Supplementary Materials and methods

Schepetilnikov et al.

Plasmid construction

The construct pbiGUS was describe in Bonneville et al. (1989). Plasmids pGEX-RISP, pGFP, pTAV, pGAD-eIF3g, pGAD-RISP, pGBK-TAV, pGBK-MAV (aa 116-242) were described previously (Thiebeauld et al, 2009).

            TAV deletion mutants (TAVDMAV; aa 116-242, TAVDdsR; aa 136-182, TAVDKKF and TAVDNGP) fused to the BD-domain in the pGBKT7 vector (Clontech) were produced by deletion mutagenesis of the pGBK-TAV. PCR products corresponding to TOR, NTOR and CTOR were ampliphied from TOR cDNA (At1G50030) with pairs of specific primers and cloned into the pGEM-T vector system. pGAD-TOR and the pGAD-NTOR were obtained by subcloning TOR and NTOR fragments into the pGADT7 vector as in-frame fusion with the AD-domain. PCR product corresponding to S6K1 was ampliphied from S6K1 cDNA (At3G08730) with pairs of specific primers and cloned into the pGADT7 as in-frame fusion with the AD-domain and into pGEX6P1 as in-frame fusion with the GST-domain to obtain pGAD-S6K1 and pGEX-S6K1 respectively. pTOR and pRISP was produced by subcloning the TOR or RISP coding sequence under the control of 35S promotor into the pGFP vector in order to replace the GFP coding region. The pTOR (D2247A), pGAD-RISP (S267A), pGEX-S6K1 (T449A), pGEX-RISP (S267A) and pRISP (S267A) mutants were prepared by site-directed mutagenesis using DpnI-cloning protocol. pBin-GFP and pBin-RFP were done on the base of the pBin-61 binary vector by introducing GFP and RFP coding regions, respectively, upstream AscI and PacI restriction sites. These restriction sites were used to introduce NTOR and CTOR coding regions into the pBin-GFP (pBin-GFP-NTOR and pBin-GFP-CTOR) and the TAV coding region into the pBin-RFP (pBin-RFP-TAV).

Protein expression, purification and antibodies

PCR products corresponding to CaMV TAV and Arabidopsis RISP, RISP-S267A mutant, S6K1, S6K1-T449A mutant, eIF3g, full-length TOR, and its N-terminal (1-1449 aa) and C-terminal (1450-2481 aa) domains were inserted into pGEX-6 as in-frame fusions with the GST-domain. GST was removed by on-column cleavage with PreScission protease (GE Healthcare).

Rabbit polyclonal antibodies anti-eIF3g and anti-TOR were raised against the full-length recombinant protein AteIF3g or the AtTOR N-terminal polypeptide, respectively. Rabbit polyclonal anti-eIF3c antibodies were a kind gift from Prof. K. Browning (University of Texas, Austin, USA). Rabbit polyclonal antibodies raised against RISP, TAV and CaMV coat protein (CP; anti-RISP, anti-TAV and anti-CP respectively) were described previously (Thiebeauld et al, 2009).

Yeast two-hybrid assay

Constructs containing wild type and deletion mutants fused to GAL4 BD- and AD-domains were co-transformed into AH109 cells. Protein interaction was scored on selective medium followed by highly stringent selection with 5 mM 3-aminotriazol (3-AT).

b-galactosidase activity assay

To verify and quantify yeast two-hybrid interactions the liquid culture b-galactosidase test was performed according to the original Clontech protocol using ONPG (o-nitrophenyl β-D-galactopyranoside) as a substrate. The relative enzyme activity was represented in percentage. The values given are the means from more than three independent experiments.

