Fig 7. The phosphorylation of FgHapX mediated by the Ser/Thr kinase FgYak1 is required for the transcriptional activity of FgHapX.

(A) The mutations of FgHapX at 245 and 338 from Ser to Ala or loss of FgYak1 led to a decreased phosphorylation level of FgHapX. Proteins extracted from ΔFgHapX::FgHapX-mCherry, ΔFgHapX::FgHapX-C^S245A/S338A-mCherry, or ΔFgYak1-HapX::FgHapX-mCherry were subjected to Phos-tag SDS-PAGE and normal SDS-PAGE followed by immunoblotting with an anti-mCherry antibody. (B) Sensitivity of the wild type, ΔFgHapX, ΔFgHapX-C, ΔFgHapX-C^S245A/S338A and ΔFgYak1 to FeSO₄. Each strain was inoculated on MM or PDA amended without or with FeSO₄ at the indicated concentration, and then incubated at 25°C for 3 days. (C) Mycelial growth inhibition of each strain under each treatment was calculated after 3 days of incubation (B). Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (n.s. not significant, *p < 0.05, **p < 0.01). (D) Relative transcription levels of iron-related genes in the wild type, ΔFgHapX, ΔFgHapX-C^S245A/S338A, and ΔFgYak1 growth in CM for 1 day. The expression level of each gene in each mutant is the amount of mRNA relative to the same gene in the wild type. Means and standard errors of each gene were calculated from three repeats. Significance was measured using unpaired t-test (n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001). (E) FgHapX interacts with FgYak1 in the co-immunoprecipitation (Co-IP) assay. Western blots of total proteins (input) from transformants expressing the FgYak1-Flag and/or FgHapX-mCherry constructs (upper panel), and the proteins eluted from anti-Flag agarose beads (lower panel) were detected with the anti-mCherry or anti-Flag antibody. Detection with the anti-GAPDH antibody was conducted for the protein loading reference. (F) FgYak1-Flag (red) interacts with FgHapX-mCherry (green) in the nucleus in the immunofluorescence assay. Nuclei were stained with DAPI. Bar = 1 μm.