Methodological guidelines for isolation and purification of plant extracellular vesicles

ABSTRACT Plant extracellular vesicles (EVs) have become the focus of rising interest due to their important roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes to modulate pathogen virulence. However, the isolation of pure intact EVs from plants still represents a considerable challenge. Currently, plant EVs have been isolated from apoplastic washing fluid (AWF) using a variety of methods. Here, we compare two published methods used for isolating plant EVs, and provide a detailed recommended method for AWF collection from Arabidopsis thaliana , followed by EV isolation via differential ultracentrifugation. To further separate and purify specific subclasses of EV from heterogeneous vesicles, sucrose or iodixanol density-based separation and immunoaffinity capture are then utilized. We found that immunoaffinity capture provides a significant advantage for specific EV isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study guides the selection and optimization of EV isolation methods for desired downstream applications.