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Page 1: Supplemental Figure 1 - The Plant Cell · 4/6/2011  · Supplemental Figure 1. Specific interaction of B”αand B”β(88-538) with the N-terminal region of HMGR1L Supplemental Data.

GST-

NT1L

GST

kD37

26

-

-

HisB”β(88-536)

Inpu

t

GST

GST-

NT1L

kD

61-

HisB”α

Inpu

t

GST

GST-

NT1L

kD

70-

GST-

NT1L

GST

kD37

26

-

-

GST-

NT1L

GST

kD37

26

-

-

HisB”β(88-536)

Inpu

t

GST

GST-

NT1L

kD

61-

HisB”β(88-536)

Inpu

t

GST

GST-

NT1L

kD

61-

HisB”α

Inpu

t

GST

GST-

NT1L

kD

70-

HisB”α

Inpu

t

GST

GST-

NT1L

kD

70-

BD P53 laminine NT1L CD1 HMGS MVK ΔNtFPS1 SQS1

AD-AtB”α - - - + - - - - -AD-AtB”β (88-536) - - - + - - - - -AD-B”α

AD-B”β(88-536)

BD P53 laminine NT1L CD1 HMGS MVK ΔNtFPS1 SQS1

AD-AtB”α - - - + - - - - -AD-AtB”β (88-536) - - - + - - - - -AD-B”α

AD-B”β(88-536)

AD-B”α

AD-B”β(88-536)

AD

BD-NT1LHIS3 LacZ

AD-B”α

AD-B”β(88-536)

AD

BD-NT1LHIS3 LacZ

A

B

C

Supplemental Figure 1.Specific interaction of B”α and B”β(88-538) with the N-terminal region of HMGR1L

Supplemental Data. Leivar et al. Plant Cell. (2011). 10.1105/tpc.110.074278

(A) Two-hybrid analysis in the yeast strain Y190. Yeast cells were cotransformed with a pAS2.1-derivative encoding BD-NT1L and either control pACT plasmid encoding AD or pACT-derivatives encoding AD fused to the complete B”α or the truncated version of B”β (residues 88-536). Interaction between the assayed partners was confirmed by the occurrence of growth on selective medium without histidine (HIS3 lanes) and β-galactosidase activity (LacZ lanes).

(B) Two-hybrid analysis in the yeast strain CG-1945. Cells harboring the indicated plasmid combinations were plated on selective medium without histidine. The occurrence (+) or absence (-) of growth and β-galactosidase activity is indicated.

(C) In vitro GST pull-down analysis. Agarose beads carrying GST or GST-NT1L were incubated with equivalent amounts of radiolabeled in vitro-synthesized HisB”α or HisB”β(88-536). Pictures on the left and the center show fluorograms of the radiolabeled products retained by the resins, whereas the picture on the right shows a coomassie-stained gel with the GST-NT1L bait and control GST used in the assay.

Supplemental Figures page 1/4

Page 2: Supplemental Figure 1 - The Plant Cell · 4/6/2011  · Supplemental Figure 1. Specific interaction of B”αand B”β(88-538) with the N-terminal region of HMGR1L Supplemental Data.

b”β-1

wtbp

434-fgfg

Ler

b”β-1

wtbp

434-fgfg

Ler

Supplemental Figure 2.RT-PCR analysis of the b”β-1 mutant

Supplemental Data. Leivar et al. Plant Cell. (2011). 10.1105/tpc.110.074278

Total RNA was extracted from Arabidopsis Ler wt and Ler b”β-1 mutant seedlings grown in MS medium for 14 days, under long-day conditions. The presence of the B”β transcript was examined by first strand cDNA synthesis and PCR amplification with primers f and g.

Supplemental Figures page 2/4

Page 3: Supplemental Figure 1 - The Plant Cell · 4/6/2011  · Supplemental Figure 1. Specific interaction of B”αand B”β(88-538) with the N-terminal region of HMGR1L Supplemental Data.

