Appendix 1
Supporting Methods
Virus-induced gene silencing (VIGS)
The NbPES cDNA fragments were PCR-amplified and cloned into the pTV00 vector
containing part of the tobacco rattle virus (TRV) genome using the NbPES(F)-F/R,
NbPES(N)-F/R, and NbPES(C)-F/R primer sets as described in Supplementary Table
S2. For VIGS of NbBOP1 and NbWDR12, the corresponding cDNA fragments were
PCR-amplified with the NbBOP1-F/R and NbWDR12-F/R primer sets. VIGS was
performed as described (Ahn et al., 2011).
Agrobacterium-mediated transient expression
Agro-infiltration was carried out as described (Voinnet et al., 2003). Agrobacterial
cultures (GV3101) containing various constructs fused to the CaMV35S promoter were
adjusted to OD600=0.6 in MES buffer (10 mM MES [pH 7.5], 10 mM MgSO4). The
suspension was incubated with acetosyringone for 2–3 h at a final concentration of 150
μM, and infiltrated into leaves of wild-type N. benthamiana plants. In all experiments,
Agrobacterium C58C1 carrying the 35S:p19 construct (Voinnet et al., 2003) was coinfiltrated to achieve maximum levels of protein expression. Expressed proteins were
analyzed at 48 h post-infiltration.
Generation of dexamethasone (DEX)-inducible AtPES RNAi lines in Arabidopsis
1
A 350 bp AtPES cDNA fragment was PCR amplified using the AtPESi-S-F/R primer set
containing XhoI and ClaI sites for the sense construct, and the AtPESi-AS-F/R primer
set containing BamHI and SpeI sites for the antisense construct. The sense and antisense
constructs of the 350 bp cDNA regions were cloned into the 5' and 3' arms of the intron
in the pSK-int vector (Guo et al., 2003) to produce an inverted-repeat sequence
(antisense-intron-sense), which was isolated by digestion with XhoI and SpeI, and
cloned into the binary vector pTA7002 (Aoyama et al., 1997).
The recombinant
plasmid containing the AtPES RNAi construct was introduced into Agrobacterium
tumefaciens (C58C1), and transformed into Arabidopsis (Col-0 ecotype) using
Agrobacterium-mediated transformation.
Transgenic plants were selected on growth
media containing hygromycin (30 mg/L).
For induction of RNAi, dexamethasone
(Sigma) was added to the medium to a final concentration of 10 μM in ethanol (0.033%)
and tween 20 (0.01 % w/v) from 30 mM stock solution.
Generation of DEX-inducible NbPES RNAi BY-2 cell lines
A 300 bp NbPES cDNA fragment was PCR amplified using the BY2 PESi-S-F/R primer
set containing XhoI and ClaI sites for the sense construct, and BY2 PESi-AS-F/R
primer set containing BamHI and SpeI sites for the antisense construct. The sense and
antisense constructs of the 300 bp cDNA regions were cloned into the 5' and 3' arms of
the intron in the pSK-int vector (Guo et al., 2003) to produce an inverted-repeat
sequence (antisense-intron-sense), which was isolated by digestion with XhoI and SpeI,
and cloned into pTA7002 vector (Aoyama et al., 1997).
2
The recombinant plasmid
containing the NbPES RNAi construct was introduced into Agrobacterium tumefaciens
(GV3101), and transformed into tobacco BY-2 cells using Agrobacterium-mediated
transformation (Nakashima et al., 2000).
Transgenic BY-2 cells were selected on
media containing hygromycin (50 mg/L) and maintained at 25°C in the dark with
weekly subculture in modified Linsmaier and Skoog medium (Banno et al., 1993)
containing hygromycin (50 mg/L). For induction of RNAi, dexamethasone (Sigma) was
added to the medium to a final concentration of 10 μM from 30 mM stock solution.
Bimolecular fluorescence complementation (BiFC)
BiFC analyses were performed as described by Ahn et al. (2011). The coding region of
AtPES, BOP1, and WDR12 was PCR-amplified and cloned into the pSPYNE vector
containing the N-terminal region of YFP (amino acid residues 1-155).
