Supplementary Materials for

Transcription

1 Supplementary Materials for An Interaction Between BZR1 and DELLAs Mediates Direct Signaling Crosstalk Between Brassinosteroids and Gibberellins in Arabidopsis Qian-Feng Li, Chunming Wang, Lei Jiang, Shuo Li, Samuel S. M. Sun,* Jun-Xian He* *To whom correspondence should be addressed. (J.-X.H.); (S.S.M.S.) Published 2 October 2012, Sci. Signal. 5, ra72 (2012) DOI: /scisignal The PDF file includes: Fig. S1. BES1 interacts with RGA in a yeast two-hybrid assay. Fig. S2. The LHR1 domain of RGA and the BIN2 phosphorylation domain of BZR1 mediate the interaction of RGA and BZR1. Fig. S3. BiFC analysis reveals a BES1 and RGA interaction in planta. Fig. S4. The gai mutant (with the GAI 17 mutation) partially suppresses root elongation of bzr1-1d. Fig. S5. RGA and GAI over fails to alter the sensitivity of bzr1-1d to BRZ. Fig. S6. The gai mutation enhances the dwarf phenotypes of bri1-5 in a root elongation assay. Fig. S7. The RGA and GAI loss-of-function double mutant (rga-24 gai-t6) has increased sensitivity to BL but reduced sensitivity to BRZ. Fig. S8. The double mutant rga-24 gai-t6 has reduced sensitivity to BRZ in a hypocotyl elongation assay. Fig. S9. bzr1-1d reduces and bri1-5 enhances sensitivity to the GA inhibitor PAC. Fig. S10. bzr1-1d reduces and bri1-5 enhances sensitivity to PAC in a hypocotyl elongation assay. Fig. S11. DELLAs and BZR1 and BES1 have limited effects on each other s gene. Fig. S12. Quantitative analysis of the effect of GA or inhibition of GA biosynthesis or of BIN2 or PP2A activity on BZR1 abundance and phosphorylation status. Fig. S13. DELLAs inhibit growth of BZR1-overexpressing plants. Fig. S14. RGA and BIN2 do not interact. Fig. S15. RGA does not compete with PP2A for phosphorylated BZR1. Fig. S16. Inhibition of GA signaling stimulates the of PP2AB α and PP2AB β.

2 Fig. S17. GA signaling inhibits SCL3. Fig. S18. Some BZR1 target genes and RGA 17 responsive genes are common. Table S1. Primers used for plasmid construction in yeast two-hybrid assays. Table S2. Primers used for RT-PCR and qrt-pcr analyses. Table S3. Primers used for plasmid construction in other experiments.

3 Fig. S1. BES1 interacts with RGA in a yeast two-hybrid assay. Yeast cells transformed with bait (pgbkt7-bes1) and prey (pgadt7-rga) vectors were selected for growth on SD-Trp/-Leu/-His medium supplemented with 20 mm 3-AT. 1

4 Fig. S2. The LHR1 domain of RGA and the BIN2 phosphorylation domain of BZR1 mediate the interaction of RGA and BZR1. (A) Yeast two-hybrid assays for interactions between BZR1 and RGA truncations. BZR1 was used as the bait (pgbkt7-bzr1). (Left) Diagrams for RGA protein truncations and functional motifs. (Right) Yeast cells transformed with combinations of BZR1 and different RGA derivatives were selected on SD-Trp/-Leu/-His medium supplemented with 50 mm 3-AT. RGAND1 to RGAND5 indicate five RGA N-terminal deletions. RGACD1 and RGACD2 represent two RGA C-terminal deletions. RGA LHR1 is RGA with the LHR1 domain deleted. (B) Yeast two-hybrid assays for interactions between RGA and various BZR1 deletions. pgbkt7-rga was used as the bait. (Right) Diagrams for BZR1 truncations and functional motifs. (Left) Interaction of RGA and different BZR1 derivatives were tested in yeast cells grown on SD-Trp/-Leu/-His medium supplemented with 80 mm 3-AT. BZR1D1 to BZR1D4 represent four different BZR1 truncations. BZR1( PEST) and BZR1( BIN2) are BZR1 truncations with the PEST and BIN2 domain deleted, respectively. BZR1(PEST+BIN2) denotes the BZR1 protein that only contains the PEST and BIN2 domains. 2

