Identification and localization of FgDML1 in Fusarium graminearum.

(A) The phylogenetic tree was constructed based on the amino acid sequences of DML1 homologs from 10 species using the maximum likelihood method in Mega X. Bootstrap values from 1000 replications were displayed on the branches. (B) FgDML1 was localized to the mitochondria.

FgDML1 is required for both the virulence of F. graminearum.

(A) Lesion lengths on wheat coleoptiles caused by different strains. These images were taken at 14 dpi. (B) Dot plot showing the lesion lengths on wheat coleoptiles at 14 dpi tested with different strains. (C) Disease symptoms on wheat leaves inoculated with PH-1, ΔFgDML, and ΔFgDML-C. Images were taken at 5 dpi. (D) Disease area on wheat leaves inoculated with different strains at 5 dpi.

FgDML1 is required for the biosynthesis of DON toxin in F. graminearum.

(A) ΔFgDML1 exhibited decreased DON content. All strains were incubated in trichothecene biosynthesis induction (TBI) medium for 7 days. (B) Relative mRNA expression levels of FgTRI5, and FgTRI6 in the tested strains. Mycelia were harvested after 2 days of cultivation in TBI medium, and mRNA were extracted for quantification using the 2-ΔΔCT method. FgGapdh was used as the reference gene. (C) FgDML1 impaired the formation of DON toxisomes, as visualized by Tri1-GFP labeling of toxisomes. Strains were cultured in TBI for 36 hours. Bar = 10 μm (D) ΔFgDML1 exhibited reduced expression levels of Tri1-GFP. Strains were cultured in TBI for 36 hours, and mycelia were harvested for western blot analysis. Bars with the same letter indicate no significant difference according to a significant difference (LSD) test at P = 0.05.

FgDML1 modulates mitochondrial morphology via interaction with FgDnm1 and negatively regulates Acetyl-CoA and ATP synthesis.

(A) Effects of FgDML1 on mitochondrial morphology. Mycelial plugs of the wild-type strain PH-1, the ΔFgDML1 deletion mutant, and the complemented mutant ΔFgDML1-C were cultured in YEPD medium for 36 hours. Mitochondria were stained using a mitochondria-specific fluorescent dye and observed under a confocal microscope. Additionally, conidial germlings were cultured in MBL medium and subjected to the same staining and imaging procedures. Scale bar = 5 μm. (B) Ultrastructural analysis of mitochondria by transmission electron microscopy (TEM). Mitochondrial ultrastructure was further examined in PH-1, ΔFgDML1, and ΔFgDML1-C using TEM. Mitochondria are indicated by red arrows. Scale bar = 200 nm. (C) FgDML1 Deletion Reduces Acetyl-CoA Levels. Mycelial cultures of PH-1, ΔFgDML1, and ΔFgDML1-C were incubated in YEPD medium for 36 hours. Acetyl-CoA levels were quantified using a commercial assay kit, following the instructions provided by the commercial assay kit. (D) FgDML1 deletion reduces ATP levels. The ATP content of PH-1, ΔFgDML1, and ΔFgDML1-C was measured following the same culture conditions as in (C), using a commercial ATP assay kit. (E) Co-immunoprecipitation (Co-IP) confirms interaction between FgDML1 and FgDnm1. Total protein extracts from strains expressing tagged versions of FgDML1 and FgDnm1 were separated by SDS-PAGE and subjected to immunoblot analysis. Co-IP was performed using ChromoTek GFP-Trap® magnetic agarose to pull down interacting proteins. Immunodetection was carried out using polyclonal anti-Flag and monoclonal anti-GFP antibodies. Monoclonal anti-GAPDH antibody was used as a reference for protein samples.

FgDML1 modulates the sensitivity of F. graminearum to cyazofamid.

