Name: Isolation and Purification of Plant Extracellular Vesicles from Arabidopsis Leaves Using an Optimized Apoplastic Wash Collection Method (Video) | JoVE Journal
Uploaded: 2026-03-24T12:50:00.000+00:00
Duration: 9 min 38 s
Description: Learn a standardized protocol for isolating plant extracellular vesicles from Arabidopsis thaliana leaves using gentle infiltration and density gradient purification. This method minimizes contamination for reliable downstream analyses.

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5 minutes

B.D. is supported by the Pathways to the Doctorate Fellowship from Texas A&#38;M University. B.H. is supported by start-up funds from Texas A&#38;M University and the United States Department of Agriculture HATCH 7009138 project number: TEX08074.

NOTE: Conduct centrifugation steps in a refrigerated centrifuge at 4 &#176;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
<ol>
	<li>Prepare 1 L vesicle isolation buffer (VIB) consisting of 20 mM MES hydrate, 2 mM CaCl2, 0.1 M NaCl, pH 6.</li>
	<li>Harvest the distal blade zones of leaves by cutting at the base of the blade to remove the petiole. 
	&#8203;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.</li>
	<li>Wash leaves with reverse osmosis (RO) water three times. Place them on paper towels and gently pat them dry.</li>
	<li>Load approximately 50 mL of leaves into a 120 mL syringe. Lightly tap the syringe to settle the leaves.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Once leaves have been infiltrated, pull out the plunger. Pour out the excess buffer into the sink.</li>
	<li>Repeat steps 1.4-1.7 until all harvested leaves are infiltrated (Figure 1A).</li>
</ol>
2. Collection of apoplastic washing fluid (AWF)
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>Centrifuge the wrapped syringe containing infiltrated leaves at 900 &#215; g for 10 min at 4 &#176;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.</li>
</ol>
3. Arabidopsis EV isolation from AWF via differential centrifugation
<ol>
	<li>Centrifuge the collected apoplastic washing fluid at 2,000 g for 20 min at 4 &#176;C using a refrigerated centrifuge equipped with a swing-bucket rotor, with standard acceleration and deceleration settings. After centrifugation, use a 0.45 &#181;m nylon cell filter to remove large cellular debris from the supernatant. Discard the pellet. Transfer the filtered supernatant into new centrifuge tubes (Figure 2).</li>
	<li>Centrifuge the filtered supernatant at 10,000 &#215; g for 30 min at 4 &#176;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).</li>
	<li>Transfer the supernatant into 17 mL ultracentrifuge tubes and ultracentrifuge at 100,000 &#215; g for 1 h at 4 &#176;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&#39;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.</li>
	<li>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 &#181;L yields protein concentrations of approximately 1-1.5 &#181;g/&#181;L and particle concentrations approaching 109-1010 particles/mL.</li>
	<li>Store the resuspended EVs at 4 &#176;C for immediate use or at -80 &#176;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.</li>
</ol>
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.
<ol>
	<li>Top-loading sucrose gradient separation of EVs
	<ol>
		<li>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 &#176;C water bath to aid in dissolving the 70% and 90% stocks.</li>
		<li>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. 
		&#8203;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.</li>
		<li>Mix the resuspended EV pellet (~100 &#181;g 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.</li>
		<li>Centrifuge at 100,000 g for 16 h at 4 &#176;C. 
		&#8203;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.</li>
		<li>Collect six 2 mL fractions per sample by pipetting from the top. Transfer each fraction into a new 17 mL ultracentrifuge tube.</li>
		<li>Resuspend the collected fractions in a ~16 mL VIB buffer and centrifuge at 100,000 g for 1 h at 4 &#176;C.</li>
		<li>Discard the supernatant, then resuspend each pellet in 50 &#181;L of VIB. 
		&#8203;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 &#181;L for higher concentration).</li>
	</ol>
	</li>
	<li>Bottom-loading sucrose gradient separation of EVs
	<ol>
		<li>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 &#176;C water bath to aid in dissolving the 70% and 90% stocks.</li>
		<li>Mix the resuspended EV pellet (~100 &#181;g of EV protein) with the 1 mL of 90% sucrose solution. Then, layer this mixture as the first layer of a discontinuous layer.</li>
		<li>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.</li>
		<li>Centrifuge at 100,000 g for 72 h at 4 &#176;C. 
		&#8203;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.</li>
		<li>Collect six 2 mL fractions per sample by pipetting from the top. Transfer each fraction into a new 17 mL ultracentrifuge tube.</li>
		<li>Resuspend the collected fractions in a ~16 mL of VIB buffer and centrifuge at 100,000 g for 1 h at 4 &#176;C.</li>
		<li>Discard the supernatant, then resuspend each pellet in 50 &#181;L of VIB buffer. 
		&#8203;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 &#181;L for higher concentration).</li>
	</ol>
	</li>
</ol>
5. Use of size exclusion chromatography (35 nm column) to purify EVs
<ol>
	<li>Buffer preparation and column equilibration
	<ol>
		<li>Prepare VIB and all additional buffers fresh. Filter all buffers through a 0.22 &#181;m membrane and degas in ultrasonic bath prior to use to prevent air bubble formation and pore blockage. 
		&#8203;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.</li>
		<li>Allow the column and buffers to equilibrate to room temperature (18-24 &#176;C) before removing column caps. 
		&#8203;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.</li>
		<li>Stand the column in an upright position and carefully remove the top cap of the column.</li>
		<li>Remove the bottom cap and allow the buffer to drain completely out of the column. 
		&#8203;NOTE: Ensure that the storage buffer has fully drained from the column before equilibration.</li>
		<li>Equilibrate the column by flushing with three column volumes (25.5 mL) of degassed 0.22 &#181;m VIB buffer.</li>
	</ol>
	</li>
	<li>EV loading and sample collection
	<ol>
		<li>Use 100 &#181;g of total EV protein for each SEC purification. Prior to loading onto the SEC column, adjust EV samples to a final volume of 500 &#181;L using VIB.</li>
		<li>Apply the 500 &#181;L EV suspension directly to the loading frit at the top of the column.</li>
		<li>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.</li>
		<li>After the EV sample (500 &#181;L) 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 &#181;L of VIB and collecting the eluate, repeating this process to obtain fractions 1-6 in 1.5 mL microcentrifuge tubes. 
		&#8203;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.</li>
	</ol>
	</li>
	<li>Column cleaning and storage
	<ol>
		<li>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. 
		&#8203;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.</li>
		<li>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. 
		&#8203;NOTE: Rinsing the column with deionized water prior to ethanol storage prevents salt crystallization within the resin bed, which preserves column performance.</li>
	</ol>
	</li>
</ol>
6. EV validation, quantification, and size/morphology assessment
<ol>
	<li>Nanoparticle tracking analysis (NTA)
	<ol>
		<li>Measure the EV size distribution and particle concentration by NTA.</li>
		<li>Dilute the EV samples at 1:100 in sterile, 0.2 &#181;m-filtered VIB to achieve a particle concentration within the optimal detection range (approximately 10&#8311;-10&#8313; particles/mL).</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Western blot analysis of EV markers
	<ol>
		<li>For Western blot analysis, wash and resuspend the EV fractions obtained after sucrose gradient separation in 50 &#181;L of VIB. Mix an aliquot of 10 &#181;L from each fraction with SDS loading buffer, denatured, and resolved on a 12% Tris-glycine gel prior to immunoblotting with EV marker TET8 antibodies.</li>
		<li>For SEC-derived EV fractions, first concentrate 400 &#181;L of each fraction to 50 &#181;L using a 100 kDa molecular weight cutoff centrifugal filter. Then, use 10 &#181;L of the concentrated sample for Western blot analysis as described above.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Deposit 5 &#181;L of EVs suspended in VIB onto glow-discharged carbon-formvar 300-mesh grids. Then, wick off the excess sample using filter paper.</li>
		<li>Then, negatively stain the grid with 3 &#181;L of electron microscopy stain, wick off with filter paper, and allow it to air dry before imaging with a 300 kV TEM.</li>
	</ol>
	</li>
</ol>