5 Fig. S3. BiFC analysis reveals a BES1 and RGA interaction in planta. The indicated constructs were cotransformed into N. benthamiana leaves and the YFP fluorescence resulted from BES1 and RGA interaction was detected with a confocal microscope. The image of YFP fluorescence (upper panel) and that merged with the light view (lower panel) are shown. Fig. S4. The gai mutant (with the GAIΔ17 mutation) partially suppresses root elongation of bzr1-1d. Col is the wild type of bzr1-1d and Ler is the wild type of gai. 3

6 Fig. S5. RGA and GAI over fail s to alter the sensitivity of bzr1-1d to BRZ. (A) Hypocotyl lengths of 6-day-old dark-grown Col, bzr1-1d, gai bzr1-1d, gai and Ler plants in the presence of 1 µm BRZ or the mock-treated plants (DMSO). Error bars represent SD (n>20 seedlings). (B) Comparison of relative BRZ responses between 6-day-old dark-grown bzr1-1d and 35S::RGA 17/bzr1-1D transgenic seedlings. The data of each genotype represents the ratio of hypocotyl length of plants under BRZ (2 µm) treatment relative to that of the mock control. Data were analyzed by one-way ANOVA followed by Bonferroni correction. Error bars represent SD (n>20 seedlings). * P<0.05; **P<0.01; NS, not significant. (C) Hypocotyl lengths of Col, bzr1-1d and the 35S::RGA 17/bzr1-1D transgenic seedlings in (B). Error bars represent SD (n>20 seedlings). 4

7 Fig. S6. The gai mutation enhances the dwarf phenotypes of bri1-5 in a root elongation assay. WS is the wild type of bri1-5. Fig. S7. The RGA and GAI loss-of-function double mutant (rga-24 gai-t6) has increased sensitivity to BL but reduced sensitivity to BRZ. (A) Primary root phenotypes of rga-24 gai-t6 plants under BL treatment. 3-day-old rga-24 gai-t6 and Ler seedlings were treated on MS media containing 10-10, 10-8, or 10-6 M BL for seven days in the light. (B) Comparison of relative BL responses between rga-24 gai-t6 and its wild-type Ler. The data of each genotype represent the ratios of primary root lengths under BL treatment relative to that of the mock-treated (DMSO) control. Data were analyzed by ANOVA with Bonferroni correction. Error bars represent SD (n>20 seedlings). * P<0.05; **P<0.01. (C) Primary root lengths of plants in (A). (D) Primary root phenotypes of rga-24 gai-t6 plants under BRZ (2 µm) treatment. (E) Comparison of relative BRZ response between rga-24 gai-t6 and Ler. The data of each genotype represents the ratio of the primary root length under BRZ treatment relative to that of the mock control. Data were analyzed by ANOVA with Bonferroni correction. Error bars represent SD (n>20 seedlings). * P<0.05; **P<0.01. (F) Primary root lengths of plants in (D). 5

8 Fig. S8. The double mutant rga-24 gai-t6 has reduced sensitivity to BRZ in a hypocotyl elongation assay.6-day-old dark-grown Col, bzr1-1d, Ler, and rga-24 gai-t6 seedlings were treated with the indicated concentrations of BRZ. Error bars represent SD (n>20 seedlings). 6

