Fungal Genetic Manipulation Protocol

M. oryzae wild-type Guy-11 [40] and all its derivative transformants and mutants strains were routinely cultured on complete medium (CM) [41] (link) at 28°C for 3–14 days [42] (link). For genomic DNA isolation, mycelia were cultured in liquid CM for 3 days. Lipid medium, glucose medium and sodium acetate medium were prepared as described previously [33] (link). All fungal transformants were generated by Agrobacterium tumefaciens-mediated transformation (AtMT) as described previously [43] (link). CM plates containing 250 μg/ml hygromycin B (Roche, Mannheim, Germany), 200 μg/ml glufosinate–ammonium (Sigma, St Louis, MO, USA) or 800 μg/ml G418 (Sigma) and defined complex medium (DCM; 0.16% yeast nitrogen base without amino acids, 0.2% asparagine, 0.1% ammonium nitrate and 1% glucose, pH 6.0 with Na₂HPO₄) [8] (link) containing 100 μg/ml chlorimuron ethyl (Sigma) were used for screening corresponding transformants.