonies and time required for their appearance. Various concentrations of some nutrients were compared based upon their effects on growth of the pathogen strains and 34 contaminants from rice materials. Tryp-tone enhanced the growth of X. c. oryzae more than that of many contaminants, including Erwinia herbicola . Peptone stimulated growth of X. c. oryzae without promoting excessive contamination. When compared with other media used for X. c. oryzae , the new culture medium enriched with tryptone and peptone gave the highest recovery and earliest appearance of colonies of Chinese strains of this bacterium.     

Integron variability in Xanthomonas arboricola pv. juglandis and Xanthomonas arboricola pv. pruni strains

The integron platform and the gene cassette arrays of 34 Xanthomonas arboricola pv. juglandis and of 47 Xanthomonas arboricola pv. pruni strains isolated from different geographical areas were screened to check their variability. Genetic variability of the strains was also tested by means of BOX-PCR. For two representative strains of the two pathovars, the integrase gene intI and part of the flanking gene ilvD were also cloned and sequenced. Whereas X. a. pv. pruni strains did not show relevant variability, six X. a. pv. juglandis strains isolated in Australia showed some differences in the gene sequences. The CLUSTALW algorithm indicated that the majority of the X. a. pv. juglandis strains are closely related to X. a. pv. pruni, whereas the X. a. pv. juglandis strains isolated in Australia were more similar to Xanthomonas hortorum pv. pelargonii. Similarly, the gene cassette array pattern of the Australian strains, as well as that of the oldest strain maintained in culture, was different from the other strains. Also, three X. a. pv. pruni strains showed a different cassette array pattern when compared with the majority of other strains but no relationships with geographical area of isolation or host plant was revealed. This study confirmed that in addition to species, integrons may generate diversity also within two X. arboricola pathovars.     

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.     

Pathogenicity, Non-Host Hypersensitivity and Host Defence Non-Permissibility Regulatory Gene hrpX is Highly Conserved in Xanthomonas Pathovars

Xanthomonas campestris pv. campestris and X. oryzae pv, oryzae contain the 1428 base pair hrpX gene whose product is involved in the regulation oi hrp genes required for pathogericity, non-host hypersensitivity and non-permissibility of compatible host defence responses. Previous Southern blot hybridization studies have suggested that hrpX is conserved in several X. campestris pathovars and X. oryzae. strains. We have confirmed and extended these findings using hrpX gene amplification by polymerase chain reaction, coupled with Southern blot hybridization analyses. Sixteen distinct pathovars of X. campestris and 12 strains of X. oryzae pv, oryzae were shown to contain homologs of hrpX which were not apparent in heterologous bacteria such as Agrobacterium tumefaciens, A. rhizogenes, Erwinia carolovora ssp. carotovora, Pseudomonas syringae pv, glycinea. P. syringae pv, labaci , and Escherichia coli. The hrpX gene is therefore highly conserved among Xanthomonas species and its gene product strongly resembles positive regulatory proteins of the AraC protein family,     

Restoration of pathogenicity of avirulent Xanthomonas oryzae pv. oryzae and X. campestris pathovars by reciprocal complementation with the hrpXo and hrpXc genes and identification of HrpX function by sequence analyses.

The molecular basis of pathogenesis by Xanthomonas oryzae pv. oryzae has been partly elucidated by the identification of a gene, hrpXo, required for bacterial blight on rice. A mutation in hrpXo results in the loss of pathogenicity on rice and the loss of hypersensitivity on nonhosts such as Datura stramonium and radishes. Pathogenicity and its ability to cause the hypersensitive reaction is restored by complementing the mutant with the heterologous hrpXc gene derived from X. campestris pv. campestris. Conversely, hrpXo complements nonpathogenic mutants of X. campestris pv. campestris and X. campetstris pv, armoraciae. Mutants bearing the heterologous hrpX gene are restored in their abilities to cause diseases typical of their chromosomal background and not the hypersensitive reaction on their respective hosts. The hrpXo and hrpXc genes are therefore functionally equivalent, and this functional equivalence extends into X. campestris pv. armoraciae and possibly into other X. campestris pathovars, since this gene is highly conserved among eight other pathovars tested. Sequence analyses of hrpXo revealed an open reading frame of 1,452 bp with a coding capacity for a protein of 52.3 kDa. The protein contains a consensus domain for possible protein myristoylation whose consequence may result in a loss of recognition by host defense and surveillance systems.     

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.     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyribonucleic acid relatedness of 21 strains of Xanthomonas species and pathovars

The relationship of 17 Xanthomonas campestris pathotype strains, three additional X. campestris strains, and the type strain of Xanthomonas albilineans were examined by DNA-DNA hybridization tests. The results coupled with those of a previous study (Hildebrand et al. 1990) support the hypothesis that X. campestris does not constitute a single bacterial species. There were low levels of DNA-DNA reassociation among many of the different pathovars examined. Six clusters of related pathovars were discerned. In addition, four of the pathovars were only distantly related to each other and to the six clusters. Xanthomonas albilineans was not closely related to any of the other xanthomonads tested.
Mapping and superimposing the botanical families of the host plants upon a three-dimensional genomic distance matrix of the xanthomonads confirms previous observations that pathovars that infect plants of the same botanical family do not necessarily belong to the same genomic group. Six legume-infecting pathovars cluster within one genomic group, but one pathovar, X. cam. pv. pisi is only distantly related to this group. There was also no genomic relationship between X. cam. pv. oryzicola and X. albilineans both of which infect Gramineae. Consequently, pathogenicity toward members of the same plant family is not a good indicator of the genomic relationships among xanthomonads nor is it a good taxonomic determinant.     

Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzae and the closely related X. oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice, respectively. Although many rice resistance (R) genes and some corresponding avirulence (avr) genes have been characterized for bacterial blight, no endogenous avr/R gene interactions have been identified for leaf streak. Genes avrXa7 and avrXa10 from X. oryzae pv. oryzae failed to elicit the plant defense-associated hypersensitive reaction (HR) and failed to prevent development of leaf streak in rice cultivars with the corresponding R genes after introduction into X. oryzae pv. oryzicola despite the ability of this pathovar to deliver an AvrXa10:Cya fusion protein into rice cells. Furthermore, coinoculation of X. oryzae pv. oryzicola inhibited the HR of rice cultivar IRBB10 to X. oryzae pv. oryzae carrying avrXa10. Inhibition was quantitative and dependent on the type III secretion system of X. oryzae pv. oryzicola. The results suggest that one or more X. oryzae pv. oryzicola type III effectors interfere with avr/R gene-mediated recognition or signaling and subsequent defense response in the host. Inhibition of R gene-mediated defense by X. oryzae pv. oryzicola may explain, in part, the apparent lack of major gene resistance to leaf streak.     

Rapid Identification of Xanthomonas campestris pv. graminis and X.c. pv. phlei by Fatty Acid Profiling

Xanthomonas campestris pv. graminis and X. campestris pv. phlei isolated from different grass-species were analysed for their fatty acid content with a gas-chromatograph and a commerially-available software package. The two pathovars could be rapidly and reliably identified and separated from each other with this technique, offering alternative to time-consuming identification by biochemical and pathogenicity tests.     

Specific detection of Xanthomonas oryzae pv. oryzicola in infected rice plant by use of PCR assay targeting a membrane fusion protein gene

Successful control of Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak, requires a specific and reliable diagnostic tool. A pathovar-specific PCR assay was developed for the rapid and accurate detection of the plant pathogenic bacterium Xanthomonas oryzae pv. oryzicola in diseased plant. Based on differences in a membrane fusion protein gene of Xanthomonas oryzae pv. oryzicola and other microorganisms, which was generated from NCBI (http://www.ncbi.nlm.nih.gov/) and CMR (http://cmr.tigr.org/) BLAST searches, one pair of pathovar-specific primers, XOCMF/XOCMR, was synthesized. Primers XOCMF and XOCMR from a membrane fusion protein gene were used to amplify a 488-bp DNA fragment. The PCR product was only produced from 4 isolates of Xanthomonas oryzae pv. oryzicola among 37 isolates of other pathovars and species of Xanthomonas, Pectobacterium, Pseudomonas, Burkholderia, Escherichia coli, and Fusarium oxysporum f.sp. dianthi. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods.     

Differential expression of conserved protease genes in crucifer-attacking pathovars of Xanthomonas campestris.

Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.     

Monoclonal Antibodies Specific for Xanthomonas campestris Bacteria Pathogenic on Wheat and other Small Grains, in Comparison with Polyclonal Antisera

4 hybridoma cell lines (named F1-AA9-D9, F1-AB3-B6, F1-BC7-C1 and F2-CA7-F11) secreting monoclonal antibodies to Xanthomonas campestris pv. undulosa were produced by fusing splenocytes from immunized Lou rats with IR983F myeloma cells. Whole cells were used both as immunogen and as antigen in ELISA and indirect immunofluorescence tests.
The monoclonal antibodies produced reacted positively with X. c. pv. undulosa (38 strains), pv. translucens (3), pv. hordei (3), pv. cerealis (2) and pv. secalis (1).
Strains from other pathovars ( X. c. pv. arrhenatheri, pv. graminis, pv. manihotis, pv. oryzicola, pv. poae and pv. pruni ) and from other species ( X axonopodis, X. ampelina ) and genus ( Pseudomonas, Erwinia, Clavibacter , wheat saprophytic strains) gave a negative reaction. In comparison, seven polyclonal rabbit antisera showed to be less specific: they reacted with unrelated X. campestris pathovars as well as with Pseudomonas strains. Nevertheless, the use of phenol-treated cells in Ouchterlony double immunodiffusion could reduce the effect of cross-reaction for antisera.
The detection of X. c. pv. undulosa by indirect immunofluorescence on infected wheat seed lots has already been applied with success.     

Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli

The production of monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (sodium salt) soluble antigens of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli (fuscans strain) is described. MAbs A6-1 and A6-2 produced to Ps. syringae pv. phaseolicola are pathovar specific. Although MAb XP2 produced to X. campestris pv. phaseoli recognized surface antigens of all strains of this pathovar (including fuscans strains) it cross-reacted specifically with X. campestris pv. malvacearum; it did not react with any other known bacteria or unidentified epiphytes from navy bean seed or leaves. The isotype of both MAbs XP2 and A6-1 is IgG3 whereas that of MAb A6-2 is IgG2a. The reactive antigens are thermostable, but their chemical nature has not been determined.     

Biological role of xanthomonadin pigments in Xanthomonas campestris pv. campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.