Role of Extracellular Vesicles in Plant-Pathogen Interactions

由病毒、细菌和真菌引起的植物疾病给美国农民造成了重大经济损失，并极大地限制了全球农业生产。在撒哈拉以南非洲和印度等发展中地区，人口的快速增长和农业产量的下降是对粮食安全的重大威胁，因此也是对政治稳定的重大威胁。因此，迫切需要改善植物的免疫系统，以便它们能够抵御疾病。这个项目的重点是了解植物免疫系统中一个似乎与人类共享的未被重视的组成部分，即细胞外小泡(EVS)。电动汽车是一种微小的球形包裹，可以将信息从一个细胞带到有机体内的另一个细胞。最近在包括人类在内的动物系统中的研究表明，这种信息可以重新编程接收细胞，使接收细胞改变其产生的蛋白质，从而改变细胞的特性，在某些情况下，改变细胞防止病毒感染的能力。Innes实验室最近的研究表明，电动汽车可能在植物中发挥类似的作用。这项研究项目旨在解决有关植物电动汽车是如何生产的、它们携带什么以及它们如何有助于植物免疫的基本问题。这些问题的答案将有助于抗病作物的发展。此外，该项目将为小学生、高中生和本科生提供植物科学实践活动，特别关注代表性不足的少数民族。本项目的重点是细胞外小泡(EVS)在植物免疫系统中的起源和作用。虽然已知植物细胞释放EV是由真菌和细菌感染刺激的，但对它们的功能知之甚少。在过去的两年里，Innes实验室开发了一种从拟南芥叶片中提纯和量化电动汽车的方法。对纯化的EVS的分析表明，它们携带microRNAs，并高度富含与生物和非生物应激相关的蛋白质，表明EVS可能参与细胞间的通讯。本提案中描述的实验将解决以下问题：1)植物EVS的蛋白质和RNA含量是否随着特定的生物胁迫而变化？2)植物EVS是产生植物EVS所必需的已知内膜运输蛋白吗？3)EVS是否是寄主诱导的真菌病原体基因沉默(HIGS)所必需的？问题1将通过蛋白质组学和RNA-SEQ分析从经过各种生物胁迫处理的拟南芥叶片中纯化的EV来解决。问题2将通过量化缺乏特定内膜运输蛋白的突变拟南芥品系中的EV产生来解决。问题3将使用正向遗传学筛选来识别不能对真菌禾谷镰刀菌中的基因进行HIGS的拟南芥突变体。这一筛选将揭示HIGS所需的拟南芥基因，并将确定EVS是否将小RNA从宿主运输到病原体。该奖项由综合组织系统分部的植物生物相互作用计划和分子和细胞生物科学部的细胞动力学和功能计划共同资助。

Plant diseases caused by viruses, bacteria, and fungi cause major economic harm to U.S. farmers and greatly limit agricultural production worldwide. In developing regions such as sub-Saharan Africa and India, the combination of rapid population growth and declining agricultural production is a major threat to food security, and hence to political stability. There is thus an urgent need to improve the immune system of plants so that they can defend themselves against disease. This project is focused on understanding an underappreciated component of the plant immune system that appears to be shared with humans, extracellular vesicles (EVs). EVs are microscopic spherical packages that can carry information from one cell to another within an organism. Recent work in animal systems, including humans, has revealed that this information can reprogram recipient cells so that the receiving cell changes the proteins it produces, and hence the properties of the cell, and in some cases, the ability of a cell to prevent infection by a virus. Recent work in the Innes laboratory indicates that EVs may serve a similar role in plants. This research project aims to address basic questions about how plant EVs are produced, what they carry, and how they may contribute to plant immunity. The answers to these questions will facilitate development of disease resistant crops. In addition, this project will provide hands on plant science activities to elementary, high school and undergraduate students, with a special focus on underrepresented minorities. This project focuses on the genesis and roles of extracellular vesicles (EVs) in the context of plant immune systems. Although EV release by plant cells is known to be stimulated by fungal and bacterial infection, nothing is known about their function. Over the last two years, the Innes laboratory has developed a method for purifying and quantifying EVs from Arabidopsis leaves. Analysis of purified EVs revealed that they carry microRNAs and are highly enriched in proteins associated with response to biotic and abiotic stress, suggesting that EVs may participate in intercellular communication. The experiments described in this proposal will address the following questions: 1) Does the protein and RNA content of plant EVs change in response to specific biotic stresses? 2) Are known endomembrane trafficking proteins required for production of plant EVs? 3) Are EVs required for host-induced gene silencing (HIGS) of genes in a fungal pathogen? Question 1 will be addressed using proteomic and RNA-seq analysis of EVs purified from Arabidopsis leaves following treatment with various biotic stresses. Question 2 will be addressed by quantifying EV production in mutant Arabidopsis lines deficient in specific endomembrane trafficking proteins. Question 3 will be addressed using a forward genetics screen to identify Arabidopsis mutants that are unable to perform HIGS of genes in the fungus Fusarium graminearum. This screen will uncover Arabidopsis genes required for HIGS, and will establish whether EVs transport small RNAs from the host to the pathogen. This award is co-funded by the Plant Biotic Interactions program in the Division of Integrative Organismal Systems division and the Cellular Dynamics and Function program in the Molecular and Cellular Biosciences division.