Supplementary Figure 1. BES1 specifically inhibits ABA responses in early seedling development. a. Exogenous BR application overcomes the hypersensitivity of bzr1-1d seedlings to ABA. Seed germination rates of wild type, bzr1-1d and transgenic bzr1-1d mutants (35S-bzr1-1D-HA and pbzr1-bzr1-1d-ha) grown on 1 µm ABA with 0, 10, 50 or 100 nm epi-bl were measured at 4 days after plating (error bars indicate S.E., n=3; over 100 seeds were tested in each independent experiment). b. Exogenous BR application barely rescues ABA-mediated post-germinative developmental arrest in bes1 ko mutants. The representative phenotypes of 10-day-old wild-type (Col-0) and bes1 ko seedlings grown on 1 µm ABA with 50 nm epi-bl were presented (left). The percentage of fully germinated seedlings of 10-day-old Col-0 and bes1 ko grown on 1 µm ABA with 0, 10, 50, or 100 nm epi- BL was presented (right, n=3, over 50 seedlings were analyzed in each independent experiment. Error bars indicate S.E.; ** : p<0.01).
Supplementary Figure 2. BES1 interacts with TPL and TPRs. a. The bes1-d interacted with TPL via the EAR motif, but not with SAP18 and SIN3. b. A dominant tpl-1 (N176H) 3 mutation disrupted the interaction with BES1 in yeast. c. BES1 interacted with TPR2 via the EAR motif, but not with TPR3. d. N-terminal domain (1-200 amino acids) of TPL, TPR1, 2, or 4 (TPL-N, TPR1-N, TPR2-N, or TPR4-N) interacted with BES1. Transgenic yeast cells expressing the indicated gene sets were selected in synthetic medium lacking Leu, Trp and His (-LTH), but containing 1 mm of 3AT.
Supplementary Figure 3. BR enhances the interaction of TPL with BES1 in the nucleus. a. TPL interacted with both phosphorylated and dephosphorylated BES1 proteins. BES1-HA was co-transfected with BRI1-HA (left) or BIN2-myc (right) into protoplasts, and incubated for 6 h. Protoplasts overexpressing BRI1-HA and BES1-HA were further incubated with 1 µm BR for 1 h. Protoplast lysates were incubated with purified GST or GST-TPL-N proteins, and pulled down with glutathione sepharose 4B. GST-TPL-N-bound proteins were visualized with anti-ha HRP antibodies. GST protein was included as a negative control. b. The interaction between TPL and BES1 was increased by BR treatment in the nucleus. BiFC assays using TPL-nV (nvenus) and cv (cvenus)-tagged BES1 in the presence or absence of BR were performed. ARR2-mRFP 1, 2 was used as a nuclear marker. Scale bar, 10 µm.
Supplementary Figure 4. The EAR motif of BES1 is essential for BR responses and repression of ABI3. a. The protein expression levels of bes1-d-ha and bes1-dmear-ha (#1-4) overexpressing lines were determined using a monoclonal anti-ha antibody. Actin was used as an internal control. b, c. 35S-bes1-DmEAR-HA lines failed to increase BR responses. Four-week-old Col-0, 35S-bes1-D-HA and 35S-bes1-DmEAR-HA lines were shown (b). The wild-type (Col-0) and bes1-d, 35S-bes1-D-HA and 35S-bes1-DmEAR-HA transgenic plants were grown in the dark with 1 µm BRZ for 5 days (upper, c). The hypocotyl length was measured (lower panel, c; error bars indicate S.E., n=10). d. Four-dayold seedlings of Col-0, 35S-bes1-D and 35S-bes1-DmEAR (#1 and #2) were transferred onto 1/2 B5 agar plates containing 0, 2.5 or 5 µm ABA, and root length was measured after 5 days. Relative growth compared to respective growth on ABA-free medium is presented (error bars indicate S.E., n=30; * : p<0.05, ** : p<0.01).