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<li>Baldrich, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+extracellular+vesicles+contain+diverse+small+RNA+species+and+are+enriched+in+10-+to+17-nucleotide+%26%2334%3Btiny%26%2334%3B+RNAs%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Plant extracellular vesicles contain diverse small RNA species and are enriched in 10- to 17-nucleotide &#34;tiny&#34; RNAs.</a> Plant Cell. 31 (2), 315-324 (2019).</li>
<li>Regente, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicular+fractions+of+sunflower+apoplastic+fluids+are+associated+with+potential+exosome+marker+proteins%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins.</a> FEBS Lett. 583 (20), 3363-3366 (2009).</li>
<li>Rutter, B. D., Innes, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growing+pains%3A+Addressing+the+pitfalls+of+plant+extracellular+vesicle+research%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">Growing pains: Addressing the pitfalls of plant extracellular vesicle research.</a> New Phytol. 228 (5), 1505-1510 (2020).</li>
<li>Thieron, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Practical+advice+for+extracellular+vesicle+isolation+in+plant-microbe+interactions%3A+Concerns%2C+considerations%2C+and+conclusions%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Practical advice for extracellular vesicle isolation in plant-microbe interactions: Concerns, considerations, and conclusions.</a> J Extracell Vesicles. 13 (12), e70022(2024).</li>
<li>Borniego, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+plant+Rnas+coat+Arabidopsis+leaves+and+are+distinct+from+apoplastic+RNAs%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Diverse plant Rnas coat Arabidopsis leaves and are distinct from apoplastic RNAs.</a> Proc Natl Acad Sci U S A. 122 (1), e2409090121(2025).</li>
<li>Chen, A., He, B., Jin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+extracellular+vesicles+from+Arabidopsis%5BTitle%5D)+AND+%22Curr+Protoc%22%5BJournal%5D)">Isolation of extracellular vesicles from Arabidopsis.</a> Curr Protoc. 2 (1), e352(2022).</li>
<li>Koch, B. L., Rutter, B. D., Innes, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arabidopsis+produces+distinct+subpopulations+of+extracellular+vesicles+that+respond+differentially+to+biotic+stress%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Arabidopsis produces distinct subpopulations of extracellular vesicles that respond differentially to biotic stress.</a> bioRxiv. , (2024).</li>
<li>Hamby, R., Cai, Q., Jin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+communication+between+organisms+inspires+innovative+eco-friendly+strategies+for+disease+control%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">RNA communication between organisms inspires innovative eco-friendly strategies for disease control.</a> Nat Rev Mol Cell Biol. 26 (2), 81-82 (2025).</li>
<li>Hou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+phytophthora+effector+suppresses+trans-kingdom+RNAi+to+promote+disease+susceptibility%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">A phytophthora effector suppresses trans-kingdom RNAi to promote disease susceptibility.</a> Cell Host Microbe. 25 (1), 153-165.e5 (2019).</li>
</ol>

