Supporting Information Vert et al. 10.1073/pnas.0803996105 Fig. S1. ARF2 interacts with BIN2 but not BES1 or BZR1. Shown is a representative example of staining for lacZ reporter expression for a variety of plasmid combinations. Staining is not detected for any of the plasmids on their own. Yeast transformed with BIN2 and ARF2 are the only tested strains to show detectable reporter activity. A–D show increasing strength of interactions for well characterized control proteins. Vert et al. www.pnas.org/cgi/content/short/0803996105 1of28 Fig. S2. ARF2 protein accumulation is not altered by BR treatment. Western blot using anti-ARF2 antibodies on total extracts from plants without or with 1 ␮M BL. The nonspecific band indicated with an asterisk (Lower) serves as a loading control. Vert et al. www.pnas.org/cgi/content/short/0803996105 2of28 Fig. S3. BIN2 can phosphorylate and reduce DNA binding of BES1. (A) Our preparation of BIN2 kinase can phosphorylate BES1. Upper shows Coomassie blue staining, and Lower shows Western blot using anti-MPB antibody. (B) In vitro DNA binding assays show that phosphorylation of BES1 by BIN2 greatly reduces BES1 binding to DNA, consistent with the results reported by Vert and Chory (1). Equivalent amounts of protein were incubated with DNA-coated beads. Proteins bound to the DNA were then pulled down, released by boiling, and run on an SDS/PAGE gel. Western blots using indicated antibodies are shown. Very little to no DNA is recovered when BIN2 is added to the reaction in the absence of BES1. 1. Vert G, Chory J (2006) Downstream nuclear events in brassinosteroid signalling. Nature441:96–100. Vert et al. www.pnas.org/cgi/content/short/0803996105 3of28 Fig. S4. arf2 resistance to BRZ is temperature-sensitive. At 20°C, arf2 mutants respond normally to BRZ treatment. If plants are grown at 22°C, hypocotyl elongation in arf2 mutants is dramatically less sensitive to reduced BR levels than what is observed in wild-type plants. Vert et al. www.pnas.org/cgi/content/short/0803996105 4of28 Table S1. Gene expression in response to the following treatments: mock (m), brassinolide (b), auxin (a), or the combination of both hormones (c) cm1 cb1 ca1 cc1 cm3 cb3 ca3 cc3 cm6 cb6 ca6 cc6 At4 g08950 0.4 1.0 0.7 1.3 0.4 1.5 0.6 1.8 0.8 1.4 0.9 1.6 SE 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.4 At3 g45970 0.9 1.0 1.0 1.5 0.4 1.0 0.6 1.3 0.6 0.8 0.6 1.1 SE 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.3 At1 g62440 0.1 0.4 0.3 0.4 0.2 1.2 0.3 2.2 0.4 0.7 0.3 2.6 SE 0.1 0.1 0.0 0.1 0.0 0.1 0.1 0.1 0.2 0.3 0.0 0.5 At1 g29490 0.2 0.6 0.7 1.7 0.2 0.7 0.2 1.7 0.4 0.3 0.3 1.6 SE 0.2 0.3 0.1 0.2 0.2 0.2 0.1 0.2 0.3 0.3 0.0 0.4 At4 g30290 0.6 0.4 0.5 0.5 0.3 0.6 0.3 1.2 0.6 0.7 0.4 1.7 SE 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.0 0.3 At1 g52830 0.3 0.7 1.6 2.5 0.1 0.5 0.4 1.6 0.2 0.3 0.4 1.0 SE 0.1 0.2 0.2 0.3 0.1 0.1 0.1 0.1 0.2 0.2 0.0 0.3 At3 g55840 0.4 1.3 0.6 1.7 0.2 1.2 0.4 1.8 0.4 1.0 0.4 1.9 SE 0.0 0.2 0.1 0.2 0.1 0.1 0.2 0.1 