Proteomic analysis to classify extracellular vesicles and plasmodesmata in Arabidopsis cell culture supernatants

The plant apoplast contains diverse proteins. In this study, we analyzed the extracellular vesicle proteome using cell culture supernatants. Unexpectedly, many plasmodesmata proteins were identified in the extracellular vesicle fraction. The comparison between the cell culture EV proteome and the plasmodesmata proteome revealed that EV and plasmodesmata proteins were indistinguishable. Highly sensitive imaging analyses showed that PDLP3-GFP, a representative plasmodesmata marker, was localized to plasmodesmata, but it was also present as small granules in apoplast regions. Next, we attempted to distinguish EV proteins from plasmodesmata proteins. The step-wise density gradient ultracentrifugation and clustering analysis separated EV protein clusters (represented by tetraspanins and membrane trafficking proteins) from plasmodesmata clusters (represented by PDLPs and PDCBs). These results provide the basis for future explorations of plant plasmodesmata and EVs.