葡萄霜霉菌诱导的气孔免疫分子机制研究

Downy mildew, one of the most destructive disease for grapevine, is caused by an obligate oomycete called Plasmopara viticola. Stomata in the plant leaf epidermis, formed by pairs of guard cells, are the main route exploited by P. viticola for penetration and sporulation to spread the disease. Stomatal closure to prevent pathogen invasion, namely stomatal immunity, is a focus of recent research on plant innate immunity. Currently, stomatal immunity induced by oomycete remains largely unknown. In the present study, we aim to clarify the molecular basis underlying stomatal immunity induced by P. viticola using grapevine as host plant, P. viticola as oomycete pathogen and β-1,3 glucan, the main structural component of oomycete cell wall, as a mimic of oomycete attack. Approaches, such as patch clamp and in situ guard cell immunohistochemical staining, will be applied to clarify the regulation by P. viticola and glucan of plasma membrane ion channels and H+-ATPases in grapevine guard cells. Omics approaches using guard cells will be performed to comprehensively understand the guard cell signaling triggered by P. viticola and glucan, and identify critical signaling components. These studies are expected to shed light on the oomycete-induced stomatal immunity, and inspire new approaches to improve the grapevine resistance against downy mildew by manipulation of stomatal immunity.

葡萄霜霉病是危害葡萄生产最严重的病害之一，是由专性寄生的卵菌葡萄霜霉菌的感染引起的。气孔存在于叶片表皮，是由一对保卫细胞围成的小孔，既是霜霉菌入侵葡萄的主要入口也是形成孢子进行传播的出口。气孔关闭以阻止病原菌的入侵，即气孔免疫是近年来植物先天免疫的研究热点之一。目前，我们对卵菌诱导的气孔免疫知之甚少。本项目以阐明霜霉菌诱导的气孔免疫分子机制为目的，选用葡萄作为宿主、霜霉菌作为病原卵菌和卵菌的细胞壁主要成分β-1,3葡聚糖作为卵菌入侵模拟物进行研究。本研究将通过膜片钳、原位保卫细胞免疫染色等技术手段阐明霜霉菌及葡聚糖对葡萄保卫细胞质膜离子通道及质子泵的调控。并通过保卫细胞组学分析，从整体上认识霜霉菌及葡聚糖诱导的保卫细胞信号通路，挖掘关键信号通路因子。这些实验结果将深化我们对卵菌诱导的气孔免疫分子机制的认识，并为开发通过控制气孔来增强葡萄霜霉病抗性的策略提供理论依据。

植物气孔是植物免疫病原菌入侵的重要关口。葡萄霜霉病是葡萄产业的世界性病害，其致病菌葡萄霜霉菌是一类专性活体寄生的卵菌。这类卵菌通过气孔入侵葡萄并通过气孔来释放孢子进行传播。但是目前，我们对气孔在免疫葡萄霜霉菌过程中的作用及机理知之甚少。通过本项目的执行，我们明确了气孔是决定葡萄霜霉菌侵染成功与否的重要关口，气孔的关闭能够显著抑制葡萄霜霉菌的侵染。保卫细胞转录组、代谢组学等工作显示植物激素脱落酸在葡萄霜霉菌诱导的气孔关闭过程中扮演重要角色。进一步实验表明脱落酸信号通路的多个核心元件以及多个转运蛋白在葡萄霜霉菌诱导气孔关闭过程中扮演重要角色。进一步的实验表明，葡萄霜霉菌细胞壁组分是葡萄感知霜霉菌诱导气孔关闭的重要信号分子。另一方面，葡萄霜霉菌能够抑制气孔关闭来促进入侵过程。前期的工作已经发现葡萄霜霉菌能够分泌RXLR类效应蛋白来抑制植物免疫。通过酵母双杂筛选实验，本项目找到了4个葡萄霜霉菌RXLR效应蛋白靶向气孔关闭核心元件。进一步生化以及遗传学实验表明这些RXLR能够抑制气孔关闭，暗示葡萄霜霉菌能够分泌RXLR类来抑制气孔免疫。这一系列的工作初步阐明了葡萄气孔免疫霜霉病机理，找到了关键抗性基因，为葡萄抗病育种提供了理论基础与新的思路。