Arabidopsis Produces Distinct Subpopulations of Extracellular Vesicles That Respond Differentially to Biotic Stress, Altering Growth and Infectivity of a Fungal Pathogen

Extracellular vesicles (EVs) secreted by mammalian cells are highly heterogeneous in content and function. Whether this is also true for EVs secreted by plant cells is not yet known. To address this, we used high-resolution density gradient ultracentrifugation and total internal fluorescence microscopy (TIRF-M) to purify and distinguish distinct subpopulations of Arabidopsis EVs. The EV marker protein TETRASPANIN 8 (TET8) was detected specifically in medium-density EVs. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted in mostly separate EV populations using TIRF-M, while PEN1 was co-secreted with PENETRATION 3 (PEN3) much more often. Secretion of EV subpopulations marked by TET8, PEN1 and RPM1-INTERACTING PROTEIN 4 (RIN4) into the apoplast and onto the leaf surface was induced by phytohormones, changes in temperature and infection with fungal pathogens. Treatment of Arabidopsis seedlings with plant EVs delayed the progression of fungal infection by altering fungal germ tube development and fungal morphology. Significantly, extracellular RNAs, including miRNAs and siRNAs, did not co-fractionate with TET8-labeled EVs, and instead, co-fractionated with extravesicular ARGONAUTE proteins in high-density fractions. Together, these data indicate that Arabidopsis EVs are highly heterogeneous and contribute to immunity but are unlikely to mediate cross-kingdom RNA interference.