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Figure S1. Purification of OsCYP18-2 from E. coli. (a) Recombinant His-tagged OsCYP18-2 was cloned into pET28a. Following transformation of E. coli, OsCYP18-2 expression was induced with isopropyl b-d-1-thiogalactopyranoside (IPTG) and the protein purified on a nickel-NTA agarose column. The samples were separated using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue. The purified protein is indicated with an arrow. (b). Representative immunoblot (IB) of the expressed OsCYP18-2-His protein probed with the monoclonal anti-His antibody.
(a)
Purified fr
action
OsCYP18-2-His
IB : anti-His
70 -
40 -
30 -
50 -
20 -
100 - (kDa)
15 -
Insoluble f
raction
No Inducti
on
Induction
Soluble fr
action
(b)
IB: anti-AD
IB : anti-BD
70 -
50 -40 -
30 -
20 -
100 -
(kDa)
BD-OsCYP18-2
AD-OsSKIP Full AD-OsSKIP C-term
AD-OsSKIP N-termAD-OsSKIP N 1-55AD-OsSKIP B 56-95AD
40 -
AD-OsSK
IP Full
AD-OsSK
IP C-term
AD-OsSK
IP N-term
AD-OsSK
IP N 1-55
AD-OsSK
IP B 56-95
AD
BD-OsCYP18-2 Bait :
Prey :
(b)
(a)
pGBKT7 OsCYP18-2 GAL4 BD
pGADT7 OsSKIPsGAL4 AD
SmaI SalI
SmaI XhoI
PADH1
PADH1
Figure S2. Immunoblot analysis of extracts prepared from yeast for the two-hybrid assay. (a) Two sets of plasmids were constructed carrying OsCYP18-2 fused to the GAL4 BD (BD-fusion) and the indicated segments of OsSKIP fused to the GAL4 AD (AD-fusion). The yeast strain AH109 was transformed with different combinations of OsCYP18-2 and OsSKIP plasmids. (b) Immunoblot analysis to assay the expression levels of each fusion protein in whole-cell extract. The immunoblot was probed with anti-BD or anti-AD antibodies.
SD-LTH+3AT
SD-LT
BD-OsCYP18-2
AD AD-OsS
KIP B
56-9
5
AD-OsS
KIP B
56-9
5
( E
79A)
AD-OsS
KIP B
56-9
5
(E7
9A, H
81A)
SD-LTH+3AT
SD-LT
BD-OsCYP18-2
AD OsSKIP
OsRBM
42
OsPRPF8
OsSF3
B3
OsHDAC8
HsCypD-His
GST-HsBcl2
IB : anti His
IB : anti GST
+ - CsA20 -
(kDa)
15 -
50 -
40 -
Input Bound fraction
+ -
Figure S3. The effect of cyclosporin A (CsA) on recombinant His-tagged HsCypD. The interaction between HsCypD and HsBcl2 was significantly reduced in the presence of CsA. HsCypD-His was immobilized on Ni-NTA agarose beads and incubated with GST-fused HsBcl2. HsCypD was pre-incubated with excess amounts of CsA for 1 h. Immunoblots were probed using anti-His and anti-GST antibodies.
Figure S4. Yeast two-hybrid analysis of the interaction between OsCYP18-2 and OsSKIP B56-95 mutated at the sites E79A or E79A and H81A.
Figure S5. Yeast two-hybrid analysis of interactions of OsCYP18-2 with homologues of candidates PPiL1 interactor (OsRBM42, OsSF3B3, OsPRPF8, OsHDAC8). Information was obtained from IntAct (http://www.ebi.ac.uk/intact/) or BioGRID (http://thebiogrid.org/) databases.
(b)
Full
N-term
B 56-95
N 1-55
1 71-78 190 356 406 428 563-568 607
1 156
1 55
56 95
1 164
OsCYP18-2-CE-YFP
NE-
YFP-
OsS
KIP
CE-YFPHA
NE-YFP
NE-YFP
NE-YFP
NE-YFPc-myc
SKIP/SNW domain
Glycine-rich box
Bipartite NLS
MonoExtC NLS
CYP domain
(c) OsCYP18-2-CE-YFP
Brig
htYF
P/Ch
loro
plas
t
NE-YFP-Ran1
RanBP1-CE-YFP
NE-YFP-OsSKIP Full
NE-YFP-OsSKIP N-term
NE-YFP-OsSKIP B56-95
NE-YFP-OsSKIP C-term
(d)
70 - 50 -40 -30 -20 -
100 - (kDa)
15 -
IB: anti-HA
B 56
-95
C-te
rm
N-te
rm
Full NE-Y
FP-R
an1
NE-YFP-OsSKIP Ran
BP1
-CE-
YFPOsCYP18-2-CE-
YFP
OsCYP18-2-CE
RanBP1-CE
70 - 50 -40 -
30 -
20 -
100 - (kDa)
15 -
B 56
-95
C-te
rm
N-te
rm
Full
NE-OsSKIP FullNE-OsSKIP C-term
OsSKIP B N-term
OsSKIP B 56-95
NE-YFP-OsSKIP
OsCYP18-2-CE-YFP
NE-Ran1
IB: anti-c-Myc
Ran
BP1
-CE-
YFP
NE-Y
FP-R
an1
OsS
KIP
C-te
rm-G
FP
(a) GFP DAPI Chloro-plast
Merged Bright
OsC
YP18
-2-G
FP
Figure S6. Subcellular localization of OsCYP18-2-GFP and OsSKIP C-term-GFP, and analysis of the interaction between OsCYP18-2 and OsSKIP. (a) Subcellular localization of OsCYP18-2-GFP (upper panel) and OsSKIP C-term-GFP (lower panel) in leaf epidermal cells or protoplast cells from transiently transformed Nicotiana benthamiana plants using the agroinfiltration method. (b) For the bimolecular fluorescence complementation (BiFC) assay, plasmids were constructed containing OsCYP18-2 fused to pSPYCE and different fragments of OsSKIP fused to pSPYNE. Different combinations of plasmids were introduced into N. benthamiana leaves using Agrobacterium GV3101 infiltration. (c) BiFC visualization of OsCYP18-2 and OsSKIPs interactions. (d) The expression level of each fusion protein in N. benthamiana leaf extract was assayed using immunoblot analysis with anti-HA or anti-c-Myc antibodies. A combination of RanBP1-CE-YFP and NE-YFP-Ran1 was used as a positive control. Scale bars: 20 μm.
