(A) Localization of MoOsm1, MoOsm1Y173D, and MoOsm1Y173A in conidia. MoOsm1-GFP, MoOsm1Y173D-GFP, MoOsm1Y173A-GFP, and H1-RFP were observed by confocal fluorescence microscopy. Bars = 5 μm. (B) Nuclear/cytoplasmic proteins harvested from conidia of ∆Moosm1/MoOsm1-GFP, ∆Moosm1/MoOsm1Y173D-GFP, and ∆Moosm1/MoOsm1Y173A-GFP strains were separated by SDS-PAGE. The intensity of MoOsm1 was compared among total proteins, nuclear proteins, and cytoplasmic proteins. ‘Y’ represents MoOsm1, ‘D’ represents MoOsm1Y173D, and ‘A’ represents MoOsm1Y173A. Bars denote standard errors from three independent experiments. Asterisks indicate significant differences (Duncan's new multiple range test p<0.01). (C) Fluorescence intensity of MoOsm1-GFP/H1-RFP in ∆Moosm1/MoOsm1-GFP, ∆Moosm1/MoOsm1Y173A-GFP, and ∆Moosm1/MoOsm1Y173D-GFP strains. The number represents the quantification of GFP and RFP signals by ImageJ. (D) Co-IP assay. Western blot analysis of total proteins (T) extracted from various transformants, suspension proteins (S), and elution proteins (E) eluted from anti-GFP beads. MoOsm1-GFP, MoOsm1Y173D-GFP, MoOsm1Y173A-GFP, and MoOsm1-FLAG were detected with respective antibodies. (E) Immunoblot analysis of proteins extracted from MoOsm1-GFP/∆Moosm1, MoOsm1Y173A-GFP/∆Moosm1, and MoOsm1Y173D-GFP/∆Moosm1 strains. (F) Gel-filtration chromatography assay of MoOsm1 dimerization. MoOsm1 was separated by the AKTA protein purification system (GE healthcare). Four protein peaks (I, II, III, and IV) were detected by western blot analysis. The brown line represents the ion peak. (G) BiFC assays for MoOsm1-nYFP and cYFP-MoOsm1 interactions. Conidia were treated with H2O2. MoOsm1Y173A-nYFP and cYFP-MoOsm1Y173A were used as control. (H) In vivo phosphorylation analysis of MoOsm1. Nuclear and cytoplasmic MoOsm1 phosphorylation was analyzed by Mn2+-Phos-tag SDS-PAGE and normal SDS-PAGE, respectively. Total proteins treated with alkaline phosphatase (phosphatase) and phosphatase inhibitor (inhibitor) were used as control.