Supplementary Figures Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus. Wang C, Zhu H, Jin L, Chen T, Wang L, Kang H, Hong Z, Zhang Z. 5 UTR CDS 3 UTR TCTCAACCATCCTTTGTCTGCTTCCGCCGCATGGGTGAGGTCATTTTGTCTAGATGACGTGCAATTTACAATGA TACTTCGTATTTAGCTTCAGTCTCTCATTCATCTTCTAACTCCGTTCCGCGCCGCGATTCCAATCAGAGACAAA ACTCCCTTCTCCTCCTCCTTCTCCAATTCCCAAAACCCTACATTCGCCGCAGCTCAACCAGTTCACTTCACTTC CCTTCTTCCTCGTCAATTCAGGGTAAGGAGTTTAAGGCTCTGAGTGAGACTAGAAGAGATGGAGGGTGGTGA AAAACCGGTGGATAATATGGAAATTGTTGATGATGATACGACTTTGCTACCCCAGAATTCTGACAAGGATCA GAATGCGGGTTCCTCTCCTATGGTGCTTGCTCCCAATTCTCTTGCTCAGATACCAAAGGATGAAGTTATGGTT GATCAGGGGGATGATGATAACAGAATTGGGGATTCTACTCCGAATAACATGCTAGAAGTCAAAACAGGTAGT GAGAATCAGCTTGAGCTTGAAGATGTGAAAACGCCTTTGCATCAGGAATTGGTCACTCCAAAGTCCAGAGAA AGGAATGTCAGGGAAATGAAGAGCGTGCTAAACGATACTGAGGTGGTTGATTATGATGAGCCTGGTGCGTCT CTGGAGCGAGAAGCATTTATGAAAGAGCTCGAGAATTTCTACAGAGAAAGGTCCTTGGAATTTAAGCCTCCTAA GTTTTATGGAGAGCCACTAAATTGCCTGAAGTTATGGAGAGCTGTTATCAGACTTGGTGGCTATGATGTGGTAA CTGGATCCAAGTTGTGGCGCCAAGTAGGAGAGTCTTTCCACCCCCCAAAGACTTGCACAACAGTCTCATGGACA TTTAGAATCTTCTATGAGAAGGCACTTTTGGAGTATGAAAAGCATAAGAGAGAAATTGGGGAGTTACAGCTCCC TGTTGGAGTATTCCCTCAACCGTCAAGTGTGGAAAAAGAGACCACTGTTTATCAGGCTCCAGGCTCAGGTAGG GCGCGGAGGGATGCGGCAGCGCGTGCAATGCAAGGTTGGCATGCTCAGCGCCTTCTTGGTTACGGTGAGGTT GCTGAGCCAGTTATTAAGGATAAGAACTTCAACCCTACAACAAAGCGTGAAAAGAACCTCAAAAGTATTGGT GCGATCAATAAACAGAGGACACCGTCTGTTCTTGAGCATGTTGAAAAAGCTGCAAACATCGACGGAGATAGG CAGTTGGTCACAGCAGTAGTGGACGTTGGACCCCCAGCTGACTGGGTGAAGATCAACGTGCGGGAAACCAAA GATTGTTTTGAAGTGTATGCGCTAGTTCCTGGGCTCCTCCGTGAGGAGGTACGAGTCCAATCAGATCCAGTTGG ACGTCTTGTTATAACTGGTATGCCAGAACATATTGACAACCCATGGGGAATCACCCCCTTCAAAAAGGTTGTGAA CTTACCTGCAAGAATTGATCCCCTTCAAACATCTGCAGTTGTTAGTTTGCATGGTAGACTATTTGTTCGGGTTCC TTTTGAGCAGGGAGCCGTGTAAATTTTCCTCCCTCCAATGGTTGGTTTCTCAAATTTCCTCAAAAGGAGTTACA GTTTTCTGAAAAATCAGGGTGGTTATGCTAGGAACTAGTCTAGCCCTGTCAGATTAGTTGAAACTATTAATTT TTTAATTCTCTTTAGGTAAAATACCGCATTTAACTTATGCAATATGCTTGATTTTCCTGTGTTAAATTCCCTTTT ACCTTCATACCCAAGAAATGGAATTTTTATGTATTCATTTTGCATTGGGGGATATTCATTAGTAACAGTGTGT AGGATTCAAATAAGATACTATAGCATATGTTTCCATACCCGTACTTTTAAAAAATTACTTGTATCAAGACCGA T A T C T G T G T A T T A T G T A T C T C T A T T A T T T A T A C T T T A G T C RNAi-2 RNAi-1 Supplementary Fig. 1 Sequence of the SIP1L cdna and the fragments used for RNAi constructs. The start (ATG) and stop (TAA) codons are highlighted in blue. The length of the coding sequence is 1278 bp in SIP1L and 1227 bp in SIP1S. The additional cdna sequence derived from exon 12 of SIP1L is shown in green. Sequences corresponding to ARID and Hsp20 domains are italicized. The two cdna fragments used for RNAi-1, and RNAi-2 are shaded in red. 1
