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 addition to exosomes and microvesicles, mammalian cells also produce apoptotic bodies, which are released during programmed cell death⁶. These vesicles have been implicated in immune modulation, tumor progression, and therapeutic delivery, making them a subject of intense biomedical research^7,8. In plants, EV classification is less well defined than in animals. Studies in Arabidopsis thaliana have identified multiple plant EV subtypes based on differences in protein markers, size, and putative biogenesis pathways. These include tetraspanin (TET)-positive EVs, which are considered exosome-like vesicles derived from multivesicular bodies^9,10, syntaxin protein Penetration 1(PEN1)-positive EVs¹¹, exocyst-positive organelle (EXPO)-derived EVs^12,13, as well as other EV populations such as autophagy-related EVs¹⁴ and pollensomes¹⁵.

Increasing evidence suggests that plants secrete EVs as part of their defense strategy against pathogens. Plant EVs deliver diverse molecular cargoes, including small RNAs, mRNAs, and other regulatory molecules, that suppress the virulence of fungal and oomycete pathogens^{10,16,17,18,19}. These findings highlight EVs as vehicles of "RNA warfare", enabling plants to directly suppress pathogen gene expression, a mechanism with profound implications for crop protection²⁰. Beyond immunity, plant EVs may also contribute to cell wall remodeling, stress responses, and developmental signaling²¹. Unlike animal EV research, where standardized isolation and characterization protocols are widely established²², the plant EV field is still in its formative stages. This limits reproducibility and comparability across studies, creating an urgent need for robust, standardized methodologies to isolate and study these vesicles.

Isolating plant EVs presents unique technical challenges not encountered in animal systems. Damaged plant tissues contain abundant secondary metabolites, cell wall debris, and organellar fragments that can co-purify with EVs, complicating downstream characterization²³. Moreover, the apoplastic washing fluid (AWF), which is commonly used as the starting material for plant EV isolation and purification, often yields a heterogeneous mixture of vesicles and contaminants²⁴. While differential centrifugation remains a widely used foundational approach, it cannot fully resolve distinct EV subpopulations or separate EVs from co-isolated macromolecular complexes^25,26. Recent reviews and methodological studies have provided important guidance and best-practice considerations for plant EV isolation and characterization^{27,28,29,30,31}. Nevertheless, practical implementation and integration of these approaches remain challenging, and additional purification steps such as density gradient centrifugation and size-exclusion chromatography are often required to improve EV purity and reproducibility across studies.

Here, we present a streamlined protocol for isolating EVs from Arabidopsis apoplastic washing fluid using a multi-step purification strategy. In contrast to commonly used plant EV isolation approaches that rely on whole-plant infiltration with vacuum pumps¹¹, this protocol avoids inclusion of petioles and vascular tissues, which can increase contamination from vascular structures. Instead, isolated leaf blades and a gentle syringe-based infiltration method are used to improve apoplast collection while minimizing tissue disruption. Careful leaf handling, orientation, and centrifugation conditions further reduce contamination from intracellular components, providing a cleaner starting material for EV isolation. Following differential centrifugation, EVs can be further purified using either sucrose density gradient fractionation or size-exclusion chromatography to achieve higher vesicle purity and resolution. The resulting EV preparations are well-suited for downstream applications, including RNA sequencing, proteomic profiling, and functional assays such as pathogen interaction and EV uptake studies, thereby enhancing reproducibility and interpretability in plant EV research.

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: At least 100 four-week-old plants per treatment or genotype are recommended to ensure sufficient EV yield. Arabidopsis plants were grown under 8 h light/16 h dark cycles. Do not use leaves that show visible signs of stress, such as leaf bleaching and shriveling.
3. Wash leaves with reverse osmosis (RO) water three times. Place them on paper towels and gently pat them dry.
4. Load approximately 50 mL of leaves into a 120 mL syringe. Lightly tap the syringe to settle the leaves.
5. Add approximately 60 mL of VIB into the syringe until the leaves are fully immersed. Expel trapped air by gently pressing the plunger. Press a piece of parafilm folded four times on the tip of the syringe to prevent leakage.
   NOTE: When performing syringe-based vacuum infiltration, ensure that the syringe and plunger are securely assembled before applying vacuum. Apply force gradually and maintain a firm grip on the syringe to prevent sudden plunger movement or loss of control.
6. Infiltrate leaves by slowly pulling the plunger outward to create a vacuum. Hold the plunger in the extended position for 5 s, then slowly release. Repeat this infiltration cycle 2-3 times until leaves appear darker and translucent, indicating successful infiltration.
7. NOTE: Pulling the plunger requires moderate force due to vacuum resistance. The syringe should be held firmly while the plunger is withdrawn slowly to maintain controlled infiltration.
8. Once leaves have been infiltrated, pull out the plunger. Pour out the excess buffer into the sink.
9. Repeat steps 1.4-1.7 until all harvested leaves are infiltrated (Figure 1A).

