OsNPR1介导水稻细胞程序性死亡和免疫分子机制研究

Programmed cell death ( PCD ) plays a critical role in defense responses in plant. Our preliminary studies showed that OsCUL3a interacted with OsNPR1 and promoted its degradation via 26S proteasome pathway. Lesion mimic phenotype and defense response were suppressed significantly in oscul3a by knocking out OsNPR1, indicating that OsNPR1 plays a critical role in regulating PCD and immunity in rice. Based on those, OsNPR1 overexpression (OsNPR1-OE) and knock out transgenic lines will be constructed for OsNPR1 functional analysis. At the same time, we are going to perform a Y2H library screening and immunoprecipitation assay for OsNPR1 interacting proteins (NIPs) screening. Reverse mutation also will be carried out for suppressors screening. Then the NIPs and suppressors overexpression and knock out transgenic lines will be constructed for phenotype identification and functional analysis. Subsequently, the regulation relationships between OsNPR1 and NIPs and the suppressors will be studied by crossing those lines with OsNPR1-OE and osnpr1 knock out mutant. The derived materials will be used for phenotype analysis including blast resistance evaluation and PCD identification. Those experiments are expected to shed light on the molecular mechanism and regulatory signal network for OsNPR1 regulating PCD and immunity response in rice.

细胞程序性死亡(Programmed cell death, PCD)在植物防卫应激反应过程中发挥重要作用。本研究前期发现OsCUL3a与OsNPR1在体内互作并介导其经由26S蛋白酶体途径被降解。敲除OsNPR1能显著抑制oscul3a类病斑表型和防卫反应，说明OsNPR1在调控水稻PCD和免疫应激反应过程中发挥关键作用。以此为基础，本研究拟构建OsNPR1过表达和敲除转基因家系以研究其功能；利用酵母双杂交及免疫沉淀质谱技术筛选OsNPR1在水稻体内的互作蛋白；构建回复突变体库筛选OsNPR1的抑制子；构建互作蛋白和抑制子的敲除和过表达家系并分别与OsNPR1过表达以及敲除材料杂交创制遗传材料；鉴定以上各材料的表型，研究OsNPR1互作蛋白和抑制子的功能并解析其与OsNPR1间的调控机制。通过以上实验初步阐明OsNPR1调控水稻PCD和免疫应激反应的信号网络及分子机制。

水稻病害是制约其安全生产的重要影响因素，挖掘更多的抗病基因，系统阐述水稻抗病的分子机制是开展水稻抗病分子育种，减轻水稻病害损失的根本举措。但受制于抗源选择面较单一，直接通过抗性品种而克隆新抗病基因的成功概率极低。由过敏反应引起的细胞程序性死亡是抗病过程中最直观的一种表现形式。以免疫自激活造成细胞死亡失控的类病斑突变体为载体，为反向溯源性分离上游抗病基因或正向深入研究调控水稻免疫的分子机制提供了极佳的突破口。我们的前期研究结果显示，OsCUL3a通过降解OsNPR1而负调控水稻细胞死亡和免疫。以此为基础，通过本项目的实施，我们对OsNPR1的互作蛋白及oscul3a的抑制子开展筛选及相关基因的功能研究，结果显示，过表达OsNPR1能显著提升水稻对稻瘟病的抗性水平；OsNPR1与OsNPR2及OsNPR3在体内互作，OsNPR2及OsNPR3在oscul3a细胞死亡调控通路中发挥相反的作用；oscul3a的类病斑表型完全依赖于SC1，其编码一个典型的CC-NBS-LRR类抗病蛋白，敲除SC1后导致oscul3a系统获得抗性不能正常激活。综合以上结果表明，SC1是一个新的水稻抗病基因，具有一定的水稻抗病分子育种应用潜力。