The authors disclose no conflict of interest.

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 capsici10,...

Extracellular vesicles (EVs) are lipid bilayer-bound membrane particles secreted by a wide range of organisms, from bacteria to humans1,2. 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 research7,8. 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 bodies9,10, syntaxin protein Penetration 1(PEN1)-positive EVs11, exocyst-positive organelle (EXPO)-derived EVs12,13, 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 pathogens10,16,17,18,19. These findings highlight EVs as vehicles of &#34;RNA warfare&#34;, 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 complexes25,26. Recent reviews and methodological studies have provided important guidance and best-practice considerations for plant EV isolation and characterization27,28,29,30,31. 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.

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

isolation and purification of plant extracellular vesicles from arabidopsis leaves using an optimized apoplastic wash collection method

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

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.

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...

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

Extracellular vesicles (EVs) are membrane-bound nanoparticles secreted by diverse organisms and cell types and are increasingly recognized as important ...

Our research focus on plant extracellular vesicles and their function in plant microbe interaction. The major limitation in our plant extracellular vesicles research field is the isolation methods vary across different labs which cause inconsistent results. Well, this work will provide a standardized, reliable, and reproducible workflow to isolate plant extracellular vesicles.To begin, cut the Arabidopsis thaliana plant shoots from the soil. Harvest the distal blade zones of leaves by cutting at the base of the blade to remove the petiole. Wash the leaves three times with RO water.Place the leaves on paper towels and gently pat dry. Then load the leaves into a 120-milliliter syringe, and tap gently to settle them till the 50-milliliter mark. Add approximately 60 milliliters of vesicle isolation buffer, or VIB, into the syringe until leaves are fully immersed.Gently press the plunger to expel trapped air. Seal the syringe tip with a piece of parafilm folded four times to prevent leakage. To infiltrate leaves, pull the plunger outward to create a vacuum.Now hold the plunger in the extended position for five seconds, then slowly release. Repeat the process two to three times until leaves appear darker and translucent indicating successful infiltration. Perform the next steps for the collection of the apoplastic wash fluid, or AWF, collaboratively to expedite completion and minimize stress to the leaves.On a clean surface, cut a 12-inch strip of scotch tape, and place its sticky side up. Wrap one end of the tape around an empty one-milliliter needleless syringe just below the wide end. Lay 50 to 70 infiltrated leaves on the tape overlapping each other with bases toward the syringe and tips upward.Wrap the tape-bound infiltrated leaves tightly around the syringe. Secure the top with two strips of tape. Place the wrapped syringe into a 50-milliliter conical tube with the cut leaf ends and syringe base facing down.Now, spin the conical tube containing the wrapped syringe in a refrigerated centrifuge equipped with a fixed-angle rotor. Then collect the supernatant or the AWF in a new 15-milliliter conical tube and place the sample on ice. Centrifuge the AWF using a swing bucket rotor with standard acceleration and deceleration settings.To remove large cellular debris, filter the supernatant through a 0.45-micron nylon cell filter into clean labeled centrifuge tubes. Centrifuge the filtered supernatant using a refrigerated centrifuge equipped with a fixed-angle rotor, applying standard acceleration and deceleration settings. Transfer the supernatant into 17-milliliter ultracentrifuge tubes, and spin the samples in an ultracentrifuge equipped with a swing bucket rotor.After centrifugation, discard the supernatant and wash the pellet in approximately 16 milliliters of VIB. Repeat the ultracentrifugation washing cycle as demonstrated to collect the extracellular vesicles or EVs. After discarding the supernatant, gently scrape the bottom of the tube in 50 microliters of VIB to recover the EV pellet.Keep 10 to 90%sucrose gradient stalks in fixed concentrations, freshly prepared and ready to use. To prepare the top-loading discontinuous sucrose gradient, begin by taking a clean 17-milliliter ultracentrifuge tube. Using a pipette, carefully add one milliliter of the most concentrated sucrose solution to the bottom of the tube.Next, gently layer one milliliter of the next lower concentration sucrose solution on top of the first layer. Continue layering using descending