Expression of Arabidopsis Extracellular Vesicle Protein Markers in Nicotiana benthamiana Reveals Distinct Vesicle Subpopulations

Mammalian extracellular vesicles (EVs) are heterogeneous in nature based on their protein content, RNA content, density, size, and functions. In contrast, our understanding of plant EV diversity is quite limited. Multiple plant EV protein markers have been identified. Two of these, TETRASPANIN 8 (TET8) and PENETRATION 1 (PEN1), appear to mark distinct subpopulations of plant EVs. To further assess the diversity of plant EV subpopulations, we purified EVs from N. benthamiana transiently expressing multiple EV marker proteins and then assessed colocalization of these markers using high resolution Total Internal Reflection Fluorescence Microscopy (TIRF-M). We confirmed that TET8 and PEN1 indeed mark distinct EV populations, as they colocalized only 4.7% of the time. This value was nearly identical to that found for EVs purified from transgenic Arabidopsis co-expressing these two markers, demonstrating that transient expression of Arabidopsis EV proteins in N. benthamiana can be used to assess EV subpopulations, bypassing the requirement of generating transgenic plants for every marker combination of interest. We then used the N. benthamiana system to assess colocalization of PEN1 and TET8 with the EV markers PATELLIN1 (PATL1), ANNEXIN2 (ANN2), and RPM1-INTERACTING PROTEIN4 (RIN4). PATL1 and ANN2 colocalized with PEN1 56.6% and 46.6% of the time, respectively, whereas they colocalized with TET8 only 28.4% and 30.8% of the time, respectively. In contrast to PATL1 and ANN2, the RIN4 protein colocalized with TET8 more frequently than with PEN1 (30% versus 13%). Together, these results indicate that plant EVs are heterogeneous in their protein cargos and that TET8 marks a distinct subpopulation of EVs. PEN1, PATL1, and ANN2 commonly mark the same EV population that is distinct from TET8-labeled EVs, while RIN4 more often associates with TET8-labeled EVs. These findings suggest that plants possess at least two different pathways for EV biogenesis and secretion.