A multidisciplinary approach to elucidate virulence functions of oomycete effetor proteins

为了在寄主植物上致病，植物病原菌必须抑制植物的先天免疫。细菌和丝状植物病原体通过将特殊的效应蛋白输送到宿主细胞中来实现这一点，宿主细胞操纵植物S的免疫反应。这项建议旨在揭示侵染拟南芥的卵菌病原菌阿拉伯透明孢子菌(HPA)的效应蛋白的毒力功能。我将使用结合结构生物学、分子生物学和植物病理学的多学科方法来揭示卵菌效应器是如何抑制植物免疫的。这项建议的第一个轨道采用X射线结晶学的结构生物学方法来阐明抑制植物防御的HPA效应器的三维结构。Track II的重点是识别这些效应器的宿主目标。在酵母双杂交(Y2H)筛选中已经确定了一些候选效应靶点。这些相互作用将在体外和植物细胞中得到验证。对于Y2H中没有结合相互作用蛋白的效应物，将采用亲和纯化拟南芥组织中的HPA效应物和质谱学鉴定共纯化蛋白的方法。这些联合努力将使人们深入了解HPA如何操纵植物防御系统，并将识别植物免疫信号的新节点。

In order to cause disease on host plants, plant pathogens have to suppress plant innate immunity. Bacterial and filamentous plant pathogens accomplish this by transporting specialised effector proteins into host cells that manipulate the plant s immune response. This proposal aims to unravel the virulence functions of effector proteins from the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), that infects Arabidopsis thaliana. I will use a multidisciplinary approach combining structural biology, molecular biology and plant pathology to reveal how oomycete effectors suppress plant immunity. Track I of this proposal takes a structural biology approach by X-ray crystallography to elucidate the threedimensional structures of Hpa effectors that suppress plant defence. Track II focusses on identification of host targets of these effectors. A number of candidate effector targets have been identified in yeast-two-hybrid (Y2H) screens. These interactions will be validated in vitro and in plant cells. Affinity purification of Hpa effectors from Arabidopsis tissue and identification of co-purifying proteins by mass spectrometry will be employed for those effectors that did not pick up interacting proteins in Y2H. These combined efforts will give insights into how Hpa manipulates the plant defence system and will identify new nodes of plant immune signalling.