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.

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)

I2-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.

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.

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.

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.

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.

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.

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.

9

Supplementary Table S1. Close homologs of EAT1.

Taxonomic

Group Species ID

Accession

Number

Protein Accession

Number Tissues expression

Bryophyta PhypXP_001784001 XP_001784001

Physcomitrella

patens PhypXP_001767478 XP_001767478

Pteridophyte SelmXP_002961225 XP_002961225

SelmXP_002969202 XP_002969202

Selaginella

moellendorffii SelmXP_002964440 XP_002964440

Angiospermae

Monocots BradXP_003580474 XP_003580474

Brachypodium

distich BradXP_003567568 XP_003567568

Oryza sativa EAT1 LOC_Os04g51070 NP_001053749 Anther, Inflorescence,

Root, Leaf, Seed

OsbHLH142 LOC_Os01g18870 NP_001042795

Anther, Inflorescence,

Pistil, Lemma, Palea,

Root, Leaf, Seed

Zea mays ZmLOC100282922 NP_001149299

ZmLOC100283549 NP_001149921

Sorghum

bicolor Sb04g030850 Sb04g030850 XP_002452697

Sb03g011940 Sb03g011940 XP_002457706

Eduicots Glycine max GlymXP_003532668 Glyma08g09420 XP_003532668 Pod, Flower, Nodule,

SAM, Root and Leaf

GlymXP_003524131 Glyma05g26490 XP_003524131 Nodule, Flower, Pod,

SAM, Root and Leaf

MedtXP_003638306 XP_003638306 Pod, Bud, Nodule,

Flower, Root and Leaf

Medicago

truncatula MedtXP_003638303 XP_003638303

Pod, Bud, Nodule,

Flower, Root and Leaf

Populus

trichocarpa PoptXP_002314221 XP_002314221

Male and Female

Catkin, Seedling, Root,

Leaf and Xylem

PoptXP_002323376 XP_002323376

Male and Female

Catkin, Seedling, Root,

Leaf and Xylem

PoptXP_002308327 XP_002308327

Male and Female

Catkin, Seedling, Root,

Leaf and Xylem

Arabidopsis

lyrata AralXP_002879311 XP_002879311

AralXP_002892324 XP_002892324

Arabidopsis

thaliana AtbHLH091 At2g31210 NP_180679

Flower and Floral

organ

10

AtbHLH089 At1g06170 NP_172107 Flower, Floral organ

and Apex

AtbHLH010 At2g31220 NP_180680 not present on array

11

Supplementary Table S2. Peptide sequences used for constructing the NJ tree.

ID Peptides Sequences (N - C)

AtbHLH010 RKGRGSRKSRTSPTERERRVHFNDRFFDLKNLIPNPTKIDRASIVGEAIDYIKELLRTIEEFKMLV

EKKRCGRFRSKKRARVGEGGGGEDQEEEEDTVNYKPQSEVDQSCFNKNNNNSLRCSWLKRKS

KVTEVDVRIIDDEVTIKLVQKKKINCLLFTTKVLDQLQLDLHHVAGGQIGEHYSFLFNTKICEGS

CVYASGIADTLMEVVEKQYMEAVPSNGY

AralXP_002879313 RKGRGSRKSRTFPTERERRVHFNDRFFDLKNLIPNPTKIGRASIVGEAIDYIKELLRTIEEFKMLV

EKKRCGRFRSKKRARVGEGGGEDQEEEEDTVNYKPQSEVDQSGFNKNNNTSLRCSWLKRKSK

VTEIDVRIIDDEVTIKLVQKKKINCLLFTTKVLDQLQLDLHHVAGGQIGEHYSFLFNTKICEGSC

VYASGIADTVMEVVEKQYMEAVPTNGY

AtbHLH089 RKGRGSKKRKIFPTERERRVHFKDRFGDLKNLIPNPTKNDRASIVGEAIDYIKELLRTIDEFKLLV

EKKRVKQRNREGDDVVDENFKAQSEVVEQCLINKKNNALRCSWLKRKSKFTDVDVRIIDDEV

TIKIVQKKKINCLLFVSKVVDQLELDLHHVAGAQIGEHHSFLFNAKISEGSSVYASAIADRVMEV

LKKQYMEALSANNGYHCYSSD

AralXP_002892324 RKGRGSRKRKVFPTERERRVHFKDRFGDLKNLIPNPTKNDRASIVGEAIDYIKELLRTIDEFKLL

VEKKRTKQRNREGDDVIDENFKAQSEVVEQCLINKKNNALRCSWLKRKSKFTEVDVRIIDDDV

TIKIVQKKKINCLVFVSKVVDQLQLDLHHVAGAQIGEHHSFLFNAKICEGSSVYASAIADRVME

VLEKQYMEALSTNNGYHCYSSD

AtbHLH091 RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPSPSKGDRASILQDGIDYINELRRRVSELKY

