Secreted Protein Extraction and Western Blotting

A trichloroacetic acid (TCA) precipitation method was used to extract the fungal secreted proteins. The fungal isolates were cultured in YG liquid medium for 3 days at room temperature. The resulting fungal culture was centrifuged at 13,000 g for 10 min and filtered through a 0.22 μM filter to remove hyphae and conidia. TCA was added to a final concentration of 10%, and the mixture chilled at 4°C overnight. The solution was centrifuged at 13,000 g for 10 min, and the resulting pellets were washed with ice-cold acetone at least twice. The final protein pellets were dissolved in 50 mM Tris–HCl buffer (pH = 7.5), and the protein concentration was determined using a Qubit 3.0 Fluorometer with the Qubit^TM Protein Assay Kit (Thermo Fisher Scientific).

Western blotting was conducted with a total of 5 μg of the secreted proteins which were separated by 10% SDS-PAGE gel electrophoresis and stained with Coomassie staining solution (0.1% Coomassie Brilliant Blue R-250, 50% methanol, and 10% glacial acetic acid). The proteins in the SDS-PAGE gel were transferred to a nitrocellulose filter membrane (GE Healthcare, Chicago, IL) using the Mini Trans-Blot^® Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, United States). Anti-GFP antibody (Rockland Immunochemicals, Gilbertsville, PA, United States) and an ECL chemiluminescent detection kit (GE Healthcare) were used to detect the resulting proteins.