Supplementary Information. Supplementary Figure S1. Phenotypic comparison of the wild type and mutants.

Transcription

1 Supplementary Information Supplementary Figure S1. Phenotypic comparison of the wild type and mutants. Supplementary Figure S2. Transverse sections of anthers. Supplementary Figure S3. DAPI staining and TUNEL assay of anthers. Supplementary Figure S4. Analysis of the expression of tapetum-expressed genes. Supplementary Figure S5. Sequence analysis of EAT1 and its homologs. Supplementary Figure S6. Expression analyses of OsAP25 and OsAP37. Supplementary Figure S7. Analysis of the EAT1 antibody and the ChIP-PCR control. Supplementary Table S1. Close homologs of EAT1. Supplementary Table S2. Peptide sequences used for constructing the NJ tree. Supplementary Table S3. Synthesized coding sequences of EAT1 and OsAP25 and OsAP37. Supplementary Table S4. Primer sequences used in this study. 1

2 Supplementary Figures and Legends Fig.S1 Supplementary Figure S1. Phenotypic comparison of the wild type and mutants. (a) Whole plants. (b) Spikelets after the removal of the lemma. (c) Anthers. (d) I 2 -KI-stained pollen grains. (e) Phenotypic segregation ratio of F1 plants from genetic crosses among eat1-1, eat1-2 and eat1-3. Bars = 20 cm in (a), 2 mm in (b) and (c), and 200 µm in (d). pa, palea; gl, glume; st, stamen. 2

3 Fig.S2 Supplementary Figure S2. Transverse sections of anthers. The wild-type (a) and eat1-1 (b) anthers. S7, stage 7; S8a, stage 8a; S8b, stage 8b; S9, stage 9; S11, stage 11; E, epidermis; En, endothecium; Ml, middle layer; T, tapetum; MMC, microspore mother cell; Tds, tetrads; Msp, microspore; DMsp, degraded microspore. Bars = 50 µm. 3

4 Fig.S3 Supplementary Figure S3. DAPI staining and TUNEL assay of anthers. (a) DAPI (4,6-diamidino-2-phenylindole) staining of microspore mother cells. (b) TUNEL assays. Red signal is propidium iodide (PI) staining, and yellow fluorescence is the merged signal from TUNEL (green) and PI. Arrows, TUNEL-positive signal. Bars = 5 µm in (a) and 50 µm in (b). S8a, stage 8a; S8b, stage 8b; S7 to 12, stage 7 to 12; T, tapetum; MMC, microspore mother cell; Tds, tetrads; Msp, microspore; DMsp, degraded microspore. 4

5 Fig.S4 Supplementary Figure S4. Analysis of the expression of tapetum-expressed genes. (a) to (g) Expression levels detected by quantitative RT-PCR (qrt-pcr) in the wild-type and eat1-1 anthers. (h) Expression of EAT1 in gamyb-4, udt1, tdr and ptc1 mutants relative to the wild type. One-way ANOVA test was used. Results are presented as mean ± standard error. S8a, stage 8a; S8b, stage 8b; S7 to 12, Stage 7 to 12. 5

6 Fig.S5 Supplementary Figure S5. Sequence analysis of EAT1 and its homologs. Sequences from the HLH and DUF domains from EAT1 and homologous genes were used for tree construction. At, Arabidopsis thaliana; Aral, Arabidopsis lyrata; Popt, Populus trichocarpa; Medt, Medicago truncatula; Glym, Glycine max; Sb, Sorghum bicolor; Zm, Zea mays; Os, Oryza Sativa; Brad, Brachypodium distachyon; Selm, Selaginella moellendorffii; Phyp, Physcomitrella patens. 6

7 Fig.S6 Supplementary Figure S6. Expression analyses of OsAP25 and OsAP37. (a) OsAP25 is highly expressed in undifferentiated callus (UDc) or differentiating callus (DC). (b) Expression levels of OsAP25 and OsAP37 in the wild-type, eat1-1, gamyb-4, udt1, tdr and ptc1 anthers at stage 9 and stage 10. (c) In situ hybridization analysis of OsAP25 and OsAP37 in wild-type anther at stage 10. Bars = 50 µm. One-way ANOVA test was used. Results are presented as mean ± standard error. 7

8 Fig.S7 Supplementary Figure S7. Analysis of the EAT1 antibody and the ChIP-PCR control. (a) Western blot analysis of EAT1 in the nuclear protein extracts of the wild-type and eat1-1 flowers at stage 9. (b) qrt-pcr analysis of OsAP19 in wild-type and eat1-1 anther. (c) Presence of the E-box motifs in the promoters of OsAP37 and OsAP19 (left) and qchip-pcr results (right) showing that the promoter fragments containing the E-box motifs, E1 to E3, E5 to E8 on OsAP37 and E1 to E4 on OsAP19 can t be amplified by EAT1 antibody immunoprecipitation (IP), compared with no antibody (No Ab). FRAG, fragment. One-way ANOVA test was used. Results are presented as mean ± standard error. 8

