Fig. 4

Verticillium dahliae VdPKS9 is a negative regulator of both MS development and melanin biosynthesis. A Analyses of MS formation. The conidia suspensions (1.0 × 10⁶ conidia/mL, 60 μL) of wild-type strain AT13 (WT), two VdPKS9 deletion strains (ΔVdPKS9), and two corresponding ectopic transformants (EC^ΔVdPKS9) were spread on the BMM medium plates that pre-covered with cellophane membranes. The development of MS was observed at 3, 5, 7, and 14 days after incubation at 25 °C in the dark and photographed with a stereoscope. The experiment was replicated three times independently for each strain, and at least three plates were observed each time. Scale bar =100 μm. B Quantification of melanin deposition in visual field by color density. Melanin accumulation level was evaluated by the density of gray color using ImageJ scanning. Three random visual fields were selected for color density scanning, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 (one-way ANOVA). C Statistics on the numbers of mature MS. VdPKS9 deletion strains (ΔVdPKS9), corresponding ectopic transformants (EC^ΔVdPKS9), and the wild-type strain AT13 (WT) was incubated in BMM medium, and the number of MS was calculated 7 days after incubation on 25 °C in dark. Six independent visual fields of 0.5 mm squared were used in calculations. Error bars represent the standard deviation, and each experiment performed three repeats, ^***P < 0.001 (one-way ANOVA). D Analyses of the relative expression of melanin biosynthesis genes during the MS development among VdPKS9 deletion strains (ΔVdPKS9), corresponding ectopic transformants (EC^ΔVdPKS9), and the wild-type strain AT13 (WT). Samples were collected indicated time-point after incubation in BMM medium at 25 °C in the dark. The expression levels of each gene were calculated by RT-qPCR for 3 repetitions using the 2^−ΔΔCT method. The heatmap of melanin biosynthesis gene expression patterns were normalized by the value Log₂ (fold-change)