0.2 0.2 0.1 0.4 At2 g23170 0.0 0.1 2.2 1.8 0.0 0.0 2.8 2.5 0.0 0.0 2.0 1.9 SE 0.1 0.2 0.2 0.1 0.1 0.1 0.3 0.1 0.1 0.1 0.1 0.4 At4 g32280 0.4 0.7 2.3 2.0 0.4 0.3 1.3 1.2 0.2 0.1 0.8 0.6 SE 0.2 0.3 0.3 0.2 0.2 0.2 0.2 0.1 0.2 0.3 0.1 0.3 At4 g14560 0.2 0.3 1.7 1.2 0.4 0.3 1.3 1.0 0.3 0.3 1.2 0.9 SE 0.1 0.2 0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.3 At5 g02760 0.7 0.9 1.7 1.6 0.6 0.8 0.9 1.4 0.5 0.6 0.8 1.4 SE 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.3 At5 g47370 0.6 0.9 2.5 2.2 0.5 0.6 1.0 1.3 0.4 0.6 1.3 1.2 SE 0.1 0.2 0.3 0.2 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.3 Expression was analyzed by quantitative RT-PCR, and all data are normalized to the expression of At1 g13320, a gene with constant expression. Numbers refer to the number of hours seedlings were exposed to each treatment (e.g., 1 ϭ 1 h). The average expression level for three replicate treatments is shown on the first line with the standard error (SE) shown on the second line. Vert et al. www.pnas.org/cgi/content/short/0803996105 5of28 Table S2. One hundred most significant genes differentially expressed under BRZ treatment in wild-type Col-0 plants (genes were ranked by adjusted P value). Average expression for each condition is given. A:arf2, C: Col, B: BRZ, M: mock. Ratios for arf2 on BRZ vs. arf2 on mock (AB/AM) and Col on BRZ vs. mock (CB/CM) are also shown. Adjusted log Affy AGI P value (odds) AB AM CB CM AB/AM CB/CM Annotation 262682࿝at At1 g75900 1.4E-11 16.2 130 1006 130 943 0.7 0.7 unknown 263890࿝at At2 g37030 2.1E-11 15.9 140 21 138 25 6.5 5.5 SAUR46 267101࿝at At2 g41480 1.3E-10 14.5 35 61 42 153 0.6 0.3 PER25 peroxidase 249894࿝at At5 g22580 1.3E-10 14.5 2010 233 1890 254 1.5 1.4 unknown 256145࿝at At1 g48750 2.4E-10 14.0 1224 463 1112 351 1.2 1.3 lipid transfer protein 245891࿝at At5 g09220 3.2E-10 13.7 605 380 773 147 1.1 1.4 amino acid transport protein AAP2 253627࿝at At4 g30650 4.9E-10 13.3 20 141 16 58 0.3 0.3 RCI2E low temperature and salt responsive protein 264007࿝at At2 g21140 5.9E-10 13.2 509 2426 756 4335 0.8 0.8 unknown 247162࿝at At5 g65730 6.2E-10 13.1 65 25 119 27 2.1 1.9 AtXTH6 263443࿝at At2 g28630 6.2E-10 13.1 120 550 108 278 0.7 0.7 fatty acid elongase 263098࿝at At2 g16005 6.9E-10 13.0 29 28 29 390 1.1 0.3 unknown 254809࿝at At4 g12410 7.0E-10 13.0 192 52 340 46 1.9 2.2 SAUR35 255177࿝at At4 g08040 7.3E-10 13.0 111 18 131 23 3.5 2.6 AtACS11 264217࿝at At1 g60190 7.6E-10 12.9 165 325 138 258 0.8 0.8 Plant U-box protein, AtPUB19 267457࿝at At2 g33790 7.8E-10 12.9 215 21 80 21 4.2 3.5 unknown 264968࿝at At1 g67360 1.4E-09 12.4 173 236 99 276 0.9 0.7 unknown 246250࿝at At4 g36880 1.4E-09 12.4 465 1818 206 910 0.8 0.8 cysteine proteinase 261881࿝at At1 g80760 1.5E-09 12.3 112 47 103 44 1.9 1.9 Delta tonoplast integral protein 260070࿝at At1 g73830 2.0E-09 12.1 370 137 431 129 1.2 1.3 BEE3, bHLH050 250475࿝at At5 g10180 2.0E-09 12.1 1597 425 496 82 1.2 1.4 sulfate transporter 251768࿝at At3 g55940 2.1E-09 12.0 35 98 29 121 0.6 0.4 phosphoinositide-specific