LVERKRCGGRHKNNEVDDNNNNKNLDDHGNEDDDDDDENMEKKPESDVIDQCSSNNSLRCS

WLQRKSKVTEVDVRIVDDEVTIKVVQKKKINCLLLVSKVLDQLQLDLHHVAGGQIGEHYSFLF

NTKIYEGSTIYASAIANRVIEVVDKHYMASLPNSNY

AralXP_002879311 RRKGRGKRKNKPFTTERERRCHLNERYEALKLLIPNPSKGDRASILQDGIDYINELRRRVSELKY

LVERKRCGGRHKNNELDNNINNNNSNDHDNDEDDIDDENMEKKPESDVVDQCSSNNSLRCSW

LQRKSKVTEVDVRIVDDEVTIKVVQKKKINCLLLVSKVLDQLQLDLYHVAGGQIGEHYSFLFN

TKIYEGSTIYASAIANRVIEVVDKHYMAALPINY

PoptXP_002308327 GKGIRKSGKVTKHFATERQRREHLNGKYTALRNLVPNPSKNDRASVVGDAINYIKELLRTVEE

LKLLVEKKRNGRERIKRRKPEEDGGVDVLENSNTKVEQDQSTYNNGSLRSSWLQRKSKHTEV

DVRLIEDEVTIKLVQRKKVNCLLSVSKVLDELQLDLHHAAGGLIGDYYSFLFNTKINEGSCVYA

SGIANKLLEVVDRQYASSTSVPAASC

PoptXP_002323376 GKGIKKTGKVTKHFATERQRREHLNGKYTALRNLVPNPSKNDRASVVGEAIDYIKELLRTVQE

LKLLVEKKRCGRERSKWRKTEDDGGVEVLDNSDIKVEPDQSAYSNGSLRSSWLQRKSKDTEV

DVRLIEDEVTIKLVQRKRVNCLLYVSKVLDELQLDLHHAAGGLIGDYYSFLFNTKINEGSCVYA

SAIANRLIEVVDRQYASSTTTVPAAGSCY

MedtXP_003638303 GKGKGKATEHLTTEKQRREQLKGRYKILRSLIPNSTKDDRASVVGDAIEYLRELIRTVNELKLL

VEKKRHEIEICKRHKTEDYAAESCHMKPFGDPDGSIRTSWLQRKSKDSEVDVRIIDDDVTIKLFQ

RKKVNCLLFVSKVLDELQLELNHVAGGHVGEYCSFLFNSKVIEGSSVHASAIANRVIDVLDTQY

AAVVPHNRM

MedtXP_003638306 GKGRGGKATKHFATEKQRREQLNGKYKILRDLIPSPTKTDRASVVGDAIEYIRELIRTVNELKLL

VEKKRHGREMCKRLKTEDDAAESCNIKPFGDPDGSIRTSWLQRKSKDSEVDVRIIDDDVTIKLF

QRKKVNCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKVNEGSSVYASAIANRVIDVMDT

QYAAGLPHISRL

12

GlymXP_003524131 GKGRGGKATKHFATEKQRREQLNGKYKILRNLIPSPTKLIGWVWFNTDDRASVVGDAIDYIRE

LIRTVNELKLLVEKKRYAKERYKRPKTEEDAAESCNIKPFGDPDGGIRTSWLQRKSKDSEVDVR

IIDDDVTIKLFQRKKINCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKGLVSLRIMEGSSV

YASAIANRVIDVLDSQYTAAVPHTNSY

GlymXP_003532668 GKGRRGKATKHFATEKQRREQLNGKYKILRNLIPSPTKLVGFVLTQTDRASVVGDAIDYIRELI

RTVNELKLLVEKKRYAKDRCKRPKTEEDAAESCNIKPFGDPDGGIRTSWLQRKSKDSEVDVRII

DDDVTIKLFQRKKINCLLFVSKVLDELQLELHHVAGGHVGEYCSFLFNSKGLVSLRIMEGSSVY

ASAIANRVIDVLDSQYAAAVPHTNSY

PoptXP_002314221 LIAFAIELQRRGQLNDNYKTLRDLIKNPSTKEDRATVIRDAIKYIIQLIRTVYELKQLVEKTRGKK

LDTIGGVDVFTKPVVRKSQYSHSYNDVSSGPSFSKKHSSVQRKSKDTEIDVRIIGDEVTIKVLRR

RKKNDYCLLFVSRVLDELHMDLHFVSSCYIGYETYYFQFKTKINGGPSSDNAHTIADKLIEVLD

SSCSI

Sb03g011940 RKRGRALGGGFHAGLANGVEKKEKQRRQRLTEKYTALMHLIPNVTKPDRATVISDAIEYIQEL

GRTVEELTLLVEKKRRRRELQGDVVDAAPTAVVVAAAATGGEAESSEGEVAPPPPPhypAVQR

QPIRSTYIQRRSKDTSVDVRIVEEDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDC

HIYMFNTKIHKGSSVFASAVASRLMEVVDEY

ZmLOC100283549 RKRGRALGGGFHAVLANGVEKKEKQRRLRLTEKYTALMHLIPNVTKTDRATVISDAIEYIQEL

GRTVEELTLLVEKKRRRRELQGDVVDAAPAAVVAAAGEAESSEGEVAPPPhypVPRQPIRSTYIQ

RRSKDTSVDVRIVEEDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDCQIYMFNTKI

HKGSSVFASAVAGRLMEVVDEY

OsbHLH142 RKRSRATAGFHGGGPANGVEKKEKQRRLRLTEKYNALMLLIPNRTKEDRATVISDAIEYIQELG

RTVEELTLLVEKKRRRREMQGDVVDAATSSVVAGMDQAAESSEGEVMAAAAMGAVAPPPRQ

APIRSTYIQRRSKETFVDVRIVEDDVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDC

