A Ubiquitin Ligase of Symbiosis Receptor Kinase Involved in Nodule Organogenesis

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1 Supplemental Tables and Figures to: A Ubiquitin Ligase of Symbiosis Receptor Kinase Involved in Nodule Organogenesis Yuan S, Zhu H, Gou H, Fu W, Liu L, hen T, Ke D, Kang H, Xie Q, Hong Z, Zhang Z Table S1. Nodulation phenotypes of SIE3 OX hairy roots. Number of nodules per plant ± SE ontrol SIE3 OX P Value Exp. I 7.6 ± 0.7 (n = 20) 13.3 ± 0.7 (n = 49) 2.4 x 10-5 Exp. II 4.0 ± 0.6 (n = 10) 10.2 ± 1.0 (n = 16) 1.5 x 10-4 Transgenic hairy roots of L. japonicus expressing pmub:sie3 (SIE3 OX ) or the empty vector pu1301 (ontrol) were inoculated with M. loti. Two independent experiments (Exp. I and II) were performed. Mean numbers of nodules per plants with standard deviation (SE) are shown. The numbers of scored plants are indicated in parentheses. 1

2 Table S2. Nodulation phenotypes of SIE3-RNAi hairy roots. Number of nodules per plant ± SE ontrol SIE3-RNAi P Value Exp. I 9.6 ± 0.8 (n = 20) 4.8 ± 0.5 (n = 39) 1.0 x 10-4 Exp. II 8.8 ± 0.6 (n = 13) 4.0 ± 0.8 (n = 25) 1.5 x 10-4 Exp. III 7.5 ± 0.5 (n = 22) 1.8 ± 0.4 (n = 35) 0 Transgenic hairy roots of L. japonicus expressing psie3:rnai (SIE3- RNAi) or the empty vector pambia s-int-t7 (ontrol) were inoculated with M. loti. Three independent experiments (Exp. I - III) were performed. Mean numbers of nodules per plants with standard deviation (SE) are shown. The numbers of scored plants are indicated in parentheses. 2

3 Table S3. Phenotypes of rhizobial infection in SIE3-RNAi hairy roots. Number of ITs with their tips at Infection events Nodule per root Samples Root hair Root epidermis Root cortex primordia segment (n) ontrol 0.79± ± ± ± ± RNAi 0.31± ± ± ± ± Hairy roots of L. japonicus were generated using Agrobacterium rhizogenes carrying the empty vector pambiai s-int-t7 (control) or the SIE3-RNAi construct. Hairy roots were inoculated with M. loti strain MAFF that constitutively expressed the β-galactosidase (lacz) marker. Eight days after rhizobial inoculation, hairy roots were stained in X-gal solution and examined under microscope. 3

4 Table S4. Primers used in this study. Primer names Used for Primer sequences (from 5 - to -3 ) SIE3-Sense-F RNAi AATGAGTATTTAAGTGT SIE3-Sense-R RNAi GTTAGATTATGATAAGAATGGT SIE3-As-F RNAi TGGGTAAGTGTAAAATAT SIE3-As-R RNAi GGGATTTATGATA AGAAT Ub-F RT-PR TTATTGTGTGTTT Ub-R RT-PR AAAAAGAAAAGAAAT Enod40-2-F RT-PR AAAATGTTATGTTGGG Enod40-2-R RT-PR AT AAAGGA AGAAGA AA NIN-F RT-PR AATATGGAAAAGGTGTTT NIN-R RT-PR TATTGGGAATGTATTAGTAGA SIE3-F RT-PR AGAAG AGGAAT GGAGA TATG SIE3-R RT-PR GAGGTTTTGGAATG TTG Lb1-F RT-PR TAAGATGTGAAAA Lb1-R RT-PR TGGATTGAAGTGTATT SymRK-PK-yF Expr. in yeast GGGATATGATGGAGAGGTAAAAATTG SymRK-PK-yR Expr. in yeast AAAGAATTTTGGTGTGGGTGAG SymRK-E-yF Expr. in yeast GGTGAATATGATGAAGGGTTTGAG SymRK-E-yR Expr. in yeast GGGGAATTTATGTGGATTAT NFR5-PK-yF-AD Expr. in yeast GGGAATTATGGAGAAAGAAGGT NFR5-PK-yR-AD Expr. in yeast GGGTGAGTTAAGTGAGTAATGG NFR5-PK-yF-BD Expr. in yeast GGGAATTATGGAGAAAGAAGGT NFR5-PK-yR-BD Expr. in yeast GGGGTGATTAAGTGAGTAATGG NFR1-PK-yF-BD Expr. in yeast AAAGAATTTTTAGTTGGATAATAAAATT NFR1-PK-yR-BD Expr. in yeast GAGTGAAATTATTAAGAAGTAA LHKI-PK- yf-bd Expr. in yeast GGAATTATGAGGATTTAAAAGTAAAG LHKI-PK- yr-bd Expr. in yeast AGTGATAAATGTTTTAAATG LHKI-E- yf-bd Expr. in yeast AAGGATAGTTTGGGAAAGAATGTG LHKI-E- yr-bd Expr. in yeast AGTGATAGAGGTTGGATTGAAG SIE3 RING- yf Expr. in yeast GGAATTATATGATGGAGTTAGTA SIE3 RING-yR Expr. in yeast GGAATTTGGAGTTGTGTAGT 4

