a, Candidates of RNA binding proteins detected in plant EVs. Proteins in red were chosen for further experimental analysis. b, RNA binding proteins AGO1, RH11, RH37, ANN1 and ANN2 can be detected from purified EVs by western blot. EVs were isolated from the untreated (Mock) and B. cinerea infected (Bc) wild-type, RH11-CFP, RH37-CFP, ANN1-YFP, ARA6-GFP and CFP-SYP61 plants. AGO1, AGO2, AGO4, AGO5 ANN2 and TET8 were detected by western blot using antibodies against AGO1, AGO2, AGO4, AGO5, ANN2, and TET8, respectively. RH11-CFP, RH37-CFP, ANN1-YFP, ARA6-GFP and CFP-SYP61 were detected by western blot using antibodies against GFP. TET8, ARA6, and SYP61 were used as EV, MVB, and Trans Golgi Network markers, respectively. 20 μg of total and 10 μg of EV proteins were used to perform the western blot. Ponceau-S staining of Rubisco was used as the loading control. c, Trypsin digestion of plant EVs. EVs were isolated from wild-type, RH11-CFP, RH37-CFP, ANN1-YFP and ANN2-CFP plants and incubated with 1% Triton X-100, with 10 μg/ml trypsin, or a combination of both, for 30 min at 37 °C. Samples were analyzed by western blot with antibodies against AGO1, TET8 and GFP, respectively. The experiments in Fig. 1b, c were repeated three times with similar results. Source data are provided as a Source Data file.