Functional Identification Of XC3605 In Xanthomonas Campestris PV. Campestris

Xanthomonas campestris pv. campestris (Xcc) is the causative agent of crucifer black rot disease and is one of the model bacteria for studying the plant-microbial interaction. It causes severe losses in agricultural yield world-wide. Xcc infects the plants in any period. Xcc first enters into by the hole or the wound, then extends along the edges and black vasular bundle cells, and finally forms a "V" shaped black rot spot. The pathogenesis mechanism of Xcc is not clear, so the research on molecular mechanism of the interaction of plant pathogentic bacteria and host is still a hot topic in the related field now.A Tn5gusA5 insertion mutant library was constructed for systematic research on the function of the genes of Xcc 8004 in our laboratory.16 genes which were annotated as hypothetical protein were selected to study their function by constructing deletion mutants and their complemented strains. The XC0007, XC0022, XC0048, XC0070, XC0130, XC0225, XC0250, XC0252, XC0288, XC0386, XC0675, XC3080, XC3605, XC3782, XC3939 and XC4051 genes of Xcc 8004 were mutated by the suicide plasmid pK18mobsacB, and generated the mutants D0007, D0022, D0048, D0070, D0130, D0225, D0250, D0252, D0288, D0386, D0675, D3080, D3605, D3782, D3939 and D4051.The extracellular protease, extracellular cellulase, extracellular amylase, exopolysaccharide, motility and pathogenicity of these mutants were detected. As a result, these mutants have no influence on the detected phenotypes, but the mutant D3605. The exopolysaccharide production, mobility and virulence on the host plant were decreased in mutant D3605, while biofilm formation was stimulated. The growths of mutant D3605 are not affected in NYGB medium and MMX medium, and D3605 has no significant effect on the HR in pepper ECW-10R.XC3605 protein contains two domains, Glyco_tran_WbsX (o-antigen biosynthesis related glycosyl transferase), and RgpF (rhamnose-glucose assembly related protein factor F).Both Glyco_tran_WbsX and RgpF are associated with the synthesis of lipopolysaccharide (LPS), and they are relatively conserved in Xanthomonas.The ORF and promoter of XC3605 was cloned to pLAFRJ and then introduced into the mutant D3605 by triparental conjugation, yielding the complement strain C3605. The exopolysaccharide production, motility, virulence and biofilm of C3605 could be restored, which suggests XC3605 gene is related to the exopolysaccharide, motility, virulence and biofilm. These results indicated that XC3605 gene plays an important role in the pathogenicity of Xcc 8004.