Supplemental Figure 1. Immunoblot analysis of wild-type and mutant forms of JAZ3
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1 Supplemental Data. Chung and Howe (2009). A Critical Role for the TIFY Motif in Repression of Jasmonate Signaling by a Stabilized Splice Variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis. Supplemental Figure 1. Immunoblot analysis of wild-type and mutant forms of JAZ3 and JAZ10.1. Protein was extracted from yeast strains used for the yeast two-hybrid assays shown in Figures 2E and F. Expression of wild-type (WT) JAZ3-AD and JAZ10.1- AD fusion proteins, as well as mutant derivatives harboring the indicated point mutation in the TIFY motif, was detected with an anti-ha antibody. 1
2 Supplemental Figure 2. Yeast two-hybrid analysis of homo- and heteromeric interactions between Arabidopsis ZIM, ZIM-like1 (ZML1), and ZIM-like2 (ZML2) proteins. Yeast strains co-transformed with empty or ZIM-, ZML1-, and ZML2-containing BD (pgilda) and AD (pb42ad) vectors were plated on media containing X-gal. The photograph was taken after 48 h of incubation of the plate at 30 C. Blue color formation is indicative of a positive protein-protein interaction. 2
3 Supplemental Figure 3. Detection of transcripts encoding JAZ10.3 and JAZ10.4 using transcript-specific primers. (A) Schematic diagram of transcripts encoding JAZ10.3 and JAZ10.4. Left-pointing arrows denote the location of oligonucleotide primers that are specific for each transcript. Primer sequences are listed in Supplemental Table 1. The gene-specific primer for JAZ10.4 hybridizes to a sequence created from splicing of the alternate donor site in exon 3 to the acceptor site in exon 4. (B) Result of an RT-PCR experiment using total RNA isolated from wounded (6 hr post wounding) Arabidopsis leaves and the transcript-specific primers indicated in (A). PCR products were visualized by agarose gel electrophoresis and ethidium bromide staining. 3
4 Supplemental Figure 4. Yeast two-hybrid analysis of the effect of I107A and G111A TIFY mutations on the interaction of JAZ10.4 with other members of the JAZ family. Yeast strains were co-transformed with plasmids encoding an AD-fusion of JAZ10.4 (or the JAZ10.4 I->A and JAZ10.4 G->A mutants; left column) and a BD-fusion of one of the indicated (top row) twelve JAZ family members. Co-transformed strains were plated on medium containing X-gal. The photograph was taken after 48 h of incubation of the plate at 30 C. Blue color formation is indicative of a positive protein-protein interaction. 4