Supplementary Figure 5. The BES1-TPL-HDA19 repressor complex formation is essential for BR responses and repression of ABI3. a, b. BR related activity of bes1- DmEAR is completely recovered by its fusion with SRDX, TPL or HDA19. The wild-type (Col-0), and 35S promoter-driven bes1-d-ha, bes1-dmear-srdx, bes1-dmear-tpl and bes1-dmear-hda19 transgenic plants were grown in the dark with 1 µm BRZ for 5 days (lower panel, a). The hypocotyl length of wild-type and transgenic plants was measured (upper panel, a, error bars indicate S.E., n=10). The representative four-week-old Col-0 and the transgenic lines were shown (b). c. Four-day-old seedlings of Col-0, 35S-bes1-DmEAR- TPL, 35S-bes1-DmEAR-HDA19 and 35S-BES1-HA were transferred onto 1/2 B5 agar plates containing 0, 2.5, or 5 µm ABA, and root length was measured after 5 days. Relative growth compared to respective growth on ABA-free medium is presented (error bars indicate S.E.;
n=20). d. Relative fold change of indicated gene expression in Col-0, 35S-bes1-D, 35S-bes1- DmEAR and 35S-bes1-DmEAR-HDA19 lines was determined by a real-time qrt-pcr (mean ± S.E., n=4; * : p<0.05, ** : p<0.01).
Supplementary Figure 6. The expression of ABI3 and ABI5 is up-regulated in tpl-1 seedlings. The expression levels of ABI3 and ABI5 in 3-day-old heterozygous tpl-1 and wildtype (Ler) seedlings were determined by real-time qrt-pcr (error bars indicate S.E., n=4). Asterisks indicate statistically significant differences analyzed by Student's t-test (** : p<0.01,* : p<0.05)
CTAATTTTATTAAATAGGCTGAAAACCATAAATTATTTGGAGCCAAGATTAAATTATGTTACATAAATCCTTTTCTCGTCATCAATACTCAAATTTAAAT TTTTTTATTTTCATAGATATTTTTGGGAATTTAAAATCCTTCCAAATAAATTTGGGAAAATAATAGATGTTTTAAAATATTTTTGTTATTTTCGAGTAGT TTACACTGATTTTACATCACTTTATGGATCAAGACCATAAAATTTAACTACATTTATTATAACTTATTTGAAAAGACATGAAACTTTCATTAGAGTTTAG GAAAATACGGCAAACTATTAGATTATCACCCTCTTGTGACTAATGTTAATACTATTTGGTAAAGCAAATAATCCCATAACTAATTTTATTTTGATTTGTT #1 CACTATCAATTGTATCATAGACGATCCGATGATTAATGTACCTAAATTTTGATTTAAATTGTATTCCTTGTAGTACGCATAGTAAAACAAAGTTCACATG TCTTTTACAATTGGTTTTCTAACATTTTAGATTTCATTTTAAACTAAAGAAAAAGGGTTTTTCGATCCCAACAATTTTTAAAGTATGTTTTTGAATTTC #1 #2 AACAAACGACTAGTACTGATATATACATCAATGCTTACAAAAATCAAATTATCTATCTCAACGTTTACTTTTGTACGTAATTACAAGTCAATATTAACAA ATATTAGTCAACTGTAACGGTTGTTGACTCAATTGATACGTGTACGTTTAGGTGGCATGTTTTTTAATAAAATAATTTTGTTTTATGTTTTGGGATTAAA AATATTTACTTTTAAAAAACTAAACCGAAAAACTGGAACCCCCAACCTCACATATTCATCACCATCGTATCCACAAACATTATCGAGACATAAATAATAA #2 TTTATAGATATATAGCAACAAATTGGAATTAAATTAATCAAACAATGTCATTTAGAATTGGGGAAATCCTAACCGACGTTATATGATTAATTTTAATTCC #3 AATTTATGTATGTGTATATATCTATAAATTATTATTTATATATTGATGATGTTCGTGCATGATGGTAATGGTAACCTTTAAAACTTTTGAATCTCAAAAC GTAAAACAACGTCATTTGGTTTGAAGAACATGCCATCTCGACGTACAAGTGTTAGCTGATTCAACAACCGTTACTGTTAATTACCATAACTAACGTTGAC TCATAATTATGTACAAAAGTAAGAGTTGAGGTAGATAATTTGAATTTTTTAAATGTTAAAGTATGAACATTACTAATTGTTTGTTCAGATTTATAAAACA #3 CAGTACCTATTTTGATGTGTTAAAATGTAATCGGGAAAATAGGAATTTTCCCAAGAAAAAAAAATACACTAAAATTTTGATGTGTTAAAATGTAATAAAT #4 ACAAGTACCTATTTCTTTCTACAACAGGTATCACATGATCTGCTCAATCTCATTTTGAAGGCAAGGTCGAACCCTGTTTGTAACTAAATATTTTACAAAT #4 ACACTAAAAGTTTTGTTTTGTTTTCCTCATGCAGAAAAAAGAAAAGAAAGAAGAAGAAAAAAAACTCACAAAAACATACATTGTTCTACAATATGTTAAA GTAATTGTTCTAGGGTAATAAAATCCGTGTCTGCCTCTTCCACTTCAATAGGAAAGTGTAAGTTTTTCGAATCTTTTTGGCCAGATAATTTAAAACCGAC CGAGGAAACTTACTTTTGTAAAAGTGACCATTTGCACATAGGAAGTAGAAAGTTGATAATTGGAAAATTTATAATATTTTATACAAAAGAAGTGTTTAAG AACCACCGCTTGGTCTCACCACTCAAATTCGAACGTATGGCTCGTGGGCACAAACTCCTGTACCAACCTTTAGATACTAGCGGCACGAGGAGACTTCTAT TTTCTCTTTGTTTTTAAGTTTTTTTATCGACTTAAATATGGCTTTTTGCACCATTTAATTTCAAAACCAATGTCTTTCTCTCGAGGAACTTTGTTTTTAT TTCTTAGAAGATGAGGGGAGATTTACTATCTAAATAAAATTTTAAATGTTTGTAAGTATTATGAGCTCAACAATTTTGTCAATAGTGCCACAAATTTAAA CGTTTGCTTTTTGTCTTCTTTGAAAAATCAAATGCTGAAAAACTGTTACATCTCTTTTTCTTAAAAACTCTTGTCTCTCTCCTTTTCCTTCTGCTGAGGT #5 AATTGAATGCTGCAAAGAGAAAGAGAATAACTTAAACCCAAAATTACACTTACCGCCAGAAAAAAAAAAGAGTTAGTTTAATCTAACATATTTTATACAA TACAATTGAATTATATTAGTAAAAAAAAAACTTCCATATAAATCATGGAACAAACTGGAACACATGGGCTCTCTTATTTTAATTTATTTTCTTTTTTGAG GGATTTAACCATGTTTATTATATAGTTTTATAAATATATATATACCATCTCTCCATAATTTATAAAATGAAAGAAAGAGAGAGTCTTCTTGTTGGAGTAA #5 ACCCAAACGGTTTTAGATTACTTATTAGCTGTTCATCAGTTCTTCCTCTCTAAAAGAGTAAAACCTAAACATCTCTCTCTGTTCTATTAGAACCAAAGAC #6 CAATCTTTGTGAACAAAACACATCTCGTATACTTCAGATCTAGACTCGAAAATTTTAGACCTCTTTACAATTGGTCTTTGTTCATCTGAAGTTGGAGAAA ATAGTTAGCTTAGGTCGGATCTTTTCATATGCTTTGGATCCTCCTTCGTCTCTTTTGTATAATTTTAACCTTATCAAGAGTTCTTTTTGAATCTCAAAAG ATTATATAGTAGTATAGAAGGTTTATATGTATATGTATAGCCAGATAGTTTATGTTGTTTAAAGATTCGATGATAGCCAAGTTGGGTTAACTTTCTTTTT CCTTGCCTCCTTACTCACATACAAACCCTATCTGTCCGTACAAAATACTAAAAACCCTAACTTTTCTCTCTCCACCAATCTAGTTTATTGTTTCATTTCC #7(+1) ACTTCAACG ATGAAAAGCTTGCATGTGGCGGCCAACGCCGGAGATCTGGCTGAGGATTGTGGAATACTCGGTGGAGACGCTGATGATACTGTTTTG #6(-1) GATGGAATTGATGAAGTTGGTAGAGAGATCTGGTTAGATGACCATGGAGGAGATAATAATCATGTTCATGGTCATCAAGATGATGATTTGATTGTTCA #7 Supplementary Figure 7. The ABI3 promoter sequence. The CAnnTG sequences are highlighted as putative BES1-binding motifs by green (mutated BES1 target sites for generating the pabi3m construct in this study) and red colors. Red character indicates 5 - UTR region and purple characters are primer sets for a ChIP assay. ATG in #7 forward primer region indicates a translational initiation site (+1).