BD-PR65

BD-NT1S

BD-NT1L

AD-B”α AD-B”α(1-397)His3 His3LacZ LacZ

BD-PR65

BD-NT1S

BD-NT1L

AD-B”α AD-B”α(1-397)His3 His3LacZ LacZ

Supplemental Figure 3.Two-hybrid analysis of B”α(1-397)

Supplemental Data. Leivar et al. Plant Cell. (2011). 10.1105/tpc.110.074278

Yeast Y190 cells were co-transformed with a pAS2.1-derivatives encoding BD-PR65, BD-NT1S or BD-NT1L and pACT2-derivatives encoding AD-B”α or AD-B”α(1-397). The absence of growth (HIS3 lane) and staining (LacZ lane) indicates failure of interaction between the assayed proteins. BD-PR65 corresponds to the A2 (pDF1) variant of PR65.

Supplemental Figures page 3/4

Page 4: Supplemental Figure 1 - The Plant Cell · 4/6/2011  · Supplemental Figure 1. Specific interaction of B”αand B”β(88-538) with the N-terminal region of HMGR1L Supplemental Data.

wt Ler b”β-1

0

5

10

15

20

25

wt b”β-1Ler

Seed

l. es

tabl

ish.

(%

) *

Supplemental Figure 4.Seedling establishment analysis of the b”β-1 mutant

Supplemental Data. Leivar et al. Plant Cell. (2011). 10.1105/tpc.110.074278

Seedling establishment assays were performed to compare the HMGR activity of the b”β-1 mutant and its wild type parental line (Ler) in vivo. Seeds were vernalized at 4º C for three days and germinated on polyester filters layered on MS medium containing 4 µM mevinolin or 4 µM lovastatin. The appearance of true leaves was evaluated after 16 days of growth at 22º C, under long day conditions. The graph shows the average and SD of three independent assays with 50 plants per assay and genotype. Under the experimental conditions, nearly no Ler plant could develop true leaves, whereas about 20 % of b”β-1 plants overcame the statin blockage. The asterisk indicates thelevel of statistical significance as determined for Student’s t test: *, P < 0.005 for b”β-1 versus wt.

Supplemental Figures page 4/4

Page 5: Supplemental Figure 1 - The Plant Cell · 4/6/2011  · Supplemental Figure 1. Specific interaction of B”αand B”β(88-538) with the N-terminal region of HMGR1L Supplemental Data.

Supplemental Data. Leivar et al. Plant Cell. (2011). 10.1105/tpc.110.074278

Supplemental Table 1: PCR Primers

18F  5’‐gatccagctctagctcg^gagg‐3’ 18R  5’‐gatgacctggcaacttctccttg‐3’ 22F  5’‐ggctgtttccactgag^gtggc‐3’ 22R  5’‐gtcagacaacttctttaccccag‐3’ 25GSP1  5’‐aggctcgttgactactttg‐3’ 25GSP2  5’‐ttagaaggggctctctgtttg‐3’ 25F‐XhoI  5’‐ccgctcgagccatggtggatacggttattccc‐3’ 25R‐XhoI  5’‐ccgctcgagttcttttgttgacttgctacctc‐3’ 25totF  5’‐gaagactagattcgatgtgttg‐3’ 25totR  5’‐ttcttttgttgacttgctacctc‐3’ 264F  5’‐gagctgaa^gtggcttccatgac‐3’ 264R  5’‐ggtccgacatacccatgatcc‐3’ 29aF1  5’‐ggaagagtcggctgtttcagac‐3’ 29aR1  5’‐ccggtactcctccagttt^cttc‐3’ 764F  5’‐catctatcgag^gtggggacag‐3’ 764R  5’‐gctcctttaactccgagcagg‐3’ a  5’‐ctcaatgttgcttgcgggagtg‐3’ b  5’‐ggaattcctgaggaatacacc‐3’ c  5’‐gagattcgagtggttcatcc‐3’ e  5’‐aggtgtattcctcaggaattcc‐3’ f  5’‐ggctgtctcatttccttgttcgg‐3’ g  5’‐tcttggtgaaagaggaggagcg‐3’ h  5’‐ttcctcattcgtcag^gagcgc‐3’ i  5’‐aacatcttcttccattgatagccg‐3’ E5‐1  5’‐gtcagatatccatggaaatcgatggtggaaacgat‐3’ E5‐2  5’‐cagtgatatcaatactcaagactaggctcagatg‐3’ H1.2R  5’‐gtcgctagcctcctttgcgttc‐3’ H1.3F  5’‐gctagcactaacagaggctgca‐3’ H2.3R  5’‐gatcctttcacaccgagtag‐3’ H2.4F  5’‐ctgtatccgaggtttgcgtg‐3’ HMGS‐F  5’‐cgaacatatggcgaagaacgttgggattttg ‐3’ HMGS‐R  5’‐ttcagaattcagtgtccattggctacagatcc‐3’ K2F  5’‐agctggagcacaacag^gcgg‐3’ K2R  5’‐ttcaggccggtcttgtccttc‐3’  NT1LF‐EcoRI  5’‐ggaattcatgaagaaaaagcaagctggtcc‐3’ NT1LR‐BamHI  5’‐gtgcggatcctggagggaatgaataatctctcc‐3’ NT1NF‐NcoI  5’‐gacatgccatggatctccgtcggaggcc‐3’ NT1NR‐BamHI  5’‐gtgcggatcccgcgtcggatgctttcggtg‐3’ NT2F‐NcoI  5’‐gacatgccatggaggatctccgtcgtagatttc‐3’ NT2R‐BamHI  5’‐gtgcggatcccgcgtcagaggctttacgaagag‐3’ NTGSTF‐HindIII  5’‐tacccaagcttatgtcccctatactaggttattg‐3’ pACTBF  5’‐ataccactacaatggatgatg‐3’ pASR  5’‐taaaacctaagagtcac‐3’ pGEX3’R  5’‐ggcagatcgtcagtcagtcac‐3’ SQS1F  5’‐ggaacatatggggagcttggggacgatg‐3’ SQS1R  5’‐acatggatccctcagtttgctctgagatatgc‐3’ T7  5’‐gtaatacgactcactataggg‐3’

