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

本项目研究分泌的RNA在植物免疫系统中的作用。 Innes和Meyers实验室最近发现，植物叶片在细胞之间的空间和表面积累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-CoV 2刺突蛋白），一些RNA具有其他功能。 对植物胞外RNA的碱基序列的分析表明，这些RNA在序列上是多样的，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要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必须以某种方式有益于植物。 该项目将测试分泌的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，拟议的研究将能够产生具有更好的免疫系统的作物，这些作物对疾病更具抵抗力。 Innes和Meyers实验室最近发现，拟南芥叶片的质外体含有丰富的长非编码RNA，包括环状RNA和小RNA。这些RNA与蛋白质颗粒结合，保护它们免受降解。值得注意的是，这种胞外RNA（exRNA）高度富集转录后修饰N6-甲基腺嘌呤（m6 A）。这些发现提出了关于植物生物学的基本问题：是否存在在植物物种中广泛保守的特定exRNA？exRNA是如何分泌的，转录后修饰对这一过程至关重要吗？为什么植物会产生exRNA？它们在植物-微生物相互作用中发挥着重要作用吗？为了回答这些问题，exRNA将从七个不同物种的质外体和叶表面纯化：拟南芥、大豆、番茄、莴苣、菠萝、水稻和玉米，这些物种是基于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。将使用RNA-seq和sRNA-seq分析这些exRNA，这将允许鉴定物种之间保守的RNA。为了评估分泌是否需要m6 A或其他修饰，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分泌的额外要求，将分析在已知RNA结合蛋白和分泌途径基因中具有突变的拟南芥和水稻植物中的exRNA含量。最后，为了评估exRNA是否有助于免疫，exRNA分泌受损的突变体将测试对真菌和细菌病原体的抗性。该奖项是共同的，由植物基因组研究计划和植物生物相互作用计划在综合有机体系统部资助。该奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准。

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.