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Figure 2. Heterogeneous populations of EVs isolated from plants. There are at least four known EV populations that have been isolated from plants: TET-positive EVs, PEN1-positive EVs, autophagy-related EVs, and pollenosomes. They have different sizes, densities, cargoes, and intracellular origins. Pathogen infection induces secretion of both TET-positive EVs and PEN1-positive EVs^[56]. In the process of EV isolation, final centrifugation of 40,000 × g (P40) pellets larger and heavier vesicles such as PEN1-positive EVs and large EVs, non-vesicular free RNA, and RNA-protein complexes^[30,79]. Small EVs, such as TET-positive EVs, are mainly present in the supernatant after 40,000 × g centrifugation and require a higher speed of ultracentrifugation at 100,000 × g for collection (P100)^[30,56]. Autophagy-related EVs marked with ATG8a were collected using 100,000 × g from plants during the autophagy process within cells^[64]. Pollenosomes secreted during pollen germination and pollen tube growth were collected at 100,000 × g from in-vitro pollen gemination media^[59]. EXPO-derived EVs originate from the plant-specific organelle EXPO, are marked by the protein Exo70E2, and have not yet been isolated from plants^[71]. This figure was created with https://www.biorender.com/.