5 Supplemental Table 1. List of constructs and primers used in this study. Clone Name(s) Forward Primers Reverse Primers Vector Cloning site Y2H analysis AD-JAZ1 and BD-JAZ1 a pb42ad or pgilda AD-JAZ2 and BD-JAZ2 5'-GAATTCATGTCGAGTTTTTCTGCCGAGTGT-3' 5'-CTCGAGTTACCGTGAACTGAGCCAAGCTGG-3' pb42ad or pgilda EcoR1-Xho1 AD-JAZ3 and BD-JAZ3 5'-GAATTCATGGAGAGAGATTTCCTCGGGTTG-3' 5'-GAATTCGGTTGCAGAGCTGAGAGAAGAACT-3' pb42ad or pgilda EcoR1-Xho1 AD-JAZ3ΔN 5'-GAATTCTTGGTAGGAGTTCCTATCATG-3' 5'-GAATTCGGTTGCAGAGCTGAGAGAAGAACT-3' pb42ad or pgilda EcoR1-Xho1 AD-JAZ3Jas 5'-GGATTCATGCCTCAAGTCTTTTCG-3' 5'-GAATTCGGTTGCAGAGCTGAGAGAAGAACT-3' pb42ad or pgilda EcoR1-Xho1 AD-JAZ3ΔJas 5'-GAATTCTTGGTAGGAGTTCCTATCATG-3' 5'-CTCGAGCTAAGCCATTACATTGGTAGA-3' pb42ad AD-JAZ3 T180A 5'-TCA CCT GCG CAG TTG GCG ATC TTT TAT GCC-3' b 5'-GGCATAAAAGATCGCCAACTGCGCAGGTGA-3' b pb42ad AD-JAZ3 I181A 5'-CCTGCGCAGTTGACGGCCTTTTATGCCGGTTC-3' b 5'-GAACCGGCATAAAAGGCCGTCAACTGCGCAGG-3' b pb42ad AD-JAZ3 F182A 5'-GCGCAGTTGACGATCGCTTATGCCGGTTCAGT-3' b 5'-ACTGAACCGGCATAAGCGATCGTCAACTGCGC-3' b pb42ad AD-JAZ3 Y183A 5'-CAGTTGACGATCTTTGCTGCCGGTTCAGTTTG-3' b 5'-CAAACTGAACCGGCAGCAAAGATCGTCAACTG-3' b pb42ad AD-JAZ3 G185A 5'-ACGATCTTTTATGCCGCTTCAGTTTGTGTTTA-3' b 5'-TAAACACAAACTGAAGCGGCATAAAAGATCGT-3' b pb42ad AD-JAZ4 and BD-JAZ4 5'-GAATTCATGGAGAGAGATTTTCTCGGGCTG-3' 5'-GAATTCTCATGTCACAATGGGGCTGGTTTC-3' pb42ad or pgilda EcoR1-EcoR1 AD-JAZ5 and BD-JAZ5 5'-GAATTCATGTCGTCGAGCAATGAAAATGCT-3' 5'-CTCGAGCTATAGCCTTAGATCGAGATCTTT-3' pb42ad or pgilda EcoR1-Xho1 AD-JAZ6 and BD-JAZ6 5'-GAATTCATGTCAACGGGACAAGCGCCGGAG-3' 5'-CTCGAGCTAAAGCTTGAGTTCAAGGTTTTT-3' pb42ad or pgilda EcoR1-Xho1 AD-JAZ7 and BD-JAZ7 5'-GAATTCATGATCATCATCATCAAAAACTGC-3' 5'-CTCGAGCTATCGGTAACGGTGGTAAGGGGA-3' pb42ad or pgilda EcoR1-EcoR1 AD-JAZ8 and BD-JAZ8 5'-CTCGAGATGAAGCTACAGCAAAATTGTGAC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACGGTGA-3' pb42ad or pgilda Xho1-Xho1 AD-JAZ9 and BD-JAZ9 a pb42ad or pgilda AD-JAZ10.1 T106A 5'-GGAACTGTTCCTATGGCGATTTTCTACAATG-3' b 5'-CATTGTAGAAAATCGCCATAGGAACAGTTC-3' b pb42ad AD-JAZ10.1 I107A 5'-ACTGTTCCTATGACGGCTTTCTACAATGGAAG-3' b 5'-CTTCCATTGTAGAAGCCGTCATAGGAACAGT-3' b pb42ad AD-JAZ10.1 F108A 5'-GTTCCTATGACGATTGCCTACAATGGAAGTGT-3' b 5'-ACACTTCCATTGTAGGCAATCGTCATAGGAAC-3' b pb42ad AD-JAZ10.1 Y109A 5'-CCTATGACGATTTTCGCCAATGGAAGTGTTT-3' b 5'-AAACACTTCCATTGGCGAAAATCGTCATAGG-3' b pb42ad AD-JAZ10.1 G111A 5'-ACGATTTTCTACAATGCAAGTGTTTCAGTTTT-3' b 5'-AAAACTGAAACACTTGCATTGTAGAAAATCGT-3' b pb42ad AD-JAZ10.3 and BD-JAZ10.3 5'-GAATTCATGTCGAAAGCTACCATAGAACTC-3' 5'-GAATTCTTACTCTCCTTGCGCTTCTCGAG-3' pb42ad or pgilda EcoR1-EcoR1 AD-JAZ10.4 and BD-JAZ10.4 5'-GAATTCATGTCGAAAGCTACCATAGAACTC-3' 5'-GAATTCCTAATCTCTCCTTGCGCTTCTTGA-3' pb42ad or / pgilda EcoR1-EcoR1 AD-JAZ10.4 I->A 5'-ACTGTTCCTATGACGGCTTTCTACAATGGAAG-3' b 5'-CTTCCATTGTAGAAGCCGTCATAGGAACAGT-3' b pb42ad AD-JAZ10.4 G->A 5'-GTTCCTATGACGATTGCCTACAATGGAAGTGT-3' b 5'-ACACTTCCATTGTAGGCAATCGTCATAGGAAC-3' b pb42ad AD-JAZ11 and BD-JAZ11 5'-GAATTCATGGATTGGTCATTCTCAAGCAAA-3' 5'-GAATTCTTAGTGCAGATGATGAGACTGGAGG-3' pb42ad or pgilda EcoR1-EcoR1 AD-JAZ12 and BD-JAZ12 5'-CCCGGGAATGACTAAGGTGAAGATGA-3' 