Arabidopsis produces distinct subpopulations of extracellular vesicles that respond differentially to biotic stress

Extracellular vesicles (EVs) secreted by mammalian cells are highly heterogenous in contents and function. Whether this is also true for EVs secreted by plant cells is not yet known. To address this knowledge gap, we used high-resolution density gradient ultracentrifugation to separate distinct subpopulations of Arabidopsis EVs. We analyzed the protein content, morphology, and purity of these subpopulations, confirming the presence of three distinct EV subpopulations. The EV marker protein TETRASPANIN 8 (TET8) was detected only in medium-density EVs and was not associated with cell wall nanofilaments, which was unique among EV proteins. TET8 and PENETRATION 1 (PEN1) were confirmed to be secreted on mostly separate EV populations using total internal fluorescence microscopy. We found that EV marker proteins are differentially secreted in response to phytohormones, changes in growth temperature, and infection with fungal pathogens Colletotrichum and Golovinomyces cichoracearum. EV subpopulations marked by TET8, PEN1, and RPM1-INTERACTING PROTEIN 4 (RIN4) were highly increased as soon as one day after fungal infection, while other EV populations remained unaffected. Together these data indicate that Arabidopsis EVs are highly heterogenous and suggest that specific EV subpopulations may contribute to plant immunity.