MARVEL-ous Extracellular vesicles carry RXLR effectors into host plant cells

2009年，世界粮食安全首脑会议确定了一项全球挑战，即到2050年将作物产量增加60%，以满足预期的粮食需求。害虫和病原体造成的损失占全球五种最重要粮食作物产量的23%，对粮食安全构成重大威胁。卵菌植物病原体对许多主要粮食作物造成严重损害。例如，马铃薯和番茄晚疫病病原体致病疫霉每年造成的全球损失估计超过100亿美元。植物病原体如致病疫霉通过在感染过程中向植物组织中递送称为效应子的毒力蛋白质库来引起疾病。其中有数百种所谓的RXLR效应物，它们被传递到活的植物细胞内，它们通常靶向宿主蛋白质以抑制免疫力。植物-微生物相互作用领域的一个主要科学挑战是了解RXLR效应物是如何分泌和递送到植物细胞中的。我们已经取得了一些关键突破，为这一提议奠定了基础。我们已经生成的数据显示，RXLR效应子与膜结合的细胞外囊泡（EV）一起分泌。我们已经发现了与这些EV相关的其他蛋白质，包括具有包含多个跨膜螺旋的MARVEL结构域的蛋白质。这些MARVEL蛋白与囊泡中的RXLR效应物共定位，我们假设它们是与RXLR效应物递送相关的EV的新标记物。在我们的提案中，我们的目标是确认MARVEL蛋白是否是与RXLR相关的EV的标志物，我们将使用尖端的超分辨率显微镜来详细深入地研究它们在致病疫霉及其宿主之间的界面上的分泌和递送。我们将通过基因沉默和CRISPR/cas 12敲除技术去除MARVEL蛋白，以确定它们对效应子分泌的重要性，从而确定它们对感染的重要性。最后，我们将标记MARVEL蛋白，并使用它们来捕获在培养基中生长和感染期间的EV，并研究EV蛋白质组，以确定参与EV生物发生，结构和功能的因素，并确定在感染过程中传递到宿主的毒力蛋白的货物。这项工作将提供一个关键的基本效应传递机制的深入理解，可能是跨卵菌植物病原体共享，可以通过农业化学或生物技术手段来预防作物病害。

In 2009, the World Summit on Food Security set a global challenge of 60% increase in crop production by 2050 to meet predicted demands for food. Damage from pests and pathogens causes losses of up to 23% of global production of the five most important food crops, representing a significant threat to food security. Oomycete plant pathogens cause severe damage to many major food crops. For example, the potato and tomato late blight pathogen Phytophthora infestans causes annual global losses estimated to exceed $10 bn. Plant pathogens such as P. infestans cause disease by delivering an arsenal of virulence proteins, called effectors, into plant tissues during infection. Amongst these are hundreds of so-called RXLR effectors that are delivered inside living plant cells where they often target host proteins to suppress immunity. A major scientific challenge in the plant-microbe interaction field is to understand how RXLR effectors are secreted and delivered into plant cells.We have made some key breakthroughs forming the basis for this proposal. We have generated data showing that RXLR effectors are secreted in association with membrane-bound extracellular vesicles (EVs). We have discovered other proteins associated with these EVs, including proteins with MARVEL domains containing multiple transmembrane helices. These MARVEL proteins co-localise with RXLR effectors in vesicles, and we hypothesise that they are novel markers of EVs associated with RXLR effector delivery. In our proposal we aim to confirm whether MARVEL proteins are markers of EVs associated with RXLRs and we will use cutting-edge super-resolution microscopy to provide a detailed and deep investigation of their secretion and delivery at the interface between P. infestans and its hosts. We will remove the MARVEL proteins by gene silencing and CRISPR/cas12 knockout techniques to determine their importance to effector secretion and thus to infection. Finally, we will tag the MARVEL proteins and use them to capture EVs following growth in media and during infection and study the EV proteome to identify factors involved in biogenesis, structure and function of EVs, and to determine the cargo of virulence proteins delivered to hosts during infection.This work will provide a deep understanding of a key fundamental effector delivery mechanism likely to be shared across oomycete plant pathogens which can be targeted by agrochemical or biotechnological means to prevent crop disease.