9 Fig. S9. bzr1-1d reduces and bri1-5 enhances sensitivity to the GA inhibitor PAC. (A) Hypocotyl elongation assay of 6-day-old bzr1-1d, bri1-5 and rga-24 gai-t6 mutants in response to GA treatment. Bar = 5 mm. (B) Comparison of relative GA 3 responses between each mutant and its wild-type control. The data of each genotype represents the ratio of hypocotyl lengths under GA 3 treatment relative to that of the mock control. (C) Hypocotyl lengths of the plants in (A). (D) Primary root phenotypes of bzr1-1d and Col in PAC and mock (methanol, MetOH)-treated plants. (E) Comparison of relative PAC responses between bzr1-1d and Col. The data of each genotype represents the ratio of primary root lengths under PAC treatment relative to that of the mock control (MetOH). (F) Primary root lengths of the plants in (D). (G) Primary root phenotypes of bri1-5 and WS in response to PAC treatment. (H) Comparison of relative PAC responses between bri1-5 and WS. The data of each genotype represents the ratio of primary root lengths in response to PAC treatment relative to that of the mock control. (I) Primary root lengths of the plants in (G). In D and G, the PAC treatment was done by vertically growing the 3-day-old seedlings on MS media containing 1 μm PAC for 7 days in the light. Data in B, E, and H were analyzed by ANOVA with Bonferroni correction. Error bars represent SD (n>20 seedlings). * P<0.05; **P<

10 Fig. S10. bzr1-1d reduces and bri1-5 enhances sensitivity to PAC in a hypocotyl elongation assay. Six-day-old dark-grown Col, bzr1-1d, WS, bri1-5, Ler, and rga-24 gai-t6 seedlings treated with different concentrations of PAC (0, 0.025, 0.05, 0.1 and 0.2 µm). Error bars represent SD (n>20 seedlings). Fig. S11. DELLAs and BZR1 and BES1 have limited effects on each other s gene. Trancript abundance of RGA, GAI, BZR1, and BES1 genes were measured by qrt-pcr analyses. (A) Relative of RGA and GAI under BL treatment (10-6 M, 2 hours). (B) Relative of BZR1 and BES1 under GA 3 treatment (50 µm, 2 hours). (C) Relative of RGA and GAI in bzr1-1d mutant. (D) Relative of BZR1 and BES1 in gai mutant. UBC was used as an internal control for normalization of data in (A) and (C). EF-1a served as the internal control for normalization of data in (B) and (D). Error bars represent SD (n = 3 experiments). Two-sample t tests were performed to compare of the tested genes under a treatment or in a mutant with that of mock control or wild-type plant. * P<0.05; **P<0.01. See table S2 for primers. 8

11 9

12 Fig. S12. Quantitative analysis of the effect of GA or inhibition of GA biosynthesis or of BIN2 or PP2A activity on BZR1 abundance and phosphorylation status. (A) Quantitation of GA 3 (50 µm) effects on the abundance of the phosphorylated BZR1 (pbzr1) and dephosphorylated BZR1 (BZR1) in Fig. 3H. (B) Quantitation of PAC (1 µm) effects on the abundance of pbzr1 and BZR1 or pmbzr1 and mbzr1 in Fig. 3I. (C) Quantitation of the effects of LiCl and GA treatments on the abundance of pbzr1 and BZR1 in Fig. 3J. (D) Quantitation of the effects of OA and GA treatments on the abundance of pbzr1 and BZR1 in Fig. 3K. (E) Quantitation of the effects of combination treatments with LiCl, OA, and GA on the abundance of pbzr1 and BZR1 in Fig. 3L. Each band was normalized for loading on the basis of Rubisco. The relative abundance of pbzr1 normalized to Rubisco in the first sample of each blot was set to 1. Error bars represent SD (n = 3 experiments). (F) Representatives of original blots for Western analyses in Figs. 3H to 3L. Arrows indicate the pbzr1 and BZR1 bands, respectively; * denotes a non-specific band detected from plant materials with anti-gfp. 10

