. Supplementary Information

Transcription

1 . Supplementary Information Supplementary Figure S1. Mature embryo sac observations. Supplementary Figure S2. STT observations. Supplementary Figure S3. Comparison of the PTB1 cdna with that of the mutant. Supplementary Figure S4. PSSR decrease was associated with PTB1 expression down-regulation in a dwarf mutant. Supplementary Figure S5. Over expression of PTB1 improved rice PSSR. Supplementary Figure S6. Tissue localization of PTB1 in Arabidopsis. Supplementary Figure S7. Sub-cellular localization of PTB1. Supplementary Figure S8. A neighbour-joining tree of PTB1 and its homologues. Supplementary Figure S9. Differentially expressed genes between the WT and ptb1 mutant. Supplementary Table S1. Supplementary Table S2. PSSR of the Reciprocal crossing. Segregations of the mutation in different populations. Supplementary Table S3. PSSR and PTB1 expression levels in complementary plants. Supplementary Table S4. Suplementary Table S5. Natural variations of PSSR and relative expression level of PTB1. Information on putative homologous rice genes involved in female sterility, pollen tube growth or callose deposition. Supplementary Table S6. Primers for map-based cloning and functional analysis of PTB1.

2 Supplementary Figures Supplementary Figure S1. Mature embryo sac observations. (a) Mature embryo sac of WT. (b) Mature embryo sac of ptb1. Mature embryo sacs were observed with the WE-CLSM method, showing that ptb1 had complete inner components, including one egg cell (E), two synergid cells (S), one central cell with two polar nuclei (P), and a group of antipodal cells (A), just as observed in wild-type. Scale bars, 100 µm.

3 Supplementary Figure S2. STT observations. (a-d) WE-CLSM observations of the style surface (a, b) and its inner structure (c, d), showing that the overall cellular structure of the STT in ptb1 mutants (b, d) is comparable to WT (a, c). Scale bars, 100 µm. (e-l) Paraffin section observations of the STT and its ECM. Transverse (e-h) and longitudinal (i-l) sections were performed, showing that the mutant STT and its ECM (f, h, j, l) were morphologically normal as in WT (e, g, i, k), for similarity degree of Alcian blue staining was detected (wathet blue regions) around the well-arranged STT both in the mutant and in WT. Scale bars, 50 µm in (e-h) and 500 µm in (i-l).

4 Supplementary Figure S3. Comparison of the PTB1 cdna with that of the mutant. The grey box indicates the mrna sequence deleted in the ptb1 mutant, the purple arrow indicates the resulting premature stop codon, the amino-acid residues in the cyan background indicate the predicted transmembrane region, the green background region indicates a conserved C 3 H 2 C 3 type RING domain, and the conserved amino acids are displayed in a blue background.

5 Supplementary Figure S4. PSSR decrease was associated with PTB1 expression down-regulation in a dwarf mutant. (a) PSSR comparison of the WT plants and c1352 mutant plants. (b) q-pcr analysis of the transcript of PTB1. Values are mean ± s.e.m. (n=3). All P values are based on two-tailed t-tests. C1352-W, wild-type of c1352 dwarf mutant.

6 Supplementary Figure S5. Over expression of PTB1 improved rice PSSR. (a) PSSR comparison of the WT plants and the PTB1 over expressing plants. (b) q-pcr analysis of the transcript of PTB1. Values are mean ± s.e.m. (n=3). All P values are based on two-tailed t-tests. PTO, PTB1 over expressing plants of Kasalath.

7 Supplementary Figure S6. Tissue localization of PTB1 in Arabidopsis. GUS activity in various organs of P PTB1 -GUS transformed Arabidopsis plants showed preferential PTB1 expression in the leaf vein (a, b) and young silique ends (g), with weak expression in the root vasculature (c), petal and stamens (f), a lack of expression in the maturing silique (h), and strong expression in the stigma and style (d, e). Scale bars, 1 cm in (h), 2 mm in (a, b, g), 200 µm in (d, e, f) and 500 µm in (c).

8 Supplementary Figure S7. Sub-cellular localization of PTB1. Transient expression in rice protoplast cells showed that the PTB1-YFP fusion protein was localized preferentially to the cytoplasm, and weakly expressed on the membrane. Scale bars, 10 µm.

