Culturing and Genetic Manipulation of Magnaporthe oryzae

M. oryzae wild type Guy11 [47 ] and all transformants were routinely cultured on complete medium (CM) at 28°C for 3 to 14 days [48 (link),49 (link)]. To isolate genomic DNA, the fungus was cultivated in liquid CM for 3 days. Lipid medium, glucose medium and sodium acetate medium were prepared as described [34 (link)]. All fungal transformants were generated by Agrobacterium tumefaciens-mediated transformation (AtMT) as described [50 (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) [32 (link)] containing 100 μg/ml chlorimuron ethyl (Sigma) were used for screening the corresponding transformants. Cell wall integrity was tested by growing the strains on CM supplemented with 100 μg/ml Congo red. The tolerance of the strains to ROSs was evaluated by the growth on CM containing 2.5 or 5.0 mM H₂O₂ or 1 mM methyl viologen.