9 Taxonomic Group Species Supplementary Table S1. Close homologs of EAT1. Accession Protein Accession ID Number Number Bryophyta Physcomitrella PhypXP_ XP_ patens PhypXP_ XP_ Pteridophyte SelmXP_ XP_ Selaginella SelmXP_ XP_ moellendorffii SelmXP_ XP_ Angiospermae Monocots Brachypodium BradXP_ XP_ distich BradXP_ XP_ Oryza sativa EAT1 LOC_Os04g51070 NP_ OsbHLH142 LOC_Os01g18870 NP_ Zea mays ZmLOC NP_ Sorghum bicolor ZmLOC NP_ Sb04g Sb04g XP_ Sb03g Sb03g XP_ Eduicots Glycine max GlymXP_ Glyma08g09420 XP_ Medicago truncatula Populus trichocarpa Arabidopsis lyrata Arabidopsis thaliana GlymXP_ Glyma05g26490 XP_ MedtXP_ MedtXP_ PoptXP_ PoptXP_ PoptXP_ AralXP_ AralXP_ XP_ XP_ XP_ XP_ XP_ XP_ XP_ AtbHLH091 At2g31210 NP_ Tissues expression Anther, Inflorescence, Root, Leaf, Seed Anther, Inflorescence, Pistil, Lemma, Palea, Root, Leaf, Seed Pod, Flower, Nodule, SAM, Root and Leaf Nodule, Flower, Pod, SAM, Root and Leaf Pod, Bud, Nodule, Flower, Root and Leaf Pod, Bud, Nodule, Flower, Root and Leaf Male and Female Catkin, Seedling, Root, Leaf and Xylem Male and Female Catkin, Seedling, Root, Leaf and Xylem Male and Female Catkin, Seedling, Root, Leaf and Xylem Flower and Floral organ 9

10 AtbHLH089 At1g06170 NP_ Flower, Floral organ and Apex AtbHLH010 At2g31220 NP_ not present on array 10

11 Supplementary Table S2. Peptide sequences used for constructing the NJ tree. ID Peptides Sequences (N - C) AtbHLH010 RKGRGSRKSRTSPTERERRVHFNDRFFDLKNLIPNPTKIDRASIVGEAIDYIKELLRTIEEFKMLV EKKRCGRFRSKKRARVGEGGGGEDQEEEEDTVNYKPQSEVDQSCFNKNNNNSLRCSWLKRKS KVTEVDVRIIDDEVTIKLVQKKKINCLLFTTKVLDQLQLDLHHVAGGQIGEHYSFLFNTKICEGS CVYASGIADTLMEVVEKQYMEAVPSNGY AralXP_ RKGRGSRKSRTFPTERERRVHFNDRFFDLKNLIPNPTKIGRASIVGEAIDYIKELLRTIEEFKMLV EKKRCGRFRSKKRARVGEGGGEDQEEEEDTVNYKPQSEVDQSGFNKNNNTSLRCSWLKRKSK VTEIDVRIIDDEVTIKLVQKKKINCLLFTTKVLDQLQLDLHHVAGGQIGEHYSFLFNTKICEGSC VYASGIADTVMEVVEKQYMEAVPTNGY AtbHLH089 RKGRGSKKRKIFPTERERRVHFKDRFGDLKNLIPNPTKNDRASIVGEAIDYIKELLRTIDEFKLLV EKKRVKQRNREGDDVVDENFKAQSEVVEQCLINKKNNALRCSWLKRKSKFTDVDVRIIDDEV TIKIVQKKKINCLLFVSKVVDQLELDLHHVAGAQIGEHHSFLFNAKISEGSSVYASAIADRVMEV LKKQYMEALSANNGYHCYSSD AralXP_ RKGRGSRKRKVFPTERERRVHFKDRFGDLKNLIPNPTKNDRASIVGEAIDYIKELLRTIDEFKLL VEKKRTKQRNREGDDVIDENFKAQSEVVEQCLINKKNNALRCSWLKRKSKFTEVDVRIIDDDV TIKIVQKKKINCLVFVSKVVDQLQLDLHHVAGAQIGEHHSFLFNAKICEGSSVYASAIADRVME VLEKQYMEALSTNNGYHCYSSD AtbHLH091 RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPSPSKGDRASILQDGIDYINELRRRVSELKY LVERKRCGGRHKNNEVDDNNNNKNLDDHGNEDDDDDDENMEKKPESDVIDQCSSNNSLRCS WLQRKSKVTEVDVRIVDDEVTIKVVQKKKINCLLLVSKVLDQLQLDLHHVAGGQIGEHYSFLF NTKIYEGSTIYASAIANRVIEVVDKHYMASLPNSNY AralXP_ RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPNPSKGDRASILQDGIDYINELRRRVSELKY LVERKRCGGRHKNNELDNNINNNNSNDHDNDEDDIDDENMEKKPESDVVDQCSSNNSLRCSW LQRKSKVTEVDVRIVDDEVTIKVVQKKKINCLLLVSKVLDQLQLDLYHVAGGQIGEHYSFLFN TKIYEGSTIYASAIANRVIEVVDKHYMAALPINY PoptXP_ GKGIRKSGKVTKHFATERQRREHLNGKYTALRNLVPNPSKNDRASVVGDAINYIKELLRTVEE LKLLVEKKRNGRERIKRRKPEEDGGVDVLENSNTKVEQDQSTYNNGSLRSSWLQRKSKHTEV DVRLIEDEVTIKLVQRKKVNCLLSVSKVLDELQLDLHHAAGGLIGDYYSFLFNTKINEGSCVYA SGIANKLLEVVDRQYASSTSVPAASC PoptXP_ GKGIKKTGKVTKHFATERQRREHLNGKYTALRNLVPNPSKNDRASVVGEAIDYIKELLRTVQE LKLLVEKKRCGRERSKWRKTEDDGGVEVLDNSDIKVEPDQSAYSNGSLRSSWLQRKSKDTEV DVRLIEDEVTIKLVQRKRVNCLLYVSKVLDELQLDLHHAAGGLIGDYYSFLFNTKINEGSCVYA SAIANRLIEVVDRQYASSTTTVPAAGSCY MedtXP_ GKGKGKATEHLTTEKQRREQLKGRYKILRSLIPNSTKDDRASVVGDAIEYLRELIRTVNELKLL VEKKRHEIEICKRHKTEDYAAESCHMKPFGDPDGSIRTSWLQRKSKDSEVDVRIIDDDVTIKLFQ RKKVNCLLFVSKVLDELQLELNHVAGGHVGEYCSFLFNSKVIEGSSVHASAIANRVIDVLDTQY AAVVPHNRM MedtXP_ GKGRGGKATKHFATEKQRREQLNGKYKILRDLIPSPTKTDRASVVGDAIEYIRELIRTVNELKLL VEKKRHGREMCKRLKTEDDAAESCNIKPFGDPDGSIRTSWLQRKSKDSEVDVRIIDDDVTIKLF QRKKVNCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKVNEGSSVYASAIANRVIDVMDT QYAAGLPHISRL 11