Supplementary Figure 8. BES1 binds to the ABI3 promoter. Electrophoretic mobility shift assay (EMSA) with GST-BES1 proteins using radiolabeled ABI3 promoter probes. The promoter regions of ABI3 (-2545 to -2486; refered to pabi3 #1, -344 to -285; refered to pabi3 #6) were used. Unlabeled pabi3 #1 or pabi #6 were used as competitor DNA. The GST-BES1 complexes are indicated by an arrow.
a #1 TCTATTCAAAATCTAACAAGTCTACTTTCACCAGCTAGAAGCTCAACGACAAACAAGTGTTTACAAACTTTGAC TATTTCTTTCATAACATCTTGAATCTAACCTAAATATGTTTTTACCAAAGTAAATCAGTATTGATCAATCAAATTAATCCGTCAATTTCACAAA ACAAATCATGAACCAATATGTAAAAAAAGATCACACTTTAATTCGCTCTTCTAATCCAAATTTCACGTTCTTATTATCTTATATATGCTTAAAA GAGAGAAAATCATGTCTAGTTTTAGACGTAAAAAAACATTTGTGTAGCCGAAGTCACACGTGTCGAGCCTGTGAGAAGACACTGCAAGAAACAG AAGAGAGACACGTGCAGGACACGTGTCGTCCGCAGCCGAACGGATTCTTTGTCTCTGATCATGGGCCTGGACCTGGACCTGTCTAAGTTAGCAT #1 TCCATTGGTCGAAAGATAACTTTAGACAAAAGATGTTGACCTTCACGCCTCTCTTCTTAGACCCCACGCGCCACACTCTCACGGTGAGAACATA AATATCAATCTCTCTTTTACCGCCTCCTACCCATTTATCTCTCTCTTTCTCAAAACCTTTCAGTCAAAATTCTCCGGCGGCTTTTAAACTATGT GAAGGAGGAGAACCTCCATAACAAGAAGCGGATTCTCTCAGTTTTCCGGCGGCGGAGGAACACAAAGCCACCGGTTTTTAGACACACAGATTTC ATTTTCAGGTTAACACTTTTCTTGTTTTATAATTTTTCAAGATCTTGGATTAGAAGACTGTTAAATAAATCTATCTCTTTATTTATAAGTTTAG GTCTTCGTTTTCTGGTCCTTACTTATCTGCGTATTACCAAATATTAAACTTTGTGGTAAAGTGATACTTAGTAATAGACGGATATAATGATGAC CCAAGCTAATGATTAATTTATTTTACTAATCATCAGGTTAAGCTCTAGATTATCTTTTGTTTAAAACCGTATTAATAAATGATTAAAGAGGAAA AAAATCTTGTGTTGATAAGTTCGCTTTAAGAAGAAAACGAGTGGGTTAAGATATTTTCCTCTTTTGGTAACGAAAACTTTATTGGAAGAGTAAA #2 TTATTAGTTTATTTATTGCTAATTTTAGTATCGTTTGTCGCTGTCACGATGTGGACCGTTCTTCTTTATTTTCACGTGGACCGACTTAATTACC TTCTTTCATTCTTGTTATTTCAATATTTTTTACGTCATTTAAACTCCACTTTACCAAAATAATTTATTTCATTCTTTGAGAGCATCAATTATCA AATAATTCAATTGTCTTTTTTCGTGATAGTTGAATAAGGGACAACTATTAAGTGAGTTTCGTGAATAGCTGAACAGGGACAAGTAACTGAAGTT #2 TGGTCAAAATAATACGATGAATTTAATATATAAGAGACAAAATTGTTATTGTCGTTTAGGTGGTATCCATATTTCAATATAGTTTGTGTGAATA CAGATATAAAAATGGTTATTGTTGTGTATATGATGCAGTTGTTAA Supplementary Figure 9. ABI5 is not directly regulated by BES1. a. The ABI5 promoter sequence. The CAnnTG sequences are highlighted by yellow color as putative BES1 binding E-boxes. Purple characters are primer sets for a ChIP assay. b. BES1 is not directly bound to the promoter of ABI5. The regions containing E-boxes of the ABI5 promoter in wild-type and pbes1-bes1-d-ha transgenic plants were analyzed by ChIP assays with anti-ha antibodies. The fold enrichment is presented as a relative level to wild-type (error bars indicate S.E.; n=3).
