Biology subjects

Birch, P. R.

Publications and source records attributed to Birch, P. R..

4 recordsLinked to original sources

bioRxiv2025

The RXLR-EER Motif Determines an Unconventional Secretion Pathway Associated with Extracellular Vesicle Production

Phytophthora infestans, the cause of potato late blight disease, delivers a suite of RXLR effectors into host plant cells to subvert immunity, whereas apoplastic effectors act extracellularly. Although the RXLR-EER motif in these effectors is critical for host translocation and is cleaved prior to secretion, the relevance of this processing is poorly understood. Prior evidence suggests RXLR effectors utilize a distinct, unconventional secretion pathway, raising the question of whether the RXLR-EER motif influences selection of the secretion route. Here, we combined genetic, molecular and cell biology approaches to investigate the secretion pathway of RXLR effectors. Confocal microscopy revealed that RXLR and apoplastic effectors localize to distinct vesicular compartments in cultured hyphae. Moreover, fusing the ER retention signal KDEL to RXLR effectors did not impair their secretion, in contrast to apoplastic effectors, which were retained in the endomembrane system, indicating that RXLR effectors bypass the canonical ER-to-Golgi pathway. Importantly, RXLR effectors associate with extracellular vesicles (EVs), whereas RXLR-EER motif mutants show reduced EV association and are rerouted through the ER-to-Golgi secretion pathway. These findings demonstrate that the RXLR-EER motif governs effector sorting into an unconventional, EV-linked secretion route. This study sheds light on the molecular basis of effector trafficking in P. infestans and underscores the potential role of EVs in delivering virulence factors during host colonization.

microbiology↗

bioRxiv2025

Identification of MARVELlous Protein Markers for Phytophthora infestans Extracellular Vesicles

Extracellular vesicles (EVs) are released from cells by unconventional secretion, but little is known about the biogenesis routes, composition or cargoes of EVs from fungal or oomycete plant pathogens. We investigated the proteome of EV-associated proteins secreted by the oomycete Phytophthora infestans, cause of potato late blight disease. We found that vesicle-associated proteins, transmembrane proteins and RxLR effectors, which are delivered into host cells to suppress immunity, were enriched in the EV proteome. By contrast, the EV-independent secreted proteome was enriched in cell wall modifying enzymes and apoplastic effectors which act outside plant cells. Two proteins each containing two tetraspanning MARVEL domains, PiMDP1 and PiMDP2, were associated with P. infestans EVs. PiMDP1 and PiMDP2 were co-buoyant with RxLR effectors in sucrose density fractions containing EVs and co-localised frequently with each other and with RxLRs at vesicles within pathogen hyphae grown in vitro and during infection. Interestingly, PiMDP2, which is up-regulated during the early biotrophic phase of infection, accumulates at the haustorial interface, a major site of effector secretion during infection. We argue that PiMDP1 and PiMDP2 are molecular markers that will facilitate studies of the biogenesis and secretion of infection-associated P. infestans EVs.

plant biology↗

bioRxiv2024

Proteolytic processing of both RXLR and EER motifs in oomycete effectors

Arg-any amino acid-Leu-Arg (RXLR) effectors are central oomycete virulence factors that target diverse host proteins and processes to suppress plant immunity. Relatively little is known about how they are processed post-translationally before delivery into host cells. Proteolytic cleavage at the RXLR motif was observed to occur prior to secretion in all Phytophthora infestans effectors tested, suggesting it is a general rule, and was observed to occur between the leucine and the second arginine. There was no cleavage of a naturally occurring second RXLR motif in a structured region of Pi21388/AvrBlb1, or one introduced at a similar position in effector Pi04314, in keeping with the motif being positionally constrained, potentially to disordered regions closely following the signal peptide. Remarkably, independent proteolytic cleavage of the Glu-Glu-Arg (EER) motif, often found immediately downstream of the RXLR, was also observed in diverse effectors, occurring immediately after the arginine. Expression of full-length effectors in host plant Nicotiana benthamiana revealed that, although secreted, they were poorly processed, suggesting that RXLR and EER cleavage does not occur in all eukaryotic cells. Our observations indicate that, whether possessing both RXLR and EER, or either motif alone, these effectors are likely proteolytically processed prior to secretion in all cases.

plant biology↗

bioRxiv2023

The WY domain of an RxLR effector drives interactions with a host target phosphatase to mimic host regulatory proteins and promote Phytophthora infestans infection.

Plant pathogens manipulate the cellular environment of the host to facilitate infection and colonization, often leading to plant diseases. To accomplish this, many specialized pathogens secrete virulence proteins called effectors into the host cell, which subvert processes such as immune signalling, gene transcription, and host metabolism. Phytophthora infestans, the causative agent of potato late blight, employs an expanded repertoire of RxLR effectors with WY domains to manipulate the host through direct interaction with protein targets. However, our understanding of the molecular mechanisms underlying the interactions between WY effectors and their host targets remains limited. In this study, we performed a structural and biophysical characterization of the P. infestans WY effector, Pi04314, in complex with the potato Protein Phosphatase 1-c (PP1c). We elucidate how Pi04314 uses a WY domain and a specialised C-terminal loop carrying a KVxF motif that interact with conserved surfaces on PP1c, known to be used by host regulatory proteins for guiding function. Through biophysical and in planta analyses, we demonstrate that Pi04314 WY or KVxF mutants lose their ability to bind PP1c. The loss of PP1c binding correlates with a reduced capacity to re-localize PP1c from the nucleolus and a decrease in lesion size in plant infection assays. This study provides insights into the manipulation of plant hosts by pathogens, revealing how effectors exploit key regulatory interfaces in host proteins to modify their function and facilitate disease.

plant biology↗