12 12 GlymXP_ GKGRGGKATKHFATEKQRREQLNGKYKILRNLIPSPTKLIGWVWFNTDDRASVVGDAIDYIRE LIRTVNELKLLVEKKRYAKERYKRPKTEEDAAESCNIKPFGDPDGGIRTSWLQRKSKDSEVDVR IIDDDVTIKLFQRKKINCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKGLVSLRIMEGSSV YASAIANRVIDVLDSQYTAAVPHTNSY GlymXP_ GKGRRGKATKHFATEKQRREQLNGKYKILRNLIPSPTKLVGFVLTQTDRASVVGDAIDYIRELI RTVNELKLLVEKKRYAKDRCKRPKTEEDAAESCNIKPFGDPDGGIRTSWLQRKSKDSEVDVRII DDDVTIKLFQRKKINCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKGLVSLRIMEGSSVY ASAIANRVIDVLDSQYAAAVPHTNSY PoptXP_ LIAFAIELQRRGQLNDNYKTLRDLIKNPSTKEDRATVIRDAIKYIIQLIRTVYELKQLVEKTRGKK LDTIGGVDVFTKPVVRKSQYSHSYNDVSSGPSFSKKHSSVQRKSKDTEIDVRIIGDEVTIKVLRR RKKNDYCLLFVSRVLDELHMDLHFVSSCYIGYETYYFQFKTKINGGPSSDNAHTIADKLIEVLD SSCSI Sb03g RKRGRALGGGFHAGLANGVEKKEKQRRQRLTEKYTALMHLIPNVTKPDRATVISDAIEYIQEL GRTVEELTLLVEKKRRRRELQGDVVDAAPTAVVVAAAATGGEAESSEGEVAPPPPPhypAVQR QPIRSTYIQRRSKDTSVDVRIVEEDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDC HIYMFNTKIHKGSSVFASAVASRLMEVVDEY ZmLOC RKRGRALGGGFHAVLANGVEKKEKQRRLRLTEKYTALMHLIPNVTKTDRATVISDAIEYIQEL GRTVEELTLLVEKKRRRRELQGDVVDAAPAAVVAAAGEAESSEGEVAPPPhypVPRQPIRSTYIQ RRSKDTSVDVRIVEEDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDCQIYMFNTKI HKGSSVFASAVAGRLMEVVDEY OsbHLH142 RKRSRATAGFHGGGPANGVEKKEKQRRLRLTEKYNALMLLIPNRTKEDRATVISDAIEYIQELG RTVEELTLLVEKKRRRREMQGDVVDAATSSVVAGMDQAAESSEGEVMAAAAMGAVAPPPRQ APIRSTYIQRRSKETFVDVRIVEDDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDC HIYMFNTKIHSGSPVFASAVASRLIEVVDEY BradXP_ RKRNRGSRAAGGPAHGGVEKKEKQRRLRLTEKYTALMLLIPNRTKEDRATVISDAIEYIQELGR TVEELTLLVGKKRRRNGAGEHHLHQGDVVDAAPAVGAAGELVLAAESSEGEVQAPLAALQPI RSTYIQRKSKETFVDVRIVEDEVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDCHIY MFNTKIHQGSPVFASAVASKLIEVVDEY Sb04g RRQKGGFGKGKGKANFATERERREQLNVKYGALRSLFPNPTKNDRASIVGDAIDYINELNRTV KELKILLEKKRNSTDRRKILKLDDEAADDGESSSMQPVSDDQNNQMNGAIRSSWVQRRSKECD VDVRIVDDEINIKFTEKKRANSLLCAAKVLEEFRLELIHVVGGIIGDHHIFMFNTKIPKGSSVYAC AVAKKLLEAVEIKKQALNIFN ZmLOC RKQKGGFGKGKGKANFATERERRQFNVKYGALRSLFPNPTKNDRASIVGDAIEYINELNRTVK ELKILLEKKRNSADRRKILKLDEEAADDGESSSMQPVSDDQNQMNGTIRSSWVQRRSKECDVD VRIVDDEINIKFTEKKRANSLLCAAKVLEEFHLELIHVVGGIIGDHHIFMFNTKIPKGSSVYACAV AKKLLEAVEIKKQAYNIFN EAT1 (OsbHLH141) RRGKGEFGKGKGKANFATERERREQLNVKFRTLRMLFPNPTKNDRASIVGDAIEYIDELNRTV KELKILVEQKRHGNNRRKVLKLDQEAAADGESSSMRPVRDDQDNQLHGAIRSSWVQRRSKEC HVDVRIVDDEVNIKLTEKKKANSLLHAAKVLDEFQLELIHVVGGIIGDHHIFMFNTKVSEGSAV YACAVAKKLLQAVDVQHQALDIFN BradXP_ RRGKGEFGKGKGKANFATERERREQLNVKYKTLKDLFPNPTKSDRASVVGDAIEYIDELNRTV KELKILVEQKWHGNKRTKIIKLDEEVAADGESSSMKPMRDDQDNQFDGTIRSSWVQRRSKECH IDVRIVENEVNIKLTEKKKVNSLLHAARVLDEFQLELIHAVGGIIGDHHIFMFNTKVSEGSSVYA CAVAKRLLQAVDAQHQAINIFH PhypXP_ KGEPRGVNHFATERQRREYLNEKYQTLRSLVPNPTKADRASIVADAIEYVKELKRTVQELQLL VQEKRRAAGDSSGAKRRRSLDATDTYPGACTPENASNGHLVMQKGNDTFSADGSQLRSSWLQ RTSQNGTHVDVRIVHDEVTIKVNQRRGKTCLVFDVISVLQELQLDLLQASGATIGEHDVFLFNT