Supplementary Figure 10. BES1-binding E-box motifs on the ABI3 promoter are essential for BR-mediated repression of ABI3 expression. a. E-box motifs in both #1 and #6 regions are required for BES1-mediated repression of ABI3. Protoplasts were cotransfected as indicated with 35S-Renilla (an internal control), and pabi3-luc, pabi3m1- LUC (mutated at -2496~2502 of #1 region), pabi3m6-luc (mutated at -278~284, -321~327, -336~342 of #6 region) or pabi3m-luc (mutated at both #1 and #6 regions), or the bes1-d effector construct, and incubated for 6 h. The relative pabi3-luc/renilla activity was measured (n=4). b, c and d. The BES1-binding E-box motifs on the ABI3 promoter are only required for BR-mediated repression, but not for ABA response. Four-day-old seedlings of pabi3-gus and pabi3m-gus transgenic plants were incubated with 5 µm ABA (b) or 1 µm epi-bl (BR, c, d) for 3 hr. The relative transcript fold changes of GUS, CPD and endogenous ABI3 in the ABA or BR treated transgenic seedlings were determined by a real-time qrt- PCR (mean ± S.E.; n=4; * : p<0.05).
Supplementary Figure 11. BES1-binding E-box motifs on the ABI3 promoter are essential for BR-mediated repression of ABA response. a. The ABA insensitiveness of abi3-8 in seed germination is completely recovered to wild type level by pabi3(m)-gabi3- FLAG expression. Five-day-old seedlings of abi3-8, Col-0, two independent abi3-8 pabi3- gabi3-flag lines (#1 and #6) and pabi3m-gabi3-flag lines (#1 and #9) grown on 1 µm ABA were presented. b. BRs barely suppress ABA responsiveness of abi3-8 pabi3m-gabi3-
FLAG lines in seed germination, but that of abi3-8 pabi3-gabi3-flag lines. Seed germination (radicle emergence) rates were determined in the wild-type (Col-0), abi3-8 pabi3-gabi3-flag and abi3-8 pabi3m-gabi3-flag lines grown on 0 (Mock) or 1 µm ABA with 0 (ABA) or 100 nm epi-bl (ABA + BL) for 3 days after plating (the error bars indicate S.E.; n=3; over 100 seeds were tested in each independent experiment; ** : p<0.01). c. The histone H3 acetylation states of the ABI3 locus is rarely altered in bes1 ko in response to exogenous BR. ChIP assays using an anti-acetyl-histone H3 antibody were performed with 4- day-old wild-type (left) and bes1 ko (right) seedlings treated with or without 1 µm epi-bl for 3 h, and the fold enrichments were normalized with mock treatment control (error bars indicate S.E.; n=4).
