Isolation of Extracellular Vesicles from Arabidopsis.

Extracellular vesicles (EVs) in plants have emerged as key players in cell-to-cell communication and cross-kingdom RNAi between plants and pathogens by facilitating the exchange of RNA, proteins, and other molecules. In addition to their role in intercellular communication, plant EVs also show promise as potential therapeutics and indicators of plant health. However, plant EVs exhibit significant heterogeneity in their protein markers, size, and biogenesis pathways, strongly influencing their composition and functionality. While mammalian EVs can be generally classified as exosomes that are derived from multivesicular bodies (MVBs), microvesicles that are shed from the plasma membrane, or as apoptotic bodies that originate from cells undergoing apoptosis, plant EVs remain poorly studied in comparison. At least three subclasses of EVs have been identified in Arabidopsis leaves to date, including Tetraspanin-positive exosomes derived from MVBs, Penetration 1 (PEN1)-positive EVs, and EVs derived from exocyst-positive organelles (EXPO). Differences in the plant starting material and isolation techniques have resulted in different purities, quality, and compositions of the resulting EVs, complicating efforts to better understand the role of these EVs in plants. We performed a comparative analysis on commonly used plant EV isolation methods and have identified an effective protocol for extracting clean apoplastic washing fluid (AWF) and isolating high-quality intact and pure EVs of Arabidopsis thaliana. These EVs can then be used for various applications or studied to assess their cargos and functionality in plants. Furthermore, this process can be easily adapted to other plant species of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of EVs from the apoplastic fluid of Arabidopsis thaliana Basic Protocol 2: Density gradient fractionation of EVs Basic Protocol 3: Immuno-isolation of EVs using Arabidopsis tetraspanin 8 (TET8) antibody.