13 KASQMMILNVNTMKCYDLGTYSISQHRTLTSYYLSPKLICRVRK PhypXP_ SelmXP_ SelmXP_ SelmXP_ UDT1 (OsbHLH164) 1 KGEPRGVNHFATERQRREYLNEKYQTLRSLVPNPTKADRASIVADAIEYVKELKRTVQELQLL VQEKRRAAGDSSGGKRRRSMDDADNYAGSCTTENASNGHLVMQKGNDTFSTDGSQLRSSWL QRTSQNGTHVDVRIVHDEVTIKVNQRRGKNCLVFDVIAVLQELQLDLLQASGATIGEHDVFLF NTKILEGSSTFAGYIAVKLLDALDRHLDITIDDSNR KGAESRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIDYVKELKRTVQELQLL VEEKRRGSNKRCKASPDDPSATDVESTTAMQQPGGTRVSKETTFLGDGSQLRSSWLQRTSQMG THIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLDELRLDLLHANGASIGEHHIFMFNTKVVLAP SSFLLSLFLFYFFLFSSALILPR KGAENRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIEYVKELKRTVQELQLL VEEKRRGSNKRRCKASPDNPSEGGGATDMESSSAIQPGGTRVSKETTFLGDGSQLRSSWLQRTS QMGTQIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLNELHLDLLHANGASIGEHHIFMFNTKIM EGTSTFAGQVATKLIDALGK KGAENRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIEYVKELKRTVQELQLL VEEKRRGSNKRRCKASPDNPSEGGGVTDMESSSAIQPGGTRVSKETTFLGDGSQLRSSWLQRTS QMGTQIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLNELHLDLLHANGASIGEHHIFMFNTKIM EGTSTFAGQVATKLIDAVDRHITLASSGL QAAAAAMGKEFKSKNLEAERRRRGRLNGNIFALRAVVPKITKMSKEATLSDAIEHIKNLQNEV LELQRQLGDSPGEAWEKQCSASCSESFVPTENAHYQGQVELISLGSCKYNLKIFWTKRAGLFTK VLEALCSYKVQVLSLNTISFYGYAESFFTIEVKGEQDVVMVELRSLLSSIVEVPSI HLH domains and DUFs were predicted by SALAD ( 53 Homologs were retrieved by blastp searches using bhlh domains and DUF from NCBI. 13

