Collaborative Research: RESEARCH-PGR: Extracellular RNA Produced By Plants: What, Where, How, Who, and Why?

本项目研究分泌RNA在植物免疫系统中的作用。Innes和Meyers的实验室最近发现，植物的叶子在细胞之间的空间和表面上积累RNA。虽然我们通常认为RNA是一种可以指导细胞合成特定蛋白质的分子（例如，COVID疫苗中的mRNA指导我们的细胞制造SARS-CoV2刺突蛋白），但一些RNA还有其他功能。对植物细胞外rna碱基序列的分析表明，这些rna序列多样，但似乎不编码蛋白质。植物细胞外非编码RNA的发现提出了本项目将解决的两个基本问题：1)植物如何分泌RNA？2)这种RNA的功能是什么？细胞分泌RNA需要大量的能量，因此这种分泌的RNA一定在某种程度上对植物有益。该项目将验证分泌RNA功能保护植物免受真菌和细菌感染的假设。如果这一假设是正确的，那么拟议中的研究将使人们能够培育出免疫系统更好、对疾病更有抵抗力的作物。这些作物需要以可持续的方式养活不断增长的全球人口，同时减少农业对环境的影响。Innes和Meyers的实验室最近发现，拟南芥叶片的外质体中含有大量的长链非编码rna，包括环状rna和小rna。这些rna与蛋白质颗粒结合，保护它们不被降解。值得注意的是，这种细胞外RNA （exRNA）在转录后修饰n6 -甲基腺嘌呤（m6A）中高度富集。这些发现提出了关于植物生物学的基本问题：是否存在特定的exrna在植物物种中广泛保守？exrna是如何分泌的？转录后修饰是否在这一过程中起核心作用？为什么植物会产生exrna ？它们在植物与微生物的相互作用中起着重要作用吗？为了回答这些问题，我们将从拟南芥、大豆、番茄、生菜、菠萝、水稻和玉米等7个不同物种的外质体和叶片表面纯化exRNA，这些物种是根据它们的系统发育多样性、基因组资源、作为作物的重要性和生理多样性选择的。这些exrna将使用RNA-seq和sRNA-seq进行分析，这将允许鉴定物种之间保守的rna。为了评估分泌是否需要m6A或其他修饰，我们将测试表达缺乏修饰位点的exrna的转基因植物的分泌效率。为了研究exRNA分泌的额外需求，我们将分析已知RNA结合蛋白和分泌途径基因突变的拟南芥和水稻植株的exRNA含量。最后，为了评估exRNA是否有助于免疫，将测试exRNA分泌受损的突变体对真菌和细菌病原体的抗性。该奖项由植物基因组研究计划和植物生物相互作用计划共同资助，该计划隶属于综合生物系统部门。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project investigates the role of secreted RNA in the immune system of plants. The Innes and Meyers laboratories recently discovered that the leaves of plants accumulate RNA in the spaces between cells and on their surfaces. Although we usually think of RNA as a molecule that can direct cells to synthesize specific proteins (e.g., the mRNA in COVID vaccines directs our cells to make SARS-CoV2 spike protein), some RNAs serve other functions. Analysis of the base sequences of plant extracellular RNAs revealed that these RNAs are diverse in sequence, but do not appear to encode proteins. The discovery of extracellular non-coding RNA in plants raises two fundamental questions that this project will address: 1) how do plants secrete RNA? and 2) what is the function of this RNA? It takes a large amount of energy for cells to secrete RNA, thus this secreted RNA must benefit the plant in some way. This project will test the hypothesis that secreted RNA functions to protect plants from infection by fungi and bacteria. If this hypothesis is correct, the proposed research will enable generation of crop plants with improved immune systems that are more resistant to disease. Such crops are needed to feed a growing global population in a sustainable manner, while reducing the environmental impacts of agriculture.The Innes and Meyers laboratories recently discovered that the apoplast of Arabidopsis leaves contains abundant long non-coding RNAs, including circular RNAs, as well as small RNAs. These RNAs are bound to protein particles, which protects them against degradation. Notably, this extracellular RNA (exRNA) is highly enriched in the post-transcriptional modification N6-methlyadenine (m6A). These discoveries raise fundamental questions about plant biology: Are there specific exRNAs that are broadly conserved across plant species? How are exRNAs secreted, and are post-transcriptional modifications central to this process? And why do plants produce exRNAs? Do they play a fundamental role in plant-microbe interactions? To answer these questions, exRNA will be purified from the apoplast and leaf surfaces of seven diverse species: Arabidopsis, soybean, tomato, lettuce, pineapple, rice, and maize, which were chosen based on their phylogenetic diversity, genomic resources, importance as crops, and diversity in physiology. These exRNAs will be analyzed using both RNA-seq and sRNA-seq, which will allow identification of RNAs that are conserved between species. To assess whether m6A or other modifications are required for secretion, transgenic plants that express exRNAs that lack modification sites will be tested for their secretion efficiency. To investigate additional requirements for exRNA secretion, the exRNA content in Arabidopsis and rice plants with mutations in known RNA binding proteins and secretory pathway genes will be analyzed. Lastly, to assess whether exRNAs contribute to immunity, mutants compromised in exRNA secretion will be tested for resistance to fungal and bacterial pathogens.This award was co-funded by the Plant Genome Research Program and the Plant Biotic Interactions Program in the Division of Integrative Organismal Systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.