HIYMFNTKIHSGSPVFASAVASRLIEVVDEY

BradXP_003567568 RKRNRGSRAAGGPAHGGVEKKEKQRRLRLTEKYTALMLLIPNRTKEDRATVISDAIEYIQELGR

TVEELTLLVGKKRRRNGAGEHHLHQGDVVDAAPAVGAAGELVLAAESSEGEVQAPLAALQPI

RSTYIQRKSKETFVDVRIVEDEVNIKLTKRRRDGCLAAASRALDDLRLDLVHLSGGKIGDCHIY

MFNTKIHQGSPVFASAVASKLIEVVDEY

Sb04g030850 RRQKGGFGKGKGKANFATERERREQLNVKYGALRSLFPNPTKNDRASIVGDAIDYINELNRTV

KELKILLEKKRNSTDRRKILKLDDEAADDGESSSMQPVSDDQNNQMNGAIRSSWVQRRSKECD

VDVRIVDDEINIKFTEKKRANSLLCAAKVLEEFRLELIHVVGGIIGDHHIFMFNTKIPKGSSVYAC

AVAKKLLEAVEIKKQALNIFN

ZmLOC100282922 RKQKGGFGKGKGKANFATERERRQFNVKYGALRSLFPNPTKNDRASIVGDAIEYINELNRTVK

ELKILLEKKRNSADRRKILKLDEEAADDGESSSMQPVSDDQNQMNGTIRSSWVQRRSKECDVD

VRIVDDEINIKFTEKKRANSLLCAAKVLEEFHLELIHVVGGIIGDHHIFMFNTKIPKGSSVYACAV

AKKLLEAVEIKKQAYNIFN

EAT1 (OsbHLH141) RRGKGEFGKGKGKANFATERERREQLNVKFRTLRMLFPNPTKNDRASIVGDAIEYIDELNRTV

KELKILVEQKRHGNNRRKVLKLDQEAAADGESSSMRPVRDDQDNQLHGAIRSSWVQRRSKEC

HVDVRIVDDEVNIKLTEKKKANSLLHAAKVLDEFQLELIHVVGGIIGDHHIFMFNTKVSEGSAV

YACAVAKKLLQAVDVQHQALDIFN

BradXP_003580474 RRGKGEFGKGKGKANFATERERREQLNVKYKTLKDLFPNPTKSDRASVVGDAIEYIDELNRTV

KELKILVEQKWHGNKRTKIIKLDEEVAADGESSSMKPMRDDQDNQFDGTIRSSWVQRRSKECH

IDVRIVENEVNIKLTEKKKVNSLLHAARVLDEFQLELIHAVGGIIGDHHIFMFNTKVSEGSSVYA

CAVAKRLLQAVDAQHQAINIFH

PhypXP_001767478 KGEPRGVNHFATERQRREYLNEKYQTLRSLVPNPTKADRASIVADAIEYVKELKRTVQELQLL

VQEKRRAAGDSSGAKRRRSLDATDTYPGACTPENASNGHLVMQKGNDTFSADGSQLRSSWLQ

RTSQNGTHVDVRIVHDEVTIKVNQRRGKTCLVFDVISVLQELQLDLLQASGATIGEHDVFLFNT

13

KASQMMILNVNTMKCYDLGTYSISQHRTLTSYYLSPKLICRVRK

PhypXP_001784001 KGEPRGVNHFATERQRREYLNEKYQTLRSLVPNPTKADRASIVADAIEYVKELKRTVQELQLL

VQEKRRAAGDSSGGKRRRSMDDADNYAGSCTTENASNGHLVMQKGNDTFSTDGSQLRSSWL

QRTSQNGTHVDVRIVHDEVTIKVNQRRGKNCLVFDVIAVLQELQLDLLQASGATIGEHDVFLF

NTKILEGSSTFAGYIAVKLLDALDRHLDITIDDSNR

SelmXP_002964440 KGAESRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIDYVKELKRTVQELQLL

VEEKRRGSNKRCKASPDDPSATDVESTTAMQQPGGTRVSKETTFLGDGSQLRSSWLQRTSQMG

THIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLDELRLDLLHANGASIGEHHIFMFNTKVVLAP

SSFLLSLFLFYFFLFSSALILPR

SelmXP_002969202 KGAENRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIEYVKELKRTVQELQLL

VEEKRRGSNKRRCKASPDNPSEGGGATDMESSSAIQPGGTRVSKETTFLGDGSQLRSSWLQRTS

QMGTQIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLNELHLDLLHANGASIGEHHIFMFNTKIM

EGTSTFAGQVATKLIDALGK

SelmXP_002961225 KGAENRGINHFATERQRREYLNEKYQTLRSLVPNPSKADRASIVADAIEYVKELKRTVQELQLL

VEEKRRGSNKRRCKASPDNPSEGGGVTDMESSSAIQPGGTRVSKETTFLGDGSQLRSSWLQRTS

QMGTQIDVRIVDDEVNIKLTQRRRRNYVLLAVLRSLNELHLDLLHANGASIGEHHIFMFNTKIM

EGTSTFAGQVATKLIDAVDRHITLASSGL

UDT1 (OsbHLH164)1 QAAAAAMGKEFKSKNLEAERRRRGRLNGNIFALRAVVPKITKMSKEATLSDAIEHIKNLQNEV

LELQRQLGDSPGEAWEKQCSASCSESFVPTENAHYQGQVELISLGSCKYNLKIFWTKRAGLFTK

VLEALCSYKVQVLSLNTISFYGYAESFFTIEVKGEQDVVMVELRSLLSSIVEVPSI

HLH domains and DUFs were predicted by SALAD

(http://salad.dna.affrc.go.jp/CGViewer/SALADonARRAYs/cgv_search_pr.html)53

Homologs were retrieved by blastp searches using bHLH domains and DUF from NCBI.

14

Supplementary Table S3. Synthesized coding sequences of EAT1 and OsAP25 and OsAP37.

ID Sequences (5' - 3')

