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1 Supplementary Information Rice APC/C TE controls tillering through mediating the degradation of MONOCULM 1 Qibing Lin 1*, Dan Wang 1*, Hui Dong 2*, Suhai Gu 1, Zhijun Cheng 1, Jie Gong 2, Ruizhen Qin 1, Ling Jiang 2, Gang Li 3, Jiu Lin Wang 1, Fuqing Wu 1, Xiuping Guo 1, Xin Zhang 1, Cailin Lei 1, Haiyang Wang 1, Jianmin Wan 1,2 1 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing , China 2 National key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing , China 3 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut
2 Supplementary Figures Supplementary Figure S1 Protein sequence alignment of Cdh1-like proteins of S. pombe (SpSRW1), H. sapiens (HsCdh1), A. thaliana (AtCcs52A1, AtCcs52A2), M. truncatula (MtCcs52A, MtCcs52B), O. sativa (TE, OsCcs52B) and the truncated te protein. The conserved domains (seven WD-40 repeats) and motifs (C-box, CSM, IR, and RVL) are marked. In addition, the two degradation signals are also marked by two red boxes in the N-terminal region of HsCdh1. 2
3 Supplementary Figure S2 Phylogenetic analysis of TE protein. Protein sequences were aligned with CLUSTAL W in MEGA and the phylogenetic tree was analyzed by MEGA Accession numbers presented here are the following: SpSlp1, P78972; ScCdc20, P26309; SlCdc20-1, CBH19893; OsCDC20-2, BAF ; SlCdc20-2, CBH19894; MmCdc20-1, XP ; HsCdc20-1, NP ; HsCdc20-2, A56021; MmCdc20-2, NP ; RnCdc20, NP ; XlCdc20-1, AAH42288; XlCdc20-2, AAC41376; DmFzy, NP ; AtCdc20-1, At4g33260; AtCdc20-2, At4g33270; AtCdc20-3, At5g27570; ScHct1, P53197; UmCdh1, AY118173; SpSRW1, O ; 3
4 CeCdh1, NP ; XlCdh1, CAA74576; HsCdh1, NP ; HsFzr1, Q9UM11.2; MmCdh1, NP ; DmCdh1-2, NP ; OsCcs52B, LOC_Os01g74146; AtCcs52A2, At4g11920; AtCcs52A1, At4g22910; SlCcs52A, CBH19891; MtCcs52A, AF134835; TE, LOC_Os03g03150; ZmCcs52A, ACN36196; ZmCcs52B, ACG33710; AtCcs52B, At5g13840; MtCcs52B, AY357299; SpSrw1, O13286; SlCcs52B, CBH19892; LcjCcs52A, DQ ; PsCcs52A, DQ059036; MsCcs52A, AF079404; SbCcs52A, XP_ ; and SbCcs52B, XP_ Sp, S. pombe; Sc, S. cerevisiae; Sl, Solanum lycopersicum; Mm, Mus musculus; Hs, Homo sapiens; Rn, Rattus norvegicus; Xl, Xenopus laevis; Dm, D. melanogaster; At, A. thaliana; Um, Ustilago maydis; Ce, Caenorhabditis elegans; Os, Oryza sativa; Mt, M. truncatula; Zm, Zea mays; Lcj, Lotus corniculatus var. Japonicus; Ps, Pisum sativum; Ms, Medicago sativa; Sb, Sorghum bicolor. 4
5 Supplementary Figure S3 Phylogenetic and protein sequence analysis of MOC1 and OsCDC27. (a) Sequence alignment of MOC1, LAS and LS proteins. The conserved domains (LHRI, VHIID, LHRII, PFYRE and SAW) were marked. The typical degradation box (D-box) is marked by a green box in the N-terminal region of MOC1, LAS and LS. (b) Phylogenetic analysis showing that OsCDC27 is the rice homolog of HsCDC27, DmCDC27, AtCDC27a, AtCDC27b, and SpCDC27. Protein sequences were aligned with CLUSTAL W in MEGA and the phylogenetic tree was constructed by MEGA Accession numbers presented here are the following: HsCDC27, NP_ ; MmCDC27, NP_ ; RnCDC27, NP_ ; DmCDC27, NP_ ; SpCDC27, NP_ ; ScCDC27p, NP_ ; AtCDC27b, NP_ ; AtCDC27a, NP_ ; OsCDC27, NP_ ; NbCDC27b, BAF (c) OsCDC27 has the same conserved TPR domain (indicated as green boxes) as HsCDC27, DmCDC27, AtCDC27b, and SpCDC27 in their C-terminal region. 5
