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URL : https://hal.archives-ouvertes.fr/hal-01332807

, S1Complementation of the upf 1-5 line with UPF1-SF. mRNA level of SMG7, AT5G45430 and AT1G36730 NMD endogenous substrates in Col-0, upf1-5, and upf1-5 complemented with UPF1-STREP-FLAG (UPF1-SF-tag) in 14-days-old seedlings by RT-qPCR. The expression was normalized to ubiquitin mRNA and expressed relative to Col-0. Values represent a mean of three independent biological replicates with standard deviations shown by error bars

. *p-<,

, PDS-DDX5-and PDS-DDX6-co-silenced or WT N. benthamiana (non-VIGS) leaves, measured by RT-qPCR. The level of PDS mRNAs in PDS-and DDX/PDS-silenced plants are expressed relative to the corresponding values in the WT control arbitrarily set as 1. (B) VIGS silencing of N. benthamiana DDX3, DDX5 and DDX6 homologs (see Table S2, Supplementary Table S3, S4) in photobleached plants measured by RT-qPCR. For DDX3 and DDX6 all N. benthamiana homologs were tested, whereas for DDX5 four N. benthamiana homologs, which correspond to Arabidopsis RH14 and RH46 helicases, were tested. For each DDX helicase one pair of primers recognize mRNAs of two N. benthamiana homologs. The level of DDX mRNAs in DDX-PDS-silenced plants are expressed relative to the corresponding values in PDS-silenced controls arbitrarily set as 1, Supplementary Fig. S2 Silencing efficiency of DDX helicases in VIGS plants (A) The level of N. benthamiana PDS mRNA in PDS-silenced, PDS-DDX3

. *p-<,

. **p,

. ***p-<-0, UBC9 mRNA was used as a reference. Supplementary Fig. S3 Sequence alignment of plant, human and yeast DDX3, DDX5 and DDX6 helicases

R. Rh6, , vol.6

R. ,

. Dhh1, , vol.6

, Residues conserved in all homologs are highlighted in black and less conserved in grey. The alignment was created using Clustal Omega and presented using GenDoc. Supplementary Fig. S4 NMD efficiency in single RH11-and RH52-or DDX5 group VIGS-silenced N

, DDX5-silenced (B) N. benthamiana leaves that were co-infiltrated with P14 silencing suppressor and G95 NMD-insensitive or G600 NMD-sensitive substrates. PDS-silencing (PDS) and UPF1-PDS co-silencing (UPF) were used as a negative and positive control, respectively. Northern blots (left panels) showing the mRNA level were performed using GFP and P14 probes, VIGS-NMD assay in RH11-and RH52-silenced (A) or

. *p-<,

, A-C) VIGS-NMD assay in (A) DDX3-, (B) DDX5-or (C) DDX6-silenced N. benthamiana leaves that were co-infiltrated with P14 silencing suppressor and G95 NMD-insensitive or G600 NMD-sensitive substrates. PDS-silencing (PDS) and UPF1-PDS co-silencing (UPF) were used as a negative and positive control, respectively. (D) The level of reporter G95 and G600 substrates in RH12-overexpressing N. benthamiana leaves that were co-infiltrated with RH12 or U1DN constructs. Overexpression of UPF1 dominant negative form (U1DN) leads to inactivation of NMD and is used as a positive control

, Western blotting of (A) RH6, (B) RH8 and (C) RH12 proteins in extracts from 14-days-old seedlings of Col-0, rh6-1, rh8-1 and rh12-1, respectively, using specific antibodies against these helicases. For each helicase antibodies obtained from Eurogentec using two different specific peptides were used. For RH6 and RH12 both peptides recognized a specific protein of an expected size (in kDa), whereas only peptide 2 gave a specific signal for RH8. Schematic representation of RH6, RH8 and RH12 genes (on the right of the Western blot for each helicase, Supplementary Fig. S6 Expression of RH6, RH8 and RH12 in rh6-1, rh8-1 and rh12-1 mutants

, Western blot analyses of RH6, RH8 and RH helicases steady-state levels in protein extract from plants at indicated developmental stages and from various tissues. Specific antibodies against each helicase and UGP control were used as described for Supplementary Fig. S6. Supplementary Fig. S8 Interaction between RH12 helicase from the DDX6 family and NMD factors, UFP1 and UFP3. Western blot analysis of co-immunoprecipitation of RH12-GFP with UPF1-HA or UPF3-HA from 21-days-old N. benthamiana leaves expressing the combination of infiltrated constructs (as indicated) expressing encoding RH12-GFP, UPF1-HA and UPF3-HA. Input (left panels) and immunoprecipitaes (right panels) were separated on SDS-PAGE and proteins were detected by chemiluminescence using anti-HA peroxidase and anti-GFP peroxidase antibodies

, UPF3-YFP (middle panel) and RH12-CFP (DDX6) (lower panel); overlay and red fluorescence of chloroplasts (right panels), Supplementary Methods S1. Plasmid constructs The plant binary vector pGWB604-C-SF-TAP was constructed as described (Golisz et al. 2013) using pDEST/C-SF-TAP (Gloeckner et al. 2007) and pGWB604 vectors, 2009.

