Fig. 3
VdPKS9 is necessary for radial growth and negatively regulates melanin biosynthesis in Verticillium dahliae. A Investigation the colony morphology of indicated strains. Wild-type strain AT13 (WT), two VdPKS9 deletion strains (ΔVdPKS9), and two corresponding ectopic transformants (ECΔVdPKS9) were cultured on potato dextrose agar (PDA) medium. Pheotypes were photographed 7 days after incubation at 25 °C in dark. B, C Measurements of the colony diameter and melanin area of the indicated strains in panel A. Error bars represent standard errors of three independent repeated measurements. ***P < 0.001 (one-way ANOVA). B Colony diameter, C melanin area. Each experiment was replicated three times. D Transcriptome comparison of VdPKS9 deletion mutant compared to the wild-type strain. The plot indicates the DEGs, GO enrichment with significance (P < 0.005, Pearson chi-square test), melanin biosynthesis-related genes, and genes encoding PKSs in V. dahliae. Circle k represent the fold-change of DEGs, bars distribute to the top and bottom side represent up- and downregulation, respectively. E Analysis the significance GO items of VdPKS9 deletion mutant compares to wild-type strain. The significance was calculated by the values of fold-change in each GO items compare to total DEGs. **P < 0.01 (one-way ANOVA). F The levels of upregulated melanin pathway genes in ΔVdPKS9 strain. G Diagram of the melanin biosynthesis pathway in V. dahliae and the putative point at which VdPKS9 may negatively regulate the pathway. The melanin biosynthesis pathway was drawn from a previous study [12]. H Analyses of the relative expression of melanin biosynthesis genes in VdPKS9 deletion mutant were compared to those of the wild-type strain and ectopic transformants by reverse transcription-quantitative PCR (RT-qPCR). Samples were collected 5 days after incubation in Czapek medium at 25 °C in the dark. The expression levels of each gene were calculated by RT-qPCR using the 2−ΔΔCT method. The expression levels were calculated in three replicates. Error bars represent standard errors of the mean, *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA)