水稻抗白叶枯病基因Xa23抗病机理的研究

Adoption of resistance (R) genes is the most effective, economic and environment-friendly strategy to control bacterial blight (BB) and bacterial leaf streak (BLS) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) respectively. Xa23 is an excellent executor R gene against BB, activated by a transcription activator-like effector (TALE) family member avrXa23, and induces hypersensitive response in rice. However, the mechanism of hypersensitive response induction by executor R gene was unclear. Here, we planned to construct a cDNA library from CBB23 infected with PXO99A and perform a yeast two-hybrid to screen XA23-interacting proteins. Afterward, all the interactions will be further reconfirmed by bimolecular fluorescence complementation, co-immunoprecipitation and other assays. Approaches of Molecular biology and Biochemistry will be employed to identify and investigate the functions of these proteins required for Xa23-triggered defense reaction. To study and develop Xa23 resistance against BLS, here we also proposed to engineer new Xa23 genes by inserting artificial effector binding elements (EBEs) in its promoter. These EBEs can recognize conserved TALEs in Xoc strains and is supposed to confer Xa23 resistance to Xoc in the battle. The overall goal of this project is to explore the new utilization of Xa23 and associated disease resistance mechanism. This study will facilitate the characterization of executor R genes and generate scientific basis for the fair use of R genes in plant disease management.

水稻白叶枯病和水稻细菌性条斑病是水稻生产上最严重的两种细菌性病害，其病原物分别是水稻白叶枯病菌（Xoo）和水稻细菌性条斑病菌（Xoc）。生产实践中控制这两种病害最有效、最经济和最环保的措施是种植抗病品种。Xa23是属于executor类的广谱抗水稻白叶枯病基因，可通过识别Xoo来源的TALE激活自身的表达，最终触发水稻的过敏反应。但executor类基因诱导表达后触发过敏反应的生化机理尚不清楚。本项目拟利用酵母双杂交系统筛选XA23的互作蛋白，并研究该类蛋白的功能及其在Xa23介导的防卫反应中的作用；同时，在Xa23启动子区添加新的EBE序列，使其能够识别Xoc来源的保守TALEs，研究XA23能否在防治水稻细菌性条斑病方面发挥作用。本项目通过对 XA23功能及抗病生化机理的研究，解析executor类抗病基因的特征及其在植物先天免疫系统中的角色，为科学利用抗病基因提供理论与实践基础。

Executor（E）基因是一类新型植物抗病基因，目前仅克隆到六个该类基因。水稻来源的E基因已挖掘出四个，但这四个基因却在水稻白叶枯病的抗病育种中发挥了重要作用，目前该类新基因的信号转导途径和抗病机理还不清楚。本项目以抗谱最广、抗性最好的水稻抗白叶病基因Xa23为研究对象，通过调查自然变异、人工突变等方式开展功能研究，阐述了Xa23等E基因的关键功能域包括有跨膜域、RxR基序、ED基序等；还通过调取互作蛋白及互作靶标的功能研究，推测Xa23是在诱导表达后，产物形成多聚体，与内置网上的钙离子通道相互作用，将内置网的钙离子泄漏到胞质内，利用钙离子信号转导激活水稻的先天免疫的。项目通过修改Xa23启动区的病原菌感应元件（effector-binding element, EBE）创制了广谱兼抗白叶枯病、细菌性条斑病的新基因。在Xa23转基因水稻的抗性检测中我们还发现，E基因可以生物技术手段发展成为有效的抗稻瘟病基因。项目研究成果不仅丰富了对植物先天免疫系统的认识，还为水稻抗病基因资源的挖掘与科学利用提供了科学依据。