, The UPF1 promoter was cloned into XbaI site of the pGWB604-C-SF-TAP vector and UPF1 cDNA into SalI and EcoRV sites of the pENTR1A. These constructs were used for LR recombination reactions (Invitrogen). The plasmid containing the UPF1 sequence with the endogenous promoter was a kind gift from

. Kertész, For localization in N. benthamiana and Arabidopsis protoplasts UPF1, UPF3, RH11 proteins were fused to protoplasts localization-optimized variants of cyan or yellow fluorescent protein (CFP or YFP). Constructs for localization were obtained by LR recombination of pGD vector (Kremers et al. 2006) with pENTR1A plasmid carrying UPF1 or UPF3 cDNAs (a kind gift from John W. S. Brown) or pENTR1A carrying RH11, Plasmids with G-95 and G-600 constructs, formerly named as GFP and GFP-abc, respectively, were described in, 2006.

. Kertész, N. benthamiana homologs of the Arabidopsis DDX3-, DDX5-and DDX6-group helicases were identified using the Solgenomic's BLASTn tool and the predicted cDNA database, VIGS-constructs were cloned into the Bin61S agroinfiltration vector or into the derivatives of Bin61S. P14, Bintra, TRV-PDS, TRV-PDS-UPF1 clones were described previously, 2006.

, In the case of DDX3 two VIGS fragments, derived from genes Niben101Scf02006g03006 and Niben101Scf05711g00004 were required for the sufficient silencing effect. Two VIGS fragments from Niben101Scf02402g01001 gene, three from Niben101Scf06442g00013, Niben101Scf08873g01020 and Niben101Scf33352g00001 genes and one from Niben101Scf02402g01001 gene were used for efficient silencing of N. benthamiana DDX3-, DDX5-and DDX6-group helicases, Solgenomics's VIGS tool to identify the homologous regions within similar genes

. Golisz, 2013) with minor modifications. 30 g of 2-week-old seedlings was cross-linked two times for 10 min by vacuum infiltration with 1% formaldehyde in PBS, followed by addition of glycine to a final concentration of 80mM. Seedlings were frozen in liquid nitrogen, ground in a laboratory blender (Waring) with dry ice (10x30 s), mixed with an equal amount (w/v) of Extraction buffer (100mM Tris-HCl, pH 8.0, 150mM NaCl, 2mM EDTA, 2mM DTT, 1mM PMSF, 0.5% Triton X-100 and protease inhibitor cocktail) and sonicated on ice using 4x30 s bursts (Bioruptor). The homogenate was centrifuged at 20,000g for 40 min at 4ºC and the supernatant was centrifuged at 35,000g for 1 h at 4ºC. The supernatant was concentrated approximately four times by dialysis against PEG, All primers used in this study are listed in Supplementary Table S5. Affinity protein purification UPF1-SF-TAP was purified as described, p.1, 20000.

P. Edta, Protein extract was supplemented with 250

, The flow-through fraction was separated by centrifugation for 1, mg/ml RNase A (Qiagen) and incubated for 3 h at 4ºC with slow rotation with 200ml anti-FLAG-M2 resin pre-washed in Wash buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1mM EDTA, 1mM DTT, 0.1% Triton X-100)

, Bound proteins were eluted overnight by incubation with 250 mg/ml of the FLAG peptide in TBS at 4ºC. The eluate was concentrated to a final volume of 20 ml using SpeedVac (Christ)

, ml, reduced with 10mM DTT for 30 min at 56ºC and alkylated with 50mM iodoacetamide for 45 min in the dark at room temperature. The alkylating agent was eliminated using 50mM DTT

, Protein solutions were subjected to a standard procedure of trypsin digestion, during which proteins were reduced with 0.5 M (5 mM f.c.) TCEP for 1 h at 60°C, blocked with 200mM MMTS (10mM f.c.) for 10 min at room temperature and digested overnight with 10 ul of 0.1 ug/ul trypsin. The resulting peptide mixtures were concentrated and desalted on a RP-C18 pre-column (Waters, Milford, MA), and further peptide separation was achieved on a nano-Ultra Performance Liquid Chromatography (UPLC) RP-C18 column (Waters, BEH130 C18 column, 75 µm i.d., 250 mm long) using ACN gradient (0-35% ACN in 160 min) in the presence of 0.1% FA at a flow rate of 250 nl/min. The column outlet was coupled directly to the ion source of the Orbitrap Velos mass spectrometer (Thermo Electron Corp

, To reduce mass errors, the peptide and fragment mass tolerance settings were established separately for individual LC-MS/MS runs after a measured mass recalibration (Malinowska et al., 2012), resulting in values 8ppm for parent and 0.01Da for fragment ions. The ramaining search parameters were as follows: enzyme, Trypsin; missed cleavages, 1; fixed modifications, Methylthio (C); variable modifications, Oxidation (M); instrument, HCD. The statistical significance of peptide identifications was estimated using a joined target/decoy database search approach. This procedure provided q-value estimates for each peptide spectrum match (PSM) in the dataset. All PSMs with q-values >0.01 were removed from further analysis. Proteins identified by a subset of peptides from another protein were excluded from analysis. The mass calibration and data filtering were carried out with MScan software, The raw files were pre-processed with Mascot Distiller software (v. 2.6, MatrixScience, London, UK) and a search was performed with the Mascot Search Engine (MatrixScience, pp.61-81

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