Similarly,
AtPES, BOP1, and WDR12 cDNAs were cloned into pSPYCE vector containing the Cterminal region of YFP (residues 156–239). The pSPYNE and pSPYCE fusion
constructs were agroinfiltrated together into the leaves of 3-week-old N. benthamiana
plants as described (Walter et al., 2004). After 48 h, protoplasts were generated and
YFP signal was detected using a confocal laser scanning microsope (Zeiss LSM510).
Real-time quantitative RT-PCR
Real-time quantitative RT-PCR was carried out using the primers in Supplementary
Table S2 as described (Lee et al., 2009). For detection of rRNA precursors containing
ITS1 and ITS2 sequences, real-time quantitative RT-PCR was performed with ITS1 (5’-
3
ggtttcgcgtatcggcatg-3’
and
5’-cgtgccggggttttgtgata-3’)
and
ITS2
(5’-
catgcggtggtgaacttgat-3’ and 5’-tgggtcatctacagcttccg-3’) primer sets.
DAB staining
H2O2 accumulation in plant tissues was detected by 3,3’-diaminobenzidine (DAB)
staining as described (Thordal-Christensen et al., 1997). Leaves of Arabidopsis
seedlings were incubated in 1 mg/mL 3,3’-diaminobenzidine Tetrahydrochloride [pH
3.8] (Sigma) for 8 h at room temperature. Then the leaves were cleared by boiling in 96%
ethanol for 10 min. H2O2 is visualized as a reddish-brown coloration.
Deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay
Leaves from AtPES RNAi Arabidopsis plants were fixed and treated with DNase-free
RNase A (30 μg/mL; Promega) and Proteinase K (20 μg/mL; Qiagen) for 5 min before
TUNEL assay. TUNEL assay was carried out according the manufacturer’s manual
(Promega), and counterstained with DAPI solution (0.1 μg/mL DAPI, 50 mM NaCl, 5
mM EDTA, 10 mM mercaptoethylamine, and 10 mM Tris-HCl [pH 7.4]) for 5-10 min
before examination with confocal microscopy (Zeiss LSM510). For a positive control,
leaves from the ethanol-treated RNAi plants (-DEX) were treated with RQ1 RNase-Free
DNase (Promega) for 15 min at 37oC before TUNEL assay according the manual
(Promega).
Transmission electron microscopy
4
Tissue sectioning and transmission electron microscopy were carried out using the
fourth or fifth leaf above the infiltrated leaf of the VIGS lines as described by Ahn et al.
(2004).
Co-immunopreciptation
Co-immunopreciptation was performed as described (Ahn et al., 2011). Flag-tagged
AtPES and BOP1, HA-tagged WDR12, and Myc-tagged BOP1 were co-expressed in
combination in N. benthamiana leaves by agroinfiltration. After 48-h incubation, leaf
extracts were prepared in 300 μL immunoprecipitation buffer (25 mM Tris [pH 7.5],
150 mM NaCl, 1 mM DTT, 1 mM CaCl2, 5 mM NaF, 25 mM Na3VO4, 1 mM PMSF, 25
mM glycerophosphate, protease inhibitor cocktail, 0.1 % Triton X-100, and 0.5 %
NP40). After brief centrifugation to remove cell debris, protein extracts (1 mg) were
incubated with 1 μg of monoclonal mouse anti-HA antibody (ABM; IP grade) or 1 μg of
monoclonal mouse anti-Myc antibody (ABM; IP grade) at 4°C for overnight, and then
25 μL packed volume of Protein A-agarose resin (Pharmacia) was added to the mixture
for further incubation at 4°C for 4 h. After brief centrifugation, the resin was washed 3
times with the immunoprecipitation buffer, and then resuspended in 2X SDS sample
buffer. After boiling, the resin was precipitated by brief centrifugation, and the
supernatant was run on 10% SDS-PAGE. Western blotting was carried out using the
monoclonal anti-Flag antibody (ABM; 1:10,000), monoclonal anti-Myc antibody (ABM;
1:10,000), and monoclonal anti-HA antibody (ABM; 1:10,000).
