Fig. 3. The molecular mechanism of TWA1-mediated transcriptional repression.
a, TPL and TPR corepressors are required for TWA1-inhibited reporter expression. Protoplasts of tpl tpr2 tpr4 triple-mutant plants were transfected with TWA1, TPL and TPR2 effector DNA. b, JAM1 and JAM2 co-expression rescues the TWA1 response in protoplasts of jam1 jam2 jam3 plants. For a,b, ABA response analysis was performed as in Fig. 2b with 3 µg effector DNA per 105 protoplasts. c,d, Accumulation and binding of GFP–TWA1 to JAM2 and TPL in nuclear subdomains of N. benthamiana epidermal cells. GFP–TWA1 and mCherry-tagged JAM2 or TPL were transiently expressed in leaves exposed to 37 °C for 2 h or kept at 20 °C. c, GFP imaging in the nucleus (see the ‘Statistics and reproducibility’ section in the Methods). Scale bar, 5 μm; insets are 1.5-fold magnifications. d, FRET–FLIM analysis. Statistical analysis was performed using two-sided Mann–Whitney U-tests; ***P = 0.005. n = 20–30 cells. Box plots are as described in Fig. 2e. e, Simplified model of repressor complex formation. Temperature transitions from 20 °C to 30 °C induce conformational changes in TWA1 that allow JAM2 binding to the carboxy-terminal part of TWA1 and TPL binding to the amino-terminal domain through EAR motifs (red). The HVR (yellow) is integral to thermosensing. JAM2 interacts with G-box-related cis-elements as dimers. TPL is tetrameric and targets subunits of the Mediator complex33. Statistical significance details are provided as Source Data.