FIG. 5.
Phosphorylation of Os-2p MAPK of N. crassa. (A) Treatment with fungicides. Mycelia of the wild-type strain 74-OR8-1a (WT) and the os-1 mutant Y256M209 (os-1) were used for analysis. Prepared mycelia of the strain tested were incubated in liquid Vogel's medium with 10 or 100 μg/ml iprodione (Ipr) for 10 min or with 10 or 100 μg/ml fludioxonil (Flu) for 10 min. Prepared mycelia were incubated in CM without iprodione and fludioxonil as a control. Mycelia of the os-2 mutant UCLA80 (os-2) were also exposed to liquid Vogel's medium with or without 100 μg/ml iprodione. The cells were harvested, and total protein extracts were prepared as described in Materials and Methods. Protein samples (50 μg) were subjected to SDS-PAGE and blotted onto nitrocellulose membranes for Western blot analysis. Phosphorylated Os-2p was detected using anti-dually phosphorylated p38 antibody (indicated by anti-TGYp). The total amount of BmHog1p was measured using anti-Hog1 C terminus antibody (indicated by anti-Hog1). Os-2p was detected using anti-dually phosphorylated p38 antibody (indicated by anti-TGYp). The total amount of BmHog1p was measured using anti-Hog1 C terminus antibody (indicated by anti-Hog1). (B) Under osmotic stress. Mycelia of the wild-type strain 74-OR8-1a (WT) and the os-1 mutant Y256M209 (os-1) were used for analysis. Prepared mycelia of the strain tested were incubated in liquid Vogel's medium with 0.2, 0.4, or 0.8 M KCl for 10 min. Prepared mycelia were incubated in liquid Vogel's medium without KCl as a control. The procedures for Western blot analysis were the same as those described above.