The roles of extracellular vesicle transport in late blight disease development

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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.