Fig. 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 hr before crosslinking. Error bars indicate the s.d. (n=3) and **: P<0.01 by Student’s t-test.