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

小RNA（sRNA）可以在细胞间移动并触发受体细胞中的靶基因沉默。最近的研究表明，宿主sRNA可以在感染过程中易位到病原体中。跨界RNA干扰（trans-kingdom RNA interference，RNAi）是一种通过沉默特定病原体基因来增强抗病性的新的防御机制。然而，跨界sRNA运输的潜在机制在很大程度上是未知的。我的奖学金旨在确定物种间sRNA易位机制中的新组件。我将采用一个以模式植物拟南芥为宿主，丝状真核生物辣椒疫霉为病原体的致病系统。辣椒疫霉形成了一个特殊的感染结构，称为吸器，它被植物来源的吸器外膜（EHM）包裹。EHM作为分泌抗微生物化合物（可能包括sRNA）的主要途径。此外，特定的拟南芥sRNAs最近已被确定为跨界RNAi的主要执行者。因此，表征EHM相关蛋白，也与跨界sRNA相关，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。三个目标将被追求：目标1：使用RNA下拉随后质谱（RP-MS）鉴定与跨界sRNA相关的蛋白。使用基于TurboID的邻近标记随后进行MS（PL-MS）鉴定与EHM相关的蛋白质候选sRNA运输相关蛋白的验证和功能分析通过这项奖学金，我将大大拓宽我对植物-微生物相互作用的知识，获得技术和可转移技能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，以实现我作为一个独立的研究小组领导的目标。

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.