AbstractPotato blight, a ravaging disease caused by the oomycete Phytophthora infestans, is a major threat to global food security. P. infestans secretes effector proteins that are delivered inside (cytoplasmic effector) or outside (apoplastic effector) plant cells to neutralise host immunity. One of the most well characterised classes of cytoplasmic effectors are the RXLR (Arg-any amino acid-Leu-Arg) class. RXLR is a highly conserved motif in genes encoding secreted proteins across many oomycete genomes, and is required for effector translocation from pathogen into host cells. However, little is known about how and where effectors are delivered during infection and the role of the RXLR motif in the translocation process.
In the first part of this study, a set of RXLR cytoplasmic effectors were characterised in terms of host subcellular localisation and their effect on pathogen colonisation. Subsequently, two host nuclear-localised cytoplasmic effectors, Pi04314 and Pi22926, were selected and mutants created in which the RXLR motif was disabled. The wild-type and mutant forms were expressed in P. infestans. Both RXLR wildtype and RXAA mutant effector fusions were secreted from haustoria, finger-like structures that form intimate interactions with host cells. Importantly, only wildtype effectors accumulated inside the host cell nuclei, their site of activity. Strikingly, my data demonstrate that the RXLR motif determines an alternative secretion pathway from P. infestans, with secretion of RXAA-mRFP mutant effector fusions, like apoplastic effectors, cell wall degrading enzymes and PAMP-like proteins, was inhibited in vitro and in planta by Brefeldin A (BFA), which is a conventional ER-to-Golgi-mediated secretion inhibitor. Wildtype cytoplasmic effectors follow an (BFA) insensitive secretion route for delivery into plant cells.
I hypothesised that extracellular vesicles (EVs) may be the mechanism of RXLR effector translocation from pathogen into host cells. EVs are secreted by diverse organisms to facilitate intercellular and extracellular communication, promote infection and evade host immune responses. I used ultracentrifugation, followed by transmission electron microscopy to identify EVs from P. infestans culture in vitro. Immunoblotting showed that secreted EVs mediate secretion of RXLR effectors but not apoplastic effectors.
This work is a major breakthrough in the understanding of plant-pathogen interactions, providing more details about the protein weapons of P. infestans, and could be exploited to develop chemicals to disrupt the specific secretory processes and inhibit pathogen infection.
|Date of Award||2019|
|Sponsors||China Scholarship Council|
|Supervisor||Paul Birch (Supervisor), Petra Boevink (Supervisor) & Stephen Whisson (Supervisor)|