禾杆锈菌效应蛋白Avrsr35激活小麦抗性蛋白Sr35及其触发ETI分子机制的研究

Wheat stem rust is a disease caused by the fungus Puccinia graminis f. sp. Tritici (Pgt) that can inflict devastating losses in crop yield. Wheat carrying the stem rust resistance (Sr) 31 resistance gene has been widely and effectively cultivated for more than 40 years. However, the new wheat rust lineage Ug99, identified in 1999, was found to be virulent on most wheat cultivars grown globally, including those carrying Sr31 gene. In 2013, transgenic wheat expressing the Sr35 gene that confers resistance against Ug99 and other Pgt races was developed. In this wheat strain, the resistance protein Sr35 is activated by the effector protein Avrsr35 which Pgt secretes into infected plant cells. The activation of Sr35 resulting in effector-triggered immunity (ETI), thus preventing further infection. However, it is still unclear how Avrsr35 activates Sr35, and how Sr35 induces ETI. Therefore, this project aims to solve the crystal structure of Sr35, Avrsr35 and their complex using X-ray diffraction. We will also use mutations and co-immunoprecipitation to elucidate how Avrsr35 activates Sr35. The results of our project will shed new light on the mechanism by which plants prevent pathogen invasion, and will also provide guidance for design of transgenic germplasms with broad spectrum resistance.

小麦杆锈病是对小麦生产最具毁灭性的真菌病害，抗锈病基因Sr31全球性引入使小麦抗锈病被控制40年。自1999年发现对全球90%的小麦品种有毒力的Ug99小种后（包括携带Sr31基因品系）其防治问题极为紧迫。把对Ug99有抗性的基因Sr35转入小麦中能使小麦获得Ug99的抗性，其机制为效应因子Avrsr35被分泌至细胞中，激活抗性蛋白Sr35，激发效应蛋白触发的免疫反应（ETI），阻止病原菌入侵。但Avrsr35如何将Sr35激活，Sr35如何激活ETI反应的机制不清楚。本项目以Avrsr35和Sr35为研究对象，以结构生物学为主要研究手段，用X射线衍射法解析Sr35、Avrsr35及其复合物的晶体结构，结合定点突变、免疫共沉淀等方法，阐明Avrsr35激活Sr35引发ETI的分子机制。本项目的实施阐释了抗性蛋白抵御病原菌入侵的免疫机制，为通过转基因技术构建广谱抗性种质资源提供理论指导。

小麦杆锈病是对小麦生产最具毁灭性的真菌病害，面对病体的入侵，NLRs家族蛋白能够感知并激发植物免疫。虽然人们通过基因组学，植物病理学和生物化学等研究方法对效应蛋白（R蛋白）激活ETI反应机制展开研究，但由于R蛋白与效应蛋白的相互作用分子机制尚不明确，此领域内的两大科学问题依然没有得到解决：一. 在没有病原菌入侵时R蛋白是如何保持抑制状态的；二. 病原菌入侵后，R蛋白是如何被effector快速激活并启动下游信号引发ETI的。本研究选取小麦抗性基因Sr35编码的Sr35蛋白为研究对象，利用X射线晶体法解析了小麦锈菌的病原效应因子Avrsr35的晶体结构，并解析了Avrsr35:Sr35的冷冻电镜结构，发现Avrsr35的同源二聚体被Sr35富含亮氨酸重复结构域识别后，其二聚体被解离成单体，并Sr35炎性小体组装以及其免疫反应。我们的项目揭示了植物CNL的直接识别和激活机制，并可作为基于结构生物学的作物改良理论基础。