13 Fig. S13. DELLAs inhibit growth of BZR1-overexpressing plants. Phenotypes of the gai pbzr1::mbzr1-cfp double mutant (A) and 35S::RGA 17/ pbzr1::bzr1-cfp transgenic plant (B). Fig. S14. RGA and BIN2 do not interact. Yeast two-hybrid assay fails to reveal an interaction between RGA and BIN2. RGA was cloned into the bait vector pgbkt7 and BIN2 was cloned into the prey vector pgadt7. Interaction was selected on SD-Trp/-Leu/-His medium supplemented with 80 mm 3-AT. 11

14 Fig. S15. RGA does not compete with PP2A for phosphorylated BZR1. BZR1 was phosphorylated by incubating MBP-BZR1 purified from E. coli with the tag-free BIN2 in the presence of ATP for ~16 hours at 30. The mixture of GST-PP2AB α protein with GST-RGA or GST was incubated with pbzr1-mbp immobilized on amylose resins. The interaction between pbzr1-mbp and GST-PP2AB α and the interaction between pbzr1-mbp and GST-RGA were detected with anti-gst. Data are representative of at least three experiments. Fig. S16. Inhibition of GA signaling stimulates the of PP2AB α and PP2AB β. (A) Relative transcript abundance of PP2AB α and PP2AB β under PAC (1 µm) treatment were measured by qrt-pcr analysis. MetOH is the mock control. (B) Relative transcript abundance of PP2AB α and PP2AB β in the gai mutant. EF-1a was used as internal control for normalization of data in (A) and (B). Error bars represent SD (n=3 experiments). * P<0.05; **P<0.01. (Two-sample t tests). 12

15 Fig. S17. GA signaling inhibits SCL3. SCL3 transcripts were detected by qrt-pcr analysis in Col plants exposed to 100 µm GA3 for two hours or 1 µm PAC for twelve days or in gai plants. EtOH and MetOH served as the mock treatment of GA3 and PAC, respectively. EF-1a was used as an internal control for normalization of data. Error bars represent SD (n=3 experiments). * P<0.05; **P<0.01. (Two-sample t tests). Fig. S18. Some BZR1 target genes and RGAΔ17 responsive genes are common. The data of BZR1 target genes were from a chromatin-immunoprecipitation microarray (ChIP-chip) study (19), and the data for RGAΔ17 responsive genes were from a microarray analysis of transgenic plants expressing DEX-(rga-Δ17) (47). 13

16 Table S1. Primers used for plasmid construction in yeast two-hybrid assays. Primer Names Sequence ( 5' 3' ) Usage RGASmal-F TCCCCCGGGTATGAAGAGAGATCATCACCAATT C Forward primer for cloning the full-length RGA cdna into pgadt7 RGAND1Sma-F TCCCCCGGGTGTTGCTTTGAAACTCGAACAATTA G Construction of the following RGA N-terminal deletion plasmids: RGAND2Sma-F RGAND3Sma-F RGAND4Sma-F RGAND5Sma-F TCCCCCGGGTCCTCTTCCGGCGAGTTCTAACG TCCCCCGGGTGAGTCAACTCGTTCTGTTATC TCCCCCGGGTCTCTCTCCGCCGCAGAATC TCCCCCGGGTCCACCGGCGCCGGATAATTC RGAND1-pGADT7 RGAND2-pGADT7 RGAND3-pGADT7 RGAND4-pGADT7 RGAND5-pGADT7 RGAND1BH-R CGGGATCCTCAGTACGCCGCCGTCGAG RGACD1RI-F CGGAATTCATGAAGAGAGATCATCACCAATTC Amplify RGA C-terminal deletions for construction of RGACD1BH-R CGGGATCCAGAACGAGTTGACTCACCCGC RGACD1-pGADT7 and RGACD2-pGADT7 RGACD2BH-R CGGGATCCATTAAGCTCAGAGAGCATATTATC RGAM-F GAGTCAACTCGTATCTACCGTCTC Construction of RGAM-R GAGACGGTAGATACGAGTTGACTC RGALHR1D-pGADT7 14

