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

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.