^: position of an exon-exon junction

Supplemental Table 1 : PCR primers    page 1/1 

Page 6: Supplemental Figure 1 - The Plant Cell · 4/6/2011  · Supplemental Figure 1. Specific interaction of B”αand B”β(88-538) with the N-terminal region of HMGR1L Supplemental Data.

Supplemental Data. Leivar et al. Plant Cell. (2011). 10.1105/tpc.110.074278

Supplemental Table 2: Plasmid constructs

The plasmid constructs, numbered on the left, are defined by the encoded protein. The plasmids were obtained by ligation of the indicated insert and vector. Inserts were obtained from its plasmid source either directly by restriction enzyme digestion or by PCR and subsequent restriction enzyme digestion.

Construct  Insert  Vector Number  Encoded Protein  Plasmid source 

Primers for PCR  Digestion  Name*  Cloning sites

#1  AD‐B”α  Plasmid #3    BglII  pACT2  BamHI 

#2  AD‐B”α(1‐397)  Plasmid #1 E5‐1 E5‐2 

EcoRV  pACT2  SmaI 

#3  AD‐B”α(1‐538)  pACT‐B”α(1‐538) Clone of the pACT library (Kim et al., 1997) obtained by screening with the BD-NT1L bait

#4  AD‐B”β  Plasmid #6 25F‐XhoI 25R‐XhoI 

XhoI  pACT2  XhoI 

#5  AD‐B”β(88‐536)  pACT‐B”β (88‐536) Clone of the pACT library (Kim et al., 1997) obtained by screening with the BD-NT1L bait

#6  B”β  pACT library (Kim et al., 1997)

25totF 25totR 

  pGEM‐T Easy   

#7  BD‐CD1  pHMGR1 cd (Dale et al., 1995)  

NdeI EcoRI 

pAS2‐1 NdeI EcoRI 

#8  BD‐HMGS  pACT library (Kim et al., 1997)

HMGS‐F HMGS‐R 

NdeI EcoRI 

pAS2‐1 NdeI EcoRI 

#9  BD‐MVK  MVA‐K‐pFL61 (Riou et al., 1994)  

EcoRI SalI 

pAS2‐1 EcoRI SalI 

#10  BD‐NT1L  pDS6‐HMGR1L (Lumbreras et al., 1995)

NT1LF‐EcoRI NT1NR‐BamHI 

EcoRI BamHI 

pAS2‐1 EcoRI BamHI 

#11  BD‐NT1S  Plasmid  #24 NT1NF‐NcoI NT1NR‐BamHI 

NcoI BamHI 

pAS2‐1 NcoI BamHI 

#12  BD‐NT2  pDS6‐HMGR2 (Enjuto et al., 1994)

NT2F‐NcoI NT2R‐BamHI 

NcoI BamHI 

pAS2‐1 NcoI BamHI 

#13  BD‐PR65 (A2 variant, PDF1) 

cDNA (EST) clone G5C6T7 from ABRC (Kieber et al., 1993)

