pgbkt7 Anti- Myc AH109 strain (KDa) 50
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1 pgbkt7 (KDa) Anti- Myc AH109 strain Supplementary Figure 1. Protein expression of CRN and TDR in yeast. To analyse the protein expression of CRNKD and TDRKD, total proteins extracted from yeast culture were analysed by western blotting using an anti-myc antibody.
2 Anti- Penta-His CBB + Purified 6His-TDRKD (KDa) GST GFP GST BIN2 GST BIL * GST-BIN2 GST-BIL1 GST-GFP His TDRKD Supplementary Figure 2. TDRKD physically interacts with BIN2 and BIL1. Pull-down assay was performed using GST-GFP (negative control), GST-BIN2, GST-BIL1, and 6His-TDRKD produced in E. coli. Proteins eluted from glutathione sepharose column were analysed by Coomassie Brilliant Blue (CBB) staining (upper panel) and western blotting using an anti-penta-his antibody (lower panel) after SDS-PAGE. Asterisk indicates the C-terminal truncated form of BIN2, which was confirmed by mass spectrometry analysis.
3 35S-SK13-YFP 35S-SK32-YFP 35S-SK41-YFP 35S-BIL2-YFP per8-sk11-yfp 35S-SK12-YFP Control 35S-BIN2-YFP 35S-BIL1-YFP a b c d e f g h i Supplementary Figure 3. Subcellular localization of GSK3s in N. benthamiana epidermis. GSK3 proteins fused with YFP were transiently expressed under the control of 35S promoter or estradiol-inducible system in N. benthamiana. (a) p19k, (b) 35S-BIN2-YFP, (c) 35S-BIL1-YFP, (d) 35S-BIL2-YFP, (e) per8-sk11-yfp, (f) 35S-SK12-YFP, (g) 35S- SK13-YFP, (h) 35S-SK32-YFP, and (i) 35S-SK41-YFP. Scale bars indicate 100 μm.
4 per8-tdr-cfp 35S-TDR-YFP per8-tdr-cfp a b + estradiol for 24 h Time after estradiol induction c 0 h d 3 h e 6 h f 12 h g 24 h Supplementary Figure 4. Subcellular localization of TDR in N. benthamiana epidermis. (a, b) Subcellular localization of (a) 35S-TDR-YFP and (b) per8-tdr-cfp. (c-g) Time course analysis on per8-tdr- CFP expression induced by 10 μm estradiol. Images for CFP fluorescence were obtained at (c) 0 hour, (d) 3 hours, (e) 6 hours, (f) 12 hours and (g) 24 hours after estradiol induction. Scale bars indicate 100 μm for (a, b) and 50 μm for (c g).
5 WT / ptdr-bzr1-gfp Cytosol / Nucleus a ptdr-bzr1-gfp b procambium bikinin BZR1 GFP BIN2 BZR1 GFP c Time after TDIF treatment (min) d ~ ~ Time after TDIF treatment (min) Supplementary Figure 5. Effect of TDIF on BIN2 activity in procambial cells. (a) GFP fluorescence in true leaves of 5-day-old ptdr-bzr1-gfp seedlings. (b) A schematic model for the detection of BIN2 activity in procambial cells. (c, d) Time course analysis on BZR1-GFP localization driven under the TDR promoter in Arabidopsis vasculature after 5 μm TDIF treatment. (c) GFP fluorescence images obtained every 10 minutes after TDIF treatment. (d) Time course of the cytosol/nucleus ratios in the two cells indicated by red and blue arrowheads in (c). Scale bars indicate 500 μm for (a) and 100 μm for (c).
6 BIL1-Y BIL2-Y SK11-Y SK12-Y SK13-Y SK31-Y SK32-Y SK41-Y ND ND ND ND FRET efficiency (%) No peptide TDIF 2 0 TDR-C Supplementary Figure 6. FRET efficiency of TDR with various GSK3s. FRET efficiencies of TDR with various GSK3s were measured with or without TDIF peptide at 5 μm in N. benthamiana epidermis (n 10). Error bars indicate SD. ND means no data.