Supplementary Fig. 2 Western blot analysis of protein expression in yeast cells. Yeast Y187 cells carrying a Gal4 DNA-binding domain (BD) fusion construct were mated with yeast AH109 cells harboring a Gal4 activation domain (AD) fusion construct. Diploid cells were selected on SD media lacking Leu and Trp, and cultured in liquid SD selection medium broth lacking Leu, Trp. Soluble protein extracts were prepared from pelleted yeast cells and used for analysis of protein expression. Recombinant proteins expressed from pgbkt7 contained a c-myc epitope and were detected using anti-myc monoclonal antibody. Gal4 AD fusion proteins contained a hemagglutinin (HA) epitope and were identified using anti-ha monoclonal antibody. Y187 Yeast cells containing no plasmid were cultured in YPD broth used as a negative control. 2
Supplementary Fig. 3 Sequence alignment of annotated MtSIP1LL1 and cloned MtSIP1LL1-1. The annotated MtSIP1LL1 (Medtr3g116200.1 (MT3.5)) has 14 exons and encodes 379 amino acids. The cdna clone MtSIP1LL1-1 from roots does not have exon 7 and encodes a peptide that is 12 amino acids. 3
Supplementary Fig. 4 Growth phenotypes of SIP1 RNAi roots grown under non-symbiotic conditions. a Two transgenic plants from each of the control and RNAi groups were randomly selected for photograph. No significant difference in growth phenotypes was observed. Bar=1 cm. b Three weeks after transplanting, root growth and lateral root density were recorded and analyzed by Students t test for statistic differences. Root growth was measured as the ratios of entire hairy root length versus the original hair root length. Lateral root density was obtained as the ratio of the total lateral roots versus the original hairy roots. The number of hairy roots scored in each group (n) is indicated, and standard errors are shown. 4
A B C D Supplementary Fig. 5 Spatial pattern of SIP1 gene expression in non-symbiotic tissues. Transgenic hairy roots of L. japonicus expressing SIP1pro::GUS were inoculated with M. loti to induce nodulation (a-b). Constitutive expression of the SIP1 gene was detected in the root caps (c), vascular tissues (a-c) and the nodule cells that were close to the root vascular tissues (b). A root cross-section showing SIP1 gene expression in the phloem and xylem cells of vascular tissues (d). Bar = 0.2 mm. 5
A B Supplementary Fig. 6 Transgenic hairy roots of L. japonicus expressing SIP1-RNAi-1 in the presence of arbuscular mycorrhizal fungi. a Growth phenotypes of SIP1-RNAi-1 roots inoculated with Glomus intraradices. The main roots of the L. japonicus plants were removed and the plants were supported only by transgenic hairy roots. Photographs of the plants were taken three weeks after inoculation with the mycorrhizal fungus. The control hairy roots were generated using the empty cloning vector pcambia1301-35s-int-t7. No significant difference in appearance between the control and RNAi roots was observed. b Expression of the SIP1 gene in L. japonicus roots after inoculation with arbuscular mycorrhizal fungi. There was no significant change in SIP1 gene expression levels the roots inoculated with Glomus intraradices and the control roots that were not inoculated with the arbuscular mycorrhizal fungus. The expression levels of the ATPase gene served as an internal control. Vector control RNAi-1 6
