A High Throughput Protoplast System for Rice Functional Genomics and Proteomics: Protein-Protein Interactions at the Host-Pathogen Interface

水稻功能基因组学和蛋白质组学的高通量原生质体系统：宿主-病原体界面蛋白质-蛋白质相互作用水稻是世界上一半以上人口的主食。稻瘟病是由稻瘟病真菌病原体引起的，具有很强的破坏性，并继续对全球粮食安全构成重大威胁。宿主和病原体检测彼此存在并作出反应的能力是由蛋白质相互作用决定的，然而，对这些相互作用的了解充其量只是零碎的。水稻和稻瘟病菌接近完整的基因组，以及广泛的生物资源和丰富的调查历史，为研究宿主和病原体之间的相互作用提供了机会。该研究项目将开发和测试一种新的系统，该系统基于光/荧光分解片段的重组，产生融合到相关真菌和/或水稻肽的蛋白质，以直接观察活植物细胞中的蛋白质相互作用。水稻原生质体（没有细胞壁的细胞）将被使用，因为它们易于操作，适合大规模分析，并且通常模拟整个植物。作为概念验证，该系统将首先使用已知相互作用的大米蛋白进行测试。随后将使用一种已知的防御启动子（无毒）蛋白及其同源水稻抗性受体来测试该系统对水稻蛋白与稻瘟病菌蛋白相互作用的适用性。该系统最终将扩大规模，以筛选未知的真菌和植物蛋白关联，这些关联被预测会控制宿主防御的部署。高通量原生质体系统还将应用于筛选植物宿主防御反应的未知真菌启动子的可行性研究，其中少数将进入鉴定相互作用植物蛋白的新筛选。为高中生制定了广泛的教育计划，并计划开展活动，促进代表性不足群体的成员入学和参与。除了进一步加深我们对植物-病原体相互作用的基本认识外，待开发的技术将对水稻和稻瘟病外植物系统中其他蛋白质-蛋白质或蛋白质-配体相互作用的发现和评估具有深远的意义。该项目的数据将在经评审的出版物中提供。数据、方案和试剂也将通过www.fsgc.net（真菌遗传库存中心）和www.mgosdb.org（稻瘟病大病菌数据库）生成和质量检查。

A High Throughput Protoplast System for Rice Functional Genomics and Proteomics: Protein-Protein Interactions at the Host-Pathogen InterfaceRice is the food staple for over half the world's population. Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is highly devastating and persists as a major threat to global food security. The ability of host and pathogen to detect and respond to each other's presence is governed by protein interactions, however knowledge of these interactions remains fragmentary at best. Access to near complete genomes of both rice and M. grisea, along with the extensive biological resources and a rich history of investigation, provides the opportunity to investigate interactions between host and pathogen. This research project will develop and test a novel system based on the reassembly of dissected fragments of light/fluorescence generating proteins fused to associating fungal and/or rice peptides to directly visualize protein interactions in living plant cells. Rice protoplasts (cells devoid of their cell wall) will be using since they are easy to manipulate, are amenable to large-scale analyses and typically mimic whole plants. As a proof-of-concept, the system will be first tested using rice proteins that are known to interact. The applicability of the system to interrogate the interaction of proteins from rice with those of the rice blast fungus will be subsequently tested using a known defense elicitor (avirulence) protein and its cognate rice resistance receptor. The system will be finally scaled up to screen for unknown fungal and plant protein associations that are predicted to control the deployment of host defenses. The high throughput protoplast system will be also applied in a feasibility study to screen for unknown fungal elicitors of plant host defense responses, of which a select few will advance into the novel screen developed to identify interacting plant proteins. An extensive education plan for high school students and activities promoting recruitment and participation by members of underrepresented groups are planned. In addition to furthering our basic understanding of plant-pathogen interactions, the technologies to be developed will have far-reaching utility for the discovery and evaluation of other protein-protein or protein-ligand interactions in plants systems outside rice and rice blast. Data from this project will be made available in refereed publications. Data, protocols, and reagents will also be made avaiable via www.fsgc.net (Fungal Genetics Stock Center) and www.mgosdb.org (Magnaportha grisea Oriza sativa database) as they are generated and qualities checked.