Protocol for Genetic Manipulation of Fungi

A95 (CBS 123872) was used as recipient (WT) for genetic manipulation. A95 and derivatives (Tables S2 and S3) were cultivated in malt extract broth/agar (MEB/MEA)^{24 (link)} at 25 °C. Osmotically stabilised media (MEBS, MEAS) contained 10.8% (w/v) sucrose^{34 (link)}. Transformants were incubated on MEA containing 24 µg ml⁻¹ hygromycin B (HYG) (Duchefa Biochemie) and/or 5 µg ml⁻¹ nourseothricin (NAT) (Carl Roth GmbH). Basal synthetic media for growth assays were SD [2% (w/v) glucose, 0.67% (w/v) Difco Yeast Nitrogen Base without Amino Acids (BD Biosciences), 2% (w/v) agar] and SD/NO₃ [2% (w/v) glucose, 0.17% (w/v) Difco Yeast Nitrogen Base without Amino Acids and Ammonium Sulfate (BD Biosciences), 0.3% (w/v) NaNO₃, 2% (w/v) agar], which were supplemented with 50 mg l⁻¹ uracil (URA), 100 mg l⁻¹ adenine (ADE), 1 g l⁻¹ 5-fluoroorotic acid (5-FOA), 48 g l⁻¹ KClO₃ and/or 0.5 g l⁻¹ yeast extract (YE). For inoculation of growth assays, one inoculation loop of cells were taken from surface-grown colonies, transferred to 0.6 ml phosphate-buffered saline (PBS) containing 6–8 glass beads (3–5 mm), and dispersed using a TissueLyser (Retsch) (2 × 20 s at 40 Hz). Dispersed cells were transferred to fresh tubes, cell titers were determined using a Thoma cell counting chamber and adjusted with PBS to 5 × 10⁵ cells ml⁻¹. Solid media were inoculated with 10 µl droplets containing 5 × 10³ cells.