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

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.