2.3. Generation and Transformation of Fungal Protoplasts.

Fungal protoplasts were generated and transformed as described in Turgeon et al. [30]. In brief, C. heterostrophus, race T strain C4, was grown in 90 mm Petri dishes with (complete medium, xylose, CMX), at 22 °C for about 1 week, in a 12 h light/12 h dark regime. A conidial suspension from these plates was germinated overnight, as follows: the spores were inoculated to 150 mL (complete medium, CM), in a rotary shaker for 18 h at 30 °C, 200 rpm, and used for the preparation of the protoplasts. The fungal suspension was centrifuged at 8000 rpm for 10 min (Sorvall, Thermo Fisher Scientific, Waltham, MA, USA; SLA1500 rotor). The resulting mycelium sediment was suspended with 70 mL enzyme-osmoticum solution (see [30]; β-glucanase was replaced by an equivalent amount, following calibration, of VinoTaste enzyme preparation, Novozymes A/S, Bagsvaerd, Denmark) and divided into five 125 mL Erlenmeyer flasks, followed by incubation for 2.5 h at 30 °C, 70 rpm, in a rotary shaker. The protoplasts released were filtered through gauze. The filtrate was then centrifuged at 4500 rpm for 5 min at 4 °C. The protoplast pellets were washed with 10 mL 0.7 M NaCl, followed by a second wash in 10 mL sorbitol–Tris–calcium (reagents were from Sigma, Saint Louis, MO, USA, unless noted otherwise) buffer (STC, see [30]).

The resulting protoplasts were suspended in 200 µL of STC and counted. A concentration of 10⁸ cells/mL was obtained, and of these, 10⁷ protoplasts were used for the transformation. The protoplasts were incubated on ice with 25 µg DNA for 12 min (a negative control without DNA was subjected to the same procedure). Polyethylene glycol was added in three aliquots of 200, 200, and 800 µL each, and then diluted with 1 mL STC and plated for regeneration. A 1% agar overlay containing 100 µg/mL Hygromycin B (HYG, catalog number H0654, Sigma, Saint Louis, MO, USA) was added after 18 h, for selection. For the protoplast viability determination, one of the control plates was overlayed with medium without HYG.