17 GAIRI-F CGGAATTCATGAAGAGAGATCATCATCATC Construction of GAI-pGADT7 GAIBH-R CGGGATCCCTAATTGGTGGAGAGTTTCC RGL1Nde-F GGAATTCCATATGATGAAGAGAGAGCACAACCA CC Construction of RGL1-pGADT7 RGL1BH-R CGGGATCCTTATTCCACACGATTGATTCGCC RGL2NDe-F GGAATTCCATATGATGAAGAGAGGATACGGAGA AAC Construction of RGL2-pGADT7 RGL2Cla-R CCATCGATTCAGGCGAGTTTCCACGCCG RGL3Nde-F GGAATTCCATATGATGAAACGAAGCCATCAAGA AAC Construction of RGL3-pGADT7 RGL3Sac-R CGAGCTCCTACCGCCGCAACTCCGCC BZR1D1ER-F BZR1D1BH-R CGGAATTCATGACTTCGGATGGAGCTACG CGGGATCCAGAGGCAGAGAATGGCTGTTG Forward and reverse primers for construction of: BZR1D1-pGADT7 BZR1D2BH-R CGGGATCCAAACTGGTGGCGATGTGTCG BZR1D2-pGADT7 BZR1D3-pGADT7 BZR1D3BH-R CGGGATCCAGCTATCTCACCAGGTAAAGG BZR1D4RI-F CGGAATTCGGGACTTCATCTCGAGTAACTC Construction of BZR1D4-pGADT7 BZR1D4BH-R CGGGATCCTCAACCACGAGCCTTCCCATT BZR1BIN-F GGTGAGATAGCTTTTCATACCCCG Construction of BZR1BIN-R CGGGGTATGAAAAGCTATCTCACC BZR1(BIN2D)-pGADT7 BZR1PEST-F CAGTTTCATACCATGGTGCCAACC Construction of BZR1PEST-R GGTTGGCACCATGGTATGAAACTG BZR1(PESTD)-pGADT7 15

18 Table S2. Primers used for RT-PCR and qrt-pcr analyses. Primer Names Sequence ( 5' 3' ) Usage GA20ox2-RT-F GA20ox2-RT-R TCCAACGATAATAGTGGCT TTGGCATGGAGGATAATGA qrt-pcr analysis of GA20ox2 GA3ox1-RT-F GA3ox1-RT-R CCATTCACCTCCCACACTCT GCCAGTGATGGTGAAACCTT qrt-pcr analysis of GA3OX1 EF-1a-RT-F EF-1a-RT-R CCCAGGCTGATTGTGCTGT GGGTAGTGGCATCCATCTTGTT qrt-pcr analysis of EF-1a RGA-RT-F RGA-RT-R TCATGCTCGAGTCCTGATTCTATGG GACAATGATCGATCTGATTCTGC RT-PCR analysis of RGA in 35S::RGAΔ17 transgenic plants RGA-qRT-F RGA-qRT-R ACTTCGACGGGTACGCAGAT TGTCGTCACCGTCGTTCC qrt-pcr analysis of RGA UBC-RT-F UBC-RT-R TCAAATGGACCGCTCTTATC CACAGACTGAAGCGTCCAAG qrt-pcr analysis of UBC SAUR-RT-F SAUR-RT-R GAGGATTCATGGCGGTCTATG GTTAAGCCGCCCATTGGATG qrt-pcr analysis of SAUR-AC1 CPD-RT-F CPD-RT-R TTGCTCAACTCAAGGAAGAG TGATGTTAGCCACTCGTAGC qrt-pcr analysis of CPD DWF4-RT-F DWF4-RT-R CATAAAGCTCTTCAGTCACGA CGTCTGTTCTTTGTTTCCTAA qrt-pcr analysis of DWF4 16

