The roles of extracellular vesicle transport in late blight disease development

真菌和卵菌引起最具破坏性的作物病害，从而严重威胁全球粮食安全。其毒力武器库的关键组成部分是称为细胞质效应子的蛋白质，这些蛋白质在植物细胞内传递以抑制免疫力。植物-微生物相互作用领域的一个关键科学挑战是了解效应物如何分泌和转移到宿主细胞中，以便更好地开发预防疾病的新方法。我们实验室的一项令人兴奋的突破表明，来自马铃薯晚疫病病原体致病疫霉的细胞质效应物可能在细胞外囊泡（EV）中积累，这意味着这是一种传递途径。在这项由工业合作伙伴先正达（Syngenta）支持的提案中，我们将研究EV的蛋白质货物，EV是如何合成和分泌的，细胞质效应物被递送到植物细胞中到达目的地的方式，以及这些过程是否可以被抑制。为了研究细胞质效应物如何通过EV被递送到植物细胞中，我们设想了3个相互关联的目标：首先，我们将利用蛋白质组学的方法来确定疫霉分泌组，特别是EV的内容。沿着促进或阻止EV在其他系统中产生的抑制剂，以及来自先正达的改变囊泡生物学的抑制剂，将用于定义EV分泌组。疫霉属的转化和细胞生物学将用于确认特定的效应蛋白是否与EV相关，以及它们是否在病原体和宿主细胞之间的吸器界面分泌。其次，我们将研究EV生物发生的分子细胞生物学，以确定病原体分泌效应物的途径。将确定被分泌以在宿主细胞内部或外部起作用的效应物在疫霉属中的亚细胞定位，鉴定与其分泌相关的特异性内膜区室。在蛋白质组学研究中确定的关键基因的功能将通过基因沉默来研究;它们是EV生物合成所必需的吗？第三，我们将使用分子和细胞生物学方法来观察和干扰进入宿主细胞的途径，以确定细胞质效应子到达其亚细胞目的地的途径。特别是，基因沉默和生物化学抑制剂将用于减弱内吞作用，以研究其对EV的影响，从而影响效应物的摄取。该提案旨在了解丝状病原体效应物易位到植物细胞中的潜在机制，特别是EV的潜在参与。这将为探索EV分泌途径为疾病控制提供新靶点的潜力提供基础。EV参与宿主和微生物细胞之间的分子转移是医学和农业研究领域中迅速兴起和令人兴奋的领域。这一提议将使人们更深入地了解EV在将丝状病原体的效应子传递到植物细胞中的作用。

Fungi and oomycetes cause the most devastating crop diseases and thus significantly threaten global food security. Critical components of their virulence arsenal are proteins called cytoplasmic effectors that are delivered inside plant cells to suppress immunity. A key scientific challenge in the plant-microbe interaction field is to understand how effectors are secreted and translocated into host cells in order to better develop new approaches to prevent disease. An exciting breakthrough in our laboratory revealed that cytoplasmic effectors from the potato late blight pathogen Phytophthora infestans may accumulate in extracellular vesicles (EVs), implicating this as a delivery route. In this proposal, supported by industrial partner Syngenta, we will investigate the protein cargo of EVs, how EVs are synthesised and secreted, the means by which cytoplasmic effectors are delivered into plant cells to reach their destinations, and whether these processes can be inhibited.To investigate how cytoplasmic effectors are delivered into plant cells by means of EVs we envisage 3 inter-related objectives: Firstly we will use proteomic approaches to define the Phytophthora secretome, and especially the contents of EVs. Inhibitors that promote or prevent EV production in other systems, along with inhibitors from Syngenta that alter vesicle biology, will be employed to define the EV secretome. Transformation of Phytophthora and cell biology will be used to confirm whether specific effector proteins are associated with EVs and whether they are secreted at the haustorial interface between pathogen and host cells. Secondly, we will investigate the molecular cell biology of EV biogenesis to define the routes of effector secretion from the pathogen. The subcellular localisation in Phytophthora of effectors that are secreted to act either inside or outside of host cells will be determined, identifying the specific endomembrane compartments associated with their secretion. The functions of key genes identified in proteomic studies will be investigated by gene silencing; are they required for EV biosynthesis?Thirdly we will use molecular and cell biology approaches to observe and to perturb entry routes into host cells in order to define the pathway travelled by cytoplasmic effectors to reach their subcellular destination. In particular, gene silencing and biochemical inhibitors will be used to attenuate endocytosis to investigate the impact of this upon EV, and thus effector, uptake. The proposal aims to provide an understanding of the mechanisms underlying translocation of filamentous pathogen effectors into plant cells and particularly the potential involvement of EVs in this. It will provide the basis to explore the potential that the EV secretory route provides novel targets for disease control. The involvement of EVs in molecular transfer between host and microbe cells is a rapidly emerging and exciting area in medical and agricultural research fields. This proposal will provide a deeper understanding of the roles of EVs in the delivery of effectors from filamentous pathogens into plant cells.