Isolation of C. orbiculare Protoplasts

C. orbiculare protoplasts were prepared as previously described (Kubo, 1991 (link); Rodriguez and Yoder, 1987 (link); Vollmer and Yanofsky, 1986 (link)) with modifications. A frozen glycerol stock of C. orbiculare was streaked on 3.9% (w/v) potato dextrose agar (PDA) medium (Nissui) in a 90 mm dish and incubated at 25°C in the dark for 3 days. Outer edges of a colony were transferred to 20 ml of 2.4% (w/v) potato dextrose broth (BD Biosciences) and incubated for 2 days at 25°C in the dark. The proliferated mycelium was collected using a 70 µm cell strainer (Corning) and incubated in 150 ml of potato-sucrose liquid medium supplemented with 0.2% yeast extract (BD Biosciences) at 25°C with shaking at 140 rpm. The mycelium was harvested, washed with sterile water, and resuspended in 20 ml of filter-sterilized (0.2 µm pore size, GE Healthcare) osmotic medium (1.2 M MgSO₄ and 5 mM Na₂HPO₄) containing 10 mg/ml driselase from Basidiomycetes sp. (Sigma-Aldrich) and 10 mg/ml lysing enzyme from Trichoderma harzianum (Sigma-Aldrich) in a 50 ml tube (Falcon, Corning). The suspension was gently agitated in a rotary shaker at 60 rpm for 90 min at 30°C. Then, the suspension was underlaid with 20 ml of trapping buffer (0.6 M sorbitol, 50 mM Tris-HCl pH 8.0, and 50 mM CaCl₂) and centrifuged at 760 × g for 5 min using a swinging-bucket rotor (Hitachi, T4SS31). Protoplasts isolated from the interface of the two layers were pelleted, washed twice using STC (1 M sorbitol, 50 mM Tris-HCl pH 8.0, and 50 mM CaCl₂), resuspended in STC at 10⁸–10⁹ protoplasts/ml, added to a 25% volume of polyethylene glycol (PEG) solution (40% [w/w] PEG3350, 500 mM KCl, 40 mM Tris-HCl pH 8.0, and 50 mM CaCl₂), and stored at −80°C until use.