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

小RNA(SRNAs)可以在细胞间移动，并在受体细胞中触发靶基因沉默。最近的研究表明，宿主sRNA在感染过程中可以移位到病原体中。通过沉默特定的病原体基因，跨王国RNA干扰(RNAi)是一种新的防御机制，为增强抗病能力提供了机会。然而，跨王国贩运SRNA的机制在很大程度上是未知的。我的研究目的是确定物种间sRNA易位机制中的新组件。我将采用一种以模式植物拟南芥为寄主，丝状真核生物辣椒疫霉为病原的病理系统。辣椒疫霉形成一种特殊的感染结构，称为吸器，它被植物来源的吸器外膜(EHM)所包裹。EHM是分泌抗微生物化合物的主要通道，可能包括sRNA。此外，特定的拟南芥sRNAs最近被确定为跨王国RNAi的主要执行者。因此，对与跨王国sRNA相关的EHM相关蛋白的鉴定将揭示植物sRNA转移到入侵病原体的机制，这将有助于开发控制疾病的创新方法。目标1：使用RNA下拉和质谱仪(RP-MS)鉴定与跨王国sRNA相关的蛋白质目标2：使用基于TurboID的邻近标记和MS(PL-MS)鉴定与EHM相关的蛋白质(PL-MS)目标3：验证和功能分析候选的sRNA贩运相关蛋白通过这次联谊会，我将极大地拓宽我对植物-微生物相互作用、获得技术和可转移技能方面的高级培训和实践经验，扩大我的合作网络，并提高我的专业成熟度，以实现我作为独立研究小组组长的目标。

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.