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

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.

Oomycete plant pathogens cause severe damage to major food crops. The potato and tomato late blight pathogen Phytophthora infestans causes annual global losses estimated to exceed $10 bn. Late blight disease is driven by effector proteins, secreted by P. infestans, that act on (apoplastic effectors) or within (cytoplasmic effectors) host cells to facilitate infection. There is a considerable gap in our knowledge of how cytoplasmic RXLR effectors are secreted from P. infestans and delivered into plant cells. We have generated unpublished data showing that RXLR effectors associate with extracellular vesicles (EVs). The EV proteome revealed proteins containing MARVEL domains containing multiple transmembrane helices which co-associate with RXLR Pi04314 in EVs.

In our proposal we aim to confirm whether MARVEL proteins are markers of EVs associated with RXLRs and use cutting-edge super-resolution microscopy to provide a detailed and deep investigation of their secretion and delivery at the haustorial interface between P. infestans and its hosts. We will remove the MARVEL proteins by gene silencing and CRISPR/cas12 knockout to determine their importance to effector secretion and thus to infection. Finally, we will use the MARVEL proteins to immunopurify EVs following in vitro growth 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 pathogens which can be targeted by agrochemical or biotechnological means to prevent crop disease.