Fig. 2

Creation of the transgenic GhACS2- and GhACS6-overexpression lines using the expression characteristics of GhACS genes. A Heatmap generated from RNA-seq data (Genome sequencing project accession: SRP118279). The relative expression levels of GhACS2 and GhACS6 in roots of G. hirsutum at 6 h and 48 h after Vd991 inoculation. Different columns in the figure represent various samples, and rows represent genes. The color represents the level of expression of the gene (log₂ (TPM+1)) in the sample. B Expression profiles of GhACS2 and GhACS6 in roots, stems and leaves of the V. dahliae susceptible YZ1 cultivars with and without Vd991 infection as assessed by qRT-PCR. GhUBQ7 was used as the internal control to normalize all the data. Each experiment was performed using three independent biological replicates. Differences between groups were compared using Student’s t-test (*P < 0.05). C Southern blot analysis of the expression levels of GhACS2 and GhACS6 in transgenic lines GhACS2-OE and GhACS6-OE, respectively. The numbers represent the transgenic lines of GhACS2-OE and GhACS6-OE. The original image was put in the supplementary figure 3. D qRT-PCR analysis of GhACS2 and GhACS6 mRNA levels in YZ1, GhACS2-OE and GhACS6-OE. GhUBQ7 was used as the reference gene. Experiments were repeated three times with similar results. Differences between groups were compared using Student’s t-test (**P < 0.01). E HPLC analysis of ACC accumulations in roots of YZ1, GhACS2-OE and GhACS6-OE plants at 16 days after inoculation with Vd991. Experiments were repeated three times with similar results. Values are means ± SDs (Student’s t-test; *P < 0.05; **P < 0.01)