2. Collection of apoplastic washing fluid (AWF)

1. Cut a ~12-inch strip of Scotch tape and place it sticky side up. Secure one end of the tape by wrapping it around an empty, needless 1 mL syringe just below the wider end. Carefully arrange approximately 50-70 infiltrated leaves in overlapping layers along the sticky surface, positioning them so that the base of each blade faces the bottom syringe and the tips point upward for collection (Figure 1B).
   NOTE: Layering multiple leaves on one strip of tape helps prevent damage. Align all leaves with the blade facing one end and oriented toward the bottom of the collection tube to allow efficient apoplastic fluid recovery during centrifugation.
2. Wrap the tape-bound leaves securely around the syringe, then fasten the top of the bundle with two strips of Scotch tape. Insert the wrapped syringe into a 50 mL tube with the cut end of the leaves and the syringe base oriented toward the bottom of the tube. Ensure the leaves are firmly taped, as loose wrapping can cause leaf damage and release broken cells that contaminate the apoplast washing fluid (Figure 1B).
3. Centrifuge the wrapped syringe containing infiltrated leaves at 900 × g for 10 min at 4 °C using a refrigerated centrifuge equipped with a fixed-angle rotor. Collect the apoplastic washing fluid in a new 15 mL conical tube and keep the sample on ice. Repeat steps 2.1-2.3 until all the leaves have been used to collect AWF.
   NOTE: The AWF needs to be clear, not green or even light green. A green AWF indicates the presence of chloroplasts/chlorophyll and can also indicate cell contamination. To minimize leaf damage, use a fixed-angle rotor.

3. Arabidopsis EV isolation from AWF via differential centrifugation

1. Centrifuge the collected apoplastic washing fluid at 2,000 g for 20 min at 4 °C using a refrigerated centrifuge equipped with a swing-bucket rotor, with standard acceleration and deceleration settings. After centrifugation, use a 0.45 µm nylon cell filter to remove large cellular debris from the supernatant. Discard the pellet. Transfer the filtered supernatant into new centrifuge tubes (Figure 2).
2. Centrifuge the filtered supernatant at 10,000 × g for 30 min at 4 °C using a refrigerated centrifuge equipped with a fixed-angle rotor, with standard acceleration and deceleration settings. Discard the pellet and retain the supernatant for ultracentrifugation. This step further removes large cellular debris and large vesicles (Figure 2).
3. Transfer the supernatant into 17 mL ultracentrifuge tubes and ultracentrifuge at 100,000 × g for 1 h at 4 °C using an ultracentrifuge equipped with a swing-bucket rotor, with standard acceleration and deceleration settings. After centrifugation, discard the supernatant and resuspend the pellet in approximately 16 mL of VIB buffer for washing. Repeat the ultracentrifugation round to collect EVs (Figure 2).
   NOTE: Centrifugation at 100,000 x g is done for the sedimentation of small vesicles such as exosomes. Subsequent washing steps help eliminate non-EV proteins by resuspending the pellet in buffer. Ultracentrifuge tubes must be carefully balanced by mass according to the manufacturer's specifications before centrifugation. Inspect tubes and rotors for defects prior to use and ensure that the rotor is properly secured before starting the run.
4. Discard the supernatant and resuspend the pellet in VIB. Use a pipette tip to gently scrape the bottom of the tube to facilitate complete recovery of the EV pellet (Figure 2).
   NOTE: The EV pellet is often translucent and may not be visually detectable once resuspended. Gently scraping the bottom of the tube facilitates the EV resuspension process. Adjust the resuspension volume to achieve the desired EV concentration, as smaller resuspension volumes generally result in more concentrated EV preparations. Typically, resuspending EVs in 50 µL yields protein concentrations of approximately 1-1.5 µg/µL and particle concentrations approaching 10⁹-10¹⁰ particles/mL.
5. Store the resuspended EVs at 4 °C for immediate use or at -80 °C for long-term storage without flash freezing in liquid nitrogen.
   NOTE: Long-term storage may reduce EV integrity. Use freshly prepared EVs whenever possible.