14 Supplementary Table S3. Synthesized coding sequences of EAT1 and OsAP25 and OsAP37. ID EAT1 OsAP25 Sequences (5' - 3') ATGATTGTTGGGGCTGGTTACTTTGAGGATTCCCACGATCAAAGTCTCATGGCAGGATCTTTGATCCAT GACTCAAATCAAGCTCCTGCAAGCAGTGAAAACACAAGCATTGATTTGCAGAAATTCAAAGTGCACCC GTACTCAACAGAAGCTCTCTCGAATACGGCCAATCTAGCTGAAGCTGCAAGAGCAATTAACCACCTTCA ACATCAACTAGAAATTGATTTGGAGCAAGAGGTTCCCCCAGTAGAAACTGCAAACTGGGATCCAGCTA TCTGCACTATACCAGATCATATCATCAACCATCAGTTTAGCGAAGATCCACAAAACATATTGGTGGAGC AACAGATCCAGCAGTATGATTCTGCACTTTATCCAAATGGTGTTTACACACCTGCACCAGATCTCCTTA ATCTTATGCAGTGCACAATGGCTCCAGCATTCCCGGCAACGACATCCGTATTCGGTGACACAACACTGA ATGGTACTAACTATTTGGATCTTAACGGTGAACTTACAGGAGTAGCAGCGGTTCCAGACAGTGGGAGT GGGTTGATGTTTGCTAGTGATTCAGCTCTCCAGTTAGGGTACCATGGCACCCAGTCGCATCTGATTAAA GATATCTGCCATTCGCTGCCGCAGAATTATGGCCTGTTTCCGAGCGAAGATGAACGCGATGTGATTATT GGCGTGGGCAGCGGCGATCTGTTTCAGGAAATTGATGATCGCCAGTTTGATAGCGTGCTGGAATGCCGC CGCGGCAAAGGCGAATTTGGCAAAGGCAAAGGCAAAGCGAATTTTGCGACCGAACGCGAACGCCGTG AACAGCTGAATGTGAAATTTCGCACCCTGCGCATGCTGTTTCCGAATCCGACCAAAAATGATCGCGCGA GCATTGTGGGCGATGCGATTGAATATATTGATGAACTGAATCGCACCGTGAAAGAACTGAAAATTCTG GTGGAACAGAAACGCCATGGCAATAATCGCCGCAAAGTGCTGAAACTGGATCAGGAAGCGGCGGCGG ATGGCGAAAGCTCAAGCATGCGCCCGGTGCGCGATGATCAGGATAATCAGCTGCATGGCGCGATTCGC AGCAGCTGGGTGCAGCGCCGCAGCAAAGAATGCCATGTGGATGTGCGCATTGTGGATGATGAAGTGAA TATTAAACTGACCGAAAAGAAAAAGGCGAATAGCCTGCTGCATGCGGCGAAAGTGCTGGATGAATTTC AGCTGGAACTGATTCATGTGGTGGGCGGCATTATTGGCGATCATCATATTTTTATGTTTAATACCAAAG TGAGCGAAGGCAGCGCGGTGTATGCGTGCGCGGTGGCGAAGAAACTGCTGCAGGCGGTGGATGTGCAG CATCAGGCGCTGGATATTTTCAACTAA CTCGAGGAATTCACCGGTTCCATGGCGGCGACCACCACCATCCCGCTGCTGCTGCTGCTGCTGGCGGCG ACCGTGGCGGCGGCGGCGGCGGAACTGAGCGTGTATCATAATGTGCATCCGAGCAGCCCGAGCCCGCT GGAAAGCATCATCGCGCTGGCGCGCGATGATGATGCGCGCCTGCTGTTTCTGAGCAGCAAAGCGGCGA CCGCGGGCGTGAGCAGCGCGCCGGTGGCGAGCGGCCAGGCGCCGCCGAGCTATGTGGTGCGCGCGGGC CTGGGCAGCCCGAGCCAGCAGCTGCTGCTGGCGCTGGATACCAGCGCGGATGCGACCTGGGCGCATTG CAGCCCGTGCGGCACCTGCCCGAGCAGCAGCCTGTTTGCGCCGGCGAATAGCAGCAGCTATGCGAGCC TGCCGTGCAGCAGCAGCTGGTGCCCGCTGTTTCAGGGCCAGGCGTGCCCGGCGCCGCAGGGCGGCGGC GATGCGGCGCCGCCGCCGGCGACCCTGCCGACCTGCGCGTTTAGCAAACCGTTTGCGGATGCGAGCTTT CAGGCGGCGCTGGCGAGCGATACCCTGCGCCTGGGCAAAGATGCGATCCCGAATTATACCTTTGGCTG CGTGAGCAGCGTGACCGGCCCGACCACCAATATGCCGCGCCAGGGCCTGCTGGGCCTGGGCCGCGGCC CGATGGCGCTGCTGAGCCAGGCGGGCAGCCTGTATAATGGCGTGTTTAGCTATTGCCTGCCGAGCTATC GCAGCTATTATTTTAGCGGCAGCCTGCGCCTGGGCGCGGGCGGCGGCCAGCCGCGCAGCGTGCGCTAT ACCCCGATGCTGCGCAATCCGCATCGCAGCAGCCTGTATTATGTGAATGTGACCGGCCTGAGCGTGGGC CATGCGTGGGTGAAAGTGCCGGCGGGCAGCTTTGCGTTTGATGCGGCGACCGGCGCGGGCACCGTGGT GGATAGCGGCACCGTGATCACCCGCTGGACCGCGCCGGTGTATGCGGCGCTGCGCGAAGAATTTCGCC GCCAGGTGGCGGCGCCGAGCGGCTATACCAGCCTGGGCGCGTTTGATACCTGCTTTAATACCGATGAA GTGGCGGCGGGCGGCGCGCCGGCCGTGACCGTGCATATGGATGGCGGCGTGGATCTGGCGCTGCCGAT GGAAAATACCCTGATCCATAGCAGCGCGACCCCGCTGGCGTGCCTGGCGATGGCGGAAGCGCCGCAGA ATGTGAATAGCGTGGTGAATGTGATCGCGAATCTGCAGCAGCAGAATATCCGCGTGGTGTTTGATGTGG CGAATAGCCGCGTGGGCTTTGCGAAAGAAAGCTGCAATGCTAGCGAACAGAAGTTGATTTCCGAAGAA GACCTCGCCGAGCAGAAACTGATCAGCGAAGAGGATCTGGCGGAACAGAAGCTGATTAGCGAGGAAG ACCTGTAAACTAGTGTCGACCCCGGG 14