9 Supplementary Figure S8. A neighbour-joining tree of PTB1 and its homologues. The full-length PTB1 protein sequence was used to search available public databases and retrieved approximately 10 homologues with an intact H2-type RING domain. Multiple alignments were performed using Muscal 3.8 software. A phylogenetic tree was then constructed by a Neighbor-Jointing (NJ) method of MEGA5 software with p-distance model and pairwise deletion and bootstrap (1000 replicates, random seed).

10 Supplementary Figure S9. Differentially expressed genes between the WT and ptb1 mutant. A total of five genes were confirmed to be differentially expressed by RT-PCR (a) and q-pcr (b) analysis. Relative expression levels were measured as the mean ± s.e.m. (n=3). All P values are based on two-tailed t-tests.

11 Supplementary Tables Materials Supplementary Table S1 PSSR of the Reciprocal crossing Indica rice Seed setting rate (%) Japonica rice IR24 Gui630 Guichao2 Kasalath Zhensan97B Nipponbare Zhonghua11 Taipei309 Magu Xiangdao ptb1 ( ) WT( ) ptb1 ( ) WT ( ) , crossed as male parents;, crossed as female parents. The seed setting rate was calculated by at least 200 emasculated spikelets for each cross.

12 Supplementary Table S2 Segregations of the mutation in different populations. Cross Total plants Fertile plants Sterile plants Expected Ratio χ 2 value ptb : ptb1 Gui : ptb1 Minghui : ptb1 X : ptb1 888R : ptb1 CDR : ptb1 Duohui : ptb1 205R : ptb1 527R : ptb1 (ptb1 Minghui63) : ptb1 (ptb1 527R) : χ , 1 = 3.84,ptb1was crossed as male parents in all crosses.

13 Supplementary Table S3 PSSR and PTB1 expression levels in complementary plants Lines No. of panicles counted PSSR Relative expression level Correlation coefficient WT ± ± ** ptb ± ± S ± ± S ± ± S ± ± S ± ± S ± ± S ± ± S ± ± S ± ± S ± ± S ± ± S ± ± PSSR and relative expression levels are measured as the mean ± s.e.m. (n=3). The relative expression level of the PTB1 gene in each transgenic plant line was determined by q-pcr with the RNA isolated from flag leaves at earlier heading stage. A P <0.01 was used for correlation (Pearson correlation analysis).

14 Supplementary Table S4 Natural variations of PSSR and relative expression level of PTB1 Accessions Class No. of PSSR Relative expression level Correlation panicle coefficient counted WT Indica ± ± ** ptb1 Indica ± ± Indica ± ± C1352 Indica ± ± Xiangnuo Japonica ± ± Zhankenuo Japonica ± ± Cplso17 Japonica ± ± Guangchangai Indica ± ± R Indica ± ± R Indica ± ± Huajing3 Japonica ± ± Ka1203 Indica ± ± R Indica ± ± R1318 Indica ± ± Keqing3 Japonica ± ± Sanbang76 Indica ± ± Yangdao2 Indica ± ± Minghui63 Indica ± ± Teqing Indica ± ± Wuyunjing8 Japonica ± ± Xudao3 Japonica ± ± Zhonghua9 Japonica ± ± R Indica ± ± IR24 Indica ± ± Gui630 Indica ± ± Taishannuo Japonica ± ± Nipponbare Japonica ± ± IR1544 Indica ± ± Guichao2 Indica ± ± Shengdao301 Japonica ± ± Sidao8 Japonica ± ± Guiyangzao18 Indica ± ± Beixiang7 Indica ± ± Huanghuazhan Indica ± ± Yanjing2 Japonica ± ± Indica ± ± Nongken57 Japonica ± ± Chaoaigan Indica ± ± Hanmadao Indica ± ± Heidu4 Indica ± ± 0.306

15 PSSR and relative expression levels are measured as the mean ± s.e.m. (n=3). Relative expression level of the PTB1 gene in each accession was determined by q-pcr with the RNA isolated from flag leaves at earlier heading stage. A P <0.01 was used for correlation (Pearson correlation analysis).

