FgMyo1 is required for toxin biosynthesis.

(A) Localization of FgMyo1-RFP in hyphae of PH-1::FgMyo1-RFP growth in toxin non-inducing media PDB and MM at 28°C for 48 h. Bar = 10 μm. (B) FgMyo1-RFP was co-localized with Tri1-GFP under the toxin inducing condition. Bar = 10 μm. (C) The interaction of FgMyo1 with Tri1 was confirmed by co-immunoprecipitation (Co-IP) analysis. Total proteins (input) extracted from the strain bearing FgMyo1-3×Flag and Tri1-GFP constructs or a single construct (FgMyo1-3×Flag or Tri1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-Flag and anti-GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-Flag agarose and further detected with anti-GFP antibody (Flag pull-down panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (D) The interaction of FgMyo1 with Tri1 was confirmed by bimolecular fluorescence complementation (BiFC) analysis. The constructs of pFgTri1-YFP^N and pFgMyo1-YFP^C were co-transformed into PH-1 to generate the strain FgTri1-YFP^N+FgMyo1-YFP^C. The strains bearing a single construct (FgMyo1-YFP^C or FgTri1-YFP^N) were used as negative controls. The YFP signals in hyphae of each strain grown in the TBI medium were examined under a confocal microscope. Bar = 10 μm. (E) The sensitivity of FgMyo1 derived mutants towards phenamacril. The wild-type PH-1, FgMyo1 silencing mutant FgMyo1-S2, inducible mutant Pzear-FgMYO1, and the point mutation strain FgMyo1^E420K were incubated on PDA supplemented with 0.3 μg/ml phenamacril (left panel). For the inducible mutant, PDA was also added with (+) or without (-) the inducer 30 μg/ml β-estradiol. Mycelial growth inhibition of each strain by phenamacril was quantified (right panel). Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (F) The toxisome formation patterns in FgMyo1 derived mutants. Each strain was grown in TBI, and images were taken after incubation for 48 h (left-upper panel). The accumulation of Tri1-GFP protein in each strain was determined by western blot assay with the anti-GFP antibody. The protein samples were also incubated with the anti-GAPDH antibody as a reference (left-lower panel). The intensities of GFP signals in each strain were also quantified. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05 (right panel). (G) The DON production of FgMyo1 derived mutants. DON was extracted from mycelia of each strain grown in TBI for 7 days. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05.