Figure S2. Construction of knockout mutants and overexpression strain. from A fungal milRNA mediates epigenetic repression of a virulence gene in <i>Verticillium dahliae</i>

A: Schematic representation of homologous recombination events involved in the targeted replacement of DCL1, DCL2, AGO1, AGO2, VdHy1 and VdMILR1. B: PCR identification of the knockout mutants with the primers gene-test-F/R indicated in A. C: Colony morphology of V592 and knock out mutants ∆dc1l, ∆dcl2, ∆ago1, ∆ago2 and Δvdmilr1 on PDA plates incubated for 2 weeks. D: Small RNA gel blotting in mutant lines shows the production of VdmilR1 is independent of DCLs and AGOs. E: The expression of VdR3 in two Tef-VdR3 strains was determined by qRT-PCR. The V. dahliae tubulin gene was used as the internal control. Error bars show ±standard deviation from three replicates. Data obtained for V592 was set to 1. F：Disease symptoms and disease grades of cotton plants infected with the V592 and Tef-VdR3 strains at 18 days postinoculation (dpi). Tef-VdR3 had increased virulence in cotton plants compared to V592