(A) ΔFgDML1 exhibits reduced sensitivity to cyazofamid. PH-1, ΔFgDML1, and ΔFgDML1-C were cultured on AEA medium modified with cyazofamid, with 50 μg/mL SHAM included to inhibit the alternative oxidase pathway. (B) Deletion of FgDML1 did not affect F. graminearum sensitivity to pyraclostrobin. The tested strains were incubated on AEA medium containing pyraclostrobin and 50 μg/mL SHAM to assess their response to the fungicide. (C) FgDML1 deletion reduces complex III enzymatic activity. Mycelial cultures of PH-1, ΔFgDML1, and ΔFgDML1-C were grown in YEPD for 36 hours, then treated with 30 μg/mL cyazofamid, 1.5 μg/mL pyraclostrobin, and 50 μg/mL SHAM for an additional 12 hours. The enzymatic activity of complex III was quantified using a commercial assay kit. (D) mRNA expression levels of complex III subunits and assembly factors in PH-1, ΔFgDML1, and ΔFgDML1-C. PH-1, ΔFgDML1, and ΔFgDML1-C were incubated in liquid AEA medium for 36 hours, followed by treatment with 30 μg/mL cyazofamid and 50 μg/mL SHAM. The relative mRNA expression levels of complex III subunits and assembly factors were quantified using the 2-ΔΔCT method. “T” denotes cyazofamid treatment.

Effects of ΔFgDML1 on asexual and sexual development.

(A) Mycelial growth rate. PH-1, ΔFgDML1, ΔFgDML1-C were cultured for 3 days on PDA, CM, MM, and V8 solid media at 25°C. (B) Mycelial morphology. The mycelial morphology of PH-1, ΔFgDML1, and ΔFgDML1-C was examined on WA plates. Scale bar = 72 μm. (C) Conidial length. Conidia of ΔFgDML1 were significantly longer in length compared to PH-1 and ΔFgDML1-C. Conidia were germinated in MBL medium, stained with Calcofluor White (CFW), and observed using an Olympus IX-71 microscope. (D) Sexual reproduction. PH-1 and ΔFgDML1-C produced perithecia and asci containing ascospores after 7 or 14 days of incubation on CA medium, while the ΔFgDML1 failed to produce these structures under the same conditions. Perithecia and ascospores were observed using a Nikon SMZ25 fluorescent stereo microscope and an Olympus IX-71 inverted fluorescence microscope. Top image: perithecium, scale bar = 1 mm; bottom image: ascospores, scale bar = 50 μm. (E) Proportion of conidial septa. The proportion of conidial septa was assessed in the tested strains. (F) Conidial production. Conidia were harvested after 4 days of incubation in MBL medium, and their concentration was quantified using a hemocytometer. (G) Germination rate. Conidial germination was observed by incubating 300 conidia for 2 or 8 hours on WA plates. Germination rates were compared, with different letters indicating significant differences based on ANOVA followed by the LSD test (P = 0.05).

Response of the strains to different stresses.

(A) Reduced sensitivity to osmotic stress in ΔFgDML1. PH-1, ΔFgDML1 and ΔFgDML1-C were subjected to osmotic stress using NaCl, KCl, and Sorbitol to evaluate their sensitivity. (B) Statistical analysis of growth inhibition under osmotic stress. Growth inhibition rates of all strains under NaCl, KCl, and Sorbitol stress were statistically analyzed. (C) Sensitivity to cell wall and membrane damaging agents. The tested strains were exposed to cell wall-damaging agent Congo Red (CR) and cell membrane-damaging agent Sodium dodecyl sulfate (SDS) to assess their sensitivity. (D) Statistical analysis of growth inhibition under CR and SDS stress. Growth inhibition rates of all strains under CR and SDS stress were statistically analyzed. (E) Sensitivity to temperature stresses. The sensitivity of the tested strains to different temperature stresses was evaluated. (F) Statistical analysis of growth inhibition under temperature stress. Growth inhibition rates of all strains under various temperature stresses were statistically analyzed. Significant differences were determined using the LSD test at P = 0.05. Bars with the same letter indicate no significant difference.

Model of FgDML1 regulation of F. graminearum sensitivity to DON toxin synthesis and cyazofamid fungicide.

FgDML1 interacts with FgDnm1 to modulate mitochondrial morphology, thereby positively regulating acetyl-CoA and ATP levels, which influence toxisome formation and ultimately affect DON production. Loss of FgDML1 reduces complex III enzymatic activity, leading to the upregulation of the assembly factors FgQCR2, FgQCR8, and FgQCR9. This alteration in assembly factor expression changes the conformation of the Qi site, leading to reduced sensitivity to cyazofamid.