16 Supplementary Table S5 Information on putative homologous rice genes involved in female sterility, pollen tube growth or callose deposition. No. Gene name Putative homologous in rice Forward primer(5-3 ) Reverse primer(5-3 ) Protein function 1 NTT LOC_Os06g40960 CCGTCCCAGATCCTCATTG CGTGCTTCCGCTTGTAGTGC protein ZOS6-05-C2H2 zinc finger protein, expressed 2 chemocya LOC_Os03g50160 GCTGGCGATGGTCCTCCTCT CGGGCTGTACTTGAACACGA Protein plastocyanin-likedomain nin containing protein, putative, expressed 3 SCA LOC_Os11g02424 TGATTGTGGTGGCGATGGTG CGAGGTTGAGCGACTTGATGC protein LTPL9 - Protease inhibitor/seed storage/ltp family protein precursor, expressed 4 LURE1 LOC_Os06g48690 GCTTATGTCTGCCACTTCC CATTATCACGCATCCCTC Protein DEFL12-Defensin and Defensin-like DEFL family 5 TTS1 LOC_Os01g52660 GCTACGGGAAGAAGGTGGTG AGAAGAGGACGCTGGTGGG CDS POEI51 - Pollen Ole e I allergen and extensin family protein precursor, expressed 6 UNE1 LOC_Os10g23220 AGGCTTTCTTCCATCAGTTC CCACCGCTCATTACATAGTT protein GIL1, putative, expressed 7 UNE4 LOC_Os08g43090 CTTGCCGAGATCGCTTTG GTTAATGCTTCATTCAGGG protein bzip family transcription factor, putative, expressed 8 UNE9 LOC_Os04g33370 GAAGCCGTTCATCCACTAC CCCGTACACCGCCGAGTTG Protein cytochrome P450, Putative,expressed 9 UNE11 LOC_Os06g49760 AGCGACTTCATCCGCAAGT GCCATGTTCTCCAGGCAGT protein invertase/pectin methylesterase inhibitor family protein,putative, expressed 10 UNE14 LOC_Os04g41540 ACGACAACCATGCCGCCTG AACTCCTCGACGTCCACGC protein OsCML22 - CalmodμLin-related calcium sensor protein, expressed UNE15 LOC_Os05g46480 Protein late embryogenesis abundant 11 GGGCAGGATACCAAGGAGG CCGACCACCGTGCTCTTCA protein,group 3, putative, expressed RNS2 protein ribonuclease T2 family domain 12 LOC_Os01g67180 ATACAACGTCACGGAAAT GCAGTAGTAGTAAGGCAATC containing protein, expressed 13 HT-protei n LOC_Os06g16370 AGGAAGGGAGCGAGTG CCAGCAGGTGTCAGGAT protein CCT/B-box zinc finger protein, putative,expressed 14 STIG1 LOC_Os01g14540 TAGAAGGAGGCAGAGCAGGT T TCTTGCGGTCGCTGAGGA protein STIGL1 - Stigma-specific Stig1 family protein precursor, expressed K LOC_Os03g14615 CAAGCAAGGCTAGGCAACAG CCGCAGAGGCCCAGAAT 16 FST LOC_Os02g07430 GATGACTCGGATGAGGAACG TTTCTCGCACGGACTTTGGT 17 SLG8 LOC_Os03g35600 GACAACGGACAAGATATAGC CAAGTAGCCTGACAAGATTC Protein LTPL117-Protease inhibitor/seed storage/ltp family protein precursor Oryza sativa (japonica cμltivar-group) FST (FST) serine/threonine-protein kinase receptor precur- sor, putative, expressed