EAT1 ATGATTGTTGGGGCTGGTTACTTTGAGGATTCCCACGATCAAAGTCTCATGGCAGGATCTTTGATCCAT

GACTCAAATCAAGCTCCTGCAAGCAGTGAAAACACAAGCATTGATTTGCAGAAATTCAAAGTGCACCC

GTACTCAACAGAAGCTCTCTCGAATACGGCCAATCTAGCTGAAGCTGCAAGAGCAATTAACCACCTTCA

ACATCAACTAGAAATTGATTTGGAGCAAGAGGTTCCCCCAGTAGAAACTGCAAACTGGGATCCAGCTA

TCTGCACTATACCAGATCATATCATCAACCATCAGTTTAGCGAAGATCCACAAAACATATTGGTGGAGC

AACAGATCCAGCAGTATGATTCTGCACTTTATCCAAATGGTGTTTACACACCTGCACCAGATCTCCTTA

ATCTTATGCAGTGCACAATGGCTCCAGCATTCCCGGCAACGACATCCGTATTCGGTGACACAACACTGA

ATGGTACTAACTATTTGGATCTTAACGGTGAACTTACAGGAGTAGCAGCGGTTCCAGACAGTGGGAGT

GGGTTGATGTTTGCTAGTGATTCAGCTCTCCAGTTAGGGTACCATGGCACCCAGTCGCATCTGATTAAA

GATATCTGCCATTCGCTGCCGCAGAATTATGGCCTGTTTCCGAGCGAAGATGAACGCGATGTGATTATT

GGCGTGGGCAGCGGCGATCTGTTTCAGGAAATTGATGATCGCCAGTTTGATAGCGTGCTGGAATGCCGC

CGCGGCAAAGGCGAATTTGGCAAAGGCAAAGGCAAAGCGAATTTTGCGACCGAACGCGAACGCCGTG

AACAGCTGAATGTGAAATTTCGCACCCTGCGCATGCTGTTTCCGAATCCGACCAAAAATGATCGCGCGA

GCATTGTGGGCGATGCGATTGAATATATTGATGAACTGAATCGCACCGTGAAAGAACTGAAAATTCTG

GTGGAACAGAAACGCCATGGCAATAATCGCCGCAAAGTGCTGAAACTGGATCAGGAAGCGGCGGCGG

ATGGCGAAAGCTCAAGCATGCGCCCGGTGCGCGATGATCAGGATAATCAGCTGCATGGCGCGATTCGC

AGCAGCTGGGTGCAGCGCCGCAGCAAAGAATGCCATGTGGATGTGCGCATTGTGGATGATGAAGTGAA

TATTAAACTGACCGAAAAGAAAAAGGCGAATAGCCTGCTGCATGCGGCGAAAGTGCTGGATGAATTTC

AGCTGGAACTGATTCATGTGGTGGGCGGCATTATTGGCGATCATCATATTTTTATGTTTAATACCAAAG

TGAGCGAAGGCAGCGCGGTGTATGCGTGCGCGGTGGCGAAGAAACTGCTGCAGGCGGTGGATGTGCAG

CATCAGGCGCTGGATATTTTCAACTAA

OsAP25 CTCGAGGAATTCACCGGTTCCATGGCGGCGACCACCACCATCCCGCTGCTGCTGCTGCTGCTGGCGGCG

ACCGTGGCGGCGGCGGCGGCGGAACTGAGCGTGTATCATAATGTGCATCCGAGCAGCCCGAGCCCGCT

GGAAAGCATCATCGCGCTGGCGCGCGATGATGATGCGCGCCTGCTGTTTCTGAGCAGCAAAGCGGCGA

CCGCGGGCGTGAGCAGCGCGCCGGTGGCGAGCGGCCAGGCGCCGCCGAGCTATGTGGTGCGCGCGGGC

CTGGGCAGCCCGAGCCAGCAGCTGCTGCTGGCGCTGGATACCAGCGCGGATGCGACCTGGGCGCATTG

CAGCCCGTGCGGCACCTGCCCGAGCAGCAGCCTGTTTGCGCCGGCGAATAGCAGCAGCTATGCGAGCC

TGCCGTGCAGCAGCAGCTGGTGCCCGCTGTTTCAGGGCCAGGCGTGCCCGGCGCCGCAGGGCGGCGGC

GATGCGGCGCCGCCGCCGGCGACCCTGCCGACCTGCGCGTTTAGCAAACCGTTTGCGGATGCGAGCTTT

CAGGCGGCGCTGGCGAGCGATACCCTGCGCCTGGGCAAAGATGCGATCCCGAATTATACCTTTGGCTG

CGTGAGCAGCGTGACCGGCCCGACCACCAATATGCCGCGCCAGGGCCTGCTGGGCCTGGGCCGCGGCC

CGATGGCGCTGCTGAGCCAGGCGGGCAGCCTGTATAATGGCGTGTTTAGCTATTGCCTGCCGAGCTATC

GCAGCTATTATTTTAGCGGCAGCCTGCGCCTGGGCGCGGGCGGCGGCCAGCCGCGCAGCGTGCGCTAT

ACCCCGATGCTGCGCAATCCGCATCGCAGCAGCCTGTATTATGTGAATGTGACCGGCCTGAGCGTGGGC

