CaWRKY40介导辣椒耐高温高湿及抗青枯病分子机制解析

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.

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。

本研究通过酵母单杂交以及双杂交系统分别分离和克隆了辣椒抗病与耐高温相关转录因子CaWRKY40的上游调控因子及其互作蛋白，并研究上述调控因子以及互作蛋白在辣椒应答青枯病、高温高湿中的功能及其与CaWRKY40之间的互作方式。研究结果如下：（1）酵母单杂交系统我们分离获得了CaWRKY40上游可能的调控因子，它们分别是CabZIP63以及CaWRKY6；通过染色质免疫共沉淀技术确定了CabZIP63和CaWRKY6结合CaWRKY40启动子的活性；（2）通过酵母双杂交分离获得了多个CaWRKY40可能的互作蛋白，并进一步通过蛋白质免疫共沉淀（co-IP），双荧光互作实验（BiFC）以及微量热涌动仪（MST）验证了它们之间的互作；（3）在本生烟草叶片亚细胞定位实验表明CabZIP63和CaWRKY6均为核蛋白，而CaVAMP7在未收逆境处理的烟草叶片中定位于细胞膜，在青枯菌接种或高温高湿逆境处理下逐渐从细胞膜转移到细胞核；(4)瞬间超表达实验结果表明CabZIP63, CaWRKY6以及CaVAMP7在辣椒叶片超表达均可以产生明显的过敏性细胞坏死，并且伴随着过氧化氢的积累，电解质的渗漏以及多个辣椒抗病或耐高温相关标记基因的上调表达；进一步通过上述基因病毒介导的基因沉默（VIGS）发现 CabZIP63, CaWRKY6以及CaVAMP7的沉默均可降低辣椒对于青枯菌接种的抗性和对高温的耐性；促进接种青枯菌的生长，降低抗病或耐高温相关基因的表达；（5）结合荧光定量PCR，染色质免疫共沉淀以及瞬间表达等方法研究了CabZIP63，CaWRKY6以及CaVAMP7与CaWRKY40之间表达和功能关联，发现CabZIP63，CaWRKY6和CaVAMP7的表达增强可提高CaWRKY40的表达，而CaWRKY40的表达增强也可以显著提高CabZIP63，CaWRKY6以及CaVAMP7的转录水平；CaVAMP7与CaWRKY40互作或同时超表达可较单个基因超表达提高激活过敏反应的活性和相关标记的表达，相反，这两个基因同时沉默比单个基因沉默导致抗病或耐高温水平变得更低；此外，我们还发现CaVAMP7在高温或青枯菌接种下由细胞膜到细胞核的转运受到CaWRKY40的调控，当CaWRKY40沉默，CaVAMP7不能进入细胞核。以上结果表明CabZIP63, CaWRKY6直接调节CaWKR