Fig 1.
Deletion of FgAtm1 led to iron accumulation.
(A) Domain analyses of Atm1 orthologs from F. graminearum and other eukaryotic species. The domain analysis was performed with InterPro Scan program of the Interpro protein database (http://www.ebi.ac.uk/interpro). (B) Colocalization of FgAtm1 or FgAtm1N1-111 (truncated FgAtm1 containing only the N-terminal 111 amino acids) with mitochondrial dye MitoTracker. The plasmid FgAtm1- or FgAtm1N1-111-GFP was ectopically transformed into ΔFgAtm1, and the resulting strain was then examined with a fluorescent microscope after MitoTracker staining. Bar = 10 μm. (C) Sensitivity of the wild-type strain PH-1, ΔFgAtm1 and the complemented transformant ΔFgAtm1-C to iron chelating agent BPS and FeSO4. A 5-mm mycelial plug of each strain was inoculated on MM or PDA without or with 0.3 mM BPS or FeSO4 at the indicated concentration, and then incubated at 25°C for 3 days. (D) Iron content in mitochondria and whole cell of the wild type, ΔFgAtm1 and ΔFgAtm1-C was determined by a laser scanning microscope with 5 μΜ fluorescent iron-binding dye FeRhoNox-1 (upper panel) or colorimetric ferrozine-based assay (lower panel) after culture in CM at 25°C for 36 hours. Bar = 20 μm. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (n.s. not significant, **p < 0.01). (E) Conidial germination of the wild type, ΔFgAtm1 and ΔFgAtm1-C in trichothecene biosynthesis induction medium (TBI) or iron-depleted TBI. Bar = 20 μm. Percentage of germinating conidia of each strain was calculated after 4-, 24- and 48-hour-incubation. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (n.s. not significant, **p < 0.01).
Fig 2.
FgAtm1 regulates the activity of cytosolic Fe-S proteins.
(A) Determination of FgLeu1, FgAco1 and FgFum1 activities in mitochondria and cytosol of the wild type, ΔFgAtm1 and ΔFgAtm1-C. The FgLeu1, FgAco1 and FgFum1 activity of the wild type were set as 1. Each strain was cultured in CM at 25°C for 36 hours before activity determination. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (n.s. not significant, **p < 0.01, ***p < 0.001). (B) The growth and microscopic observation of colony edge of the wild type and ΔFgAtm1 on MM with or without 10 mM leucine, 70 mM NH4Cl, 10 mM sodium glutamate or 10 mM uric acid sodium at 25°C for 3 day or 7 days. Bar = 100 μm. (C) The content of total glutathione in mitochondria and whole cell of the wild type, ΔFgAtm1 and ΔFgAtm1-C. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (n.s. not significant, *p < 0.05).
Fig 3.
The reduced activity of cytoplasmic Fe-S proteins activates transcription of the FgAreA-HapX cascade.
(A) Sensitivity of the wild type, ΔFgNiiA, ΔFgXdh and ΔFgAreA 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 < 0.05, **p < 0.01). (B) Relative transcription levels of FgAREA treated by NaNO3 or hypoxanthine, or deletion of FgATM1, FgNIIA or FgXDH. PH-1 was treated with MM-N+70 mM NaNO3 or MM-N+1 mM hypoxanthine for 4 hours after culture in CM at 25°C for 1 day. The expression level in the wild type without treatment was set as 1. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (*p < 0.05). (C) The enrichment of FgAreA-GFP at the promoter of FgHAPX was induced by the treatment with NaNO3 or hypoxanthine, or deletion of FgATM1. Each strain was treated with MM-N+70 mM NaNO3 or MM-N+1 mM hypoxanthine for 4 hours after culture in CM at 25°C for 1 day. ChIP- and input-DNA samples were quantified by quantitative PCR assay. A control reaction was processed in parallel with rabbit IgG and PH-1 transformed only with GFP used as a negative control for detecting GFP enrichment at the FgHAPX promoter. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (n.s. not significant, *p < 0.05). (D) Relative transcription levels of FgHAPX in the wild-type PH-1 and ΔFgAreA under a non-preferred nitrogen source or in the background of FgATM1 deletion. Each strain was treated with MM-N+70 mM NaNO3 or MM-N+1 mM hypoxanthine for 4 hours after culture in CM at 25°C for 1 day. The expression level in the wild type without treatment was set as 1. 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).
