Isolation and Purification of Plant Extracellular Vesicles from Arabidopsis Leaves Using an Optimized Apoplastic Wash...

Extracellular vesicles (EVs) are membrane-bound nanoparticles secreted by diverse organisms and cell types and are increasingly recognized as important mediators of intercellular and cross-kingdom communication. While EV biology is well established in mammalian systems, plant EV research is comparatively recent and rapidly expanding, driven in part by evidence that plant EVs can deliver small RNAs, mRNAs, proteins, and other cargoes involved in immunity and plant-microbe interactions. However, isolating high-purity plant EVs remains technically challenging due to rigid cell walls, abundant secondary metabolites, and the risk of cytosolic contamination during apoplastic fluid collection. This protocol outlines a streamlined workflow for the isolation and purification of plant EVs from Arabidopsis thaliana leaf tissue. A gentle syringe-based infiltration step is used to minimize cellular damage and cytosolic leakage, while using individual leaf blades (without petioles/vascular tissue) helps reduce contamination from vascular-associated and intracellular contents. EVs are initially enriched by differential centrifugation and can be further purified using either sucrose density gradient fractionation or size-exclusion chromatography (SEC). This protocol yields EVs with preserved structural integrity and reduced cellular contamination by minimizing cell damage and cytoplasmic leakage during apoplastic fluid collection, as assessed by nanoparticle tracking analysis and transmission electron microscopy. These EV samples are suitable for downstream applications, including RNA profiling, proteomics, and functional assays for plant-microbe interaction studies. Overall, this method provides a reproducible and adaptable approach for obtaining high-quality plant EVs.