Supplementary Fig. 7 Assays for specific binding of SIP1L to the NIN promoter in vitro. The NIN promoter was amplified by PCR as described elsewhere (Zhu et al. 2008). GST-SIP1L was purified using glutathione Sepharose 4B beads (GE Healthcare). Electrophoretic mobility shift assay (EMSA) was performed in a binding buffer (20 mm Tris-HCl, ph 7.9, 1 mm EDTA, 60 mm KCl, 1 mm DTT and 5% glycerol, in the presence of 1.25~2.5 µg GST-SIP1L and nonlabeled NIN promoter DNA fragment. GST protein was used to replace GST-SIP1L in the control. The reactions were allowed to proceed at 28 o C for 20 min, and the reaction products were resolved in a 5% native polyacrylamide gel with ice-cooled running buffer (10 mm Tris-Cl, ph 8.5, 76 mm glycine and 2.6 mm EDTA). The gel was stained with ethidium bromide and imaged under UV light. 7
A B Supplementary Fig. 8 Analysis of the transcript levels of the SIP1 and NIN genes in SIP1-RNAi and SIPL/S over-expression transgenic hairy roots. a RNA was isolated from individual root systems of the control (ck) and SIP1-RNAi-1 hairy roots 8 and 12 days post inoculation (dpi) with Rhizobium. Real-time RT-PCR (qrt-pcr) analyses were performed to assess the transcript levels of the NIN, SIP1 and Ubiquitin genes. The transcript level of the ATPase gene was used as an internal reference. The results revealed that SIP1 was effectively suppressed in RNAi roots, whereas the transcript level of the NIN gene was not changed in the SIP1-RNAi hairy roots, suggesting that down-regulation of the SIP1 gene expression by SIP1-RNAi is not sufficient for suppression of the NIN gene expression in planta. Other regulatory elements may participate in the regulation of the NIN gene expression during nodule organogenesis. Expression level of ubiquitin in Line1 was missing due to an experimental mistake. SIP1 1000 and NIN 100 indicate 1000- and 100-fold of the original expression levels. b RT-PCR analyses were performed to assess the transcript levels of SIP1L, SIP1S and internal reference ATPase of transgenic hairy roots that overexpressing SIP1L and SIP1S, suggesting SIP1L and SIP1S were effectively over-expressed. 8
Supplementary Table 1 Database accession numbers and the peptide length of plant SIP1L-like sequences. SIP1L-like ID Amino acid residues MtSIP1LL1 Medtr3g116200.1 (MT3.5) 379 GmSIP1LL1 Glyma06g01640.1 391 GmSIP1LL2 Glyma04g01560.1 451 RcSIP1LL1 GenBank: EEF52922.1 449 PtSIP1LL1 GenBank: EEE79891 307 PtSIP1LL2 GenBank:EEE94817 302 VvSIP1LL1 GenBank: CBI40776.3 449 VvSIP1LL2 XP_002273480.1 446 AtSIP1LL1 AT1G76510.1 434 AtSIP1LL2 AT1G20910 398 SbSIP1LL1 