The PathEVome project consisted of three inter-related and technically ambitious work packages (WPs):
WP1: What proteins are found in EVs and how do EVs traffic between pathogen and host cells?
We optimised extracellular vesicle (EV) isolation from Phytophthora and validated vesicle integrity using nanoparticle tracking, transmission electron microscopy, sucrose gradients, and detergent sensitivity assays. We generated a detailed EV proteome, revealing enrichment of >35 RXLR effectors in EVs, while apoplastic effectors were absent. Two infection-induced EV markers, PiMDP1 and PiMDP2 (each containing eight transmembrane domains), were identified and characterised.
Genetic and cell biological analyses demonstrated that RXLR effectors are secreted via a Golgi-bypass pathway and co-localise with EV markers in hyphal vesicles. In contrast, apoplastic effectors follow the conventional ER-to-Golgi secretion route. Addition of a KDEL motif blocked apoplastic effector secretion but not RXLR secretion, confirming unconventional trafficking of RXLRs. Super-resolution microscopy further resolved effector-containing vesicles accumulating at haustoria and confirmed co-association of RXLRs with EV markers. PiMDP2 was shown to accumulate at the extrahaustorial membrane, consistent with deposition during EV secretion.
WP2: How are EVs formed and how are effectors packaged into them?
Density gradient fractionation of enriched EVs demonstrated that RXLR effector Pi04314 co-fractionates with EV marker PiMDP2, indicating co-association with EVs.
Using proximity labelling, we identified proteins associated with effectors during secretion. We further showed that RXLRs co-localise with ATG9 and Rab7, but not with the Golgi-to-MVB marker BP80, refining understanding of the unconventional secretion route. Silencing ATG9 compromised virulence, demonstrating its requirement for infection.
We established that the RXLR and associated EER motifs undergo proteolytic processing prior to secretion. Mutation of the RXLR–EER motif blocked unconventional secretion, EV association, and host cell translocation of RXLRs. Finally, we implemented CRISPR-Cas12 technology to generate single and double knockouts of EV markers PiMDP1/2, enabling ongoing functional analysis of EV biogenesis.
WP3: What are the routes for uptake of cytoplasmic effectors into host cells and how do they reach their destination?
Purified EVs were shown to trigger immune responses in plant leaves, consistent with the presence of PAMPs on the EV surface. This suggests EV targeting to plant cells may occur via immune receptor recognition.
We made a major breakthrough by demonstrating that RXLR effectors enter plant cells via clathrin- and Ara6-mediated endocytosis. Gene silencing confirmed the requirement for clathrin-mediated pathways. Purified endosomes isolated via density gradients and immunocapture of Ara6-GFP and clathrin-GFP contained RXLR effectors during infection, providing direct proteomic evidence for endocytic uptake.
Our results have been disseminated in high-impact publications and presented at international congresses. We generated multiple transgenic Phytophthora lines co-expressing effectors and EV markers to dissect secretion pathways, which we are investigating in a newly-granted project. We also made transgenic Phytophthora lines expressing secretion pathway markers that we have shared with industrial partners for their in-house research.
