Mechanisms of siRNA mediated broad-spectrum resistance to eukaryotic pathogens

农业的可持续性受到病原体的威胁，病原体对作物产量和食品安全造成重大损害。虽然植物已经进化出无数的免疫机制来保护自己，但成功的病原体可以克服这种监视系统并引起疾病。病原体毒力因子与宿主先天免疫之间的动态相互作用决定了疾病是否会发生。为了保护作物免受病原体的侵害，必须确定新的防御机制并了解其功能的分子基础，以便设计创新的方法来提高抗病性。最近的研究发现了植物中基于小RNA （sRNA）的防御机制。小RNA是短RNA分子（长度通常为20-24个核苷酸），基于序列互补指导抑制靶基因的表达。由sRNAs触发的基因沉默或RNA干扰（RNAi）是真核生物中一种基本的、普遍的调控机制，影响着广泛的生物过程。在宿主-病原体相互作用过程中，已经观察到一种生物产生的sRNAs会影响对立生物的基因表达。尽管这种“跨物种”RNAi是一个令人兴奋的概念，代表了宿主-病原体军备竞赛的新视角，但仍存在许多挑战。一个基本的挑战是确定哪些sRNAs在入侵的病原体中执行靶基因沉默。此外，还缺乏直接的实验证据来证明植物sRNAs在入侵病原体中引导基因沉默。在将srna有效地应用于疾病控制之前，需要填补这些主要的知识空白。在本项目中，我们旨在研究植物sRNAs在自然侵染过程中病原基因沉默的机制。这个项目建立在我们最近的发现上，表明一个特定的植物sRNAs家族赋予对丝状真核病原体的抗性。这些sRNA是通过一种独特的生物发生途径产生的，该途径导致多种sRNA池的产生，这有可能在广泛的真核病原体中沉默靶基因。因此，我们确定了一个指定的“抗菌”sRNAs家族，可能赋予广谱抗性。我们将结合遗传学、分子生物学、生物化学、合成生物学和植物病理学等方法，以及拟南芥-辣椒疫霉病理系统模型来研究这一特定sRNAs家族在植物免疫中的功能。跨物种基因沉默将通过检测在入侵病原体中引导基因沉默的蛋白质复合体中的植物sRNAs来监测。我们将通过检测sRNA产生缺陷的植物突变体对其他真核病原体的易感性来验证这种新的防御机制赋予广谱抗性的假设。我们将进一步探索如何编辑srna产卵序列以产生定制的srna，从而增加病原体基因沉默并提高耐药性。该项目的结果将为植物免疫的基本原理提供新的见解，并为发展可持续的疾病控制策略提供新的机会，这些策略对广泛的病理系统具有深远的影响。

The sustainability of agriculture is threatened by pathogens, which cause substantial damage to crop yield and food safety. Although plants have evolved a myriad of immunity mechanisms to defend themselves, successful pathogens can overcome this surveillance system and cause disease. The dynamic interactions between virulence factors of pathogens and the innate immunity of a host determine whether disease will occur. To protect crops from pathogens, it is essential to identify new defense mechanisms and understand the molecular basis of their functions in order to design innovative approaches to elevate disease resistance.Recent research discovered a small RNA (sRNA)-based defense mechanism in plants. Small RNAs are short RNA molecules (usually 20-24 nucleotide in length) that guide the inhibition of target gene expression based on sequence complementarity. Gene silencing or RNA interference (RNAi) triggered by sRNAs is a fundamental and universal regulatory mechanism in eukaryotes that impacts a wide range of biological processes. During host-pathogen interactions, sRNAs produced from one organism have been observed to affect gene expression in the opposing organism. Although this "trans-species" RNAi is an exciting concept that represents a new perspective in host-pathogen arms race, many challenges remain. A fundamental challenge is to identify which sRNAs in plant execute target genes silencing in invading pathogens. Furthermore, direct experimental evidence demonstrating gene silencing guided by plant sRNAs in invading pathogens is lacking. These major gaps of knowledge need to be filled before sRNAs can be effectively deployed in disease control. In this project, we aim to investigate the mechanism underlying pathogen gene silencing by plant sRNAs during natural infection. This project builds on our recent discoveries suggesting that a specific family of plant sRNAs confer resistance to a filamentous eukaryotic pathogen. These sRNAs are produced from a unique biogenesis pathway that leads to the generation of a diverse sRNA pool, which has the potential to silence target genes in a broad range of eukaryotic pathogens. As such, we identified a designated family of "antimicrobial" sRNAs that may confer broad-spectrum resistance.We will use a combination of genetics, molecular biology, biochemistry, synthetic biology, and plant pathology approaches and the model Arabidopsis thaliana-Phytophthora capsici pathosystem to investigate the function of this specific family of sRNAs in plant immunity. Trans-species gene silencing will be monitored by detection of plant sRNAs in the protein complex that guides gene silencing in the invading pathogen. We will test the hypothesis that this novel defense mechanism confers broad-spectrum resistance by examining plant mutants defective in sRNA production for susceptibility to additional eukaryotic pathogens. We will further explore how sRNA-spawning sequences could be edited to produce bespoke sRNAs that can increase pathogen gene silencing and elevate resistance. The outcome of this project will be to provide novel insight into fundamental principles of plant immunity and offer new opportunities for the development of sustainable disease control strategies with far-reaching implications to a broad range of pathosystems.