Optima xpn

Membrane-associated proteins were purified following a previously established protocol [16 (link)] with modifications. In summary, frozen mycelium samples (2–3 g, press-dried) from fungal cultures grown for 6, 24, and 48 h were mechanically disrupted by griding using the mortar and pestle tools while immersed in liquid nitrogen. The resulting material was then resuspended in a 20 mM HEPES buffer with a pH of 7.6, supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), and 150 mM NaCl. Subsequently, the samples underwent a centrifugation step at 500×g for 5 min. The resulting supernatant was collected and further centrifuged at 5000×g for 20 min. The remaining supernatants were subjected to a final centrifugation step (~ 85,000×g) for 120 min using a Beckman Coulter Optima XPN centrifuge. The obtained precipitate was resuspended in a solution containing 100 mM of triethylammonium bicarbonate and 1% sodium deoxycholate detergent. This resuspended mixture was vigorously mixed using a vortex mixer and incubated in a thermomixer at 95 °C and 800×g for 5 min. The Pierce™ BCA Protein Assay Kit was used to quantify the protein associated content of the plasma membrane fraction.

EVs were isolated from broth cultures using methods adapted from Thery et al. and Rodrigues et al. [59 (link), 60 ]. Z. tritici cells were removed from broth cultures by centrifugation at 4500×g for 25 min in a benchtop centrifuge. Culture supernatant was centrifuged at 15,000×g (4 °C) for 45 min in an Avanti J series High Performance centrifuge (Beckman coulter) with a JA 14.50 rotor to remove cell debris. Supernatant was carefully removed and passed through a 0.45 µm MF-Millipore membrane filter using vacuum filtration. EVs were pelleted from the culture filtrate by ultracentrifugation for 75 min at 100,000×g and 4 °C in open-top Ultra Clear 38.5 mL tubes (Beckman coulter, 344058) using an SW 32 Ti swinging bucket rotor and Optima XPN ultracentrifuge (Beckman Coulter). Supernatant was removed completely and the EV pellet washed with 1X phosphate buffered saline (PBS), pH 7.4 (filter sterilised with 0.22 µm membrane), by ultracentrifugation at 100,000×g (75 min, 4 °C). The EV pellet was resuspended in 50 to 100 µL of 1X PBS, pH 7.4 and protein content measured using the Qubit™ protein assay kit. EV samples were flash-frozen with LN₂ and stored at − 80 °C. This process was repeated with a Fries3-only control to confirm EV-like particles were not an artefact of the growth medium.

Exosome isolation was prepared as described [47 (link)]. Before exosome isolation, BMSCs were cultured in the exosome-free medium to avoid interference from bovine exosomes. After 72 h, cell culture medium was collected, and exosomes were isolated from the supernatant by serial centrifugation of 300× g for 10 min, 2000× g for 15 min and 12,000× g for 30 min to remove floating cells and cellular debris. The supernatant was then passed through a 0.22-μm filter (Millipore, USA). The percolate was collected and centrifuged at 150,000× g for 90 min at 4 °C (Optima™XPN, Beckman Coulter) to obtain the exosome precipitate. The pellet was resuspended in 1 mL of sterile PBS and filtered through a 0.22-μm syringe-driven filter unit (Millipore). Finally, exosomes were stored in aliquots of 200 μL at −80 °C until being used.