Plant Growth Regulation

Plant Growth Regulation

SUPPLEMENTARY MATERIAL (R2)

A novel GRAS protein gene MtSymSCL1 plays a role in

regulating nodule number inMedicago truncatula

Goon-Bo Kim and Young-Woo Nam*

Department of Life Science, Sogang University, Seoul 121-742, Korea

*To whom correspondence should be addressed.

Tel: +82-2-705-8792; fax: +82-2-704-3601.

E-mail address: (Y.-W. Nam).

______

Electronic Supplementary Data

Supplementary Table S1.Oligonucleotide primers used to clone promoters and coding sequences.

Supplementary Table S2.Oligonucleotide primers used for the preparation of RNAi constructs targeting nodulation-related genes of M. truncatula.

Supplementary Table S3.Oligonucleotide primers used for quantitative real-time PCR analysis.

Supplementary Table S4.Oligonucleotide primers used for PCR-genotyping of Ri-transformed roots.

Supplementary Figure S1.Multiple alignment of Class V GRAS proteins.

Supplementary Figure S2. Expression pattern of MtSymSCL1 in various organs of M. truncatula.

Supplementary Figure S3.Gene Expression Atlas (GEA) of MtSymSCL1 and other nodulation-related genes.

Supplementary Figure S4.Gene Expression Atlas (GEA) of five symbiosis-related GRAS protein genes.

Supplementary Figure S5.Control images of NLS-RFP and MtSymSCL1-GFP fusion proteinslocalized inM. truncatula roots..

Supplementary Figure S6.Map of the genomic region of MtSymSCL1.

Supplementary Figure S7.Transcript levels of nodulation signaling-related genes in RNAi roots.

Supplementary References

Supplementary Tables

Supplementary Table S1. Oligonucleotide primers used to clone promoters and coding sequences

Amplified region / Accession / Forward primer /
Reverse primer (5'  3')a / Reference
MtSymSCL1-cds b / KC292521 / AAAAAGCAGGCTTCACCATGATGAAAGTAGGATTTGAAG/
AGAAAGCTGGGTGCCCTAGAAGTTCTACAAGTCC / This study
MtSymSCL1-proc / KC292522 / AAAAAGCAGGCTCGTGTTTCAACGTCCCGTAAGAC /
AGAAAGCTGGGTTCAAATCCTACTTTCATCAT / This study
MtSymSCL1-pro+ORFd / KC292522 / AAAAAGCAGGCTCGTGTTTCAACGTCCCGTAAGAC /
AGAAAGCTGGGTGCCCTAGAAGTTCTACAAGTCC / This study
MtREM1.1-pro / NW_003763420.1 / AAAAAGCAGGCTGATTAATCACCAAAAACTATGTACCT /
AGAAAGCTGGGTTTTTGTTTTCTTTCTCTTTCTAAG / Kim et al (2013)

aEach primer carries Gateway recombination sites, AAAAAGCAGGCTnn (forward) and AGAAAGCTGGGTn (reverse).

b Coding sequence

c Promoter

dOpen reading frame

Supplementary Table S2.Oligonucleotide primers used for the preparation ofRNAi constructs targeting nodulation-related genes of M. truncatula

Name / Target gene / Accession / Reference / Forward primer /
Reverse primer (5' 3')a
GUSi / GUS / AF485783 / pBI121 / AAAAAGCAGGCTTGAACTGTGCGTCACAGCCA /
AGAAAGCTGGGTGGTAAGTGCGCTTGCTGAG
SymSCL1i / MtSymSCL1 / KC292521 / this study / AAAAAGCAGGCTCGGATTTGGACATCATGCAAGG /
AGAAAGCTGGGTCTAGCACTCATTGGAACCTGC
HAP2-1i / MtHAP2-1 / EF488826 / Combier
et al (2006) / AAAAAGCAGGCTTAATGGCTATGCAACCTGTTTATC /
AGAAAGCTGGGTCTTATGGTTCCAGCAGCTGTCT
STR2i / MtSTR2 / FJ659116 / Zhang et al (2010) / AAAAAGCAGGCTCGACAGGTTATGCACCAAAGGGT /
AGAAAGCTGGGTCGCCCTAATCTGAAATCAGCAGC
Cbf1ib / MtCbf1 / JQ918298 / Hogekamp et al (2011) / AAAAAGCAGGCTATATGAGACAAGCAGGTGCATATTC /
AGAAAGCTGGGTCTATACTTTCCATTTGCATGAGAGTGGTGGC
SymSCL3i / MtSymSCL3c / XM_003608835.1 / GenBank / AAAAAGCAGGCTCGCGCGATAAATCACACGCTTCG /
AGAAAGCTGGGTCTGAGAGTTCTTCACCAATGACT

aEach primer carries Gateway recombination sites, AAAAAGCAGGCTnn (forward) and AGAAAGCTGGGTn (reverse).