17 serine/threonine-protein kinase receptor 18 SLG9 LOC_Os07g36544 GCTCCCCTCTTCCTTTTC GAGAGATGAGTTGGCAAGA precur- sor, putative, expressed CCTCCACGAAGATTCAAGGTT receptor protein kinase, putative, 19 SLG29 LOC_Os01g57510 TTGGGCACCATGTTTCTGTCT T expressed S-domain receptor-like protein kinase, 20 SLG40 LOC_Os01g66640 CACCAGAGATGTAACCAATG GAGAACAAGTCCACCAATG putative 1,3-beta-glucan synthase component 21 LOC_Os06g51270 CTATGGATGTCCCTACGA CTTGTCAATGTCTGTCCC domain containing protein, expressed 1,3-beta-glucan synthase component 22 LOC_Os06g08380 TGGCGTATTGTTAGGTTG AGAGTACAGGAGCCCAGA domain containing protein, expressed 23 LOC_Os06g02260 GGGGCTACTGAAGGCTAT AATGTATGGTGCGAAGAG callose synthase, putative, expressed 24 LOC_Os03g03610 GAATGGGATGAGGAACTT TATGTCTTGGGCATTAGG 1,3-beta-glucan synthase component domain containing protein, expressed 25 LOC_Os03g02756 TTTGAATGGCAGAAGGTC AACCGAATACCAGTAAGAGG 1,3-beta-glucan synthase component, putative, expressed 26 LOC_Os01g34880 AGGTCGTGGATTTGTTGT ATTGAAGATAAAGGGTGC callose synthase, putative, expressed 27 LOC_Os02g58560 CAGAATGTTGTCCTGCTA TCAGTACAAAGTCGCTCA protein 1,3-beta-glucan synthase component, putative, expressed 28 LOC_Os02g14900 GTTGCCTCGCTTTCATAC AGTGAACCCATTCCGTAG protein 1,3-beta-glucan synthase component domain containing protein, expressed 29 LOC_Os01g48200 GGTGGCTGCTATTGTTCT ATCCTCGTCTGCATCTCC callose synthase, putative, expressed 30 LOC_Os01g34930 TTTGGCATTCATCCCTAC CCGATCTTGCGGAGCACA callose synthase, putative, expressed 31 LOC_Os01g34890 TTTGTTTGATTGGCTCTG CAAATGTAAATATCGGCAC protein GLUCAN SYNTHASE-LIKE protein, putative 32 LOC_Os01g55040 ACCGAAGTTTCGATAGGG ACCACCAGGTCAGCACAT protein 1,3-beta-glucan synthase component, putative, expressed We searched the Rice Genome Browser database ( by querying sequences of some previously published genes relevant to the ptb1 phenotype, such as those genes related to PT growth, female sterility and self-incompatibility. This search identified a total of 20 putative homologue genes in rice genome. The callose synthesis and metabolism related genes (No ) were searched by a putative function search tool (

18 Supplementary Table S6 Primers for map-based cloning and functional analysis of PTB1 Name Forward primer(5-3 ) Reverse primer(5-3 ) Application RL31 AAACGAGCATAACTAAAGAG ACAAGTCCCACTGCCTAT Fine mapping RL85 GGTTGCCCTATAGCTTAT GATGTATGCGTTCTTGAG Fine mapping RL33 CAATCCCGACACGATAAT TCCCGTAAAGTGATAAAATA Fine mapping RL60 CCCGCACTCCGCTCACTT ACGCCGTACTCCCTCCTCC Fine mapping RD19 ATGTGGCATCCAAAGTGT AACGTTAGGGAAAAGTCC Fine mapping RD20 CAAAGACCGAGATGCTAT GAAACACGAACGGGAGGC Fine mapping RL167 CCCCAGCCATCACAACTC GCCTTTCCTTTCCCTCCC Fine mapping D PTB1 ATGAGGATTAGGGTTCTG CACTGGGTTTAGTTCTTTT DNA deletion confirming CD PTB1 CACCAGCCTAATCATTGTCTC TTCCCTGTTGTGCCCTCT RNA deletion confirming P PTB1 AAACTGCAGAACATCTAATCTCATTAAATTTAAC GCCGGATCCGGCCTGCTAGCTAGATCTGGC Promoter clone C PTB1 GCCGGATCCATGGCGATGCGGGGGGTCGA GGACTAGTGCGATCCGGCAGCTGATGC CDS clone a RT PTB1 ATTATTGTCTTGTCGGTTCTTGTG GTATTCCCTGTTGTGCCCTCT Expression analysis RING PTB1 GCCGGATCCGGCCAAGATACTGCATATCATCC CCCAAAGCTTCATCGTCGCCGCTGTCACCTC RING domain clone Actin CTTCATAGGAATGGAAGCTGCGGGT CGACCACCTTGATCTTCATGCTGCTA Internal reference a, One of the RT PTB1 primer pair was designed located in the deletion region of mutant PTB1 to exclude the influence of mutant background.