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

Summary

This protocol describes an optimized method for isolating extracellular vesicles (EVs) from Arabidopsis thaliana leaves to support downstream molecular and functional analyses.

Abstract

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.

Introduction

Extracellular vesicles (EVs) are lipid bilayer-bound membrane particles secreted by a wide range of organisms, from bacteria to humans^1,2. In animal systems, EVs are commonly classified into exosomes, microvesicles, and apoptotic bodies based on their biogenesis pathways and size³. Exosomes (~30-150 nm in diameter) originate from the fusion of multivesicular bodies (MVBs) with the plasma membrane, leading to the release of intraluminal vesicles⁴, while microvesicles (~100-1000 nm in diameter) bud from the plasma membrane directly⁵. In addi....

Protocol

NOTE: Conduct centrifugation steps in a refrigerated centrifuge at 4 °C to preserve EV integrity. Sterile tubes should be used. All sample handling steps should be performed rapidly and efficiently, ideally by multiple people working in parallel, to minimize tissue damage and prevent contamination that can compromise EV quality.

1. Leaf preparation and infiltration

1. Prepare 1 L vesicle isolation buffer (VIB) consisting of 20 mM MES hydrate, 2 mM CaCl₂, 0.1 M NaCl, pH 6.
2. Harvest the distal blade zones of leaves by cutting at the base of the blade to remove the petiole.
   ​NOTE:....

Results

To evaluate whether the optimized AWF collection protocol reduces tissue damage and cytoplasmic contamination, the AWF obtained using our method (Method 1) was directly compared with an alternative method (Method 2) based on whole-plant vacuum pump infiltration¹¹. Cell damage following infiltration was assessed by Trypan Blue staining, which revealed markedly reduced staining in leaves processed using Method 1, indicating lower levels of cell rupture (Figure 3A). To f.......

Discussion

Plant EVs have emerged as pivotal players in plant immunity, particularly in mediating cross-kingdom communication with fungal pathogens³². Increasing evidence shows that plants secrete EVs into the apoplast, where they function to deliver small RNAs (sRNAs), mRNAs, and proteins that can suppress virulence in invading pathogens, such as Botrytis cinerea, Rhizoctonia solani, and oomycete pathogen Phytophthora capsici^10,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45 µm nylon filters	VWR	76479-032
1 ml needless syringe	BD Biosciences	309659
100 kDa ultra centrifugal filter	Amicon	UFC 910008
100 ml needless syringe	BD Biosciences	551721050
15 ml conical tubes	VWR	89039-664
50 ml conical tubes	VWR	89039-656
Arabidopsis thaliana (Col-0)	laboratory-maintained lines	Not applicable
Carbon-formvar 300-mesh grids	TED PELLA	01753-F
Clear tape	Scotch Magic Tape	S-9783
Parafilm	Fisher Scientific	13-374-12
PBS buffer	Made in house	Not applicable
qEV original column Gen2 35 nm	IZON Science	ICO-35
Refrigerated centrifuge	Eppendorf	5810R
Refrigerated centrifuge fixed-angle rotor	Eppendorf	F-34-6-38
Refrigerated centrifuge swing-bucket rotor	Eppendorf	A-4-62
Sucrose	VWR	470302-810
Ultracentrifuge	Beckman Coulter	XPN-100
Ultracentrifuge tubes	Beckman Coulter	337986
UranyLess stain	Electron Microscopy Sciences	250218-01