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Do Pathogen Extracellular Vesicles Deliver Crop Disease?

Periodic Reporting for period 2 - PathEVome (Do Pathogen Extracellular Vesicles Deliver Crop Disease?)

Reporting period: 2020-04-01 to 2021-09-30

Currently, almost 1 billion people are undernourished. In 2009, the World Summit on Food Security set a global challenge of 60 % increase in crop production by 2050. One of the major constraints to achieving food security is crop loss due to pests and diseases; up to 50% of crop losses in developing countries are due to these causes. Whilst human diseases are mainly caused by bacteria and viruses, the majority of plant diseases are caused by fungi and oomycetes. The major diseases on cereals (wheat, barley, rice etc) are caused by fungi. Arguably, oomycetes such as Phytophthora species are the major causes of disease on dicotyledonous crops (e.g. potato, tomato, soybean, pumpkin etc). Breeding to introduce disease resistance from wild plant species into crops has met limited success and is often overcome by rapidly evolving pathogen species. Oomycete and fungal diseases are thus generally controlled by application of fungicides, which potentially carry environmental as well as financial costs. To meet the ambitious targets for crop production innovative approaches are needed to prevent such diseases, which rely on a detailed and deep understanding of the processes and mechanisms by which oomycetes and fungi infect crop plants.

Pathogenic fungi and oomycetes secrete ‘effector’ proteins that act either outside or are delivered to the inside of living plant cells to facilitate disease. Effector proteins manipulate host processes, often by directly targeting host proteins, and are responsible for suppressing plant immunity. Over the past 15 years our understanding of filamentous plant pathogen intracellular effectors has advanced, largely driven by early breakthroughs in the identification of so-called RXLR effectors in oomycetes. A key objective to prevent crop diseases is to understand the means by which oomycete and fungal pathogens deliver effectors into plant cells. Prior to this project we were the first research group to demonstrate the secretion and delivery of RXLR effectors into plant host cells. Preliminary evidence was obtained that revealed the potential association of RXLR effectors with membrane-bound extracellular vesicles (EVs) that may be trafficked between pathogen and host cells.

The objectives are to determine:
1) What proteins are found in EVs and how do EVs traffic between pathogen and host cells?
2) How are EVs formed and how are effectors packaged into them?
3) What are the routes for uptake of effectors into host cells and how do they reach their destination?
Experiments that have been performed to date, and the results achieved, are organised in three interdependent blocks of work known as 'Work Packages'.

Work Package 1 (WP1): A key aim of this WP is to identify proteins that are secreted from Phytophthora under different conditions. Conditions include the use of inhibitors to perturb secretory pathways, and the use of approaches to purify extracellular vesicles (EVs). We have also been transforming Phytophthora to express secreted proteins, or proteins involved in secretion.

WP2: In meeting the objectives of this WP we have been transforming Phytophthora to express a range of mRFP fusions to proteins that are markers of endocytic pathways. We have transformed Phytophthora to successfully express Golgi, actin, microtubule, ER and autophagy markers. In each case western blotting has confirmed expression of the fusion proteins and confocal microscopy is being used to study the localisation of each protein inside Phytophthora.

WP3: The aim of this WP is to determine whether endocytosis is involved in RXLR effector uptake. Good progress has been made in the first 18 months to show that endocytosis is required for RXLR effector uptake into plant cells.
The following exciting progress has been made, and will be published in the coming period:
• We have confirmed that Phytophthora RXLR effectors are cleaved during secretion
• We have demonstrated that the RXLR motif is required for non-conventional secretion of these effectors
• We have developed methods for EV purification and shown that RXLRs are associated with them whereas conventionaly secreted proteins are not.
• We have demonstrated that endocytosis is required for RXLR effectors to be taken into host plant cells following their secretion from haustoria.
• We have developed Phytophthora lines expressing endocytic pathway markers and secreted proteins for use in studying oomycete secretion pathways and processes during infection.

The generation of marker lines and a deeper understanding of how oomycete plant pathogens deliver effector proteins to host plant cells will provide many novel avenues to develop new strategies to prevent economically imporetant crop diseases. In particular, the Ag-Chem industry is keen to develop new, more environmentally friendly and sustainable chemical control agents to prevent Phytophthora diseases. We have thus discussed the possible uptake of tools and approaches developed in this project with potential industrial partners, for the use in developing new chemical control agents.
PathEVome Work Packages: 3 interdependent pieces of work structured to achieve project objectives.