Suppression of GhMC4 gene expression in cotton decreases the resistance to V. dahliae. (A) Disease symptoms of pTRV: 00 plants and GhMC4-silenced cotton lines (pTRV: GhMC4-3 and pTRV: GhMC4-12) at 0- and 14  DPI with Vd991. The pathogen inoculation was performed using the root-dipping method. The experiment was repeated three times. (B) Expression of GhMC4 gene in cotton plants inoculated with Vd991. (C) Relative transcript levels of immune-related genes in pTRV: 00 plants and GhMC4-silenced cotton lines (pTRV: GhMC4-3 and pTRV: GhMC4-12) at 14  DPI with Vd991. Total RNAs were extracted from cotton roots after infected with Vd991. Gene expression levels were normalized using ubiquitin gene as the control according to the 2^−ΔΔCt method. Data represent means and SE (n = 12) from three independent biological replicates. Statistical significance was calculated using a Student’s t-test. Asterisks indicate significant differences (**P < 0.01). (D) Western blot analysis of GhMC4-MYC protein abundance in tobacco leaves. YFP and YFP–VdPHB1 co-transformed tobacco leaves with MYC-GhMC4, respectively. Proteins were detected with anti-YFP and anti-MYC antibodies. (E) ROS accumulation after transient expression of Control (MYC), GhMC4-MYC and GhMC4-MYC/VdPHB1–YFP in N. benthamiana leaves. ROS were detected using 3,3ʹ-diaminobenzidine (DAB) staining. Scale bar = 1 cm. (F) Active oxygen after transient expression of Control (MYC), GhMC4-MYC and GhMC4-MYC/VdPHB1–YFP in N. benthamiana leaves were determined by luminol method. Statistical significance was calculated using a Student’s t-test. Asterisks indicate significant differences (*P < 0.05; **P < 0.01).