Diverse plant RNAs coat Arabidopsis leaves and are distinct from apoplastic RNAs

Abstract

Transgenic expression of a double-stranded RNA in plants can induce silencing of homologous mRNAs in fungal pathogens. Although such host-induced gene silencing is well-documented, the molecular mechanisms by which RNAs can move from the cytoplasm of plant cells across the plasma membrane of both the host cell and fungal cell are poorly understood. Indirect evidence suggests that this RNA transfer may occur at a very early stage of the infection process, prior to breach of the host cell wall, suggesting that silencing RNAs might be secreted onto leaf surfaces. To assess whether Arabidopsis plants possess a mechanism for secreting RNA onto leaf surfaces, we developed a protocol for isolating leaf surface RNA separately from intercellular (apoplastic) RNA. This protocol yielded abundant leaf surface RNA that displayed an RNA banding pattern distinct from apoplastic RNA, suggesting that it may be secreted directly from the leaf surface rather than exuded through stomata or hydathodes. Notably, this RNA was not associated with either extracellular vesicles or protein complexes; however, RNA species longer than 100 nucleotides could be pelleted by ultracentrifugation. Pelleting was inhibited by the divalent cation chelator EGTA, suggesting that these RNAs may form condensates on the leaf surface. These leaf surface RNAs are derived almost exclusively from Arabidopsis, but come from diverse genomic sources, including rRNA, tRNA, mRNA, intergenic RNA, microRNAs, and small interfering RNAs, with tRNAs especially enriched. We speculate that endogenous leaf surface RNA plays an important role in the assembly of distinct microbial communities on leaf surfaces. Significance StatementPlant leaves are colonized by a complex community of microbes that is shaped by host genetics. Although secreted metabolites are thought to mediate this effect, we investigated whether plants might also secrete RNA that could potentially structure microbial communities via cross-kingdom RNA interference. Here we report that Arabidopsis leaves are covered with diverse RNAs of plant origin, including abundant tRNAs and tRNA fragments. This leaf surface RNA is not associated with extracellular vesicles or protein complexes; however, it is less degraded than RNA found inside the extracellular spaces of leaves, suggesting that leaf surface RNA is secreted directly rather than exuded through stomata or hydathodes. We propose that this RNA plays a direct role in shaping the leaf microbiome.