The mechanism of trans-kingdom sRNA trafficking in plant-pathogen interactions

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Small RNAs (sRNAs) can move cell-to-cell and trigger target gene silencing in the recipient cells. Recent studies suggest that host sRNAs can translocate into pathogens during infection. By silencing specific pathogen genes, trans-kingdom RNA interference (RNAi) is a novel defense mechanism that offers opportunities to enhance disease resistance. However, the mechanisms underlying trans-kingdom sRNA trafficking is largely unknown. My fellowship aims to identify novel components in the machinery of inter-species sRNA translocation. I will employ a pathosystem with the model plant Arabidopsis thaliana as the host and the filamentous eukaryote Phytophthora capsici as the pathogen. P. capsici forms a specialized infection structure, called haustorium, which is enveloped by the plant-derived extra-haustorial membrane (EHM). EHM functions as a major gateway to secrete antimicrobial compounds, presumably including sRNAs. Furthermore, specific Arabidopsis sRNAs have recently been identified as major executors of trans-kingdom RNAi. Hence, characterization of EHM-associated proteins that are also associated with trans-kingdom sRNAs will uncover mechanisms by which plant sRNAs are translocated to invading pathogens, which will aid in developing innovative approaches to control disease.Three objectives will be pursued:Objective 1: Identification of proteins associated with trans-kingdom sRNAs using RNA pull-down followed by mass spectrometry (RP-MS)Objective 2: Identification of proteins associated with EHM using TurboID-based proximity labelling followed by MS (PL-MS)Objective 3: Validation and functional analysis of candidate sRNA trafficking-related proteinsThrough this fellowship, I will greatly broaden my knowledge of plant-microbe interactions, gain advanced training and practical experience in technical and transferable skills, expand my collaborative network, and enhance my professional maturity to achieve my goal as an independent research group leader.