19 BZR1-RT-F BZR1-RT-R GCAGATGTCTCCAAATACTGCTG GACATGCCATTTGGGTTTGCCTAG qrt-pcr analysis of BZR1 BES1-RT-F BES1-RT-R GCAATTGTCTCCAAACACAGCAG CTCCAATCCTTCCTTCCGACATG qrt-pcr analysis of BES1 KOR-RT-F KOR-RT-R CAAGCTTGCTGGTGCTCAGTTG TGCTGGTCTGATTGTGGAAGGTC qrt-pcr analysis of KOR CESA6-RT-F CESA6-RT-R ACCTTGGCCCGGTAATAGTGTG CGTCACTTCCAAGGAAGACCTG qrt-pcr analysis of CESA6 EXP1-RT-F EXP1-RT-R TGCTACCCTTGGAGCAATGACG ACAAGCACCTCCCATTGTGC qrt-pcr analysis of EXP1 XTH23-RT-F XTH23-RT-R TGGAACCCACAACGCATCATATTC TGTGGCCCATTCTTCAGCGTTC qrt-pcr analysis of XTH23 EXPL2-RT-F EXPL2-RT-R GCGCCGACAGAGATCTTCTCAAAC ACCGTAATCGCAAGGAACTCTCC qrt-pcr analysis of EXPL2 XTH17-RT-F XTH17-RT-R GTGGTACAAGCTTTGCGTTCTTAC AGCTACCCGCATAGACATGCAC qrt-pcr analysis of XTH17 SCL3 flc SCL3 rlc AACAACAATGGGTATAGCC TGCTGCGTAGGTGTAA qrt-pcr analysis of SCL3 17

20 Table S3. Primers used for plasmid construction in other experiments. Primer Names Sequence ( 5' 3' ) Usage BZR1Smal-F TCCCCCGGGATGACTTCGGATGGAGCTACG Construction of BZR1-pMN6 and bzr1-1d-pmn6 plasmids for protoplast BZR1Kpn-R GGGGTACCTCAACCACGAGCCTTCCCATT transient assay of RGA transcriptional activity RGASmal-F TCCCCCGGGATGAAGAGAGATCATCACCAATT C Construction of RGA-pMN6 for RGA transcriptional activity assay RGAKpn-R GGGGTACCTCAGTACGCCGCCGTCGAG BIM1Sma-F TCCCCCGGGATGGAGCTTCCTCAACCTCG Construction of BIM1-pMN6 for BZR1 BIMKpn-R GGGGTACCCTACTGTCCCGTCTTGAGCC transcriptional activity assay RGABH-F RGASal-R CGCGGATCCATGAAGAGAGATCATCACCAATT C TTAGTCGACTCAGTGCGCCGCCGTCGAGAGTT TC Construction of GST-RGA and MBP-RGA for pull-down assay and overlay assay of RGA and BZR1 interaction SAURproXho-F CCGCTCGAGGGATTGTCTTTAATTGTAAGTTAG Construction of SAURpro-pGreen for SAURproNcoI-R CATGCCATGGATTTTCCCTTATGTTTTCCTGAA G transcriptional activity assay SCL3proXho-F CCGCTCGAGAAATCCCACACCCAAGCCTC Construction of SCL3pro-pGreen for SCL3proNcoI-R CATGCCATGGTGAAGGCCAAAAGCTTGATTTT G transcriptional activity assay BZR1GW-F CACCATGACTTCGGATGGAGCTAC Cloning of full-length BZR1 into the gateway BZR1GW-R ACCACGAGCCTTCCCATTTC TOPO entry vector 18

21 RGAGW-F CACCATGAAGAGAGATCATCACCAATTC Cloning of full-length RGA into the gateway TOPO entry vector RGAGW-R TCAGTACGCCGCCGTCGAG Bin2BH-F CGGGATCCATGGCTGATGATAAGGAGATG Construction of ppal7-bin2 for expressing tag-free BIN2 in E. Coli Bin2RI-R CGGAATTCTTAAGTTCCAGATTGATTCAAG BZR1ER-F CGGAATTCATGACTTCGGATGGAGCTACG Construction of MBP-BZR1 for pull-down assay and overlay assay of RGA and BZR1 BZR1BH-R CGGGATCCTCAACCACGAGCCTTCCCATT interaction 19