Fig. 9

Characterization of FfH2A.Z knock-down and overexpression in fungal development. (A) Radial hyphal growth assay on complete media (V8, CM) and minimal medium (ICI supplemented with 6 mM glutamine). Strains were grown for 4 days (TetOff::FfH2A.Z) and 5 days (OE::FfH2A.Z) at 30 °C, respectively. Experiments were performed in biological triplicates. Hyphal growth of Fusarium fujikuroi (FfWT) was arbitrarily set to 100%. Mean values and standard deviations are shown. For statistical analysis, a student’s t-test was performed. Asterisks above the bars denote significant differences in vegetative growth of the indicated strains compared to FfWT, *p < 0.05; **p < 0.001. (B) Expression of the conidiation-specific genes FfWET1, FfFLB3-4, and FfABA1 in FfWT and FfH2A.Z knock-down mutant were assessed 4 dpi after inoculation on V8 supplemented with doxycycline hyclate (DOX). Plates were incubated in 16 h light and 8 h dark at 20 °C. Expression levels were monitored using RT-qPCR. Mean values and standard deviations are shown and RE denotes relative expression. The conidiation assay was performed using FfWT and OE::FfH2A.Z. V8 plates inoculated with the respective strains were incubated for 7 days at an L/D rhythm before harvesting and conidia quantitation. Experiments were performed in triplicates. Conidia production of FfWT was arbitrarily set to 100%. A student’s t-test was performed to assess the significance levels. Asterisks above the bars denote significant differences in the gene expression/conidia production of the indicated strains compared to the respective wild type, *p < 0.05; **p < 0.001. (C) Heatmap of the relative SM production of FfH2A.Z knock-down and overexpression strains compared to FfWT in different nitrogen conditions. Fusarium metabolites were analyzed by LC-HRMS after cultivation in liquid ICI supplemented with 60 or 6 mM glutamine (30 °C, 180 rpm). To ensure result comparability, the analysis of all mutants and cultivation conditions was performed in three independent replicates. Determined quantities are normalized to the biomass formation (area/g dry weight). SM biosynthesis of FfWT was arbitrarily set to 100%