5 Lj Lj Lj Lj Lj Lj M E--LSTIKDAFDRVTKK QKSSSSKTQE VIDQIRQE IENVLDTMQS-VNNTDHVLD HKTVLNELK ASLLQ M D--LNPIKDAFDRVAKK QKMSGSKTQE VVAQMILE IENSLEIIKA-EHFGSEV-D KSVFGELK KKLLE M EFHLSSIKDAFDRVAKK QKTSSKSQE TVDQVGRE IEQALATLQSPQ-DPLTLAD QKSILTELK SKLNA M E--LSTVKDAFDRVVKK QKLSLSKSQE VIDLVRYE IEEALSKIQSSG-DPMSPVD QKSILTELK HKLNT M E--LSNIKDAFDRVAKK QKLSSSKSQE VIDQVGRE IEDALAKLQA---DPASPVD QKSILMELK TKLSI M E--LDSLREAFDRVIEK RASSSAKAQE VIDQIVSE VEQAITKMQMMNTDSMGTAD HSSILAELK AKLNE M E--IDSLREGFDRVAEK RSLSSAKALE VVDQIVNE IEQAIVKLQMMNTDSTGNVD HPSILAELK AKLNE M E--LKSIKDAFDRVATK QKLSYSKTNE IVHLLSQE IDKALSILQE-TPSSDTLLD HRSILADVK KVFME FDRV TAPLSQME GTQKELNVALSKYGKLLEKNFNTDISKAY R N -IDIDVHTLN Q IIANHFYRQGL F EIG DHFLS IAPLSQLE GTQKELNIALSKYPKQLEKSFNPDIAKAY R N -IDFDAHTVN Q IIAGHFYRQGL F DVG DFIN IGSLQHLE GPTKELNSSIAKYQKLTEKLLNPDISKAY R N -VEFDSHIIN Q IIASHFYRQGL F DLG DSIIN IPVNQLE GSQKELNSDLSKYPKILEKSFHPDISRTY V N -VDFDYHLVN Q IIASHFYRQGL F DLG DLIN IGSQSQLE GSQKELNMNLSKYPRLLEKSFNPDISKAY R N -VDFDFHTVN Q IIASHFYRQGL F DIG ELIN LAPLNQLE GQKELNVALSKYLKLLEKSFSPDISKAY R N -VDFEASTIN S IIANHFYRQGL F DLG DSFVR MAPLNQLE GSQKELNVALSKYLKLLEKSFNPDISKAY R N -VDFEVHTVN N IIANHFYRQGL F DLG DMFVH IAPITQLE AAEKELHAALTKYPKVLEKQLNPDISKAY R N NVEFDTHIVN Q IIANFFYRQGM F DIG DLVA VVGEPESAAVMKSPF L E MYQILEA MKNQDL E PALK W ATSN SDKL AQSGS DIVLK LHSMQFVKILQNGSR- EANVPESTAAMKSLF S E MYLILEA MKNKNL E PALN W ATAN SNKL KENGS DLLLK LHLQFVEILQGGSR- EAEESNATA-IRSNF L E MHHVIEA MRVRNL Q PALT W VSAN REKL VQIGS NLELK IHTLQYVEVVQNGTQ- EAGEPEATA-LRSQF L E LHQILDA IRAKNL A PALK W ISTN REKL MKSNS NLELK IHRLQFLEILKGGSR- EAGEPEDTA-LKSQF L E MFQILDA MKARDL E PALN W VSNN REKL KQNGS NLELK LHRLQFVEILQKGGR- EGESDGAH-LKLQF Q E MYSILEA MQVRNL Q PALS W AAKN HDQL LQNGS MLELK LHQLQFVEILTKGSR- EGELGGAS-LKLPF Q E MYAILEA MKARNL E PALS W AAKN HDQL LQNGS MLEFK LYQLQFVEILSKGSRG ETGESEST--RQSF V E MYRILEA MKRRDL E PALN W AVSN SDKL KQARS DLEMK LHSLHFLEIAQGKNS- ---DEA LHY ARTYLSPF ASSHIADIQKLMGSLLWTGKL DSSP YHALLSPSNW DRLAEELKRQF N L L G Q S ---SKA LSY VRTHISPF GANHFSEIQKLMALLWSGRL HHSP YSDLLSPTNW NVVAEELTRQF N L L G Q S ---ADA LKY SRTLAPF AKLYKDEFHKLMGLMYVGRL QNSP YAELLSPVHW EMTTEELARQF Y L M G Q S ---ADA LNY AKTYLSPF ASVHTKEFLRVIVSVWTGKL ENYP HSELLSPTHW EKLSEELTRDF N L L G Q S ---ADA LNY ARTYLAPF ASLHMDEIQKLMALLWVGRL DSSP YSELMVPSLW EKLAEELTRQF S L L G Q S ---DEA LKY ARTHLVPF ASLHKAEIQKLMALLWADRL DQSP YAEFMSSTHW EKLAEELTHQF S L L G Q S EAKDEA LLY ARTHLVPF AAVHKEEFQKLMALLWVGRL DQSP YSELMSSAHW EKLAEELTHQF S L L G Q S ---KEA INY ARKHIATF ADSLPEIQKLMSLLWNRKL DRSP YSEFLSPALW NNAVKELTRQY N L L G E S YNSPLSVTISAGVQALPP LLKFMNVMVGKKQEWQTMNQLPVPVELDSE L Q F HSI FVPVSKEQ ATEDNPP FDSPLSVTIAAGFQGLPP LLKFMNVMAGKKHEWQSMKQLPVPVELDRE F Q F HSI FVPVLKEQ STDENPP YENPLNVVFAAGIEGLPT LLKLVNVMAAKKQEWQEMKQLPVPVELGKE F Q F HSI FVPVSRDQ GSEENPP GSPLSLAISAGIDGLPT LLKLAEVMAIKKQEWQALKQLPVPVELGRE F Q F HSI FVPVSREQ GSDENPP YESPLSVAIAAGIEGLPT LLKLANVMAAKKQEWQAMKQLPVPVDLGRE F Q F HSI FVPVSRDQ GSEENPP SESPLGVAVSAGFQGLPT LLKLTTVMAAKKQEWQAMKQLPVPIDIGPE F Q Y HSV FVPVLREQ SSDENPP RESPLSVAVSAGFQGLPT LLKLTQVMAAKKQEWQVMKQLPVPIDIGPE F Q Y HSV FVPVLREQ SSDENPP SESPLSITVKAGTQALPV LLKYMNVMANKKLDWQTMEQLPVDVQLSEE F Q F HSV FVPVSKEQ SSDDNPP S/L M LMS G HVLKQSISK M SKNGSKL-FKPYPFDVDAAL K Q LYF 386 M LMQ G HVLKQSINK M SKNGSKT-FKPYPSDIDSTQ R Q LHF 385 M LLP L HVLKQSIMK L SKNSTRT-FKPYPAEATVAH R Q VFF 387 M LMP L HVLKQSMAK M SKGSSRT-FKPYPAEASIAQ R Q LFF 385 M LMP G HVLKQSIMK L SKSSTRM-FKPYPNESTVGQ M Q LYF 383 M LMP G HVVSKQSIMK L SKSSSRP-FKPYPSEAVASQ K Q LHF 386 M RMP G HVVSKQSIMK L SKSSSRA-FKPYPSEAMASH K Q LHF 390 M MMS G HVLKQTINK M SKNGSKSSFKPYPTDVDISR K Q LHF 386 H /S S/T S 5109N RanBPM-RA TLH Figure S1. Amino acid sequence alignment of SIE3-like ubiquitin ligases from plants. Lotus japonicus SIE3 (Lj) and its homologs from Populus trichocarpa (, GenBank acc. Xp_ ), Medicago truncatula (, AES63574), Oryza sativa (, Np_ ), Zea mays (, AR34292), Vitis Vinifera (, Xp_ ), Ricinus communis (, Xp_ ), and Arabidopsis lyrata (, Xp_ ) were aligned with MacVector software (Version 8.1.2). Identical residues and conserved substitutions among the eight sequences are shown in inversed and shaded letters, respectively. Underlined are conserved domains including a tetrapeptide motif (FDRV), the N-terminal part of OG5109 domain (5109N), the TLH motif, the RanBPM-RA domain and the degenerate RING finger (conserved residues are shown).

