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的经济损失，并极大地限制了世界范围内的农业生产。在撒哈拉以南非洲和印度等发展中地区，人口快速增长和农业生产下降的结合对粮食安全构成重大威胁，从而对政治稳定构成威胁。因此，迫切需要改善植物的免疫系统，使它们能够抵御疾病。这个项目的重点是了解植物免疫系统中一个未被充分认识的成分，似乎与人类共享，细胞外囊泡（EVs）。电动汽车是微小的球形包裹，可以在生物体内将信息从一个细胞传递到另一个细胞。最近在包括人类在内的动物系统中的研究表明，这些信息可以对受体细胞进行重编程，从而使受体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，还可以改变细胞预防病毒感染的能力。Innes实验室最近的工作表明，电动汽车可能在植物中起着类似的作用。本研究项目旨在解决植物电动汽车是如何产生的，它们携带什么，以及它们如何促进植物免疫的基本问题。这些问题的答案将促进抗病作物的发展。此外，该项目将为小学、高中和本科生提供实践植物科学活动，特别关注代表性不足的少数民族。本项目主要研究细胞外囊泡（EVs）在植物免疫系统中的发生和作用。虽然已知真菌和细菌感染会刺激植物细胞释放EV，但对其功能一无所知。在过去的两年中，Innes实验室开发了一种从拟南芥叶片中纯化和定量ev的方法。对纯化ev的分析显示，它们携带microrna，并且高度富集与生物和非生物应激反应相关的蛋白质，这表明ev可能参与细胞间通讯。本实验将解决以下问题：1)植物EVs的蛋白质和RNA含量是否会随着特定的生物胁迫而发生变化？2)植物电动汽车的生产是否需要已知的膜转运蛋白？3)真菌病原体中基因的宿主诱导基因沉默（HIGS）是否需要ev ？问题1将通过蛋白质组学和RNA-seq分析来解决，这些分析是从拟南芥叶片中纯化的经过各种生物胁迫处理的ev。问题2将通过量化缺乏特定膜转运蛋白的拟南芥突变系的EV产量来解决。问题3将使用正向遗传学筛选来确定无法在真菌镰刀菌中执行基因HIGS的拟南芥突变体。该筛选将揭示HIGS所需的拟南芥基因，并将确定ev是否将小rna从宿主转运到病原体。该奖项由综合有机系统部的植物生物相互作用项目和分子与细胞生物科学部的细胞动力学和功能项目共同资助。

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.