Figure 4: BZR1 interacts with TOPLESS through the EAR motif.

(a) A yeast two-hybrid assay shows the interaction between BZR1 and TPL. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (-His) plus 50 mM 3AT medium. (b) Mutation in the EAR motif abolishes the BZR1–TPL interaction. Core Leu residues of the EAR motif were substituted to Ala. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 1 mM 3AT medium. (c) BZR1 interacts with TPL and other TPRs. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 5 mM 3AT medium. TPL-N, N-terminal domain of TPL (1–344); TPR1-N, 1–343; TPR2-N, 1–335; TPR3-N, 1–335; and TPR4-N, 1–344. (d) BZR1 interacts with TPL through EAR motif in vivo. Arabidopsis mesophyll protoplasts were transfected with TPL-Myc together with bzr1-1D-GFP or bzr1-1DΔEAR-GFP, and the extracted proteins were immunoprecipitated by anti-GFP antibody. Gel blots were probed with anti-Myc or anti-GFP antibody. (e) TPL binds to DWF4 promoter through BZR1. GST-TPL-N (amino acids 1–344) was incubated with a biotin-labelled DWF4 promoter DNA (400 bps) together with MBP or MBP-BZR1 and pulled down with streptavidin agarose beads. Gel blots were probed with anti-GST or anti-MBP antibody. The full scans of the gel blots (d,e) are shown in Supplementary Fig. 4. (f) The TPL DNA binding on the promoters of CPD and DWF4 are enhanced by BR treatment. TPL DNA-binding was determined by ChIP assay using TPLp::TPL-HA transgenic plants. One-week seedlings grown on the medium containing 2 μM PPZ were treated with mock (M) or 100 nM BL for 4 h before crosslinking. Error bars indicate the s.d. (n=3) and **P<0.01 by Student’s t-test.