Sb10g024400 461 SbSIP1LL2 Sb04g029990.1 480 ZmSIP1LL1 GRMZM2G180654_T01 468 ZmSIP1LL2 GRMZM2G105807_T01 461 ZmSIP1LL3 GRMZM2G110109_T01 478 ZmSIP1LL4 GRMZM2G421899_T03 483 HvSIP1LL1 GenBank: BAJ86433.1 327 OsSIP1LL1 LOC_Os06g41730.1 461 OsSIP1LL2 LOC_Os02g48370.1 486 LjSIP1L GenBank: JN602367 425 LjSIP1S GenBank: EU559710 408 The identification (ID) information of SIP1L-like sequences was derived from http://www.plantgdb.org/. These SIP1L-like sequences were used for phylogenetic analysis shown in Fig. 4. 9
Supplementary Table 2 Primers used in this study. Primer names Used for Primer sequences (from 5 - to -3 ) SIP1L-yF Expr. in yeast AAAGGTACCCATATGGAGGGTGGTG SIP1L-yR Expr. in yeast TTTTGTCGACAAGCTTCACGGCTCCCTG SIP1Lc-yF Expr. in yeast GGGAGATCTCATATGCCTGTTGGAGTAT MtDMI2-PK-yF Expr. in yeast AACCATATGGAGCAGGCTACAGAACAGTAC MtDMI2-PK-yR Expr. in yeast AAGTCGACCTATCTCGGTTGAGGGTGTG OsSym-PK-yF Expr. in yeast AGGCATATGGCAACGTGCAACTTCAAAACC OsSym-PK-yR Expr. in yeast AAACTCGAGCTACCCCGGAAGCGAAGGCATC MtSIP1LL1-yF Expr. in yeast AGGCATATGGATCTCG TTCATTCAC MtSIP1LL1-yR Expr. in yeast AACGAATTCACACAGCTCCCTGCTC OsSIP1LL1-yF Expr. in yeast AAGCATATGATGGCCCAGTTTAGGTCTGC OsSIP1LL1-yR Expr. in yeast AGGCTCGAGTTACTTTGACTGCTCGAATGGTG SIP1L-eF Expr. in E. coli AAAGTCGACATGGAGGGTGGTGAAAAACCG SIP1L-eR Expr. in E. coli AAAGCGGCCGCTTACACGGCTCCCTGCTC SIP1S-eF Expr. in E. coli TTTCTAGACCGTCGACTCGAAATTGTTGAT SIP1S-eR Expr. in E. coli TTTAAGCTTTTACACGGCTCCCTG SIP1-rF RT-PCR AAGAACTTCAACCCTACAACAAAGCG SIP1-rR RT-PCR AGTCTACCATGCAAACTAACAACTGCAG AtSIP1LL1-rF RT-PCR AGGAGAAAGGCTTGAATTCAACC AtSIP1LL1-rR RT-PCR ATGCAGGCTCACAACCGCTG OsSIP1LL1-rF RT-PCR AAGATAAAGGGACGGTGTCTGTC OsSIP1LL1-rR RT-PCR CATGGAGAGTGACAACTGCCG MtSIP1LL1-rF RT-PCR CAGCTGTTAAGGACAAGAACTTCAG MtSIP1LL1-rR RT-PCR ACCATGCAAACTAACAACAGCAG SIP1-rtF qrt-pcr GTACGAGTCCAATCAGATCCAG SIP1-rtR qrt-pcr CTTGCAGGTAAGTTCACAAC ATPase-rtF qrt-pcr CAATGTCGCCAAGGCCCATGGTG ATPase-rtR qrt-pcr AACACCACTCTCGATCATTTCTCTG Ub-rtF qrt-pcr TTCACCTTGTGCTCCGTCTTC Ub-rtR qrt-pcr AACAACAGCACACACAGACAATC NIN-rtF qrt-pcr AATGCTCTTGATCAGGCT NIN-rtR qrt-pcr AGGAGCCCAAGTGAGTGCTA SIP1L/S-coloc-F Co-localization AAGTCATGACCATGGAGGGTGGTGAAAAACCG SIP1L/S-coloc-R Co-localization AGGACTAGTCACGGCTCCCTGCTCAA SIP1-ri1F RNAi-1 AACTGCAGGTCGACCCCGGGGCAGTTGTTAGTTTGCATGG SIP1-ri1R RNAi-1 GGTCTAGAGGATCCACGGGTATGGAAACATATGC SIP1-ri2F RNAi-2 AACTGCAGGTCGACCCCGGGTCCCTCAACCGTCAAGTGTG SIP1-ri2R RNAi-2 GGTCTAGAGGATCCTCCAACGTCCACTACTGCTG SIP1L/S-OX-F overexpression AAGTCATGAATGGAGGGTGGTGAAAAACCG SIP1L/S-OX-R overexpression AAAGGTGACCTTACACGGCTCCCTGCTC SIP1-pF Promoter TAGGGTTCGCTTGGTACGTC SIP1-pR Promoter GGGAATTCCTCTTCTAGTCTCACTCAGAG SymRK-pF Promoter AAGACGGAGAAGTAAGCTTTGAGTTGAG SymRK-pR Promoter AAGTCGACAATCTGAAGGAGAATTTACCCC NIN-pF Promoter AATCTGCAGTTACACGTGGACGCAGCTCCC NIN-pR Promoter AACGGATCCGCTAGCTGATCCAATTAAGTACC 10