4. Usage of sucrose density gradient fractionation for EV purification

NOTE: Ultracentrifugation alone co-sediments multiple types of vesicles. To achieve finer resolution, density gradient fractionation can be employed, separating vesicles according to their buoyant density and flotation properties. In this approach, sucrose gradients are used to enrich defined EV subpopulations. Alternatively, iodixanol gradients can be used.

1. Top-loading sucrose gradient separation of EVs
   1. Prepare 10-90% sucrose gradient stocks consisting of the following concentrations: 10%, 16%, 22%, 28%, 34%, 40%, 46%. 52%, 58%, 64%, 70%, and 90% using VIB. Use a 60 °C water bath to aid in dissolving the 70% and 90% stocks.
   2. Prepare a discontinuous gradient by layering 1 mL of each sucrose solution in a 17 mL ultracentrifuge tube, starting with the most concentrated solution at the bottom and proceeding sequentially to the least concentrated. Exclude the 10% sucrose layer when assembling the gradient.
      ​NOTE: Carefully dispense each density layer along the inner wall of the tube to minimize mixing between adjacent layers. An automatic pipette set to a slow dispensing speed is recommended to provide continuous, controlled flow and improve gradient stability. If a regular manual pipette is used, dispensing should be performed slowly and steadily, with the pipette tip positioned just above the interface of the previous layer, and care taken to avoid air bubble formation.
   3. Mix the resuspended EV pellet (~100 µg of EV protein) with the 1 mL of 10% sucrose solution. Then, layer this mixture as the final layer of the discontinuous layer. Total volume will be ~12.1 mL.
   4. Centrifuge at 100,000 g for 16 h at 4 °C.
      ​NOTE: For extended ultracentrifugation runs, confirm that tubes, caps, and rotors are rated for prolonged high-speed operation and that manufacturer-recommended balancing procedures are followed.
   5. Collect six 2 mL fractions per sample by pipetting from the top. Transfer each fraction into a new 17 mL ultracentrifuge tube.
   6. Resuspend the collected fractions in a ~16 mL VIB buffer and centrifuge at 100,000 g for 1 h at 4 °C.
   7. Discard the supernatant, then resuspend each pellet in 50 µL of VIB.
      ​NOTE: The EV pellet is not visible. Gently scrape the tube bottom with the pipette tip to resuspend. Adjust the buffer volume as needed (e.g., 10 µL for higher concentration).
2. Bottom-loading sucrose gradient separation of EVs
   1. Prepare 10-90% sucrose gradient stocks consisting of the following concentrations: 10%, 16%, 22%, 28%, 34%, 40%, 46%, 52%, 58%, 64%, 70%, and 90% using VIB buffer. Use a 60 °C water bath to aid in dissolving the 70% and 90% stocks.
   2. Mix the resuspended EV pellet (~100 µg of EV protein) with the 1 mL of 90% sucrose solution. Then, layer this mixture as the first layer of a discontinuous layer.
   3. Prepare the remaining discontinuous sucrose gradient by sequentially layering 1 mL of each sucrose solution on top of the EV-90% sucrose mixture, starting from 70% sucrose and proceeding stepwise to lower concentrations (64%, 58%, 52%, 46%, 40%, 34%, 28%, 22%, 16%, and 10%). Carefully dispense each layer along the wall of the tube using a pipette to minimize mixing between adjacent layers. Total volume will be ~12.1 mL.
   4. Centrifuge at 100,000 g for 72 h at 4 °C.
      ​NOTE: For extended ultracentrifugation runs, confirm that tubes, caps, and rotors are rated for prolonged high-speed operation and that manufacturer-recommended balancing procedures are followed.
   5. Collect six 2 mL fractions per sample by pipetting from the top. Transfer each fraction into a new 17 mL ultracentrifuge tube.
   6. Resuspend the collected fractions in a ~16 mL of VIB buffer and centrifuge at 100,000 g for 1 h at 4 °C.
   7. Discard the supernatant, then resuspend each pellet in 50 µL of VIB buffer.
      ​NOTE: The EV pellet is not visible. Gently scrape the tube bottom with the pipette tip to resuspend. Adjust the buffer volume as needed (e.g., 10 µL for higher concentration).