15 OsAP37 CCATGGTTCTCGAGGAATTCACCGGTATGAATGCGGCGGTGCTGCTGCTGCTGCTGGCGCTGGCGGCGC TGCCGGCGAGCTGCGCGCCGCCGCGCAGCTTTCGCCTGGAACTGGCGAGCGTGGATGCGAGCGCGGCG GATGCGGCGAATCTGACCGAACATGAACTGCTGCGCCGCGCGATCCAGCGCAGCCGCTATCGCCTGGC GGGCATCGGCATGGCGCGCGGCGAAGCGGCGAGCGCGCGCAAAGCGGTGGTGGCGGAAACCCCGATC ATGCCGGCGGGCGGCGAATATCTGGTGAAACTGGGCATCGGCACCCCGCCGTATAAATTTACCGCGGC GATCGATACCGCGAGCGATCTGATCTGGACCCAGTGCCAGCCGTGCACCGGCTGCTATCATCAGGTGG ATCCGATGTTTAATCCGCGCGTGAGCAGCACCTATGCGGCGCTGCCGTGCAGCAGCGATACCTGCGATG AACTGGATGTGCATCGCTGCGGCCATGATGATGATGAAAGCTGCCAGTATACCTATACCTATAGCGGCA ATGCGACCACCGAAGGCACCCTGGCGGTGGATAAACTGGTGATCGGCGAAGATGCGTTTCGCGGCGTG GCGTTTGGCTGCAGCACCAGCAGCACCGGCGGCGCGCCGCCGCCGCAGGCGAGCGGCGTGGTGGGCCT GGGCCGCGGCCCGCTGAGCCTGGTGAGCCAGCTGAGCGTGCGCCGCTTTGCGTATTGCCTGCCGCCGCC GGCGAGCCGCATCCCGGGCAAACTGGTGCTGGGCGCGGATGCGGATGCGGCGCGCAATGCGACCAATC GCATCGCGGTGCCGATGCGCCGCGATCCGCGCTATCCGAGCTATTATTATCTGAATCTGGATGGCCTGC TGATCGGCGATCGCGCGATGAGCCTGCCGCCGACCACCACCACCACCGCGACCGCGACCGCGACCGCG CCGGCGCCGGCGCCGACCCCGAGCCCGAATGCGACCGCGGTGGCGGTGGGCGATGCGAATCGCTATGG CATGATCATCGATATCGCGAGCACCATCACCTTTCTGGAAGCGAGCCTGTATGATGAACTGGTGAATGA TCTGGAAGTGGAAATCCGCCTGCCGCGCGGCACCGGCAGCAGCCTGGGCCTGGATCTGTGCTTTATCCT GCCGGATGGCGTGGCGTTTGATCGCGTGTATGTGCCGGCGGTGGCGCTGGCGTTTGATGGCCGCTGGCT GCGCCTGGATAAAGCGCGCCTGTTTGCGGAAGATCGCGAAAGCGGCATGATGTGCCTGATGGTGGGCC GCGCGGAAGCGGGCAGCGTGAGCATCCTGGGCAATTTTCAGCAGCAGAATATGCAGGTGCTGTATAAT CTGCGCCGCGGTCGCGTGACCTTTGTGCAGAGCCCGTGCGGCGCGCTGCGCGCTAGCGAACAGAAGTT GATTTCCGAAGAAGACCTCTAAACTAGTGTCGACCCCGGG 15

