What are the roles of oomycete RXLR effectors in the establishment of plant disease?

所有的微生物都会引发植物的免疫反应。成功的病原体通过分泌效应物来抑制这些防御，这些效应物在宿主细胞内外起作用，并且在许多情况下被认为直接与宿主防御相关蛋白靶点相互作用。细菌使用III型分泌系统（T3SS）在宿主细胞内传递效应物。它们在宿主中的靶蛋白的鉴定为细菌发病机制的进化和细菌干扰宿主防御过程的宿主模仿提供了相当大的见解。植物病理学中最重要的问题之一是：微生物真核病原体，如真菌和卵菌，如何操纵宿主的代谢和防御来建立疾病？卵菌包括疫霉（Phytophthora） 70余种和霉霉（Pythium） 90余种，是双子叶植物中最重要的经济病原菌。最近，我们已经证明含有RXLR基序的卵菌效应物被递送到寄主植物细胞内，在那里它们操纵寄主防御来建立疾病。RXLR“特征”，结合几个卵菌基因组的及时测序，提供了一个无与伦比的机会来揭示这些卵菌中的效应补体，并使用效应物作为“探针”来识别和表征与它们相互作用的关键宿主防御蛋白。我们将鉴定并比较两种亲缘关系较远的卵菌——马铃薯晚疫病病原菌疫霉（Phytophthora infestans）和拟南芥病原菌拟南芥Hyaloperonospora arabidopsidis——的RXLR效应补体靶向的宿主蛋白。这种比较方法对于揭示每种病原体效应物靶向的宿主蛋白至关重要；从而揭示了病原卵菌可能的关键植物防御靶点。我们预计这也将揭示受其他病原体（如细菌、真菌和线虫）操纵的靶点，从而为开发新的、广谱的、持久的抗病策略提供信息。我们还将在每个病原体中识别针对相同宿主蛋白的效应物。这将揭示卵菌效应补体中功能冗余的水平，表明哪些效应物如果被宿主抗性蛋白识别，可能很容易脱落以逃避检测，而不会影响致病适应性。这些知识将为育种人员提供信息，使他们能够从作物育种计划中消除这种抗性。我们对RXLR效应物宿主靶点的搜索将涉及传统的和高通量的酵母-2-杂交（Y2H）方法。后者利用了最近在Dana-Faber研究所Marc Vidal世界领先的Y2H实验室中开发的bbbb12000拟南芥蛋白的Y2H基质，我们将与他合作。利用沃里克和邓迪的互补细胞生物学和分子分析，我们将验证和定位两种病理系统中常见的效应-靶标相互作用。使用RNAi，我们将敲低相应的RXLR效应物的表达，以评估它们在毒力和功能冗余中的重要性。同样，在合作小组中使用不同的方法，我们将描述宿主靶蛋白对一系列防御机制的贡献。我们将利用由Warwick小组协调的BBSRC系统生物研究方法（SABR）倡议项目的补充数据，该项目旨在使用数学建模方法确定生物应激反应中重要的关键宿主调控节点和途径。我们怀疑效应器的共同目标将包括这样的监管“压力点”，允许在该提案中利用SABR中揭示的应激反应途径的信息。最后，我们将在芸苔和马铃薯作物中过表达关键宿主靶蛋白，寻求破坏相应效应物的正常活性，促进抗病。

All microbes trigger immune responses in plants. Successful pathogens suppress these defences by secretion of effectors, which act either outside or inside host cells and are presumed, in many cases, to interact directly with host defence-associated protein targets. Bacteria use a type III secretion system (T3SS) to deliver effectors inside host cells. The identification of their target proteins in the host has provided considerable insights into the evolution of bacterial pathogenesis and the host mimicry employed by bacteria to interfere with host defence processes. One of the most significant questions in plant pathology is: how do microbial eukaryotic pathogens, such as fungi and oomycetes, manipulate host metabolism and defences to establish disease? Oomycetes include more than 70 Phytophthora and 90 Pythium species which are amongst the most economically important pathogens of dicotyledenous plants. Recently, we have shown that oomycete effectors containing the motif RXLR are delivered inside host plant cells where they manipulate host defences to establish disease. The RXLR 'signature', in combination with the timely sequencing of several oomycete genomes, has presented an unparalleled opportunity to reveal the effector complements in these oomycetes and to use the effectors as 'probes' to identify and characterize the key host defence proteins with which they interact. We will identify and compare the host proteins targeted by the RXLR effector complements of two distantly related oomycetes, the potato late blight pathogen, Phytophthora infestans, and the Arabidopsis pathogen, Hyaloperonospora arabidopsidis. This comparative approach is vital to reveal host proteins that are targeted by effectors from each pathogen; thus revealing the likely crucial plant defence targets of pathogenic oomycetes in general. We anticipate this will also reveal targets that are manipulated by other pathogens, such as bacteria, fungi and nematodes, thus informing the development of novel, broad-spectrum, durable disease resistance strategies. We will also identify effectors within each pathogen that target the same host protein. This will reveal the level of functional redundancy within oomycete effector complements, indicating which effectors, if recognized by a host resistance protein, may be readily shed to evade detection, without compromising pathogenic fitness. This knowledge will inform breeders, allowing them to eliminate such resistances from crop breeding programmes. Our searches for host targets of RXLR effectors will involve conventional and high throughput yeast-2-hybrid (Y2H) methods. The latter exploits the recent development of a Y2H matrix of >12000 Arabidopsis proteins in Marc Vidal's world leading Y2H lab at The Dana-Faber Institute, with whom we will collaborate. Using complementary cell biological and molecular analyses at Warwick and Dundee we will verify and localize effector-target interactions that are common to the two pathosystems. Using RNAi, we will knock down expression of corresponding RXLR effectors to assess their importance in virulence and contribution to functional redundancy. Again, using different approaches in the collaborating groups, we will characterise the contributions of host target proteins to a range of defence mechanisms. We will draw on complementary data from a BBSRC Systems Approaches to Biological Research (SABR) initiative project, coordinated by the Warwick group, which aims, to use mathematical modeling approaches to identify key host regulatory nodes and pathways important in biotic stress responses. We suspect that common targets of effectors will include such regulatory 'pressure points', allowing information on stress response pathways revealed in SABR to be exploited in this proposal. Finally, we will over-express key host target proteins in Brassica and potato crops, seeking to disrupt the normal activity of corresponding effectors and promote disease resistance.