5. Use of size exclusion chromatography (35 nm column) to purify EVs

1. Buffer preparation and column equilibration
   1. Prepare VIB and all additional buffers fresh. Filter all buffers through a 0.22 µm membrane and degas in ultrasonic bath prior to use to prevent air bubble formation and pore blockage.
      ​NOTE: Incomplete degassing or use of unfiltered buffers can introduce air bubbles or particulate matter into the resin bed, leading to disrupted flow and reduced EV separation efficiency. If bubbles are observed in the column, the run should be stopped and the column re-equilibrated.
   2. Allow the column and buffers to equilibrate to room temperature (18-24 °C) before removing column caps.
      ​NOTE: The column and buffers should be fully equilibrated to room temperature before cap removal. Applying a room-temperature buffer to a cold column can cause bubble formation within the resin, resulting in distorted elution profiles and reduced reproducibility.
   3. Stand the column in an upright position and carefully remove the top cap of the column.
   4. Remove the bottom cap and allow the buffer to drain completely out of the column.
      ​NOTE: Ensure that the storage buffer has fully drained from the column before equilibration.
   5. Equilibrate the column by flushing with three column volumes (25.5 mL) of degassed 0.22 µm VIB buffer.
2. EV loading and sample collection
   1. Use 100 µg of total EV protein for each SEC purification. Prior to loading onto the SEC column, adjust EV samples to a final volume of 500 µL using VIB.
   2. Apply the 500 µL EV suspension directly to the loading frit at the top of the column.
   3. Allow the sample to fully enter the resin bed before adding VIB buffer. Maintain continuous column flow throughout the separation to ensure accurate size-based elution.
      NOTE: Column flow should remain continuous throughout the separation. Allowing the column to run dry or pausing the flow for extended periods disrupts size-based separation and can lead to fraction overlap and reduced EV-enrichment.
   4. After the EV sample (500 µL) has fully entered the resin bed, add 2 mL of VIB and allow it to elute completely. This eluate corresponds to the column dead volume and should be discarded. EV fractions were then collected by sequentially adding 400 µL of VIB and collecting the eluate, repeating this process to obtain fractions 1-6 in 1.5 mL microcentrifuge tubes.
      ​NOTE: The dead volume should not contain EVs. Based on validation using the 35 nm column, EVs are typically recovered in fractions 1-3. If needed, the dead volume can be assessed by nanoparticle tracking analysis (NTA) or immunoblotting for EV markers to confirm the absence of EVs.
3. Column cleaning and storage
   1. Immediately after fraction collection, flush the column with one column volume (8.5 mL) of 0.5 M NaOH to sanitize the resin. Then, flush the column with 17 mL of VIB to restore neutral pH. The column is then ready for loading of the next sample.
      ​NOTE: Flushing the column with buffer alone is insufficient to remove residual proteins or vesicles. The NaOH cleaning step is essential to prevent carry-over between samples, particularly for downstream nucleic acid or RNA analyses.
   2. For long-term storage, flush the column with two column volumes of deionized water after cleaning, followed by two column volumes of 20% ethanol for storage. Recap the column and store it upright.
      ​NOTE: Rinsing the column with deionized water prior to ethanol storage prevents salt crystallization within the resin bed, which preserves column performance.

6. EV validation, quantification, and size/morphology assessment

1. Nanoparticle tracking analysis (NTA)
   1. Measure the EV size distribution and particle concentration by NTA.
   2. Dilute the EV samples at 1:100 in sterile, 0.2 µm-filtered VIB to achieve a particle concentration within the optimal detection range (approximately 10⁷-10⁹ particles/mL).
   3. Gently mix the diluted samples to avoid bubble formation and load them into the instrument equipped with a 532 nm laser and a high-sensitivity sCMOS camera.
   4. Perform data acquisition and analysis using NS Xplorer software with a detection threshold of 10 nm (up to 1,000 nm), applying a dynamic observation volume-based concentration algorithm.
2. Western blot analysis of EV markers
   1. For Western blot analysis, wash and resuspend the EV fractions obtained after sucrose gradient separation in 50 µL of VIB. Mix an aliquot of 10 µL from each fraction with SDS loading buffer, denatured, and resolved on a 12% Tris-glycine gel prior to immunoblotting with EV marker TET8 antibodies.
   2. For SEC-derived EV fractions, first concentrate 400 µL of each fraction to 50 µL using a 100 kDa molecular weight cutoff centrifugal filter. Then, use 10 µL of the concentrated sample for Western blot analysis as described above.
3. Transmission electron microscopy (TEM)
   1. Deposit 5 µL of EVs suspended in VIB onto glow-discharged carbon-formvar 300-mesh grids. Then, wick off the excess sample using filter paper.
   2. Then, negatively stain the grid with 3 µL of electron microscopy stain, wick off with filter paper, and allow it to air dry before imaging with a 300 kV TEM.

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