EV isolation demonstration.

Prepare a 30 ml syringe by removing the plunger and creating additional drainage holes using heated forceps. Punch holes into a 50 ml conical tube cap and melt the inner circle to create an opening, then carefully attach the cap to the syringe barrel.

Cut six-week-old Arabidopsis plants at the root, keeping a small root segment attached. Weigh the fresh plants and gently rinse them three times with deionized water to remove soil and particulates.

Place rinsed plants into a French press with leaf bottoms facing down, cover with sterile vesicle isolation buffer, and vacuum infiltrate for 20 seconds to drive air from leaf intercellular spaces and replace it with buffer.

Lift the plunger and remove plants from the French press, then gently flick and dab them against paper towels to remove excess buffer. Carefully package the infiltrated plants into modified syringes, placing approximately three plants per syringe.

Insert the loaded syringes into 50 ml conical tubes suspended in plastic bottles, place into a centrifuge rotor, and spin at 700 G for 20 minutes at 4°C to collect the apoplastic wash fluid at the tube bottom.

Remove infiltrated plants from syringes and harvest one healthy young leaf from each biological control sample. Freeze the collected leaf tissue in liquid nitrogen for later extraction of total protein.

Aliquot approximately 500 microliters of apoplastic wash into polystyrene ultracentrifugation tubes and bring each sample volume to 3 milliliters with cold vesicle isolation buffer. Balance samples and prepare for differential ultracentrifugation to pellet extracellular vesicles.