Supplementary Figure 12. BR signaling pathway is linked to the ABI3-ABI5 module of ABA signaling. a, b. Ectopic expression of ABI5 suppresses the bes1-1d phenotypes in ABA responses. The protein expression levels of ABI5-HA in bes1-d 35S-ABI5-HA were detected with an anti-ha antibody. Notably, #12 and #18 showed the ABI5-HA expressions. Two asterisks indicate non-specific bands (a). En-2, bes1-d and 35S-ABI5-HA transgenic seeds (#12 and #18) were germinated on 1/2 B5 agar plates containing 0, 0.5 and 1 µm of ABA for 3 days and the germinated seeds (radicle emergence) were counted (b; error bars indicate S.E.; n=3; **: p<0.01,*: p<0.05; over 50 seeds were tested in each independent experiment). c, d. ABA hypersensitivity of bri1 is recovered by loss-of-function abi3-8 and abi5-7 mutations. Seed germination phenotypes of Col-0, bri1, abi3-8, abi5-7, bri1 abi3-8 and bri1 abi5-7 in the presence of ABA (c). The seeds were germinated on 1/2 B5 agar plates containing 1 µm ABA for 4 days and the germinated seeds were counted (error bars indicate S.E.; n=3; ** : p<0.01,* : p<0.05; over 50 seeds were tested in each independent experiment). Assay of
ABA-mediated root growth inhibition in bri1 abi3-8 and bri1 abi5-7 seedlings (d). Four-dayold Col-0, bri1, bri1 abi3-8 and bri1 abi5-7 seedlings were transferred onto 1/2 B5 agar plates containing 0, 1 and 2.5 µm ABA. Representative Col-0, bri1, bri1 abi3-8 and bri1 abi5-7 seedlings grown on ABA-containing medium for 5 days.
Supplementary Figure 13. DWF4 overexpression (dwf4-d) promotes seed germination in the presence of paclobutrazol (PAC). Eight-day-old seedlings of Col-0 and dwf4-d grown on 1/2 B5 medium containing 0 or 10 ppm PAC (a). Col-0 and dwf4-d seeds were germinated on 1/2 B5 agar plates containing 0, 10 or 20 ppm PAC for 14 days and the germinated seeds (radicle emergence) were counted (b; error bars indicate S.E., n=3; ** : p<0.01; over 50 seeds were tested in each independent experiment).
Supplementary Figure 14. Hypothetical model for BRs and ABA crosstalk. Upon BR stimulation, the active BES1 accumulated in the nucleus directly binds to the ABI3 promoter and recruits transcriptional co-repressor TPL-HDA19 complexes. The repressor complexes facilitate histone deacetylation and maintain the heterochromatin state of the ABI3 locus. These events repress ABI3 and subsequently reduce the expression of its downstream target genes, including ABI5.
Supplementary Figure 15. Full scan data of immunoblots. Asterisks indicate the bands shown in the figures.
Supplementary Table 1. ABA-responsive genes are downregulated in bes1-d, but not in bzr1-1d Transcript fold changes of ABA-responsive (RD29A/ B and ABI1) and BRbiosynthetic (CPD and DWF4) genes at 3h after 5 µm of ABA treatment (mean ± S.E. n=6). Transcript fold changes of ABA-responsive (RD29A/ B and ABI1) and BR- biosynthetic (CPD and DWF4) genes at 3h after 5 µm of ABA treatment (mean ± S.E. n=3).
