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Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

ABSTRACT: Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, which contained a 31-kb insert, complemented mutant NP1. A restriction map of pIH1 was constructed, and deletion analyses identified a 10-kb HindIII fragment that restored pathogenicity to NP1. Southern hybridization analysis indicated that DNA sequences in the 10-kb HindIII fragment are conserved among other X. campestris pathovars tested. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. A 2.7-kb ClaI fragment was sequenced, and two possible open reading frames (ORF1 and ORF2) were found. Results indicated that ORF2 but not ORF1 may be expressed in Escherichia coli and in X. campestris pv. glycines. The carboxy terminus of the potential polypeptide encoded by ORF2 has an amino acid sequence similar to that of the gamma subunit of oxaloacetate decarboxylase, which is involved in sodium ion transport in Klebsiella pneumoniae.

SUBMITTER: Hwang I

PROVIDER: S-EPMC205798 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

Hwang I I Lim S M SM Shaw P D PD

Journal of bacteriology 19920301 6

Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, ...[more]

PMID: 1312532

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Project description:ABSTRACT The pathogenic bacterium Xanthomonas campestris pv. campestris, the causal agent of black rot of Brassicaceae, manipulates the physiology and the innate immunity of its hosts. Association genetic and reverse-genetic analyses of a world panel of 45 X. campestris pv. campestris strains were used to gain understanding of the genetic basis of the bacterium's pathogenicity to Arabidopsis thaliana. We found that the compositions of the minimal predicted type III secretome varied extensively, with 18 to 28 proteins per strain. There were clear differences in aggressiveness of those X. campestris pv. campestris strains on two Arabidopsis natural accessions. We identified 3 effector genes (xopAC, xopJ5, and xopAL2) and 67 amplified fragment length polymorphism (AFLP) markers that were associated with variations in disease symptoms. The nature and distribution of the AFLP markers remain to be determined, but we observed a low linkage disequilibrium level between predicted effectors and other significant markers, suggesting that additional genetic factors make a meaningful contribution to pathogenicity. Mutagenesis of type III effectors in X. campestris pv. campestris confirmed that xopAC functions as both a virulence and an avirulence gene in Arabidopsis and that xopAM functions as a second avirulence gene on plants of the Col-0 ecotype. However, we did not detect the effect of any other effector in the X. campestris pv. campestris 8004 strain, likely due to other genetic background effects. These results highlight the complex genetic basis of pathogenicity at the pathovar level and encourage us to challenge the agronomical relevance of some virulence determinants identified solely in model strains. IMPORTANCE The identification and understanding of the genetic determinants of bacterial virulence are essential to be able to design efficient protection strategies for infected plants. The recent availability of genomic resources for a limited number of pathogen isolates and host genotypes has strongly biased our research toward genotype-specific approaches. Indeed, these do not consider the natural variation in both pathogens and hosts, so their applied relevance should be challenged. In our study, we exploited the genetic diversity of Xanthomonas campestris pv. campestris, the causal agent of black rot on Brassicaceae (e.g., cabbage), to mine for pathogenicity determinants. This work evidenced the contribution of known and unknown loci to pathogenicity relevant at the pathovar level and identified these virulence determinants as prime targets for breeding resistance to X. campestris pv. campestris in Brassicaceae.

Project description:UnlabelledN-Acetylglucosamine (GlcNAc), the main component of chitin and a major constituent of bacterial peptidoglycan, is present only in trace amounts in plants, in contrast to the huge amount of various sugars that compose the polysaccharides of the plant cell wall. Thus, GlcNAc has not previously been considered a substrate exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, expresses a carbohydrate utilization system devoted to GlcNAc exploitation. In addition to genes involved in GlcNAc catabolism, this system codes for four TonB-dependent outer membrane transporters (TBDTs) and eight glycoside hydrolases. Expression of all these genes is under the control of GlcNAc. In vitro experiments showed that X. campestris pv. campestris exploits chitooligosaccharides, and there is indirect evidence that during the early stationary phase, X. campestris pv. campestris recycles bacterium-derived peptidoglycan/muropeptides. Results obtained also suggest that during plant infection and during growth in cabbage xylem sap, X. campestris pv. campestris encounters and metabolizes plant-derived GlcNAc-containing molecules. Specific TBDTs seem to be preferentially involved in the consumption of all these plant-, fungus- and bacterium-derived GlcNAc-containing molecules. This is the first evidence of GlcNAc consumption during infection by a phytopathogenic bacterium. Interestingly, N-glycans from plant N-glycosylated proteins are proposed to be substrates for glycoside hydrolases belonging to the X. campestris pv. campestris GlcNAc exploitation system. This observation extends the range of sources of GlcNAc metabolized by phytopathogenic bacteria during their life cycle.ImportanceDespite the central role of N-acetylglucosamine (GlcNAc) in nature, there is no evidence that phytopathogenic bacteria metabolize this compound during plant infection. Results obtained here suggest that Xanthomonas campestris pv. campestris, the causal agent of black rot disease on Brassica, encounters and metabolizes GlcNAc in planta and in vitro. Active and specific outer membrane transporters belonging to the TonB-dependent transporters family are proposed to import GlcNAc-containing complex molecules from the host, from the bacterium, and/or from the environment, and bacterial glycoside hydrolases induced by GlcNAc participate in their degradation. Our results extend the range of sources of GlcNAc metabolized by this phytopathogenic bacterium during its life cycle to include chitooligosaccharides that could originate from fungi or insects present in the plant environment, muropeptides leached during peptidoglycan recycling and bacterial lysis, and N-glycans from plant N-glycosylated proteins present in the plant cell wall as well as in xylem sap.

Dataset Information

Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

Publications

Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

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