16 Supplementary Table S4. Primer sequences used in this study. Primer Name Sequence (5' - 3') Purpose Os409-F 5'TACTTGAGCCAACCGAGCTA 3' map-based cloning Os409-R 5'TGGAGATCGAGAGAGAATGG 3' map-based cloning Os410-F 5'TATTGTCACCTCACCCGTCG 3 map-based cloning Os410-R 5'CGTCGATCTTGTATTTTCAC 3 map-based cloning XY409-1-F 5'TGACTTAATATCAGAGCACCT 3 map-based cloning XY409-1-R 5'ACGGCACTTCTTTTTCCTTC 3 map-based cloning XY409-2-F 5'TACGACAAATCTGAACCGAG 3 map-based cloning XY409-2-R 5'GCTCTTTCTCCACCTACCAAT 3 map-based cloning XY409-3-F 5'CACAAAGAACAAACATAGGTCA 3 map-based cloning XY409-3-R 5'ATGGTGGAGATTGTTTGC 3 map-based cloning XY409-4-F 5'GCAATTGATTCCACTTCTT 3 map-based cloning XY409-4-R 5'ATAACCCGACCGCTACAT 3 map-based cloning XY409-5-F 5'TTTGGTGGATATGTATCTAGG 3 map-based cloning XY409-5-R 5'CCGTTCCACGTTGATACAG 3 map-based cloning eat1-2-f 5 ACGGCCAATCTAGCTGAAGC 3 mutant identification eat1-2-r 5 ATCTCGTTCGTCCTCACTGG 3 mutant identification eat1-3-f 5 TGGCTTGCCACCAAAGTTGG 3 mutant identification eat1-3-r 5'CCACTGCACATGCATAAACC 3 mutant identification EAT1-RT-F 5 CAGAGGAGGTCAAAGGAATG 3 qrt-pcr EAT1-RT-R 5 TCCAATCCTGGTCAAATAAG 3 qrt-pcr EAT1-pro-F 5 GGATCCTGCGAGCTCCCAGAAGTTAT 3 GUS assay EAT1-pro-R 5 CCATGGTTTGGCAAAACAGTGCTAGG 3 GUS assay EAT1-situ-F 5 CCACCTTCAACATCAACTAGA 3 in situ analysis of EAT1 EAT1-situ-R 5 CAATAATCACATCTCGTTCGT 3 in situ analysis of EAT1 MSP1-RT-F 5 CTCACAATGAAGTCCACCAC 3 qrt-pcr MSP1-RT-R 5 ACCAGAGGTCCACATTCCAT 3 qrt-pcr UDT1-RT-F 5 CCCACTGAGAACGCCCATTA 3 qrt-pcr UDT1-RT-R 5 CACCATCACAACATCCTGCT 3 qrt-pcr GAMYB-RT-F 5 CAGTGGCAATTCATTCACTGAATC 3 qrt-pcr GAMYB-RT-R 5 TCCAGATCCCATTGAAGTGCTTTG 3 qrt-pcr TDR-RT-F 5 GGAGGAGTAACAAGGACCCA 3 qrt-pcr TDR-RT-R 5 ACCTCCAGCAGCGAGTCCCT 3 qrt-pcr PTC1-RT-Probe 5 GCACGTAAGGCAAGGACGGTGAGC 3 qrt-pcr API5-RT-F 5 CCCCTTTATTTATTGGTGAC 3 qrt-pcr API5-RT-R 5 CCTCTACCACCACGGGATAA 3 qrt-pcr OsCP1-RT-F 5 CTACGGGATCAGGAACCACT 3 qrt-pcr OsCP1-RT-R 5 CTCGGTAACAGTAACAAAGATA 3 qrt-pcr TDR-1F 5 CATATGGGAAGAGGAGACCACCT 3 Y-2-H TDR-mu-R 5 GGATCCTCAGTTGTCTTCCAGCTCGT 3 Y-2-H TDR-1R 5 TCAATCAAACGCGAGGTAAT 3' Y-2-H EAT1-2F 5 GAATTCATTGTTGGGGCTGGTTA 3 Y-2-H EAT1-2R 5 GTCGACTTAGTTGAATATGTCGAGGGC 3 Y-2-H EAT1-muF 5 GGCCAATCCGGCCAAGGGTGAGTTCGGAAAGGG 3 Y-2-H EAT1-muR 5 GGCCTTAGAGGCCTTAGTTGAATATGTCGAGGGC 3 Y-2-H 16

