Screening And Functional Analysis Of Genes Involved In Resistance Against Xanthomonas Oryzae Pv.oryzicola

Rice bacterial leaf streak disease,caused by Xanthomonas oryzae pv.oryzicola（Xoc）,is an important disease in rice that results in great yield loss in China every year.The rice-Xoc interaction most probably does not obey the gene-for-gene theory.So far,no R gene conferring resistance to Xoc has been identified.No high-resistance cultivar is available in rice production,and control of this disease mainly depends on pesticides.Nonhost resistance is strong and long-lasting.The host range of Xoc is narrow,and thus nonhost resistance of plants to this pathogen widely occurs.Therefore,identification of genes involved in nonhost resistance to Xoc and elucidation of its mechanisms are valuable in both providing insights into the mechnisms underlying resistance to Xoc and breeding for cultivars with high resistance to Xoc.In this thesis research,genes involved in nonhost resistance to Xoc were screened and identified employing c DNA library VIGS in Nicotiana benthamiana,function and mechanisms of calcium channel CNGC genes in nonhost resistance to Xoc were analyzed,and regulatory roles of At WKRY6,At WKRY8 and Sl PIP1 in resistance to Xoc in their ectopic over-expression transgenic rice were studied,and the main results are as follws:（1）A set of genes involved in regulating nonhost resistance to Xoc were screened out using approach of c DNA library VIGS in N.benthamiana.The Xoc-N.benthamiana system suitable for c DNA library VIGS screening was established.Using this system,38 genes whose silenced plants showed altered Xoc-induced HR in comparison with control plants were identified from a screening of a total of 343genes.Sequencing results demonstrated that 21 genes are involved in resistance,stress response,carbohydrate recognition,ethylene signaling,protein folding,degrading,vesicle-mediated transport and acetylation,reactive oxygen species（ROS）and antioxidation,and transcription,while function of 7 genes is unknown.Function and mechanisms of these 28 genes in nonhost resistance to Xoc deserve further elucidation.（2）Functions and mechanisms of tomato CNGC family in nonhost resistance to Xoc were analyzed.Sl CNGC1,Sl CNGC11 and Sl CNGC14 negatively,while Sl CNGC16,Sl CNGC17 and Sl CNGC18 might positively regulated Xoc-induced HR in tomato as revealed by VIGS.Bacterial number counting assay showed that Sl CNGC1 and Sl CNGC14 also strongly negatively regulated nonhost resistance to Xoc.Using Aequorin transgenic plants and luminol-based quantitative detection system in combination with VIGS analyses,it was revealed that Sl CNGC1 and Sl CNGC14 could encode functional calcium channels,through which they modulated cytosol Ca²⁺concentration and thereby affected ROS accumulation,expression of a set of defense-related Ca²⁺signaling genes including Sl Ca M6,Sl CDPK10,Sl CBP60g and Sl CAMTA3,as well as callose deposition,and thereby regulated nonhost resistance to Xoc.（3）Ectopic over-expression transgenic rice of At WKRY6,At WKRY8 and Sl PIP1were constructed and regulatory roles of these genes in resistance to Xoc in their ectopic over-expression transgenic rice were studied.Fifteen lines of T₀ transgenic plants for each gene were obtained using Agrobacterium-mediated transformation method.Inoculation and bacterial number counting assays demonstrated that At WKRY6 and Sl PIP1 negatively,while At WKRY8 positively regulated rice resistance to Xoc.Expression dynamics of a set of genes involved in regulating rice resistance to Xoc in response to Xoc inoculation indicated that Sl PIP1 down-regulated PAD4,LYP6,MPK6 and consequently down-regulated PR1a,PR1b and PR10,thereby executed its negative regulatory role in resistance to Xoc;At WKRY6 down-regulated PR1a,PR1b and PR10 through unknown mechanisms,thereby fulfilled its negative regulatory role in resistance to Xoc;while At WKRY8 down-regulated WRKY45-1 and consequently up-regulated PR1a and PR1b,thereby executed its positive role in resistance to Xoc.Additionally,expression of MAPK10.2 was rapid and strong in all three types of transgenic plants in response to Xoc inoculation,implying it might be a common characteristic of rice response to Xoc inoculation.Collectively,in this study,a number of genes involved in regulating nonhost resistance to Xoc were identified.Functions and mechanisms of tomato CNGC family in nonhost resistance to Xoc were demonstrated.Roles of ectopic over-expression of At WKRY6,At WKRY8 and Sl PIP1 in rice resistance to Xoc were revealed.This study provides gene resources for further elucidating the molecular mechanisms underlying resistance to Xoc and for breeding for resistance cultivar against Xoc.