Supplementary Table 2. Details of primer combinations Supplementary material and methods.
Supplementary Methods Plasmid constructs and protoplast transient expression assay The full-length cdna of bes1-d (BES1 P233L ), and bes1-dmear-srdx, bes1-dmear-tpl and bes1-dmear-hda19 were cloned into plant expression vectors containing hemagglutinin (HA) or FLAG at the C-terminus under the control of the 35S C4PPDK promoter 1, 2. For LUC-fused reporter, 3-kb of the ABI3 promoter was cloned into a LUC containing plant expression vector 1. All mutants of pabi3-luc were generated using the QuickChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer s instructions. All cdnas and the mutations were confirmed by DNA sequencing. For the reporter assay, 2 x 10 4 protoplasts were transfected with 20 µg of total plasmid DNA composed of different combinations of the reporter (pabi3m1-luc, pabi3m6-luc, or pabi3m-luc), an effector (bes1-d) and an internal control (p35s-renilla). The transfected protoplasts were then incubated at 1 x 10 4 cells per ml for 6 h at room temperature. Protein protein interaction assays The protoplasts overexpressing BES1-HA with or without BIN2-myc were lysed using IP buffer [50 mm Tris-HCl (ph 7.5), 75 mm NaCl, 5 mm EDTA, 1 mm DTT, 1 protease inhibitor cocktail (Roche), and 1% Triton X-100]. Total protoplast lysates were incubated with recombinant GST or GST-TPL-N proteins for 1 h. The protein complexes were then precipitated with glutathione sepharose 4B (GE healthcare). Precipitated proteins were analyzed by SDS PAGE and detected using HRP-conjugated monoclonal anti-ha antibody (Roche). Electrophoretic mobility shift assay (EMSA) For EMSA assays, double-strand oligonucleotides of #1 and #6 regions containing BES1- binding E-boxes were synthesized. The sequences of oligonucleotide probes are as follows.
ABI3 #1 F: tccttgtagtacgcatagtaaaacaaagttcacatgttcttttacaattggttttctaac, R: gttagaaaaccaattgtaaaagaacatgtgaactttgttttactatgcgtactacaagga ABI3 #6 F: acaattggtctttgttcatctgaagttggagaaaatagttagcttaggtcggatcttttcatatgc R: gcatatgaaaagatccgacctaagctaactattttctccaacttcagatgaacaaagaccaattgt The double strand probes were labeled with [γ 32 P] ATP(>3000 Ci/mmol, 10 mci/ml) by T4 polynucleotide kinase (New England Biolabs) for 30 min at 37 C. The unlabeled radionucleotides were removed using Illustra MicroCpin G-25 column (Amersham). The 5 µg of GST alone or GST-BES1 proteins were incubated in the binding buffer [10 mm HEPES ph 7.9, 5 mm Tris-Cl ph 8.0, 2.5% glycerol, 0.5 mm DTT, 50 µg/ml BSA, 25 mm KCl 250 µm EDTA, 1 µg poly(di-dc)] with 20,000 cpm of double-strand oligonucleotides for 20 min in room temperature. The reaction mixture was resolved on 6% polyacrylamide gel in 0.5X TBE buffer. Supplementary References 1. Hwang, I. & Sheen, J. Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413, 383-389 (2001). 2. Ryu, H. et al. Nucleocytoplasmic shuttling of BZR1 mediated by phosphorylation is essential in Arabidopsis brassinosteroid signaling. Plant Cell 19, 2749-2762 (2007). 3. Long, J. A., Ohno, C., Smith, Z. R. & Meyerowitz, E. M. TOPLESS regulates apical embryonic fate in Arabidopsis. Science 312, 1520-1523 (2006).