Fig. 6

VdPKS9 regulates hyphal growth and MS formation in Verticillium dahliae. A VdPKS9 mediates the morphological development of hyaline-hyphae in V. dahliae. Growth phenotype of wild-type strain (AT13), transformant overexpressing VdPKS9 in strain AT13, wild-type strain (Vd991, hyaline-hyphae type, highly virulent on cotton), and its VdPKS9 deletion mutant were cultured on PDA medium. The growth phenotype and the thickness of aerial hyphae were photographed at 7 and 5 days after incubation at 25 °C in dark, respectively. B Analyses of the relative expression of melanin biosynthesis genes in the transformant overexpressing VdPKS9 in strain AT13 and the VdPKS9 deletion mutant in Vd991 strain by RT-qPCR. RNA samples were collected from the indicated strains that grow on PDA medium for 5 days, and the transcript level in two wild-type strains (AT13 and Vd991) was set as control. C Analyses of MS development of two wild-type strains of AT13 and Vd991, AT13 overexpressing VdPKS9, and the VdPKS9 deletion mutant in the Vd991 strain background. All strains were cultured on BMM medium for 7, 14, and 21 days on BMM medium, and photographed by stereomicroscope. Scale bar = 100 μm. Arrowheads indicate MS. Morphological observation showed that each strain had 3 plates and repeated 3 times independently. D Analyses of conidiation in the indicated strains. Three 0.5 mm diameter mycelial plugs were collected with a hole puncher from the edge of strains grown for 7 days, and then shaken in sterile water containing 0.1% Tween-20 for 1 min. Conidia were counted using a hemocytometer, with three repeats for each strain. E The hyaline hyphae (AH) and melanized hyphae (MH) types in V. dahliae strains. Each strain was grown on minimal media plates in the darkness at 25 °C for 7 days. F The relative expression of VdPKS9 between AH-type and MH-type strains during culture on Czapek medium. The conidia of these strains were grown at 25 °C and samples were collected at 2 and 3.5 days, the expression of VdPKS9 in MH-type strain (HB01) was set as control. Analyses of expression levels in three different experiments were conducted by RT-qPCR using the 2^−ΔΔCT method. G Relative expression of VdPKS9 in AH-type and MH-type strains under nutrition stress. The conidia of indicated strains were grown on Czapek medium at 25 °C for 2 days, then transferred to basal medium and incubated an additional 1.5 days. Samples were collected after culturing each media, and the expression levels of VdPKS9 at 2 days after incubation on Czapek medium for each strain was set as control. Analyses of expression levels in three different experiments were conducted by RT-qPCR using the 2^−ΔΔCT method. H The expression of VdPKS9 in AT13 and Vd991 strains in response to stress conditions. The conidia of AT13 and Vd991 strains were cultured on Czapek medium at 25 °C for 2 days, then transferred to basal medium or low temperature (4 °C) and incubated another 1.5 days (2–3.5 dpi), and re-transferred toCzapek medium for an additional 1.5 days. Samples at different growth time points were collected for transcript analysis, and the expression of VdPKS9 on Czapek medium at 2 days was set as control. Analyses of expression levels in three different experiments were conducted by RT-qPCR with three repetitions using the 2^−ΔΔCT method