6 Supplementary Figure S4 Ubiquitination analysis of MOC1. In vitro ubiquitination assay showing that MOC1 is poly-ubiquitinated (represented by multiple up-shifted bands) by WT plant extracts and can be detected by antibodies against MOC1 or polyub antibodies on Western blots. 6
7 Supplementary Figure S5 Mapping of the moc1-5 allele. (a) Mapping of moc1-5. (b) Western blot analysis showing the absence of MOC1 proteins in moc1-5 plants (top). Rubisco staining showing a roughly equal loading of proteins (bottom). 7
8 Supplementary Tables Supplementary Table S1 Molecular markers used for mapping of the TE gene Primer sequences (5 3 ) Marker Type Forward primer Reverse primer g41 SSR CGCTCTGAAATCCGATATTTGC GGCTCTCTCCTCCTCGTTGC RM22 SSR GGTTTGGGAGCCCATAATCT CTGGGCTTCTTTCACTCGTC RM523 SSR AAGGCATTGCAGCTAGAAGC AAGGCATTGCAGCTAGAAGC g62 InDel GCATAACATCAAAGCCATGCTA GCTCCAATTCGATCTTCGTC g105 InDel GCCCTTCTCATTGCCTCG CTCTTCTCGCTTCTTCTTTTCG g118 InDel ATGTACAATAGTATTCCCACGTC AAAACAAGGTTTAATGGTAGTG 8
9 Supplementary Table S2 Molecular markers developed in this study for mapping of the moc1-5 allele Marker Type Primer sequences (5 3 ) Forward primer Reverse primer Indel 6-9 InDel GTGCGAATTCCTCCACCTC CGACATGGCTGGTCTCTCTC xs31 InDel TGCCAATTAGCTCGATTCTG TGGTCAAAATTTGTTCAAACTCA apo1 InDel CCGGTTTTGGTTTGTCTCAG ATTCCTTTGTGGGCTCCTTT RM3628 SSR GCCCTAGACACACCCGTACC TGCCAGATCAGAAATCATGC RM162 SSR GTGGTAGGGCGAAATGATCTGC ATCACGCGCTCCTACCTCACC xs30 InDel CAGCCGATTGGACTCGAT AACACCTTGCCGAACACC xs35 InDel TAAAAGAAAGGTTACTCATGGATTTTT TAATGCCTGCTGCATCTTAGG xs40 InDel TCCTCACACACAAGCACACA TCAAATTGGCATGCATTGAT xs51 InDel AAGTTCGCCGGAGAGAGAAG CAGCGTCATGGTTTCTGAAAGCCA xs44 InDel TTAATTAATCACCAATGCAATCTATGA CCAGCAACTTTTGCTTTGTG xs55 InDel CTTAGATTTGACACAACCTGCAC ATTCTGCAGTGCACGAGCTA xs56 InDel AACTATAGATCGGGATCGCTCA TTCAAAGAAACGAAAATCAACG 9
10 Supplementary Table S3 Primers used for construction of protein expression plasmids Fragment or plasmid Forward primer (5 3 ) Reverse primer(5 3 ) TE MOC1 pmal-c2x::mbp-moc1-m CTAGGAAGATCTATGGATC ACCACCACCACCAC CGCGGATCCATGCTCCGG TCACTCCACTCC GCGGACTTGGCGCTGGCG TGCGCGGACCTG CTAGATCTCGAGTCACCGGATGT AGCTCCTAACAAATG CGCAAGCTTCTACGACGACGACG GCTGCCA CGTCGACGGCGCCGCCGCCGCA ACGGCAC 10
11 Supplementary Table S4 plasmids Primers used for construction of yeast two-hybrid assay Fragment Forward primer (5' 3') Reverse primer (5' 3') OsCDC27 AGTGAATTCATGGAAACCCTAATGGTGGAC ATAGGATCCTTAAATCTCATCATCATCAT CATCATCATCC MOC1 AGTGAATTCATGCTCCGGTCACTCCACTCCTC AATGGATCCCTACGACGACGACGGCTGC MOC1-m AGTGAATTCATGCTCCGGTCACTCCACTCCTC AATGGATCCCTACGACGACGACGGCTGC MOC1d AGTGAATTCGGCCATGGCCACGTCGTC AATGGATCCCTACGACGACGACGGCTGC TE GGAATTCCATATGATGGATCACCACCACCACCA CCTG CTAGGAAGATCTTCACCGGATGTAGCTC CTAACAAATG 11
12 Supplementary Table S5 Primers used in Real time Quantitative RT-PCR Gene Primer sequences (5 3 ) Type Name Forward primer Reverse primer Actin qrt-pcr TCGTCTGCGATAATGGAACTG CCGACAATGCTGGGGAAG TE qrt-pcr TGGTCTTCGCATAATATCCTTG TGTCCCTACAGCAAGGTGAGT OsTB1 qrt-pcr CCTACCATGAGAGAAGAGACCA GTAGTGGGCTATGATCAGATGTG OSH1 qrt-pcr CTCAACACGCTCTCCATCTC GCTTGAGCTCCTGATCCAC 12
13 Supplementary Reference: 49. Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, (2007). 13
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