Figure 7.
CDK8 interacts with TGAs and is associated with the PR1 promoter. A, CDK8 interacts with TGA5 and TGA7, but not TGA1, TGA2, TGA3, TGA4, or TGA6, in Y2H assays. Yeast strains Y187 and AH109 were transformed with pGBKT7-CDK8 and pGADT7-TGA1-7 or an EV, respectively. Selected yeast diploids from a DD plate were recultured, diluted to OD600 nm = 1, 0.1, or 0.01, and placed onto a DD plate and a TD plate. B, Co-IP assay between HA-CDK8 and TGA5-GFP or TGA7-GFP in N. benthamiana. Proteins extracted from N. benthamiana leaves transiently expressing GFP, TGA5-GFP, or TGA7-GFP and HA-CDK8 by agroinfiltration were immunoprecipitated with an anti-GFP magnetic trap and immunoblotted with an anti-GFP or an anti-HA antibody. C, CDK8 is associated with the PR1 promoter in ChIP assays. Col-0 and CDK8-MYC cdk8-1 transgenic plants were treated with 0.5 mm SA for 4 h. Chromatin was extracted, then immunoprecipitated with an anti-MYC antibody. D, CDK8 is required for the association of RNA polymerase II (Pol II) with the PR1 gene promoter and coding region. After Col-0 and cdk8-4 mutant plants were treated with 0.5 mm SA for 16 h, chromatin was extracted, then immunoprecipitated with an anti-RNA polymerase II antibody. Primers used in the qPCR (C and D) amplify the following amplicons: -2000bp, amplifies upstream of the PR1 promoter at the −2,000-bp region; as-1, amplifies the sequence containing the as-1 region at the PR1 promoter; Actin2 was used as a negative control. The results are representative data from three independent experiments. Data represent means of three independent samples with se. Asterisks indicate significant differences (Student’s t test, *, P < 0.05). The experiments were repeated at least two times with similar results.