FIG 1.

Antofine inhibits growth of F. graminearum and targets GDH1 and RRD1. (A). Wild-type F. graminearum (Fg) spores were coincubated with 100 μg/ml of antofine (A₁₀₀), tylophorine (T₁₀₀), or solvent control (−), and mycelial growth was monitored by absorbance at 600 nm (ABS₆₀₀). Results are representative of five biological replicates. The chemical structures of tylophorine and antofine are shown. (B) Growth of wild-type S. cerevisiae (Sc WT) or Scgdh1 and Scrrd1 mutants in the presence of 2 μg/ml antofine (A₂), tylophorine (T₂), or solvent control (−) was monitored by absorbance at 600 nm (ABS₆₀₀). Values represent the average of three technical replicates with standard deviation. The results are representative of four biological replicates. (C) F. graminearum GDH1 and RRD2 complement antofine hypersensitivity to the Scgdh1 and Scrrd1 mutants, respectively. Fg GDH1 (FGSG_07174) was expressed in the Scgdh1 mutant, and FgRRD1 (FGSG_09229) and FgRRD2 (FGSG_01092) were expressed in the S. cerevisiae Scrrd1 mutant. Empty vectors (EVs) were used as controls. Expression of FgGDH1 and FgRRD1 was induced by galactose and was monitored in the absence and presence of 2 μg/ml antofine. Values represent average percentage of growth with respect to growth in the absence of antofine of three technical replicates with standard deviation. (D) Thermal shift assays using 1 mg/ml of purified FgGDH1 and FgRRD2 proteins in the absence and presence of antofine (A₁₀₀), and tylophorine (T₁₀₀) at 100 μg/ml. Results are representative of two independent biological replicates with four technical replicates in each experiment.