5
Incorporation of 35S-labelled methionine
7 day-old Arabidopsis seedlings of AtPES RNAi lines were transferred to media with
ethanol or DEX (10 μM) for further growth for 4 days. Using 35S-labelled methionine,
newly synthesized proteins were detected as described (Lageix et al., 2008).
Sucrose gradient sedimentation
Sucrose gradient sedimentation was performed as described (Kwon and Cho, 2008). 0.2
g of fine frozen plant tissue powder were homogenized in 1 ml PEB buffer (40 mM
Tris–HCl [pH 8.0], 20 mM KCl, 10 mM MgCl2, 100 mg/ml heparin, 100 μg/ml
chloramphenicol, 25 μg/ml cycloheximide). Cell debris was removed by centrifugation
at 16,000 g for 15 min at 4 oC, and 0.5 ml of the supernatant was loaded onto a 4.4 ml
10-50 % sucrose cushion in PEB buffer, and then centrifuged at 45,000 rpm for 65 min
at 4 oC using a SW 55Ti rotor (Beckman). Fractions of 400 μl each were collected and
30 μl of each selected fraction was analyzed by SDS-PAGE and immunoblotting.
Immunoblotting was performed according to the manufacturer’s instructions using
monoclonal mouse antibodies against the HA tag (1:10,000 dilution; ABM), the Flag tag
(1:10,000; ABM), and the 60S ribosomal protein L10a (1:10,000; Santa Cruz), and then
with horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (Promega) and
ECL chemiluminescence kit for detection (GE Healthcare Life Sciences).
Metabolic labeling of rRNA
6
Metabolic labeling of rRNA was performed according to Lange et al. (2011). Equal
fresh weights (100 mg each) of wild-type and AtPES RNAi seedlings were incubated
for 4 h with 500 μCi [α-32P]-UTP that was diluted in 1 ml sterile water. Total RNA was
extracted with a SpectrumTM Plant Total RNA kit (Sigma), according to manufacturer
instructions. Uptake of the radioactive UTP was estimated by scintillation counting of
crude extracts. RNA samples, separated by agarose gel electrophoresis, were blotted on
Hybond NX membranes (Amersham) and analyzed with a phosphorimager (Molecular
Dynamics).
Ribosome profiling
Ribosomes were isolated from N. benthamiana leaves as described by Ahn et al. (2011).
Frozen tissues were homogenized in 2 mL PEB (200 mM Tris-HCl (pH 9.0), 200 mM
KCl, 25 mM EGTA, 36 mM MgCl2, 5 mM dithiothreitol, 50 mg/mL cycloheximide, 50
mg/mL chloramphenicol, 0.5 mg/mL heparin, 1% Triton X-100, 1% Tween 20, 1%
Brij-35, 1% deoxycholic acid, 1% Igepal CA-630, and 2% polyoxyethylene) per mL of
tissue. Cell debris was removed by centrifugation at 16,000 g for 15 min at 4°C, and the
supernatant was loaded onto an 8-mL 1.6 M sucrose cushion and centrifuged at 170,000
g for 18 h. Pellets were resuspended in 700 μL R buffer (200 mM Tris-HCl [pH 9.0],
200 mM KCl, 25 mM EGTA, 36 mM MgCl2, 5 mM DTT, 50 mg/mL cycloheximide,
and 50 mg/mL chloramphenicol) and incubated at 4°C for 1 h, as described by Zanetti
et al. (2005). The ribosomes were layered onto an 11-mL 15%–60% sucrose gradient
and centrifuged at 170,000 g for 3 h at 4°C, and the light absorbance of each fraction
was measured at 254 nm with a fraction collection system.
7
Immunofluorescence
Preparation of BY-2 cells and immunofluorescence were carried out as described by
Lee et al. (2009). For double-labeling of Flag and α-tubulin, fixed and permeabilized
BY-2 cells that express NbPES:Flag fusion proteins were immunolabeled with a 1:500
dilution of anti-Flag (IgG3) antibodies (Applied Biological Materials) and a 1:1000
dilution of anti-α-tubulin (IgG2a) antibodies (DM1A; Sigma). Next, the cells were
incubated with a 1:1000 dilution of IgG3-specific Alexa Fluor 593-conjugated antimouse IgG antibodies (Molecular Probes); and a 1:1000 dilution of IgG2a-specific
Alexa Fluor 488-conjugated anti-mouse IgG antibodies (Molecular Probes). Finally,
cells were stained with 0.2 µg/mL DAPI and observed under a confocal laser scanning
microscope (Carl Zeiss LSM 510).