Western blot assay

Rabbit anti-ARF polyclonal antibodies were a kind gift from C. Ritzenthaler (IBMP, Strasbourg, France). Polyclonal Phospho-(Ser/Thr)Akt substrate (RxRxxS/T) antibody for RISP-P detection was from Cell Signaling Technology. Polyclonal anti-TOR, anti-S6K1, anti-TOR-P-S2448 and anti-S6K1-P-T389 antibodies directed against human TOR or S6K1, or their phosphorylated form respectively were from Santa-Cruz Biotechnology. Anti-GST antibody fused to the horse-reddish peroxidase was from Sigma.

After SDS–PAGE, gels were transferred to polyvinylidene difluoride membrane PVDF with a Trans-Blot electrophoretic transfer cell (Bio-Rad). Pre-blocking was done with 1% BSA dissolved in 1.0% Tween20/PBS, and the membrane was incubated with primary antibodies (1:500 diluted in 1% BSA, 1% Tween20/PBS). After a series of washes the membrane was incubated with HRP-conjugated secondary anti-rabbit or anti-goat IgG antibodies (Sigma; 1:1000 diluted in 1% BSA, 1% Tween20/PBS).

Plants growth conditions and viral infection

The Arabidopsis T-DNA line G548 was a kind gift of Christian Meyer (Versailles, France).  The RNA interference (RNAi) line 35-7 was described previously (Deprost et al. 2007). The P6 and P6DdsR transgenic lines AT7 and AT7DdsR respectively were described (Haas et al, 2008). Plants were grown in the greenhouse under the standard conditions.

To obtained CaMV infection Arabidopsis plants were mechanically inoculated with CaMV Cabb-J1 isolate 4-5 weeks after germination. In the case of TOR-deficient line 35-7 we selected plants carrying serrated and pointed leaves (with very low or not detectable TOR levels according to qPCR and Western blot (Fig. 4C). Plant samples were collected at 0, 7, 10, 14, 17, 21 dpi and analyzed by Western blotting using anti-TAV and anti-CP antibodies.

Agroinfiltration protocol

For agroinfiltration Agrobacterium tumefaciens strain GV3101 was used. Bacteria carrying pBin61-based vectors were cultured overnight at 28°C with 10 mM 2-N-morpholino-ethanesulfonic acid (MES) and 20 mM acetosyringone. After overnight incubation cells were transferred to 10 mM MES (pH 5.85), 10 mM MgCl₂, 150 µM acetosyringone and incubated for 3 h at room temperature until 0.5 to 1.0 optical density. Infiltration was done into the lower leaf surfaces of young (six- to seven-leaf-stage) N. benthamiana plants with a needleless syringe. The plants were incubated during 24 h in a growth chamber at 24°C.

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Plant seedlings growth conditions

Arabidopsis seedlings were cultured at MS agar (Murashige and Skoog medium with MSMO-salt mixture; Sigma) for 14 days after germination (14 dag), harvested and ground in liquid nitrogen followed by homogenization in fresh ice-cold Extraction buffer (50 mM Tris pH 7.6, 150 mM NaCl, 0.1% NP-40, GM-132; Sigma, Complete protease inhibitors cocktail; Roche). Supernatant was used for immunoprecipitation assays or western blot detection.

TOR signalling inhibition in seedlings by Torin-1

For Torin-1 assay (see Fig. S2A) 14-dag transgenic seedlings TAV-overexpressing line AT7 were incubated in fresh MS medium with or without Torin-1 (200 nM; kindly provided by David Sabatini, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA) for 6 hours at 24°C under constant shacking (120 rpm) and light conditions. Seedlings were harvested and grinded in liquid nitrogen followed by homogenization in fresh ice-cold Extraction buffer. Supernatant was used for western blot detection with specific antibodies.

Arabidopsis protoplasts

Arabidopsis protoplasts, when indicated were prepared either from Arabidopsis suspension cell culture and transfected with plasmid DNA by PEG method (Yoo et al., 2007). The mono- (pGFP) and bi-cistronic (pbiGUS, Bonneville et al, 1989) reporter constructions were co-transfected with pRISP or its mutant pRISP-S267A as indicated in Fig. 6F.