 EcoRV SmaI 

pAS2‐1  SmaI 

#14  BD‐SQS1  pACT library (Kim et al., 1997)

SQS1F SQS1R 

NdeI BamHI 

pAS2‐1 NdeI BamHI 

#15  BD‐1Lextra  pDS6‐HMGR1L (Lumbreras et al., 1995)

NT1LF‐EcoRI NT1LR‐BamHI 

EcoRI BamHI 

pAS2‐1 EcoRI BamHI 

#16  BD‐ΔNtFPS1  pBlues SK+ [FPS1] (Delourme et al., 1994)  

EcoRI BamHI 

pAS2‐1 EcoRI BamHI 

#17  GST‐ B”α  Plamid #3    BglII  pGEX‐5X‐2  BamHI 

#18  GST‐NT1L  Plasmid #10   EcoRI SalI 

pGEX‐4T‐1  EcoRI SalI 

Supplemental Table 2: Plasmid constructs     page 1/2 

Page 7: Supplemental Figure 1 - The Plant Cell · 4/6/2011  · Supplemental Figure 1. Specific interaction of B”αand B”β(88-538) with the N-terminal region of HMGR1L Supplemental Data.

Supplemental Data. Leivar et al. Plant Cell. (2011). 10.1105/tpc.110.074278

Supplemental Table 2: Plasmid constructs     page 2/2 

Construct  Insert  Vector 

Number  Encoded Protein  Source Primers for PCR  Digestion  Name*  Cloning sites

#19  GST‐PR65  Plasmid #23   EcoRI XhoI 

pGEX‐4T‐1 EcoRI XhoI 

#20  HisB”α  Plasmid #1   NheI BglII 

pET‐28a(+) NheI BamHI 

#21  HisB”β  Plasmid #6 25F‐XhoI 25R‐XhoI 

XhoI  pET‐28a(+)  XhoI 

#22  HisB”β(88‐536)  Plasmid #5    XhoI  pRSET C  XhoI 

#23  HisPDF2  Plasmid #32    XhoI  pET‐28a(+)  XhoI 

#24  HMGR1S  Plasmid #25    EcoRI  pBluescript SK+  EcoRI 

#25  HMGR1S  λcAT12 Original clone obtained by screening of λgt10 cDNA library (Caelles et al., 1989)

#26  NT1L‐GST  pGEX‐4T‐1 (GST coding sequence)

NTGSTF‐ HindIIIpGEX3’R 

HindIII XhoI 

Plasmid #27 HindIII XhoI 

#27  NT1L(pET‐23d)  Plasmid #10 NT1LF‐EcoRI 

pASR EcoRI(blunt)† 

SalI pET‐23d(+) 

NcoI(blunt)†  SalI 

#28  NT1S‐GST  pGEX‐4T‐1 (GST coding sequence)

NTGSTF‐HindIII pGEX3’R 

HindIII XhoI 

Plasmid #29 HindIII XhoI 

#29  NT1S(pET‐23d)  Plasmid #11   NcoI SalI 

pET‐23d(+) NcoI SalI 

#30  NT2‐GST  pGEX‐4T‐1 (GST coding sequence)

NTGSTF‐HindIII pGEX3’R 

HindIII XhoI 

Plasmid #31 HindIII XhoI 

#31  NT2(pET‐23d)  Plasmid #12   NcoI SalI 

pET‐23d(+) NcoI SalI 

#32  PDF2(19‐588) (PR65 A3 variant) 

pACT‐PDF2 (19‐588) A clone of the pACT library (Kim et al., 1997)

#33  1Lextra‐GST  Plasmid #28 (GST coding sequence)   BamHI  Plasmid #34  BamHI 

#34  1Lextra(pET‐23d)  Plasmid #26 T7 

NT1LR‐BamHI XbaI BamHI 

Plasmid #26 XbaI BamHI 

* Vectors commercially available are as follows: pACT2 and pAS2-1, Clontech; pBluescript SK+, Stratagene; pET-23d(+) and pET-28a(+), Novagen; pGEM-T Easy, Promega; pGEX-4T-1 and pGEX-5X-2, Amersham Biosciences; pRSET C, Invitrogen-Life Technologies. † Prior to ligation, ends were blunted with Mung Bean Nuclease (Promega).