OsCYP18-2-GFP + SKIP Full(b)
Distance (µm)
Inte
nsity
Ch2 : GFPChS1 : ChloroplastChD : Bright
(d)
(f)
(c)
Inte
nsity
Distance (µm)
Ch2 : GFPChS1 : ChloroplastChD : Bright
(a) OsCYP18-2-GFP + SKIP B56-95
(e)
Figure S7. Quantification of fluorescence in the cytoplasm and nucleus. (a,b) Fluorescence and bright-field merged representative images of epidermal cells after transient expression of OsCYP18-2-GFP and SKIP B56-95/SKIP Full (NE-YFP-OsSKIP Full or AtSKIP Full-HA). (c,d) Quantitation of GFP fluorescence in the nucleus and cytoplasm along the scanned lines (red arrow) in images (a,b) using the LSM5 image program (Carl Zeiss Laser Scanning System LSM 510). The profile shows the intensity of GFP fluorescence (green) and chloroplast autofluorescence (red) along with distance. (e,f) Values are maximum fluorescence intensities in the nucleus and cytoplasm and autofluorescence in the chloroplast. All images were captured at same focal planes and with the same laser intensity.
(a)
pCAMBIA1300-OsCYP18-2
T-NOSOsCYP18-2 RBLB HPTII
SpeI KpnI
35S-P 35S-PT-NOS
OsCYP18-2
AtACT2
WT V1 OE1 OE2 OE3
AtCYP18-2
At
(b)
OsCYP18-2OsACT1
OE1 OE2 OE3
WT 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Os (T1)
WT OE1 OE2 OE3Os (T2)
OsCYP18-2
OsACT1
(c)
OsCYP18-2
WT OE1 OE2 OE3Os
Ponceau S
IB: anti-OsCYP18-2
20 -(kDa)
15 -
WT OE1 OE2 OE3At
OsCYP18-2AtCYP18-2
Ponceau S
IB: anti-OsCYP18-2
20 -(kDa)
15 -
Figure S8. Constitutive expression of OsCYP18-2 in rice and Arabidopsis. (a) Structure of the pCAMBIA1300 binary vector harbouring OsCYP18-2 under the control of the 35S promoter. (b) Ectopic and constitutive expression of OsCYP18-2 in the T1 (14 lines) and T2 (OE1, OE2, OE3) generations of transgenic rice and in T3 homozygous Arabidopsis plants was compared with that in WT plants or vector controls using semi-quantitative RT-PCR analysis. AtACT2 and OsACT1 were used as controls for mRNA normalization. V1: pCAMBIA vector control transgenic plants; At: OsCYP18-2-expressing transgenic Arabidopsis plants; WT: wild-type plants; Os: OsCYP18-2 transgenic rice plants. (c) Immunoblot analysis of the OsCYP18-2 over-expressing plants using anti-OsCYP18-2 antibody.
WT V1 OE1 OE2 OE30
50
100
150
200
250(a)
Stom
atal
den
sity
(no/
mm
²)
Vector OsCYP18-2 OE (b)
Figure S9. Stomatal density and stomatal aperture in leaves of Arabidopsis plants over-expressing OsCYP18-2 and in leaves of vector control and wild-type plants. (a) Stomatal density under normal conditions. (b) Photographs showing stomatal closure (arrowheads) under drought-stress conditions in leaf epidermal cells of OsCYP18-2 OE and vector control Arabidopsis plants. Scale bar: 20 μm.
BD
BD-OsSKIP
BD-OsSKIP C-term
BD-OsCYP 18-2
positive
0 5 10 15 20 25 30
+
+
+
+
SD/-W/X-α-gal
_
SD/-WH
BD
BD-OsSKIP
BD-OsSKIP C-term
BD-OsCYP18-2
BD-AD
0 0.5 1
(b)
α-Galactosidase activity (PNP- α-gal)
(a)SmaI SalI
pGBKT7 PADH1 GAL4 BD TADH1&T7
AD/OsCYP18-2/OsSKIP C-term/OsSKIP
Figure S10. Transcriptional activation analysis of OsCYP18-2 using a yeast one-hybrid system derived from the GAL4 two-hybrid system. (a) OsCYP18-2 and OsSKIP or OsSKIP deletion constructs were used. pGBKT7 was used as the effector plasmid and yeast strain AH109 was used as the reporter yeast. (b) Tran-scriptional activation was monitored by assaying α-galactosidase activity (left) and yeast growth on -Trp media containing X-α-gal or -Trp-His media (right). The plasmids pGBKT7-OsSKIP or pGBKT7-OsSKIP deletion forms and pGBKT7 were used as the positive and negative controls, respectively.