7 Relative expression Relative expression a 1.4 bin2 triple gsk3 quadruplet3#4 b SK11 SK * ATSK11 ATSK12 ATSK13 0 quad #1 #2 #3 #4 #5 #6 #7 #8 #9 gsk3 quadruple + 35S-SK11, SK12-RNAi Supplementary Figure 7. Expression levels of SK11, SK12, and SK13 in the gsk3 quadruple and sextuple lines. (a) Relative expression levels of SK11, SK12, and SK13 in the gsk3 quadruple mutant (line no.4 in T3 generation) were calculated by comparing with those in the bin2 triple mutant. Error bars indicate SD (n = 3). Asterisks indicate statistical differences (P < 0.001; Student s t test). Quantitative PCR was performed independently three times. (b) Relative expression levels of SK11 and SK12 in the gsk3 quadruple mutant and gsk3 sextuple mutants (lines T1#1 9) were calculated by comparing with those in the bin2 triple mutant. Sextuple mutants were produced by introducing 35S-SK11, 12-RNAi into the gsk3 quadruple mutant. However, sextuple mutants showed seedling lethal phenotype. Therefore, expression levels of SK11 and SK12 against each individual have to be examined before analysing vascular phenotype in hypocotyls.
8 Cell types (%) a b Xylem (XY) Phloem (PH) Procambium (PC) Uncharacterized cells (UC) Ws quadruple * ** n.s. n.s. XY PC PH UC Supplementary Figure 8. Cell count analysis in hypocotyls of Ws and gsk3 quadruple mutant. (a) Distinction of vascular cells, which were classified into four different parts; xylem (blue), phloem (red), procambium (orange), and uncharacterized cells (green). Scale bar indicates 50 μm. (b) Ratios of each cell type in total cell number were calculated based on data from Fig. 6e (n = 12). Error bars indicate SD. Asterisks indicate significant differences using the Student s t test (* P < 0.05, ** P < 0.005). n.s. indicates no significant difference according to the Student s t test. XY, PH, PC, and UC indicate that xylem, phloem, procambium, and uncharacterized cells, respectively.
9 gsk3 quadruple + 35S-SK11, SK12-RNAi #1 #2 #3 gsk3 quadruple #4 #5 #6 #7 #8 #9 Supplementary Figure 9. Cross sections of hypocotyls in gsk3 quadruple and sextuple mutants. Cross sections of hypocotyls of gsk3 quadruple and sextuple (T1#1 9) mutants grown on MS agar plates for 11 days were shown. Scale bars indicate 50 μm.
10 Col-0 tdr-1 tdr-1 / ptdr-tdr-gfp +TDIF +TDIF +TDIF a b c Xylem Procambium d (KDa) TDR K747E CFP TDR CFP p19k e (KDa) BIN2 YFP Anti GFP 50 Anti GFP 100 Supplementary Figure 10. Biochemical and functional analysis of BIN2 and TDR fused with fluorescence protein. (a-c) Genetic complementation assay for TDR-GFP. Leaves of (a) WT, (b) tdr-1, and (c) tdr-1/ptdr-tdr-g3gfp were cultured in liquid MS medium for 10 days with 1000 nm. Red and blue lines show procambial and xylem cells, respectively. Scale bars indicate 200 μm. (d, e) Western blot analysis on TDR and BIN2 fused with fluorescent proteins. Specific bands were detected with an anti-gfp antibody..
11 binding TDIF-dependent SK group Y2H FRET bikinin *1 decrease in FRET BIN BIL1 II BIL ATSK ATSK13 I ATSK ATSK III ATSK ATSK IV ATSK42 - ND ND Supplementary Table. 1 Summary for functional analysis of plant GSK3s. Functions of all plant GSK3s were examined based on previous reports (*1: DeRybel et al., 2009) and our results in N. benthamiana and yeast. +; effective and positive, - ; ineffective and negative, and ND indicates no data.
