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.

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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 fraction OsCYP18-2-His IB : anti-His (kDa) 15 - Insoluble fraction No Induction Induction Soluble fraction (b)

IB: anti-AD IB : anti-BD (kDa) BD-OsCYP18-2 AD-OsSKIP Full AD-OsSKIP C-term AD-OsSKIP N-term AD-OsSKIP N 1-55 AD-OsSKIP B AD 40 - AD-OsSKIP Full AD-OsSKIP C-term AD-OsSKIP N-term AD-OsSKIP N 1-55 AD-OsSKIP B AD BD-OsCYP18-2 Bait : Prey : (b) (a) pGBKT7 OsCYP18-2GAL4 BD pGADT7 OsSKIPs GAL4 AD SmaI SalI SmaI XhoI P ADH1 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-OsSKIP B56-95 ( E79A) AD-OsSKIP B56-95 (E79A, H81A) SD-LTH +3AT SD-LT BD-OsCYP18-2 ADOsSKIPOsRBM42OsPRPF8OsSF3B3OsHDAC8 HsCypD-His GST-HsBcl2 IB : anti His IB : anti GST + - CsA 20 - (kDa) 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 ( or BioGRID ( databases.

(b) Full N-term B N OsCYP18-2-CE-YFP NE-YFP-OsSKIP CE-YFP HA NE-YFP c-myc SKIP/SNW domain Glycine-rich box Bipartite NLS MonoExtC NLS CYP domain (c) OsCYP18-2-CE-YFP Bright YFP/Chloroplast 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) (kD a) 15 - IB: anti-HA B C-term N-term Full NE-YFP -Ran1 NE-YFP-OsSKIP RanBP1 -CE-YFP OsCYP18-2-CE- YFP OsCYP18-2- CE RanBP1- CE (kD a) 15 - B C-term N-term Full NE-OsSKIP Full NE-OsSKIP C-term OsSKIP B N-term OsSKIP B NE-YFP-OsSKIP OsCYP18-2-CE- YFP NE-Ran1 IB: anti-c-Myc RanBP1 -CE-YFP NE-YFP -Ran1 OsSKIP C-term -GFP (a) GFP DAPI Chloroplas t Merged Bright OsCYP18-2 -GFP 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) Intensity Ch2 : GFP ChS1 : Chloroplast ChD : Bright (d) (f) (c) Intensity Distance (µm) Ch2 : GFP ChS1 : Chloroplast ChD : 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-NOS OsCYP18-2 RB LB HPTII SpeI KpnI 35S-P T-NOS OsCYP18-2 AtACT2 WT V1 OE1 OE2 OE3 AtCYP18-2 At (b) OsCYP18-2 OsACT1 OE1 OE2 OE3 WT Os (T 1 ) WT OE1 OE2 OE3 Os (T 2 ) OsCYP18-2 OsACT1 (c) OsCYP18-2 WT OE1 OE2 OE3 Os Ponceau S IB: anti-OsCYP (kDa) 15 - WT OE1 OE2 OE3 At OsCYP18-2 AtCYP18-2 Ponceau S IB: anti-OsCYP (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 T 1 (14 lines) and T 2 (OE1, OE2, OE3) generations of transgenic rice and in T 3 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.

(a) Stomatal density (no/mm²) VectorOsCYP18-2 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.

SD/-W/ X-α-gal _ SD/-WH (b) α-Galactosidase activity (PNP- α-gal) (a) SmaI SalI pGBKT7 P ADH1 GAL4 BD T ADH1&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) Transcriptional 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.