17 EAT1-3F 5 ACTAGTGTTGAATATGTCGAGGGCCT 3 BiFC EAT1-3R 5 GTCGACGTTGAATATGTCGAGGGCCT 3 BiFC TDR-2F 5 ACTAGTATGGGAAGAGGAGACCACCT 3 BiFC TDR-2R 5 GTCGACATCAAACGCGAGGTAATGCA 3 BiFC EAT1-1F 5 GAATTCATGATTGTTGGGGCTGGTTA 3 preparation of EAT1 protein EAT1-1R 5 GTCGACTTAGTTGAATATGTCGAGGGC 3 preparation of EAT1 protein OsAP25-RT-F 5 AGCTTTCCGATCTGTGCAAT 3 qrt-pcr OsAP25-RT-R 5 GTCAAAGCTAACCATGAATACG 3 qrt-pcr OsAP37-RT-F 5 TACGACGAGCTGGTGAACGA 3 qrt-pcr OsAP37-RT-R 5 GTTGTAGAGCACCTGCATGTTC 3 qrt-pcr OsAP19-RT-F 5 ACAAACCAAGGATGGGAAAG 3 qrt-pcr OsAP19-RT-R 5 AAGGACTTGCGCCACTGAAC 3 qrt-pcr EAT1-anti-F 5 CCATGGATGATTGTTGGGGCTGGTTA 3 preparation of EAT1 antibody EAT1-anti-R 5 CTCGAGTTGTGGATCTTCGCTAAACTGA 3 preparation of EAT1 antibody OsAP25-ChIP-F 5 ACCTCGTGTTAGATTTCCTCCA 3 qchip PCR OsAP25-ChIP-R 5 AACGTGCTCACGAAAAAGAGAG 3 qchip PCR OsAP37-ChIP-F 5 ACAGCATTAACACCATTATTCTCC 3 qchip PCR OsAP37-ChIP-R 5 TAGCTAAGAGAAATGAACTATGGC 3 qchip PCR OsAP37-ChIP-SEG1-F 5 CATCCTCCATTTCTTGTATTTG 3 qchip PCR OsAP37-ChIP- SEG1-R 5 TGCTACGTGATGAACTAGGA 3 qchip PCR OsAP37-ChIP- SEG2-F 5 CATCCCACCATTTCCAAAGTA 3 qchip PCR OsAP37-ChIP- SEG2-R 5 GAACTGGTTCAACTGCCGGC 3 qchip PCR OsAP19-ChIP- SEG1-F 5 GGCAAACTAACCTGGTACTAAAC 3 qchip PCR OsAP19-ChIP- SEG1-R 5 ATGGGGTCAAGGAGAATCAGTG 3 qchip PCR OsAP19-ChIP- SEG2-F 5 CCGTTGAGATCAACGCTCTT 3 qchip PCR OsAP19-ChIP- SEG2-R 5 ATAGGTCGCGCGGCCTGAGG 3 qchip PCR OsCP1-ChIP-1-F 5 TAAAGAAGCAGTTGCCATCC 3 ChIP PCR OsCP1-ChIP-1-R 5 GAAGCGTGATGCAGTAATAC 3 ChIP PCR OsCP1-ChIP-2-F 5 GTTCCGAGCCATTACGACAC 3 ChIP PCR OsCP1-ChIP-2-R 5 GATTGGGGATTGAGGTGTTA 3 ChIP PCR OsAP25-situ-F 5 GAATTCTCGCCAAGGAGTCCTGCAACTA 3 in situ analysis of OsAP25 OsAP25-situ-R 5 AAGCTTAGCGATGCAAATGATGAAGAT 3 in situ analysis of OsAP25 OsAP37-situ-F 5 GAATTCTGCGGAGGTTCGCCTACTGC 3 in situ analysis of OsAP37 OsAP37-situ-R 5 AAGCTTCTCGTCGTACAGCGATGCCT 3 in situ analysis of OsAP37 17

18 Supplementary Reference 53 Mihara, M., Itoh, T. & Izawa, T. SALAD database: a motif-based database of protein annotations for plant comparative genomics. Nucleic Acids Res 38, (2011). 18