6 A B BD / AD SD -2x -4 RING p53 / SV40 lam / SV40 N SIE3 / SIE3 N N LjSIE3-RING HsBL-RING Figure S2. Protein structure and dimerization of Lotus japonicus SIE3. A, Three di me nsio na l model o f LjSI E3 w as pro du ce d usin g Mo de ll er at PS2 se rve r ( The RING finger and the N- and -termini are indicated. B, Self-interaction of LjSIE3 in the yeast two-hybrid system. The full-length SIE3 was fused with the Gal4 DNA binding domain (BD) in bait vector pgbkt7 or with the Gal4 transcription activation domain (AD) in prey vector pgadt7. Yeast cells harboring the constructs in the bait and prey vectors were grown on SD/-Trp-Leu/X-gal medium (SD-2x) and selected for protein-protein interaction on selective medium SD/-Trp- Leu-His-Ade (SD-4). The interaction between murine tumor-suppressor p53 and simian virus 40 large T-antigen (pgbkt7-53 and pgadt7-sv40) was used as a positive control, while the combination between human lamin (Lam) and SV40 (pgbkt7- Lam and pgadt7-sv40) served as a negative control., The 3D structure of the degenerate RING domain of LjSIE3 is similar to that of the canonical RING domain of human BL protein (GenBank Acc. AAI36464).

