A fungal ABC transporter FgAtm1 regulates iron homeostasis via the transcription factor cascade FgAreA-HapX
Fig 5
FgHapX activates iron acquisition genes via suppressing the transcriptional repressor FgSreA.
(A) Identification of FgHapX and FgSreA binding motifs (indicated by black squares) by the multiple EM for motif elicitation (MEME) program. Four iron-consuming genes and FgSREA were used for the FgHapX binding motif analysis, and five iron acquisition genes were used for the FgSreA binding motif analysis. (B) Verification of the binding of FgHapX with the promoters of four iron-consuming genes and FgSREA by electrophoretic mobility shift assay (EMSA). The promoter of each gene was incubated with purified GST-FgHapXN1–230 or GST with or without proteinase K for 20 min at 25°C. (C) Relative transcription levels of FgSREA in the wild type and ΔFgHapX cultured in CM for 1 day. The relative expression level of FgSREA in each mutant is the relative amount of mRNA in the wild type. Means and standards error of each gene were calculated from three repeats. Significance was measured using an unpaired t-test (*p < 0.05, **p < 0.01). (D) Sensitivity of the wild type and ΔFgSreA to FeSO4. A 5-mm mycelial plug of each strain was inoculated on MM or PDA without or with FeSO4 at the indicated concentration, and then incubated at 25°C for 3 days. Mycelial growth inhibition of each treatment was calculated after a 3-day-incubation. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test (**p < 0.01, ***p < 0.001). (E) The content of extra- and intracellular siderophores in ΔFgSreA was increased as compared to that in the wild type in the chrome azurol S (CAS) assay. Each strain was cultured in MM lacking Fe2+ for 8 hours after growth in CM for 36 hours. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test (*p < 0.05). (F) Total iron content of the wild type or ΔFgSreA was determined by a laser scanning microscope with 5 μΜ fluorescent iron-binding dye FeRhoNox-1 (left panel) or colorimetric ferrozine-based assay (right panel) after each strain was cultured in CM at 25°C for 36 hours. Bar = 20 μm. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test (*p < 0.05). (G) Relative transcription levels of six iron acquisition genes in ΔFgSreA cultured in CM for 1 day. The relative expression level of each gene in ΔFgSreA is the amount of mRNA relative to that of the wild type. The expression level of each iron acquisition gene in PH-1 was set to 1. Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test (*p < 0.05, **p < 0.01).