Supplemental Figure 1 - The Plant Cell · 4/6/2011 · Supplemental Figure 1. Specific interaction...
Transcript of Supplemental Figure 1 - The Plant Cell · 4/6/2011 · Supplemental Figure 1. Specific interaction...
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
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
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
wt Ler b”β-1
0
5
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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
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
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
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).