7 Figure S3. BIF assay of interaction of SIE3 with the extracellular or intracellular portion of SymRK. N. benthamiana leaves were co-transfected with Agrobacterium cells carrying constructs for expression of the full-length SIE3 and either the extracellular portion of SymRK (SymRK-E) or the intracellular protein kinase domain (SymRK-PK). SIE3 was fused with the split FP N-terminus (SN), whereas the SymRK segments were tagged with the split FP -terminus (S). The reconstituted cyan fluorescence was detected by a confocal microscope (OLYMPUS BX61WI). o-expression of Arabidopsis IPK24 (BL-interacting protein kinase 24) and calcineurin B-like (BL) served as a positive control. The split FP fragments (SN / S) were used in the negative control. Overlay images (bottom panel) were generated by superimpose of the fluorescence signal (top panel) with the bright-field images (middle panel). Bars, 20 µm for the positive and negative controls; 30 µm for the inner two columns.

8 A Days after infiltration B Days after infiltration SIE3- GFP PUb- SymRK Myc- SymRK Degr. SIE3 Loading tr. Figure S4. Time course of protein expression in N. benthamiana leaves. N. benthamiana leaves were co-infiltrated with A. tumefaciens cells carrying protein expression constructs and Agrobacterium cells expressing the gene silencing suppressor p19. At different time intervals (days) after infiltration, leaf protein extracts were resolved on 10% SDS-PAGE. Anti-GFP (a) and anti-myc antibodies (b) were used to detect recombinant proteins (Upper panels). The membranes were also stained with Ponceau dye for protein loading controls (Lower panels). Molecular masses of marker proteins are in kd. The positions of SIE3-GFP and its degraded products (Degr. SIE3) and Myc-SymRK and its polyubiquitinated products are indicated. Note that Myc-SymRK were detected as high molecular mass smear ladders in addition to the expected size of Myc-SymRK, presumably due to polyubiquitination of Myc-SymRK.

9 A Nodules per plant > B tr. SIE3OX tr. SIE3 OX Exp. I Exp. II Nodules per plant > tr. RNAi tr. RNAi tr. RNAi Exp. I Exp. II Exp. III Figure S5. Nodulation phenotypes of transgenic hairy roots with altered SIE3 transcript levels. Transgenic hairy roots were produced using Agrobacterium rhizogenes cells carrying either SIE3 OX (A) or SIE3-RNAi construct (B). Transgenic hairy roots expressing vector (pu1301 or pambia s-int-t7) served as controls. The hairy roots were inoculated with M. loti and grown in nitrogen fertilizer-free soil in order to induce nodulation. Four weeks after rhizobial inoculation, the numbers of nodules per plant were recorded. Plants were divided into different groups on the basis of nodules per plant, and the relative ratios of various groups were calculated. Two largescale experiments for SIE3 OX overexpression (Exp. I and II in A) and three large-scale experiments for SIE3-RNAi (Exp. I-III in B) were conducted and presented separately.

10 Ub SIE3 Lb Ub SIE3 Lb Ub SIE3 Lb Days post Rhizobium inoculation Figure S6. Real-time PR analysis of gene expression in SIE3-RNAi transgenic hairy roots. Total RNA was isolated from individual root systems including nodules 16, 21 and 32 days post Rhizobium inoculation. Gene expression levels of ubiquitin (Ub), SIE3, and leghemoglobin (Lb) insie3- RNAi hairy roots were assayed by real-time PR. Relative expression levels of SIE3 and Lb were expressed relative to those of Ub in the same sample.

11 Figure S7. lassification of infection threads in transgenic hairy roots. Transgenic hairyrootsoflotus japonicus were generated using Agrobacterium rhizogenesmediated transformation. The hairy roots were inoculated with M. loti strain that constitutively expressed lacz. Eight days post inoculation with the lacz-labelled M. loti cells, the hairy roots were stained with X-gal solution and observed under microscope to reveal the cellular locations of infection threads (ITs). Arrows indicate the cellular locations of infected thread tips. Bars, 30 μm.