水稻钙依赖蛋白激酶OsCDPK19介导双重抗性的分子机理研究

Plants have two types of defense mechanism to combat pathogens, namely pathogen-associated molecular pattern (PAMP)–triggered innate immunity (PTI) and effector-triggered immunity (ETI). The knowledge of molecular crosstalk for PTI and ETI in plants is rather limited. To understand the molecular regulatory network for rice and fungus (Magnaporthe oryzea) interaction, we have obtained and investigated the function of an effector-interacting calcium-dependent protein kinase OsCDPK19. By knocking-out OsCDPK19 in rice and inoculation of compatible and incompatible race of rice blast fungus, we found that the OsCDPK19 plays a positive role in PTI and a negative role in ETI. In order to study the molecular mechanism underlying the dual role of OsCDPK19 in rice innate immunity, we are trying to achieve the following results in this project: 1) To understand the molecular signal transduction mechanisms of OsCDPK19 in responses to Ca2+ signal and physical reaction to Avr-Pita/Pita using prokaryotic expressed proteins and protoplast system in vitro and in vivo. 2) To determine the key residues for OsCDPK19 in PTI and ETI pathways by sited-specific mutants. 3) To establish the OsCDPK19 signaling network by isolation of new interactors with yeast two-hybrid and co-immunoprecipitation, and to understand their biological function in resistance to rice blast. Therefore, our study will reveal molecular mechanisms for thedual role of OsCDPK19 in defense signaling pathways and offer further insights into the calcium-dependent protein kinase in response to biostresses. Besides, the results may provide theoretical basis for genetic improvement of rice resistance against rice blast in agriculture.

植物天然免疫主要有病原相关分子模式激活的PTI和病原菌效应子激活的ETI。目前，对植物PTI和ETI的分子信号相互关联认识不足。为深入研究水稻与稻瘟病菌互作分子调控网络，申请人将借助前期筛选获得的与效应子互作且具有负调控PTI、正调控ETI双重功能的水稻钙依赖蛋白激酶OsCDPK19，通过遗传、分子、生化和细胞学技术手段，全面解析OsCDPK19介导双重抗性的分子机理，预期达成以下目标：1）通过原核表达蛋白和原生质体转化，确定OsCDPK19与Ca2+信号和植病体系模型Avr-Pita/Pita的生理、生化关系。2）构建OsCDPK19点突变体，确认该蛋白在传递抗病信号中的关键位点。3）筛选OsCDPK19新互作蛋白，并验证其生物学功能，构建OsCDPK19介导双重抗病角色的信号调控网络。本研究将加深钙传感蛋白CDPK在响应生物胁迫的认识，也为遗传改良水稻提高其稻瘟病抗性提供理论依据。

水稻（Oryza sativa L.）是世界上最重要的粮食作物之一，而由病原真菌（Magnaporthe oryzae）引起的稻瘟病对水稻的生产具有严重的危害。因此，开展稻瘟病菌与水稻与的相互作用研究，对维护粮食安全重大意义。效应子Avr-Pita与抗性蛋白Pita是稻瘟菌与水稻互作的经典植病体系，Avr-Pita能被Pita识别产生ETI（Effector-triggered immunity）反应，但Avr-Pita在调节植物PTI（Pathogen-associated molecular patterns-triggered immunity）反应以及Avr-Pita和Pita在体内识别的分子机理仍知之甚少。本课题前期利用Avr-Pita筛选获得水稻中的互作蛋白钙调蛋白激酶OsCDPK19。OsCDPK19具有两种长度的蛋白：一个含有磷酸激酶活性区、连接区和Ca2+调控区，另一个只含有磷酸激酶活性区。本项目验证了OsCDPK19通过其磷酸激酶活性区分别与Avr-Pita、Pita互作，且Avr-Pita能够降解长片段OsCDPK19。基因敲除实验明确了OsCDPK19负调控水稻PTI反应，正调控ETI反应。烟草细胞死亡实验显示OsCDPK19通过其激活片段与Avr-Pita、Pita（尤其是CC结构域）互作促进细胞死亡。磷酸化实验证实OsCDPK19能够将Pita的LRD区、线粒体细胞色素c氧化酶（COX）组装蛋白OsCOX11磷酸化。这些结果暗示Avr-Pita可能通过降解全长OsCDPK19，释放其激活区从而激活OsCOX11，增强COX酶活性清除活性氧，从而负调控PTI反应；另外，OsCDPK19通过其激活区将Pita磷酸化，与Avr-Pita、Pita共同作用促进细胞死亡，从而正调控水稻ETI反应。本研究全面揭示Avr-Pita调控水稻PTI和ETI反应信号网络提供了基础，为研究植病体系的互作关系提供新思路，为水稻抗性品种的遗传改良提供新方向。