Fig. 5

Colony phenotypes and osmotic stress tolerance of ΔMr-OPY2, ΔMr-STE50 and WT. a Upper panel: colonies of the strains on PDA (Scale bar represents 10 mm). Middle panel: colonies on PDA plus 0.75 M KCl (scale bar represents 1 mm). Lower panel: bright field images of individual germlings in YE (0.01% yeast extract) plus 0.75 M KCl (Scale bar represents 10 μm). Colony pictures were taken 18 days after inoculation. Germling pictures were taken 16 h after inoculation. b Growth curves of the strains on PDA plates plus 0.75 M KCl. Growth assays were repeated three times with three replicates per repeat. Data are expressed as the mean ± SE. Note: growth of ΔMr-OPY2 and ΔMr-STE50 was greatly reduced. c Upper panel: Western blot analysis of Mr-OPY2 protein levels in ΔMr-OPY2 grown in SDY (1), WT grown in SDY (2) and in SDY plus 0.75 M KCl (3). The band intensity of WT in SDY is set at 1, and WT in SDY with KCl is relative to it; ΔMr-OPY2 is a negative control. Lower panel: a portion of the loading control gel (for the western blot analysis in the upper panel) stained with Coomassie Brilliant Blue. M: Protein ladder. The uncropped images of the Western blot and the SDS-PAGE gel are shown in Supplementary Fig. 13. d Upper panel: yeast two-hybrid confirmation of the physical interaction of Mr-SET50 with Mr-OPY2 and three MAPKKK proteins (SSK2, STE11 and BCK1). Colonies were grown in SD-His-Ade-Leu-Trp + X-α-gal + AbA (Takara, Dalian China). NC: negative control (yeast cells containing the plasmid pGADT7-T and pGBKT7-Lam). PC: positive control (yeast cells containing the plasmid pGADT7-T and pGBKT7-53). Lower panel: Mr-STE50 lacks autoactivation activity. Y2HGold cells with pGBKT7-Mr-STE50 cannot grow in SD-His-Trp-Ade + X-α-gal (Takara, Dalian China). NC and PC are the same as those in the upper panel. Images are representative of at least three independent experiments for each condition