CaWRKY40介导辣椒耐高温高湿及抗青枯病分子机制解析

辣椒是重要蔬菜和典型的茄科植物，在高温高湿下一般发病严重，剖析高温高湿下抗病机制有利于其抗病遗传改良。前期研究发现CaWRKY40对辣椒抗青枯病及耐高温高湿均有重要调节作用，并分别鉴定了其启动子应答高温元件及青枯菌侵染区域。本项目拟进一步分离并鉴定这些元件或区域特异性结合蛋白和CaWRKY40结合蛋白并通过ChIP、瞬间表达、BiFC和Co-IP等方法予以验证，分析其亚细胞定位及通过qPCR分析其编码基因对高温高湿及青枯菌侵染的应答，通过在辣椒上瞬间超表达、 VIGS及在转基因烟草中超表达，分析这些蛋白对CaWRKY40表达调节，或与CaWRKY40互作对细胞坏死、H2O2积累、耐高温高湿、抗青枯病及相关标记基因表达的影响。通过研究，提出CaWRKY40介导辣椒耐高温高湿及抗病分子机制，可为辣椒等茄科植物抗病遗传改良提供合理技术策略和具有应用价值的基因。

Pepper（Capsicum annuum）is a one of the most important vegetable crops on the globe and a typical Solanaceae species. Heavy yield losses of pepper are frequently caused by diseases, which are usually aggravated by high temperature and high humidity. A better understanding of pathogen defense mechanisms operating under high temperature and high humidity conditions will be helpful for the genetic improvement of pepper disease resistance. Our previous studies showed that in pepper CaWRKY40 acts as an important regulator resistance to the bacterial pathogen Ralstonia solanacearum and thermotolerance. The promoter regions of CaWRKY40 which are responsive to high temperature and Ralstonia solanacearum inoculation were also identified. In this project, regulatory proteins binding to these promoter regions or the CaWRKY40 protein will be identified by Yeast one-hybrid (Y1H) or Yeast two-hybrid (Y2H) sceeing, respectively. Y1H interaction results will be confirmed in planta by ChIP (Chromatin Immunoprecipitation) and transient expression in onion epidermic cells. Y2H results will be conformed in planta by BiFC (Bimolecular FluorescenceComplementation) and Co-IP(co-immunoprecipitation). The putative Y1H and Y2H interactors will be characterized by subcellular localization analysis. The transcription levels of these genes in response to high temperature, high humidity and Ralstonia solanacearum inoculation will be elucidated using quantitative real-time RT-PCR (qPCR) technology. Using transient overexpression and virus-induced gene silencing (VIGS) analysis in pepper plants as well as stable overexpression in transgenic tobacco plants, the function of promising Y1H and Y2H interactors in transcriptional regulation of CaWRKY40 and interaction with the CaWRKY40 protein, respectively, will be studied in detail. These analyses will cover a wide variety of biological conditions relevant to the roles of CaWRKY40 as a pontential mediator of crosstalk between diease resistance and thermotolerance. Conditions to be analyzed will include Ralstonia solanacearum inoculation, high temperature and high humidity stress, cell death induction and H2O2 accumulation. We will also analyze the transcriptional regulation of marker genes associated with disease resistance as well as thermotolerance. Taken together, this project will comprehensively address molecular mechanisms of CaWRKY40 in resistance of pepper to high temperature, high humidity and Ralstonia solanacearum. Our result will also serve as a foundation for the design of new technological strategies, and provide new valuable genes, for the genetic improvement of Capsicum annuum and other Solanaceae.