concentrations of sucrose omitting the 10%solution. Add 1.1 milliliters of the previously prepared mixture containing 100 microliters of EV and one milliliter of 10%sucrose solution as the final layer in the ultracentrifuge tube to yield a total volume of about 12.1 milliliters.Spin the top-loaded sucrose gradient mixture in an ultracentrifuge. After ultracentrifugation, collect two-milliliter fractions per sample by pipetting from the top, and transfer each fraction into a new 17-milliliter ultracentrifuge tube. Add about 16 milliliters of VIB to each fraction.Centrifuge them and resuspend each pellet in 50 microliters of VIB. For bottom loading, carefully load 1.1 milliliter of the prepared mixture containing 100 microliters of EV and one milliliter of 90%sucrose to the bottom of the 17-milliliter ultracentrifuge tube to form the first layer of the discontinuous sucrose gradient. Next, gently layer one milliliter of the next lower concentration sucrose solution on top of the first layer.Continue layering using descending concentrations of the sucrose. After preparing the bottom-loaded gradient, carry out the centrifugation and fraction collection steps as demonstrated for the top-loaded method. Prepare VIB and all additional buffers fresh, and filter the buffers through a 0.22 micron membrane.Degas the buffer in the ultrasonic bath before use. Set up the size exclusion chromatography column with a 35-nanometer-pore size at room temperature and open the top cap. Then remove the bottom cap and allow the buffer to drain from the column.To equilibrate the column, flush the column with three column volumes of VIB buffer. Then apply 500 microliters of VIB-adjusted EV suspension directly to the loading frit at the top of the column. Wait for it to fully enter the resin bed.Now, add two milliliters of VIB, and let it elute completely. After discarding the eluate, add 400 microliters of VIB and collect 400 microliters of the eluate as fraction one in 1.5-milliliter microcentrifuge tubes. Repeat the process to collect a total of six fractions.The preparation of leaves using the optimized protocol described in this article showed less cell damage than the existing method. Consistently, the AWF produced using the optimized protocol contained less chlorophyll levels and a weaker chloroplast marker protein signal indicating low tissue damage and cytoplasmic contamination. Sucrose density gradient centrifugation effectively enriched and purified EVs.The EVs were primarily detected in fractions four and five with fraction four showing the highest particle concentration. Transmission electron microscope analysis of fraction four confirmed vesicles of the expected size and typical cup-shaped morphology. Size exclusion chromatography effectively purified plant EVs and nanoparticle tracking analysis.And transmission electron microscope analysis of the resulting fraction confirmed vesicles of expected size with typical cup-shaped morphology. This work will provide researchers a protocol to get a pure plant extracellular vesicles, which can be used to characterize their cargoes, including RNA, protein, metabolites, and other molecular cargoes. The key challenge in plant extracellular vesicle field is how to get a pure sample that are free from contaminations from the intracellular components.This work will help researchers to get a pure plant extracellular vesicles to further study their function in plant development, in plant microbe interactions.

isolation-purification-plant-extracellular-vesicles-from-arabidopsis

Research

JoVE Journal

Biology

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Source: Rufián, J. S., et al, Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue. J. Vis. Exp. (2022)This video demonstrates a syringe-based pressure method for extracting apoplastic bacteria, which proliferate and form microcolonies within the apoplast, the intercellular space between plant cells. The method yields debris-free bacterial samples suitable for downstream single-cell analysis.

Extraction of Apoplastic Bacteria Using a Syringe-Based Pressure Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

mRNA Interactome Capture from Plant Protoplasts

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein

We describe a simple yet highly reproducible method to isolate extracellular RNA from the leaf surface and apoplast of Arabidopsis plants.

A Stepwise Guide to the Isolation and Analysis of Leaf Surface and Apoplastic RNA Using Arabidopsis Rosettes

Learn a standardized protocol for isolating plant extracellular vesicles from Arabidopsis thaliana leaves using gentle infiltration and density gradient purification. This method minimizes contamination for reliable downstream analyses.

Isolation and Purification of Plant Extracellular Vesicles from Arabidopsis Leaves Using an Optimized Apoplastic Wash Collection Method (Video) | JoVE Journal

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

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