Plant Extracellular RNA

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.

_{GFP fluorescence from GFP-PEN1 EVs using a confocal fluorescence microscope.}

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.

_{Density-purified Arabidopsis EVs.}

We will isolate and characterize exRNA from the apoplast and from the surface of leaves from diverse crop species, chosen based on their phylogenetic diversity, genomic resources, importance as crops, and diversity in physiology. These exRNAs will be analyzed using both RNAseq and sRNAseq, which will allow us to identify RNAs that are conserved between species. We will also assess how the exRNA content changes in response to pathogen perception, as we hypothesize that exRNAs play an integral role in defense. We will characterize post-transcriptional modifications on exRNA, including m6A, 5-methylcytosine, N-glycan additions, and 3’ end modification (e.g., phosphorylation and methylation). To assess whether m6A or other modifications are required for secretion, we will use mutant plants to express exRNAs that lack modification sites and examine their secretion efficiency. To investigate additional requirements for exRNA secretion, we will assess the effect of mutations in known RNA binding proteins and secretory pathway genes on exRNA content in Arabidopsis and rice. Lastly, to investigate potential roles of exRNA in pathogen defense, we will test whether Arabidopsis and rice circular RNAs can function as sponges for fungal sRNAs, and we will also test whether purified exRNA-protein complexes can confer resistance to infection by fungal and bacterial pathogens.

_{Apoplastic fluid contains long RNAs}

This project will generate a very large dataset of exRNAs found in seven diverse crop species, which will be an invaluable resource for future research on plant-microbe and plant-insect interactions. It will also provide in depth training and career development for two postdoctoral researchers and multiple undergraduates, all of whom will gain experience in science outreach by developing and implementing a hands-on plant science activity targeting third grade classrooms in Bloomington IN and East St. Louis, IL.