Fig 4.
Over-expression of FgHAPX leads to iron accumulation in ΔFgAtm1.
(A) Sensitivity of the wild type, ΔFgAtm1, ΔFgHapX and ΔFgAtm1-HapX 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 3-day-incubation. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Total iron content of the wild type, ΔFgAtm1, ΔFgHapX and ΔFgAtm1-HapX 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 culture in CM at 25°C for 36 hours. Bar = 20 μm. Means and standard errors were calculated from three repeats. Significance was measured using unpaired t-test (*p < 0.05, **p < 0.01). (C) The content of extra- and intracellular siderophores of the wild type, ΔFgAtm1, ΔFgHapX and ΔFgAtm1-HapX determined by the chrome azurol S (CAS) assay. Each strain was cultured in MM lack of Fe2+ for 8 hours after growth in CM for 36 hours. 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 the genes involved in iron homeostasis in the wild type, ΔFgAtm1, ΔFgHapX and ΔFgAtm1-HapX cultured in CM for 1 day. The expression level of each gene in each mutant is the relative amount of mRNA compared to the level in the wild type. Mean and standard error of each gene were calculated with results from three repeats. Significance was measured using unpaired t-test (n.s. not significant, *p < 0.05, **p < 0.01).
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).
Fig 6.
FgHapX interacts with FgGrx4 to regulate iron homeostasis in F. graminearum.
(A) Domain architecture of the F. graminearum FgGrx4 protein analyzed with InterPro Scan program of the Interpro protein database (http://www.ebi.ac.uk/interpro). FgGrx4 contains a thioredoxin (TRX)-like domain and a glutaredoxin (GRX)-like domain. (B) The GRX domain of FgGrx4 is required for its interaction with FgHapX in yeast two-hybrid hybridization (Y2H) assays. Serial dilutions of yeast cells (cells/ml) transferred with the bait and prey constructs were assayed for growth on SD-Leu-Trp-His plates. A pair of plasmids pGBKT7-53 and pGADT7 was used as a positive control. A pair of plasmids pGBKT7-Lam and pGADT7 was used as a negative control. (C) FgHapX interacts with FgGrx4 in the co-immunoprecipitation (Co-IP) assay. Western blots of total proteins (input) from transformants bearing the FgGrx4-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. (D) The interaction of FgHapX and FgGrx4 in the nucleus is dependent on the GRX domain of FgGrx4, but independent on FgAtm1 by bimolecular fluorescence complementation (BiFC) assay. A pair of constructs FgHapX-CYFP+NYFP, and another pair of constructs FgGrx4-NYFP+CYFP were used as negative controls. YFP signals were observed using confocal microscopy. Bar = 10 μm. (E) Sensitivity of the wild type and ΔFgGrx4 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. (F) Mycelial growth inhibition of each treatment was calculated after 3-day-incubation (E). Means and standard errors were calculated from three repeats. Significance was measured using an unpaired t-test (*p < 0.05, **p < 0.01). (G) Relative transcription levels of iron-related genes in the wild type, ΔFgHapX and ΔFgGrx4 after growth in CM for 1 day. The relative expression level of each gene in each mutant is the amount of mRNA relative to the wild type. Means and standards error of each gene were calculated from three repeats. Significance was measured using an unpaired t-test (n.s. not significant, *p < 0.05, **p < 0.01).
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-CS245A/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-CS245A/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-CS245A/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.
Fig 8.
A proposed model of iron homeostasis regulated by FgAtm1 in F. graminearum.
FgAtm1 regulates the export of GSH-linked [2Fe-2S] clusters from mitochondria to cytoplasm, subsequently affects the assembly of cytoplasmic Fe-S proteins nitrite reductase (FgNiiA) and xanthine dehydrogenase (FgXdh) that are key enzymes for non-preferred nitrogen utilization. The activated FgNiiA and FgXdh inhibit the transcription of nitrogen metabolism regulator FgAreA. FgAreA activates expression of the transcription repressor FgHapX, subsequently regulates the transcription of iron-related genes in F. graminearum. In addition, the function of FgHapX is dependent on its interacting protein FgGrx4 and phosphorylation mediated by kinase FgYak1.