Fig. 6

Silencing of cotton secretome-encoding genes by virus-induced gene silencing (VIGS) affects resistance to Verticillium dahliae. A Expression patterns of 13 selected secretome members in Gossypium hirsutum resistant cultivar Zhongzhimian No.2 (ZZM2) and susceptible cultivar Junmian No.1 after inoculation with V. dahliae strain Vd991. Values (log₂ fold change) represent the averages from three biological replicates. Transcript expression data are from the indicated cultivars at the different time points after inoculation with V. dahliae. Green shading indicates downregulation, and red shading indicates upregulation. B Silencing of selected secretome members in cv. Zhongzhimian No.2 by virus-induced gene silencing (VIGS) affects resistance to V. dahliae. Approximately 14 days after the VIGS procedure in 3-week-old ZZM2 plants, the gene-silenced and wild-type (WT) plants were inoculated with 5 mL of V. dahliae strain Vd991 conidial suspension (5 × 10.⁶ conidia/mL) or sterile water (mock) using a root-dip method. Experiments consisted of three replicates of 12 plants each arranged in a complete random block design. The Verticillium wilt phenotypes of wilting leaves and vascular discoloration were photographed 4 weeks after inoculation. Infiltration with the empty vector pTRV2 (TRV2:0) served as a positive control. C Evaluation of the disease symptoms in gene-silenced plants inoculated with V. dahliae. The disease ratings were classified as grade 0 (healthy plants), grade 1 (0–25% leaves wilting), grade 2 (25–50% leaves wilting), grade 3 (50–75% leaves wilting), and grade 4 (75–100% leaves wilting). The ratings were conducted with 12 cotton seedlings at 3 weeks post-inoculation with three replicates. All the disease index value displays significant change among the gene-silenced plants compared to the positive CLA1-silenced cotton plants (P < 0.01). D The silencing efficiency of 13 selected secretome members was determined by RT-qPCR analysis. The cotton GhUbiquitin gene was used as an endogenous control. CK represents the control infiltration with the empty vector pTRV2 (TRV2:0). The means and standard errors from three biological replicates are shown. Asterisks ** indicate significant differences (P < 0.01)