Identification And Fuctional Study Of Genes Induced In Xanthomonas Oryzae PV. Oryzae By Its Host Plant

Xanthomonas oryzae consists of two pathogenic variants, X. oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola(Xoc), which cause bacterial blight and bacterial steak in rice respectively. Xoo invades rice leaves through hydathodes while Xoc through stomatal. Both diseases are very serious in rice production in China. Currently, studies on Xoo pathogenesis have focused on the fuction of important genes, such as LuxR gene, avr gene, genes encoding quorum sensing receptor protein, genes related to typeⅢsecretion system, two-component regulation system or DSF-based quorum sensing regulation system. But researches on virulence-associated genes in the aspect of pathogen-host interaction using proteomics were fewer reported.Along with the completion of whole genome sequencing of rice and Xoo, this plant disease has become an important mode in plant-pathogen interaction research. From the pathogen-host interaction level to clone a new virulence factor in pathogen, to study its function and mechanism of pathogenicity, are quite meaningful to reveal the mechanism of pathogencity of Xoo and to design disease control strategy. So, in this study, proteomics was used to analyze the induced protein of X. oryzae pv. oryzae strain PXO99 during the different stages of interaction(3 h,6 h,12 h) with susceptible cultivar of rice IR24. Then, eight genes were identified by NCBI blast. These genes are candidate genes induced in the interaction, which are probably associated with virulence. So, we respectively constructed these eight genes disruption mutants. The results of pathogenicity test showed that 6 genes mutants reduced pathogenicity. Finally, we indentified a new virulence-associated gene, xanA(YP₀01915572.1). In order to find the function of xanA gene, we characterize the contribution of xanA gene to growth, extracellular polysaccharide, biofilm, oxidative stress and cell morphology of PXO99.(Ⅰ) The total proteins of X. oryzae pv. oryzae strain PXO99 were extracted after interaction with leaf extract of the susceptible rice IR24 for 3 h,6 h and 12 h. To analyze the differences of proteome expression between with and without leaf extract of host plant, proteome analysis was performed by two-dimensional gel electrophoresis (2-DE). The results clearly revealed that comparing to the control,29 protein spots were differentially expressed both in 3 h and 6 h while 38 protein spots in 12 h. Among them,8 protein spots had significant differences. They are phosphor-glucomutase(PGM) (gi|84622382), Arginase(gi|84622054), chaperonin GroEL (gi|21229998), septum site-determining protein MinD (gi|84624994), bacteriolferritin (gi|84623542), inositol-1-monophosphatase (gi |84624213), spermidine synthase (gi|58579842), Pirin (gi|84622991). By blasting on NCBI, we found 8 genes encode these proteins. They are xanA (YP₀01915572.1), rocF (YP₀01915898.1), groEL (YP₀01911694.1), minD (YP₀01912473.1), bfr (YP₀0 1914071.1), PXO₀0388 (YP₀01913112.1), PXO₀3650(YP₀01911408.1), PXO₀2027 (YP₀01914882.1). Then qRT-PCR (quantitative real-time PCR) was performed to analyze the expression of these genes after interaction for 1 h,3 h,6 h,12 h and 18 h. The results showed that these genes were induced in 12 h and 18 h.(Ⅱ) These eight genes were cloned from X, oryzae pv. oryzae strain PXO99, with degenerated primers by PCR. We named them xanA, rocF, groL, minD, bfr, imp, spd and pir. These 8 genes disruption mutants of PXO99 were constructed by single cross-over event. Using leaf-cutting inoculation to test the pathogenicity of eight genes mutants, the results showed that 6 genes mutants reduced pathogenicity except bfr and spd. Among them, xanA mutant strain exhibited obstructed formation of biofilm, reductive production of EPS, decreased ability to resist oxidative stress. But compared to the wild-type strain, the mutant strain had the same cell morphology and could also stimulate HR in nonhost tobacco.