Arabidopsis mesophyll protoplasts were prepared from 3- to 4-week-old plantlets (Col-0 and line 35-7) and transfected with plasmid DNA by PEG (Yoo et al, 2007). The mono- (pGFP) and bi-cistronic (pbiGUS, Bonneville et al, 1989) reporter constructions were co-transfected with pTAV and either pTOR or its “dead-kinase” mutant pTOR (D2247A) as indicated in Fig. 3B, Fig. 4D and Supplementary Fig. S4. After 20–24 h of incubation at 27 °C, protoplasts were harvested by centrifugation and protein extract was prepared by three cycles of freezing and thawing in 200 μl of GUS extraction buffer (50 mM NaH₂PO4 (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarcosyl). The aliquots were immediately taken for GUS and GFP reporter gene assays. GUS activity was measured by a fluorimetric assay (Pooggin et al, 2000). GFP activity was quantified using the fluorimetric assay. The values given are the means from more than three independent experiments.

Real-time RT-PCR Analyses

Total RNA from Arabidopsis protoplasts was extracted using Trizol (Invitrogen). RNA samples (2g of total RNA) were reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen). cDNA was quantified using a SYBR® Green qPCR kit (EUROGENTEC) and gene specific primers. To monitor biGUS mRNA levels primers were designed for 3`-region of GUS as followed: Forward 5`-CGGCAGAGAAGGTACTGGAA-3` and Reverse 5`-CCAGCCATGCACACTGATAC-3`. The primers for detection of TOR mRNA levels were taken from Deprost et al (2007). Transcript levels were normalized to that of actin (At3g18780) and GAPDH (At3g04120).

Semiquantitative RT-PCR Analyses

Total RNA from Arabidopsis protoplasts was extracted using Trizol (Invitrogen). RNA samples were reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen) with oligo-(dT)₁₈ primer (Fermentas). Semi-quantitative RT-PCR was performed with the pair of specific primers to full-length biGUS construct. The PCR conditions are as followed: 2 min, 94°C (first cycle); 30s, 94°C; 30 s, 56°C; 3 min, 72°C (20 cycles). PCR products were separated on a 1.2% agarose gel and visualized after ethidium bromide staining.

Detection of CaMV Replication by Reporter-targeted PCR

Principals and details of the experiments were described in Kobayashi and Hohn (2003). Turnip protoplasts were transfected with the following plasmids in the indicated combinations pE4Pin (viral clone with PCR reporter, 10 mg); pAATAV, Wild type (WT) or 3-aa deletion mutants, 4 mg; pGWGAG (monocistronic CaMV GAG), 4 mg; pGWPol (monocistronic CaMV Pol), 2 mg. The total amount of plasmid was adjusted to 20 mg with pBluescript. Three days post-transfection, protoplasts were harvested and low molecular weight DNA was extracted. Replication of CaMV DNA was assayed by reporter targeted PCR using PSS and LAS primers through 32 reaction cycles.

 GST pull down assay

PCR products corresponding to TAV, full-length TOR, its N-terminal (NTOR, aa 1-1449) and C-terminal (CTOR, aa 1450-2481) domains were inserted into pGEX-6P1 (Pharmacia Biotech) as in-frame fusions with the GST-domain.

Binding of TAV to GST-TOR, GST-NTOR, GST-CTOR and GST alone was carried out in buffer A-100 (50 mM Hepes, pH 7.5, 100 mM KCl, 3 mM MgAc₂) in 300 μl reaction mixture over night at 4°C with rotation. TAV-binding complexes were washed three times with buffer A-100. The presence of TAV, TOR and its deletion mutants in the bound fraction as well as 30 μl of the unbound fraction was analyzed by Western blot with anti-GST and anti-TAV antibodies.

