Fig. 4
VdZFP1 and VdZFP2 participate in microsclerotia development of Verticillium dahliae. A Microsclerotia morphology of WT, ΔVdZFP1, ECΔVdZFP1, ΔVdZFP2, ECΔVdZFP1, and ΔVdZFP1_2 strains. Each strain was cultured on BMM plates covered with cellophane membranes. The development of microsclerotia was observed at 7 and 14 dpi after incubation at 25 °C in the dark and photographed with a stereoscope. Each strain was repeated three times independently, and at least three plates were observed each time. Scale bar = 100 μm. B–D Statistics on differences in the number, volume, and melanin coverage of microsclerotia between WT, deleted mutants, and complemented strains. WT, ΔVdZFP1, ECΔVdZFP1, ΔVdZFP2, ECΔVdZFP1, and ΔVdZFP1_2 strains were cultured on BMM medium at 25 °C in the dark. After incubating for 7 days, the diameter of 30 matured microsclerotia of each strain was measured, while the number or melanin coverage of microsclerotia was calculated from 20 or 6 visual fields of 0.5 or 1 mm squared, respectively. B Microsclerotia diameter, C microsclerotia number, and D melanin coverage of microsclerotia. Error bars represent the standard deviation of each independent experiment, and all experiments performed three repeats, **P < 0.01, and ***P < 0.001 (Student’s t test). E Analyses of the relative expression of melanin biosynthesis genes during the microsclerotia development among WT, ΔVdZFP1, ECΔVdZFP1, ΔVdZFP2, ECΔVdZFP1, and ΔVdZFP1_2 strains. All strains were grown on BMM medium at 25 °C in the dark and collected at 7 dpi. Compared with WT strain, the expression level of each gene in transformants was detected by RT-qPCR for 3 repetitions using the 2−ΔΔCT method and finally presented as a heatmap. This experiment was independently repeated 3 times to determine the trend