The Functional Study Of XopR Gene In Xanthomonas Campestris Pv. Campestris In Plant Innate Immunity

Black rot was a disease caused by Xanthomonas campestris pv.campestris, causing serious impact on the cruciferous plant yield and resulting in huge losses of the agricultural economy. Xcc effector proteins were secreted through the type III secretion system into the host cell to affect host innate immunity. XopR was a candidate effector protein of Xcc strain8004, and XopR existed in almost all of Xanthomonas.In this study, the transcriptional expression and secretion of XopR were detected via molecular biological methods. We generated a XopR deletion strain (AXopR) and analyzed the pathogenicity of AXopR on cruciferous plants. In addition, we conducted researches on pathogenic mechanism of XopR in Arabidopsis, aiming to clarify the biological function of the candidate effector XopR and to provide clues for the study of mechanism of Xcc8004.The main results were as follows:1. A total of five reference genes, thy A, gyrB, rpoB, gapdh and16S rRNA, of Xanthomonas campestris pv.campestris were systematically compared during cultured in different media to identify the most suitable gene for comparative expression studies in Xcc using reverse transcription quantitative PCR (qRT-PCR). The gyrB was the most stable gene would be used for normalization of qRT-PCR experiments data using two statistical methods GeNorm and NormFinder.2. The expression and regulation of XopR gene were analyzed through qRT-PCR. The results showed that the XopR gene transcription was regulated by the type III regulator of HrpG and HrpX, and the XopR protein secretion was type III secretion system dependent, indicating that XopR was a type III effector protein of Xcc8004.3. A XopR gene deletion mutant (AXopR) was generated, and the AXopR strain caused decreased disease symptoms on Zhonggan15, Zhonggan21, Zhongbai83and Xinlimei in contrast with Xcc8004, suggesting that XopR was required for the full virulence of XccS004on cabbage host plants with variety specificity.4. The expression of PTI (PAMPs-triggered immunity) marker gene FRK1(Flagellin receptor kinasel) was measured by dual luciferase repoter system, which showed XopR in Arabidopsis protoplasts inhibited flg22-induced FRK1::LUC transcription. MAPKs (Mitogen-activated protein kinase) activity was also detected in Arabidopsis protoplasts. The results showed that XopR did not inhibit flg22-induced MAPKs activity, suggesting that XopR can inhibit PTI signaling pathway, but it was not MAPKs-dependent.5. XopR Arabidopsis transgenic plants were obtained via agrobacterium-mediated inflorescence dipping methods.Xcc8004was inoculated on the XopR Arabidopsis transgenic plants, the result showed that XopR Arabidopsis transgenic plants could promote the virulence of Xcc8004in contrast with wild type Col-0, suggesting XopR operated virulence function on Arabidopsis. XopR suppressed flg22induced reactive oxygen species (ROS) and callose deposition.6. The confocal microscopy revealed that green fluorescent protein XopR-GFP fusions localized to plant cell plasma membrane in Arabidopsis protoplasts.