In vitro kinase assay (³²P)

The assay was performed in kinase buffer (25 mM HEPES pH 7.6, 10 mM MgCl₂, 100 mM ATP) with 10 mCi gATP³² and with the aliquots of WT and AT7 extracts in the presence of recombinant S6K1, RISP and TAV as the substrates respectively. Samples were incubated at 30°C for 30 min and subsequently analyzed by SDS-PAGE.

Immunoprecipitation

Plant samples were homogenized in extraction buffer [50 mM Tris pH 7.6, 150 mM NaCl, 0.1% NP-40, GM-132 proteasome inhibitor (Sigma), Complete protease inhibitors cocktail (Roche)] and insoluble material was removed by centrifugation (30 min, 12000g, 4°C). Lysate was pre-cleaned by incubation with protein A-agarose beads (Roche) at 4°C for 30 min. The supernatant was then incubated with either Normal rabbit serum (RS, Sigma), or anti-eIF3c, or anti-TOR serum prebound to A-agarose beads overnight at 4°C. Immunoprecipitates were washed three times with the extraction buffer, eluted from the beads with 0.1 M Glycine, pH 2.5 and analyzed by Western blot.

Polyribosome isolation

For polyribosome isolation we used 7 day-old Arabidopsis Col-0 and transgenic lines 35-7, AT7 (TAV) and AT7DdsR (TAVDdsR) seedlings. After harvesting, fresh material was frozen and ground in liquid nitrogen. For the cytoplasmic extracts, 0.5 mg of powder were resuspended in the ice-cold extraction buffer (100 mM Tris-HCl, pH 8.8, 50 mM KCl, 25 mM MgCl₂, 5 mM EGTA, 15.4 units/mL^–1 heparin, 18 µM cycloheximide, 15.5 µM chloramphenicol, 0.5% NP-40, 1 mM DTT and protease inhibitors cocktail (Roche). For EDTA-treated polysomes, extraction buffer was supplemented with 50 mM EDTA. Cell debris were removed by centrifugation. Supernatants were loaded on 10 to 50% (w/w) sucrose gradients in 100 mM Tris-HCl, pH 8.8, 50 mM KCl, 25 mM MgCl₂ (supplemented with 50 mM EDTA for EDTA-treated polysomes) and centrifuged at 39000 rpm in a Beckman SW41 rotor at 4°C for 2,8 h (see Fig. 5) and 2 h (see Fig. 6F) respectively. Fractions were collected and total proteins in each fraction were precipitated with 10% TCA and analyzed by Western blot.

For polysomes dissociation experiments (Fig. 5G and H): 0.5 mg of the N₂ liquid grinded powder was resuspended in the ice-cold extraction buffer supplemented with 50 mM EDTA and incubated for 15 min on ice. Cell debris was removed by low speed centrifugation. Supernatant was loaded on 10 to 30% (w/w) sucrose gradient supplemented with 10 mM EDTA and centrifuged at 39000 rpm in a Beckman SW41 rotor at 4°C for 6 hours.

Transient expression and imaging analysis

TAV and the N- and the C-terminal domains of TOR fused to RFP and GFP, respectively, were transiently expressed in Nicotiana benthamiana epidermal cells by agroinfiltration. Fluorescence was detected using a confocal microscope Zeiss LSM510 (Jena, Germany). The quantitative colocalization analyse were performed using the NIH ImageJ software with the Colocalization Finder plugin, available at http://rsb.info.nih.gov/ij/plugins/. This software was used to determination the Rr, which describes the extent of overlap between image pairs. It is a value between –1 and +1, with –1 being no overlap and +1 being perfect overlap of two images.

Quantification of band density

To qualify the bands obtained via Western blot analysis, we applied ImageJ software based analysis (http://rsb.info.gov/ij). The area under curve (AUC) of the specific signal was corrected for the AUC of the loading control (corresponding substrate). The highest value of phosphorylation with the wild type extract was set as 100% and other conditions were recalculated.