Periodic Reporting for period 5 - PathEVome (Do Pathogen Extracellular Vesicles Deliver Crop Disease?)
Reporting period: 2024-10-01 to 2025-09-30
In 2009 the World Summit on Food Security set a global challenge of increasing crop production by >50% by 2050. Pests and diseases provide major constraints to food production and as much as 50% of crops are lost due to these challenges. Whereas most human diseases are caused by bacteria and viruses, arguably fungi and oomycetes (filamentous pathogens) are the major agents of plant diseases. Breeding to introduce natural resistance from wild relatives of crops has, in most cases, had limited long-term success, and disease control using agrochemicals is seen as both environmentally and financially costly. To provide a platform for innovative control measures, PathEVome aimed to understand the mechanisms underlying infection by filamentous pathogens.
Pathogenic oomycetes and fungi secrete effector proteins that act either outside (apoplastic) or inside (cytoplasmic) plant cells, targeting host proteins and processes to suppress immunity. Oomycete cytoplasmic effectors contain a conserved protein motif, RXLR, that is required for delivery into plant cells. Prior to PathEVome, the host targets of many RXLR effectors had been discovered, but how these effectors are delivered into plant cells was poorly understood. However, we had preliminary data that suggested these effectors could be trafficked to host cells in association with extracellular vesicles (EVs).
During the PathEVome project we used proteomics to reveal the proteins associated with EVs produced by the oomycete Phytophthora. The EV proteome was enriched with RXLR effectors and vesicle trafficking proteins. By contrast, effectors that act on the outside of plant cells were not associated with the EV proteome. We found vesicle trafficking proteins that act as EV markers and allowed us to determine the unconventional secretion pathway by which EVs are generated. Critically, we found that RXLR effectors were processed by proteases to direct them to EVs and were taken into host cells by a process called endocytosis. These landmark discoveries provide us with an understanding of filamentous pathogen virulence processes which can be targeted to prevent crop infections.
Pathogenic oomycetes and fungi secrete effector proteins that act either outside (apoplastic) or inside (cytoplasmic) plant cells, targeting host proteins and processes to suppress immunity. Oomycete cytoplasmic effectors contain a conserved protein motif, RXLR, that is required for delivery into plant cells. Prior to PathEVome, the host targets of many RXLR effectors had been discovered, but how these effectors are delivered into plant cells was poorly understood. However, we had preliminary data that suggested these effectors could be trafficked to host cells in association with extracellular vesicles (EVs).
During the PathEVome project we used proteomics to reveal the proteins associated with EVs produced by the oomycete Phytophthora. The EV proteome was enriched with RXLR effectors and vesicle trafficking proteins. By contrast, effectors that act on the outside of plant cells were not associated with the EV proteome. We found vesicle trafficking proteins that act as EV markers and allowed us to determine the unconventional secretion pathway by which EVs are generated. Critically, we found that RXLR effectors were processed by proteases to direct them to EVs and were taken into host cells by a process called endocytosis. These landmark discoveries provide us with an understanding of filamentous pathogen virulence processes which can be targeted to prevent crop infections.
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.
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.
The following exciting achievements were published during the project:
• We have confirmed that Phytophthora RXLR effectors are proteolytically processed during secretion.
• We have developed methods for EV purification and shown that RXLRs are associated with them whereas conventionally secreted proteins are not.
• We have demonstrated that the RXLR motif is required for unconventional secretion of these effectors and for association with EVs.
• We have discovered that the unconventional secretion pathway for RXLR effectors involves the trafficking markers Rab7 and ATG9
• 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 important crop diseases.
• We have confirmed that Phytophthora RXLR effectors are proteolytically processed during secretion.
• We have developed methods for EV purification and shown that RXLRs are associated with them whereas conventionally secreted proteins are not.
• We have demonstrated that the RXLR motif is required for unconventional secretion of these effectors and for association with EVs.
• We have discovered that the unconventional secretion pathway for RXLR effectors involves the trafficking markers Rab7 and ATG9
• 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 important crop diseases.