CATGCGTGGGTGAAAGTGCCGGCGGGCAGCTTTGCGTTTGATGCGGCGACCGGCGCGGGCACCGTGGT

GGATAGCGGCACCGTGATCACCCGCTGGACCGCGCCGGTGTATGCGGCGCTGCGCGAAGAATTTCGCC

GCCAGGTGGCGGCGCCGAGCGGCTATACCAGCCTGGGCGCGTTTGATACCTGCTTTAATACCGATGAA

GTGGCGGCGGGCGGCGCGCCGGCCGTGACCGTGCATATGGATGGCGGCGTGGATCTGGCGCTGCCGAT

GGAAAATACCCTGATCCATAGCAGCGCGACCCCGCTGGCGTGCCTGGCGATGGCGGAAGCGCCGCAGA

ATGTGAATAGCGTGGTGAATGTGATCGCGAATCTGCAGCAGCAGAATATCCGCGTGGTGTTTGATGTGG

CGAATAGCCGCGTGGGCTTTGCGAAAGAAAGCTGCAATGCTAGCGAACAGAAGTTGATTTCCGAAGAA

GACCTCGCCGAGCAGAAACTGATCAGCGAAGAGGATCTGGCGGAACAGAAGCTGATTAGCGAGGAAG

ACCTGTAAACTAGTGTCGACCCCGGG

15

OsAP37 CCATGGTTCTCGAGGAATTCACCGGTATGAATGCGGCGGTGCTGCTGCTGCTGCTGGCGCTGGCGGCGC

TGCCGGCGAGCTGCGCGCCGCCGCGCAGCTTTCGCCTGGAACTGGCGAGCGTGGATGCGAGCGCGGCG

GATGCGGCGAATCTGACCGAACATGAACTGCTGCGCCGCGCGATCCAGCGCAGCCGCTATCGCCTGGC

GGGCATCGGCATGGCGCGCGGCGAAGCGGCGAGCGCGCGCAAAGCGGTGGTGGCGGAAACCCCGATC

ATGCCGGCGGGCGGCGAATATCTGGTGAAACTGGGCATCGGCACCCCGCCGTATAAATTTACCGCGGC

GATCGATACCGCGAGCGATCTGATCTGGACCCAGTGCCAGCCGTGCACCGGCTGCTATCATCAGGTGG

ATCCGATGTTTAATCCGCGCGTGAGCAGCACCTATGCGGCGCTGCCGTGCAGCAGCGATACCTGCGATG

AACTGGATGTGCATCGCTGCGGCCATGATGATGATGAAAGCTGCCAGTATACCTATACCTATAGCGGCA

ATGCGACCACCGAAGGCACCCTGGCGGTGGATAAACTGGTGATCGGCGAAGATGCGTTTCGCGGCGTG

GCGTTTGGCTGCAGCACCAGCAGCACCGGCGGCGCGCCGCCGCCGCAGGCGAGCGGCGTGGTGGGCCT

GGGCCGCGGCCCGCTGAGCCTGGTGAGCCAGCTGAGCGTGCGCCGCTTTGCGTATTGCCTGCCGCCGCC

GGCGAGCCGCATCCCGGGCAAACTGGTGCTGGGCGCGGATGCGGATGCGGCGCGCAATGCGACCAATC

GCATCGCGGTGCCGATGCGCCGCGATCCGCGCTATCCGAGCTATTATTATCTGAATCTGGATGGCCTGC

TGATCGGCGATCGCGCGATGAGCCTGCCGCCGACCACCACCACCACCGCGACCGCGACCGCGACCGCG

CCGGCGCCGGCGCCGACCCCGAGCCCGAATGCGACCGCGGTGGCGGTGGGCGATGCGAATCGCTATGG

CATGATCATCGATATCGCGAGCACCATCACCTTTCTGGAAGCGAGCCTGTATGATGAACTGGTGAATGA

TCTGGAAGTGGAAATCCGCCTGCCGCGCGGCACCGGCAGCAGCCTGGGCCTGGATCTGTGCTTTATCCT

GCCGGATGGCGTGGCGTTTGATCGCGTGTATGTGCCGGCGGTGGCGCTGGCGTTTGATGGCCGCTGGCT

GCGCCTGGATAAAGCGCGCCTGTTTGCGGAAGATCGCGAAAGCGGCATGATGTGCCTGATGGTGGGCC

GCGCGGAAGCGGGCAGCGTGAGCATCCTGGGCAATTTTCAGCAGCAGAATATGCAGGTGCTGTATAAT

CTGCGCCGCGGTCGCGTGACCTTTGTGCAGAGCCCGTGCGGCGCGCTGCGCGCTAGCGAACAGAAGTT

GATTTCCGAAGAAGACCTCTAAACTAGTGTCGACCCCGGG

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

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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

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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, 835-842 (2011).