b Designed based on the full coding sequence of MtCbf1 (or MtNF-YC11)but expected to target MtCbf2 (or MtNF-YC6; GenBank accession JQ918303) as well due to close similarity in the nucleotide sequences.

c Named based on the upregulated expression pattern in arbuscular mycorrhizal roots (

Supplementary Table S3. Oligonucleotide primers used for quantitative real-time PCR analysis

GeneForward (5’  3’)Reverse (5’  3’)Accession/reference

MtACTIN2GACTCAGTACTTTCCAGCAGATGTGCAGATTATACCCTCGGATCTCACCJQ028731.1

MtDMI2TGGATCAGAGCATGTGTGCTGAAGAACCACACCAAAGCTGAJ418369

MtENOD11ACCCTTGTTCCTCTAGGGCTTGCTGCTTACGCGATGGCGGTTCCTTGTGAJ297721

MtERN1ACCGATGCATCTAGCTTGGAGCAAGGAGCACAAGGGTGGAAEU038802.2

MtHAP2-1AAACGGAGGCAGTCCAAATGTTATGGTTCCAGCAGCTGTCTC117738

MtNINCAGCTTCCCAGAGTTGAGCAGCAGAGGGTGGGGATTTCAAXM_0036186060

MtSymREM1CGAAGGAACTTCAAGCAGTGTTTGCTAGTACAGCATCTCGGTCTC96630

MtSymSCL1CATGTGCTGAACGTGTTGTTGCTATTACAACGGTTCACCGCTTCAAGthis study

Supplementary Table S4. Oligonucleotide primers used for PCR-genotyping of Ri-transformed roots

GeneForward (5’  3’)Reverse (5’  3’)Expected size

SymSCL1aSCL1-Nf:TGAGGTAAGTGATTGGGTTGAACACSCL1-Nr:CAGATTATACCCTCGGATCTCACC488 bp

GFPGACAAGCAGAAGAACGGCAT/CTTGTACAGCTCGTCCATGC252 bp

rolCGTCGACTGCCCGACGATGATG/CACTCGCCATGCCTCACCAAC332 bp

aGenomic DNA region (+183–670 nucleotide position from the start codon)encodingthe amino terminus of MtSymSCL1.

Supplementary Figure Legends

Supplementary Figure S1.Multiple alignment of Class V GRAS proteins. The predicted amino acid sequences of the two representative GRAS proteins from each of the three subclasses (V-1, V-2, and V-3) are aligned. AtSCL23 and Medtr4g076020.1 belong to subclass V-1, AtSCR and Medtr7g074650.1to subclass V-2, and GmSCL4 and MtSymSCL1 to subclass V-3. Amino acid residues conserved at least in 4 out of 6 proteins are represented in black background. Note that substantial sequence similarity only occurs in the C-terminal region. Amino acids conserved only in subclass V-3 are indicated by red arrowheads. Sequence alignment was carried out with the CLUSTALW program (Thompson et al. 1994).

Supplementary Figure S2.Expression pattern of MtSymSCL1 in various organs of M. truncatula. M. truncatula A17 plants were inoculated with S. meliloti and, at 28 dai, the transcript levels of MtSymSCL1 in uninoculated roots (R-), inoculated roots (R+), stems (T), leaves (L), flowers (F), green pods (P), and green seeds (D) were analyzed by real-time PCR. Relative transcript levels were calculated using the uninoculated roots as a reference. Error bars indicate the upper and lower limits of 95% confidence intervals.