Statistical Analyses
Two-tailed Student’s t-tests were performed using the Minitab 16 program (Minitab Inc.;
http://www.minitab.com/en-KR/default.aspx) to investigate the statistical differences
between the responses of the samples. Significant differences between control and
other samples were indicated by one (P ≤ 0.05) or two (P ≤0.01) asterisks.
8
SUPPORTING REFERENCES
Ahn, C.S., Han, J.A., Lee, H.S., Lee, S. and Pai, H.S. (2011) The PP2A regulatory
subunit Tap46, a component of the TOR signaling pathway, modulates growth and
metabolism in plants. Plant Cell 23, 185-209.
Ahn, J.W., Kim, M., Kim, G.T., Lim, J.H. and Pai, H.S. (2004) Phytocalpain controls
the proliferation and differentiation fates of cells in plant organ development. Plant J.
38, 969–981.
Aoyama, T. and Chua, N.-H. (1997) A glucocorticoid-mediated transcriptional
induction system in transgenic plants. Plant J. 11, 605-612.
Banno, H., Hirano, K., Nakamura, T., Irie, K., Nomoto, S., Matsumoto, K. and
Machida, Y. (1993) NPK1, a tobacco gene that encodes a protein with a domain
homologous to yeast BCK1, STE11, and Byr2 protein kinases. Mol. Cell Biol. 13,
4745–4752.
Guo, H.-S., Fei, J-F., Xie, Q. and Chua, N-H. (2003) A chemical-regulated inducible
RNAi system in plants. Plant J. 34, 383 –392.
Kwon, K.C. and Cho, M.H. (2008) Deletion of the chloroplast-localized AtTerC gene
product in Arabidopsis thaliana leads to loss of the thylakoid membrane and to
seedling lethality. Plant J. 55, 428-442.
Lageix, S., Lanet, E., Pouch-Pélissier, M.N., Espagnol, M.C., Robaglia, C., Deragon,
J.M. and Pélissier, T. (2008) Arabidopsis eIF2α kinase GCN2 is essential for growth
in stress conditions and is activated by wounding. BMC Plant Biol. 8, 134.
9
Lee, J.Y., Lee, H.S. Wi, S.J. Park, K.Y., Schmit, A.C. and Pai, H.S. (2009) Dual
functions of Nicotiana benthamiana Rae1 in interphase and mitosis. Plant J. 59,
278–291.
Nakashima, K., Shinwari, Z. K., Sakuma, Y., Seki, M., Miura, S., Shinozaki, K.
and Yamaguchi-Shinozaki, K. (2000) Organization and expression of two
Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydrationand high-salinity-responsive gene expression . Plant Mol. Biol. 42, 657–665.
Thordal-Christensen, H., Zhang, Z.G., Wei, Y.D. and Collinge, D.B. (1997)
Subcellular localization of H2O2 in plants: H2O2 accumulation in papillae and
hypersensitive response during the barley- powdery mildew interaction. Plant J. 11,
1187–1194.
Voinnet, O., Rivas, S., Mestre, P. and Baulcombe, D. (2003) An enhanced transient
expression system in plants based on suppression of gene silencing by the p19
protein of tomato bushy stunt virus. Plant J. 33, 949–956.
Walter, M., Chaban, C., Schütze, K., Batistic, O., Weckermann, K., Näke, C.,
Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., Harter, K. and Kudla, J.
(2004) Visualization of protein interactions in living plant cells using bimolecular
fluorescence complementation. Plant J. 40, 428-438.
Zanetti, M.E., Chang, I.F., Gong, F., Galbraith, D.W. and Bailey-Serres, J. (2005)
Immunopurification of polyribosomal complexes of Arabidopsis for global analysis
of gene expression. Plant Physiol. 138, 624–635.
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