12 name sequence object TDRK747E-f GAAATAATCGCCGTGGAAAAACTTTGGGG Mutagenesis TDRK747E-r CCCCAAAGTTTTTCCACGGCGATTATTTC Mutagenesis TDRCDS-f GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAAAAAGAAGAACATTTCTpDONR TDRCDS-r GGGGACCACTTTGTACAAGAAAGCTGGGTCCACCCCAATCCTTTGACATTTApDONR BIN2CDS-f CACCATGGCTGATGATAAGGAGATGCC BIN2CDS-r AGTTCCAGATTGATTCAAGAAGCTTAG BIL1CDS-f CACCATGACTTCGATACCATTGGGGC BIL1CDS-r GGGTCCAGCTTGAAATGGAAAG BIL2CDS-f CACCATGGCCTCATTACCATTGGGG BIL2CDS-r ACTGTTTTGTAATCCTGTGCTCATTTG AtSK11CDS-f CACCATGGCGTCAGTGGGTATAGC AtSK11CDS-r CAAACCGAGCCAAGGACAC AtSK12CDS-f CACCATGGCCTCGGTGGGC AtSK12CDS-r CAAACTGAGCCACGGAC AtSK13CDS-f CACCATGGCTTCTGTGGGAACATTACC AtSK13CDS-r GAGAGCGAGGAAGGAACATTG AtSK31CDS-f CACCATGGAGCGAGACGAAGAAG AtSK31CDS-r AAGTTTCCTTGCCGATCTG AtSK32CDS-f CACCATGAACGTGATGCGTCGTCTC AtSK32CDS-r AGAGCTACTTCCCGTTCCC AtSK41CDS-f CACCATGGCATCCTCTGGACTGG AtSK41CDS-r CGAATGCAAAGCCATGAAGAGG BZR1CDS-f CACCATGACTTCGGATGGAGCTACG BZR1CDS-r ACCACGAGCCTTCCCATTTC pbin2-f CACCGTTAAGACCCAAAGAAACATAAATGC pbin2-r GAGAGAACAAATGAGTTTCAATCAG pbil1-f CACCGTTCCAAGAAATTGATTATGATCGTC pbil1-r GTGCTTTTACAGCTCTAACTTCTC pbil2-f CACCGAATTTGTTTAAAGAAGCTGATGGATG pbil2-r GTGCTTTTTTACTCTTTTCTCTCTCAAC TDRKD-f CACCTGCTTCCAGAAAAGCTACGGAAAC TDRKD-r TCACACCCCAATCCTTTGACATTTAAC TUA4-f TCTTGAACATGGCATTCAGC Realtime PCR TUA4-r CGGTTTCACTGAAGAAGGTGT Realtime PCR ATSK11-f GGAGCAAGAGACTCTACTGGTGT Realtime PCR ATSK11-r CCACTGTCGCTTCCATTTCT Realtime PCR ATSK12-f GGCAATGTGACTGAGACTGG Realtime PCR ATSK12-r TCCGCCATGTAACTGATTGT Realtime PCR ATSK13-f CGGCGGTGACTTTTCAGA Realtime PCR ATSK13-r GCCATAGAAGAAGCTGGTAATGTT Realtime PCR BES1-semi-F CAGGTTCATGTATACTTCATCTC semi-qrtpcr BES1-semi-R GCGTATTCCTCGAAGAAGATG semi-qrtpcr BIN2Y2H-f GCCATATGATGGCTGATGATAAGGAGATGCC BIN2Y2H-r GCGAATTCTTAAGTTCCAGATTGATTCAAGAAGC TDRY2H-f GCCCATGGTCCAGAAAAGCTACGG TDRY2H-r GCGGATCCCACCCCAATCCTTTGACATTTAAC BIL1Y2H-f GCCATATGATGACTTCGATACCATTGGGGC BIL1Y2H-r GCGAATTCCTAGGGTCCAGCTTGAAATGG BIL2Y2H-f GCCATATGATGGCCTCATTACCATTGGGG BIL2Y2H-r GCGAATTCTTAACTGTTTTGTAATCCTGTGCTC AtSK11Y2H-f GAGGACCTGCATATGATGGCGTCAGTGGGTATAG AtSK11Y2H-r TTCGGCCTCCATGGCCAAACCGAGCCAAGGACA AtSK12Y2H-f GCCATATGATGGCCTCGGTGGGC AtSK12Y2H-r GCGAATTCTCACAAACTGAGCCACGG AtSK13Y2H-f GAGGACCTGCATATGATGGCTTCTGTGGGAACAT AtSK13Y2H-r TTCGGCCTCCATGGCGAGAGCGAGGAAGGAACA
13 AtSK31Y2H-f GAGGACCTGCATATGATGGAGCGAGACGAAGAAG AtSK31Y2H-r TTCGGCCTCCATGGCAAGTTTCCTTGCCGATCTG AtSK32Y2H-f GAGGACCTGCATATGATGAACGTGATGCGTCGTC AtSK32Y2H-r TTCGGCCTCCATGGCAGAGCTACTTCCCGTTCC AtSK41Y2H-f GAGGACCTGCATATGATGGCATCCTCTGGACTG AtSK41Y2H-r TTCGGCCTCCATGGCCGAATGCAAAGCCATGAAG AtSK42Y2H-f GAGGACCTGCATATGATGGAATCTCATCTGGGAAA AtSK42Y2H-r TTCGGCCTCCATGGCTATCATATATCTAACAAAGTAGT pgbkt7-f GCCATGGAGGCCGAATTC pgbkt7-r CATATGCAGGTCCTCCTCTG Supplementary Table 2. Primers used in this study
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