phospholipase C 259846࿝at At1 g72140 2.2E-09 12.0 45 56 28 103 0.9 0.5 peptide transporter PTR2-B 263809࿝at At2 g04570 2.2E-09 12.0 330 815 349 1004 0.8 0.8 GDSL-motif lipase/hydrolase 264527࿝at At1 g30760 2.3E-09 11.9 45 121 46 141 0.6 0.6 reticuline oxidase-like protein 249037࿝at At5 g44130 2.4E-09 11.9 133 446 244 1286 0.7 0.7 unknown 259616࿝at At1 g47960 2.8E-09 11.8 128 160 101 195 0.9 0.8 unknown 245422࿝at At4 g17470 3.1E-09 11.7 77 65 66 265 1.1 0.5 PPT1-like thioesterase 250646࿝at At5 g06720 3.4E-09 11.6 24 81 28 126 0.5 0.3 PER53 peroxidase 250199࿝at At5 g14180 3.6E-09 11.5 275 696 133 345 0.8 0.7 unknown 250157࿝at At5 g15180 3.9E-09 11.5 86 29 159 30 1.8 2.1 PER56 peroxidase 245506࿝at At4 g15700 5.6E-09 11.1 31 20 51 17 1.2 2.0 glutaredoxin 251072࿝at At5 g01740 6.1E-09 11.1 1741 166 1634 319 1.7 1.4 unknown 251196࿝at At3 g62950 6.3E-09 11.0 61 43 86 33 1.2 1.9 glutaredoxin 254907࿝at At4 g11190 6.8E-09 10.9 205 315 475 1704 0.9 0.8 unknown 263876࿝at At2 g21880 7.0E-09 10.9 215 332 140 351 0.9 0.8 RAS superfamily GTP-binding protein 246228࿝at At4 g36430 7.7E-09 10.8 45 177 36 252 0.5 0.4 PER49 peroxidase 252118࿝at At3 g51400 8.0E-09 10.8 216 66 199 53 1.7 2.0 unknown 257952࿝at At3 g21770 8.2E-09 10.8 432 942 349 1000 0.9 0.8 PER30 peroxidase 255549࿝at At4 g01950 1.0E-08 10.6 363 793 321 1289 0.9 0.8 mitochondrial glycerol-phosphate acyltransferase 256601࿝s࿝at At3 g28290 1.0E-08 10.6 355 306 206 41 1.2 5.0 integrin-like 259878࿝at At1 g76790 1.1E-08 10.5 256 281 226 688 1.0 0.8 catechol O-methyltransferase COMT-like8 256598࿝at At3 g30180 1.1E-08 10.5 1945 551 2143 588 1.2 1.2 BR6OX2 262427࿝s࿝at At1 g47600 1.3E-08 10.4 230 55 435 53 1.7 1.9 thioglucosidase 261327࿝at At1 g44830 1.3E-08 10.3 239 100 274 88 1.2 1.3 AP2-EREBP transcription factor 248252࿝at At5 g53250 1.5E-08 10.2 1113 2602 1105 3530 0.9 0.9 unknown 258629࿝at At3 g02850 1.6E-08 10.2 190 107 179 55 1.3 1.7 stelar Kϩ outward rectifying channel (SKOR) 245463࿝at At4 g17030 1.8E-08 10.0 89 211 102 260 0.7 0.6 AtEXLB1 254667࿝at At4 g18280 2.0E-08 9.9 139 1194 109 441 0.6 0.7 glycine-rich cell wall protein 248844࿝s࿝at At5 g46900 2.4E-08 9.8 872 1383 882 3282 0.9 0.8 extA 249719࿝at At5 g35735 2.7E-08 9.7 128 321 64 168 0.8 0.7 unknown 247723࿝at At5 g59220 2.7E-08 9.7 155 157 103 318 1.0 0.7 protein phosphatase 2C 263063࿝s࿝at At2 g18140 2.7E-08 9.6 27 47 24 86 0.6 0.4 PER14 peroxidase 259328࿝at At3 g16440 2.8E-08 9.6 217 134 205 81 1.1 1.4 lectin 267590࿝at At2 g39700 2.8E-08 9.6 257 380 346 889 0.9 0.8 AtEXPA4 249482࿝at At5 g38980 2.9E-08 9.6 513 95 175 65 1.5 1.4 unknown 250231࿝at At5 g13910 2.9E-08 9.6 189 69 165 66 1.6 1.7 AP2-EREBP transcription factor LEAFY PETIOLE 246550࿝at At5 g14920 3.3E-08 9.5 3202 1680 3397 1240 1.1 1.1 unknown 247477࿝at At5 g62340 3.5E-08 9.4 137 27 88 23 3.3 2.7 unknown 264404࿝at At2 g25160 3.6E-08 9.4 43 27 91 169 1.4 0.7 CYP82F1 253957࿝at At4 g26320 3.6E-08 9.4 133 227 135 446 0.8 0.6 unknown 245392࿝at At4 g15680 4.3E-08 9.2 29 28 82 25 1.0 2.1 glutaredoxin Vert et al.