5'-CCCGGGCTAAGCAGTTGGAAATTCCTC-3' pb42ad or pgilda Sma1-Sma1 AD-ZIM and BD-ZIM 5'-GAATTCATGTTGGTCGCCATTCGATT-3' 5'-CTCGAGTTAGTGATCACCTAACAGAT-3' pb42ad or pgilda EcoR1-Xho1 AD-ZML1 and BD-ZML1 5'-GAATTCATGGATGATCTTCATGGAAG-3' 5'-CTCGAGTCACTGTGTGTTGCTAATGTC-3' pb42ad or pgilda EcoR1-Xho1 AD-ZML2 and BDZML2 5'-GAATTCATGGATGACCTACATGGAAGC-3' 5'-CTCGAGTCACTGTGAGTTGCTTATGTC-3' pb42ad or pgilda EcoR1-Xho1 BD-COI1 c pgilda Stable transgenic lines 35S-JAZ10.1 5'-GGTACCATGTCGAAAGCTACCATAGAACTC-3' 5'-GAGCTCTTAGGCCGATGTCGGATAGTAAGG-3' pbi-ts Kpn1-Sac1 35S-JAZ10.3 5'-GGTACCATGTCGAAAGCTACCATAGAACTC-3' 5'-GAGCTCTTACTCTCCTTGCGCTTCTCGAG-3' pbi-ts Kpn1-Sac1 35S-JAZ10.4 5'-GGTACCATGTCGAAAGCTACCATAGAACTC-3' 5'-GAGCTCCTAATCTCTCCTTGCGCTTCTTGA-3' pbi-ts Kpn1-Sac1 35S-JAZ10.4 I->A 5'-ACTGTTCCTATGACGGCTTTCTACAATGGAAG-3' b 5'-CTTCCATTGTAGAAGCCGTCATAGGAACAGT-3' b pbi-ts Kpn1-Sac1 35S-JAZ10.4 G->A 5'-ACGATTTTCTACAATGCAAGTGTTTCAGTTTT-3' b 5'-AAAACTGAAACACTTGCATTGTAGAAAATCGT-3' b pbi-ts Kpn1-Sac1 35S-JAZ10.4-GUS 5'-ACGATTTTCTACAATGCAAGTGTTTCAGTTTT-3' 5'-AAAACTGAAACACTTGCATTGTAGAAAATCGT-3' pbi121 BamH1/ BamH1 35S-JAZ10.4 I->A -GUS 5'-ACTGTTCCTATGACGGCTTTCTACAATGGAAG-3' b 5'-CTTCCATTGTAGAAGCCGTCATAGGAACAGT-3' b pbi121 BamH1/ BamH1 35S-JAZ10.1-eYFP 5'-GGATCCATGTCGAAAGCTACCATAGAA-3' 5'-GGATCCGGCCGATGTCGGATAGTAAGG-3' pbi-eyfp BamH1-BamH1 35S-JAZ10.3-eYFP 5'-GGATCCATGTCGAAAGCTACCATAGAA-3' 5'-GGATCCCCTCTCCTTGCGCTTCTCGAG-3' pbi-eyfp BamH1-BamH1 35S-JAZ10.4-eYFP 5'-GGATCCATGTCGAAAGCTACCATAGAA-3' 5'-GGATCCATCTCTCCTTGCGCTTCTTGA-3' pbi-eyfp BamH1-BamH1 Transient expression 35S-eYFP 5'-GGTACCATGGTGAGCAAGGGCGAGGAG-3' 5'-GAGCTCTCACTTGTACAGCTCGTCCA-3' pbi-ts Kpn1-Sac1 35S-JAZ10.1-eYFP 5'-GGATCCATGTCGAAAGCTACCATAGAA-3' 5'-GGATCCGGCCGATGTCGGATAGTAAGG-3' pbi-eyfp BamH1-BamH1 35S-JAZ10.3-eYFP 5'-GGATCCATGTCGAAAGCTACCATAGAA-3' 5'-GGATCCCCTCTCCTTGCGCTTCTCGAG-3' pbi-eyfp BamH1-BamH1 35S-JAZ10.4-eYFP 5'-GGATCCATGTCGAAAGCTACCATAGAA-3' 5'-GGATCCATCTCTCCTTGCGCTTCTTGA-3' pbi-eyfp BamH1-BamH1 35S-JAZ10.4 I->A -eyfp 5'-ACTGTTCCTATGACGGCTTTCTACAATGGAAG-3' b 5'-CTTCCATTGTAGAAGCCGTCATAGGAACAGT-3' b pbi-eyfp BamH1-BamH1 JAZ3-nYFP 5'-GTCGACAATGGAGAGAGATTTTCTC-3' 5'-GCGGCCGCGTGGTTGCAGAGCTGAGAGA-3' psy728 Sal1-Not 1 JAZ3-cYFP 5'-GTCGACAATGGAGAGAGATTTTCTC-3' 5'-GCGGCCGCGTGGTTGCAGAGCTGAGAGA-3' psy738 Sal1-Not 1 nyfp-jaz3 5'-GTCGACAATGGAGAGAGATTTTCTC-3' 5'-GAGCTCTTAGGTTGCAGAGCTGAG-3' psy735 Sal1-Sac1 cyfp-jaz3 5'-GTCGACAATGGAGAGAGATTTTCTC-3' 5'-GAGCTCTTAGGTTGCAGAGCTGAG-3' psy736 Sal1-Sac1 Transcript-specific RT-PCR JAZ10.3 5'-GGTACCATGTCGAAAGCTACCATAGAACTC-3' 5'-GGATTGTTGAAGAATCATTAC-3' JAZ10.4 5'-GGTACCATGTCGAAAGCTACCATAGAACTC-3' 5'-ATGGGAAGATCGAAAGATCT-3' a Described in Melotto et al (2008) Plant J 55: b Primers for site-directed mutagensis c Described in Thines et al (2007) Nature 488:
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