Fig. 5

VdPKS9 functions epistatic to the characterized melanin biosynthesis pathway to negatively regulate melanin biosynthesis and MS formation. A Investigation of MS formation in VdPKS9 deletion strains (ΔVdPKS9), corresponding ectopic transformants (EC^ΔVdPKS9), and the wild-type strain AT13 (WT) inhibits the melanin biosynthesis pathway using tricyclazole. The MS induction on BMM medium contained indicated concentration of tricyclazole for 7 days and were observed using a stereoscope. B Relative expression analyses of the melanin biosynthesis genes in the indicated strains following treatment with tricyclazole. The samples in panel A in this figure were collected to extract RNA for reverse transcription-quantitative PCR (RT-qPCR) for analyses of the expression of melanin biosynthesis pathway-related genes. The wild-type strain AT13 was grown on BMM medium without supplementation as the control (WT-CK tricyclazole). The expression levels of each gene were calculated by RT-qPCR using the 2^−ΔΔCT method. Error bars represent standard errors of the mean, ^***P < 0.001 (one-way ANOVA). C Colony morphology of the VdPKS9::VdPKS1 double deletion mutant. The indicated strains were cultured on PDA medium for 7 days at 25 °C in the dark. D Analyses of MS formation in the VdPKS9::VdPKS1 double deletion mutant. All strains (VdPKS9 deletion strains ΔVdPKS9, VdPKS1 deletion strains ΔVdPKS1, additional deletion of VdPKS9 under the VdPKS1 deletion mutant ΔVdPKS9_1, and the wild-type strain AT13) were cultured on BMM medium for 7 and 14 dpi, and the MS were photographed using a stereoscope. E Analyses of the expression of the melanin biosynthesis genes in the double deletion mutant ΔVdPKS1::VdPKS9. The indicated strains were cultured on BMM medium for 7 days. Error bars represent standard errors of the mean, ^**P < 0.01, and ^***P < 0.001 (one-way ANOVA). F MS morphology of the indicated strains supplemented with the melanin biosynthesis pathway intermediate scytalone. All of the indicated strains were cultured on the BMM medium or supplemented with the scytalone (50 mg/mL) for 7 and 14 days. The MS were observed using a stereoscope, scale bar = 100 μm. G Relative expression analyses of the melanin biosynthesis genes of the indicated strains following supplementation with scytalone. The indicated strains were cultured on BMM medium for 5 days. The expression levels of each gene were calculated by RT-qPCR using the 2^−ΔΔCT method. Error bars represent standard errors of the mean, ^*P < 0.01, ^**P < 0.01, and ^***P < 0.001 (one-way ANOVA). H Relative expression analyses of melanin biosynthesis genes and VdPKS9 in the ΔVdPKS1-OE^VdPKS9 strain compared with the ΔVdPKS1 strain. Samples were collected from Czapek medium 5 days after incubating at 25 °C in the dark. The observation of MS and colony melanin, as well as quantitative detection of genes, were repeated three times independently, and three plates or samples were performed each time