[show abstract][hide abstract] ABSTRACT: Genetic polymorphism within the genomes of bacterial pathogens determines their evolutionary potential during long-term interaction with their hosts. To investigate the level of genetic variation in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of rice bacterial blight disease, three DNA marker systems, including (i) restriction fragment length polymorphism (RFLP) of the avrBs3/PthA family genes (avrXa27), (ii) RFLP of insertion (IS) elements and (iii) random amplified polymorphic DNA (RAPD) markers, were used to detect polymorphism among 32 Xoo strains that differed in their virulence patterns. All these strains contained multiple avrXa27 homologs that were variable in copy number and genomic location. RFLP of six IS elements revealed that these mobile sequences were abundant in Xoo genomes, with 150 of the total of 165 discernable markers being variable. Thirty-eight decamer primers of RAPD amplified a total of 691 bands, with 100% of them being variable. In addition, analysis of molecular variance (AMOVA) of data from RFLP analysis of IS elements and from RAPD analysis showed that most of the genetic variation residues were within Xoo populations, rather than between populations. Although all three DNA marker systems supported that substantial variation was maintained in Xoo genomes, Mantel tests did not identify significant correlation between the similarity coefficients calculated from them. The results of the present study indicated that Xoo genomes contain a high level of genetic polymorphism, which greatly facilitates the evolution of this important pathogen during interaction with its host rice plant.
Systematic and Applied Microbiology 01/2008; 30(8):587-600. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial leaf blight, a serious disease of rice worldwide. A Tn5-based transposon randomly insertional mutant library was previously constructed. By screening mutants against susceptible rice cultivar IR24, four mutants were identified with reduced virulence on rice plants and were found to have Tn5 transposon inserted at an endo-1,4-beta-D glucanase (E.C. 22.214.171.124) gene eglXoB. In planta growth analysis indicated that multiplication of the mutants in rice leaves was greatly reduced comparing to the Xoo wild-type strain. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that the expression of eglXoB was induced in planta. Genetic complementation of these mutants with a functional eglXoB gene restored both virulence and in planta growth, suggesting that the eglXoB gene was required for virulence. Ectopic expression of eglXoB in Escherichia coli demonstrated its endoglucanase activity. Otherwise, the growth of the mutants in synthetic medium containing cellulose as the sole sugar source was not affected. Data of this study suggested eglXoB gene is required for pathogenesis of rice bacterial blight disease.
[show abstract][hide abstract] ABSTRACT: A novel transposon mutagenesis system for the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. campestris (Xcc) was developed using a Tn5-based transposome. A highly efficient transformation up to 10(6) transformants per microg transposon DNA was obtained. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses of Tn5 insertion sites suggested a random mode of transposition. The transposition was stable in the transformants for 20 subcultures. Eighteen thousand and 17000 transformants for Xoo and Xcc, respectively, were generated, corresponding to 4X ORF coverage of the genomes. The libraries will facilitate the identification of pathogenicity-related genes as well as functional genomic analysis in Xoo and Xcc.