Supplementary Figure S3.Gene Expression Atlas (GEA) of MtSymSCL1 and other nodulation-related genes. (A) GEA of MtSymSCL1 (Mtr.31955.1.S1_at; blue line) and MtERN1(Mtr.7556.1.S1_at; red line). (B)GEA of MtNIN (Mtr.28094.1.S1_at; blue line), MtENOD11 (Mtr.13473.1.S1_at; red line), MtSymREM1 (Mtr.13003.1.S1_at; green line), MtHAP2-1 (Mtr.43750.1.S1_at; pink line), and MtDMI2 (Mtr.51192.1.S1_at; magenta line).GEA Data were retrieved from

Supplementary Figure S4. Gene Expression Atlas (GEA) of five symbiosis-related GRAS protein genes. Expression profiles of Mtr.24642.1.S1_at (Medtr1g086970.1), Mtr.47463.1.S1_s_at (Medtr2g089100.1), Mtr.7264.1.S1_at (Medtr3g022830.1), Mtr.36004.1.S1_at(Medtr4g104020.1; MtSymSCL3), and Mtr.31955.1.S1_at and Mtr.31954.1.S1_at(MtSymSCL1) in nodulated or arbuscular mycorrhizal roots are shown. Locus names are from Medicago truncatula genome database version 3.5 ( The locus for two Affymetrix probes, Mtr.31955.1.S1_at and Mtr.31954.1.S1_at, remains to be determined. GEA Data were retrieved from

Supplementary Figure S5.Control images of NLS-RFP and MtSymSCL1-GFP fusion proteinslocalized inM. truncatula roots.Translational gene fusions constructed under a native promoter of MtSymSCL1 were introduced into hairy roots via A. rhizogenes-mediated transformation. (A)–(C)NLS-RFP detected in nodulesfromthe composite rootstransformed with anempty vector pK7FWG-nlsR. The roots were subsequently inoculated withS. meliloti. Fluorescent signals are localized in nucleiof nodule surface cells(white arrowheads in A and C). A small surface area surrounding nodulesin (B)is magnified in(C). (D)Localization of MtSymSCL1-GFP in uninoculated roots. Note that while red fluorescent signals indicate nuclei (white arrows), no green fluorescent signals are detected.Bars at bottomindicate magnification levels. For images of MtSymSCL1-GFP localized in nodulated roots, see Fig. 4f–n.

Supplementary Figure S6.Map of the genomic region of MtSymSCL1. The positions of the sequences used for designing primers for inverse PCR (red), genotyping (green), or cloning of partial cDNAs for the preparation of RNAi constructs (blue) are shown on the 3.2-kb EcoRI fragment. Regions corresponding to the two initially identified partial cDNAs (AL386879 and AL386880) are indicated with solid lines. Primers P1f and wkR1 were used to determine the nucleotide sequence of inverse PCR products.

Supplementary Figure S7.Transcript levels of nodulation signaling-related genes in RNAi roots. A. rhizogenes–transformed GUSi and MtSymSCL1i plants grown in pots were inoculated with S. meliloti and roots were sampled at 7 and 42 dai. Relative quantities (RQ) of MtSymSCL1 (S), NIN (N), ERN1 (E), MtHAP2-1 (H), SymREM1 (R), and DMI2 (D) transcripts are plotted as fold changes in comparison to those at 0 dai. MtACTIN2(GenBank accession JQ028731.1) was used as an endogenous normalization control. Thin bars indicate the upper and lower limits of 95% confidence intervals.

Supplementary Figure S1

Supplementary Figure S2

A

B

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

Supplementary References

Combier JP, Frugier F, de Billy F, Boualem A, El-Yahyaoui F, Moreau S, Vernie T, Ott T, Gamas P, Crespi M, Niebel A (2006) MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicagotruncatula. Genes Dev 20:3084–3088

Hogekamp C, Arndt D, Pereira PA, Becker JD, Hohnjec N, Kuster H (2011) Laser microdissection unravels cell-type-specific transcription in arbuscular mycorrhizal roots, including CAAT-box transcription factor gene expression correlating with fungal contact and spread. Plant Physiol 157:2023–2043

Kim GB, Bae JH, An CS, Nam YW (2013) Single or multiple gene silencing directed by U6 promoter-based shRNA vectors facilitates efficient functional genome analysis in Medicago truncatula. Plant Mol Biol Rep (accepted)

Popp C, Ott T (2011) Regulation of signal transduction and bacterial infection during root nodule symbiosis. Curr Opin Plant Biol 14:458–467

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res22:4673–4680

Zhang Q, Blaylock LA, Harrison MJ (2010) Two Medicagotruncatula half-ABC transporters are essential for arbuscule development in arbuscular mycorrhizal symbiosis. Plant Cell 22:1483–1497

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