The roles of extracellular vesicle transport in late blight disease development

真菌和卵菌引起最具破坏性的农作物疾病，从而严重威胁全球粮食安全。它们毒力武器库的关键成分是被称为细胞质效应器的蛋白质，这些蛋白质在植物细胞内传递以抑制免疫。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是如何分泌和转运到宿主细胞中的，以便更好地开发新的预防疾病的方法。我们实验室的一项令人兴奋的突破揭示了来自马铃薯晚疫病菌的细胞质效应器可能积累在细胞外小泡(EVS)中，这意味着这是一种传递途径。在这个计划中，在工业伙伴先正达的支持下，我们将调查EVS的蛋白质货物，EVS是如何合成和分泌的，细胞质效应物是如何运送到植物细胞到达它们的目的地的，以及这些过程是否可以被抑制。为了研究细胞质效应物是如何通过EVS运送到植物细胞中的，我们设想了三个相互关联的目标：首先，我们将使用蛋白质组学的方法来定义疫霉分泌体，特别是EVS的含量。在其他系统中促进或阻止EV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，将被用来定义EV分泌组。疫霉的转化和细胞生物学将被用来确定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上分泌。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。将确定被分泌作用于宿主细胞内外的效应器在疫霉体内的亚细胞定位，以确定与其分泌相关的特定内膜室。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，以确定细胞质效应分子到达其亚细胞目的地的途径。特别是，基因沉默和生化抑制剂将被用来减弱内吞作用，以研究这对EV的影响，从而影响效应器的摄取。该提案旨在提供对丝状病原体效应分子移位到植物细胞中的潜在机制的理解，特别是EVS在这方面的潜在参与。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。EVS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一个迅速崛起和令人兴奋的领域。这一建议将使我们更深入地了解EVS在将丝状病原体的效应物运送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.