2.1 Plant materials and virus inoculation
C. chinense N.J. Jacq. PI159236 (L3L3) and various N. benthamiana transgenic plants were maintained in growth chambers at 32°C with 16 hrs of photoperiod, light intensity of 8000 lx and 70% relative humidity. For viral inoculation, first pair of the developed leaves from the plants was mechanically inoculated with purified virions at a concentration of 50µg/mL in inoculation buffer (0.02 M sodium phosphate buffer, pH 7.0), using carborundum as an abrasive. At 7 days post inoculation (dpi), samples were taken from inoculated and systemic leaves, for 14 and 21 dpi, samples from systemic leaves and for 28 dpi, samples were collected from the asymptomatic (recovered leaves) along with the symptomatic leaves.
2.2. Viral strain, purification, and viral RNA extraction
Pepper mild mottle virus - Italian strain (PMMoV-I), reported earlier, was used in this work (Wetter et al. 1984). The protocols for purification of virion and viral RNA extraction were as enumerated earlier (Alonso et al. 1991; García-Luque et al. 1990).
2.3. Isolation of CcGRXS12 and sequence analysis
Previous work in our lab through “mRNA differential PCR”, identified the Capsicum chinense (L3L3) (PI 159236)-derived transcript corresponding to GRX gene when the plants were infected with compatible (PMMoV-I) and incompatible (PMMoV-S) viral strains. The complete sequence of the gene was obtained through RACE-PCR (Chenchik et al. 1998). The chloroplast targeting region of the protein was predicted by chloroP 1.1 programme (Emanuelsson et al. 1999). Protein sequence alignment was performed by Clustal W program (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
2.4. Measurement of oxidoreductase activity
The purified native protein was used to determine the GSH-disulfide oxidoreductase activity in HED assay (Holmgren and Aslund1995). To express the protein in prokaryotic system, the truncated CcGRXS12 protein (63 amino acids in length) tagged with 6X His-tag at its N-terminal region was cloned in pQE-1 vector (obtained from Dra.Maria Teresa)and the gene insert was confirmed through sequencing. E. coli M15 (pREP4) strain was transformed with the plasmid harboring the cloned gene construct. Protein expression and purification was performed by using His-select Nickel affinity gel resin column (Sigma Aldrich, USA) according to the manufacture’s instruction. GRX activity was measured as an oxidation of NADPH in a reaction comprising 1mM GSH, 0.7mM β-hydroxy ethyl disulphide (HED), 0.25 mM NADPH and 6.4µg/mL glutathione reductase in Tris-Cl pH 7.4. The reaction mixture was incubated at room temperature for 2 min then the decrease in OD at 340 nm was recorded in a spectromax micro-plate reader for 1 min at room temperature. The decrease in absorbance at 340 nm, following the addition of His-GRX protein, was recorded. Enzyme activities were normalized by correcting for the absorbance before the addition of GRX protein. One unit of activity is defined as the consumption of 1µmol of NADPH per minute calculated from the expression (ΔA340×V)/ (min× 6.2), where V is the cuvette volume in mL and 6.2 is the mM extinction coefficient for NADPH. Three independent experiments were performed at each substrate concentration, and the apparent Km value and Kcat values were calculated by non-linear regression using the program SigmaPlot 12.0.
2.5. Transgenic plants
Nicotiana benthamiana Domin transgenic plants constitutively expressing free GFP (Nb:GFP; line 11), full length GRX (Nb:GRX; line 3), full length GRX fused with GFP (Nb:GRX-GFP; line 16) and truncated GRX (63aa) fused with GFP (Nb:Δ2MGRX-GFP; line 40) were analyzed. These transgenic lines were provided by Dr. Maria Teresa Serra Yoldi (Montes-Casado et al. 2010). The constructs were driven at their N-terminal by CaMV35S promoter and have NOS terminator at its C-termini.
2.6. Plant total protein extraction
Total protein from the fresh leaf samples (1mg) were extracted in 5 μL Laemmli buffer (Laemmli 1970). Briefly, samples were heated to 95°C for 5 min followed by sonication for 5 min and then clarified by centrifugation at 20,000 g for 5 min. Total protein extracts (5 μL) were resolved in SDS polyacrylamide gels (SDS-PAGE) using 17.5% and 4.5% polyacrylamide as solving and concentrating gels, respectively according to Laemmli (1970).
2.7. Viral coat protein analysis
Total plant protein extracts (5 μL) and a viral coat protein extracts of 5, 2.5 and 1.25 ng were electrophoresed on 17.5% and 4.5% SDS-PAGE gel. The proteins were stained with Coomassie Blue R250 and PMMoV-I coat protein was quantified using a densitometer and the Quantity One 1-D analysis software (Bio-Rad, Hercules, USA).
2.8. Immunoblot analysis
Total proteins profiled in SDS-PAGE were electrotransferred onto PVDF membranes (Amersham). The membranes were initially blocked with PBST (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.2) containing 5% skimmed milk for 30 min. For the immunodetection of GRX, GFP and viral coat protein (CP) expression, the electro transferred membranes were incubated overnight at 4°C with the specific antisera of His-GRX (1/1000; raised in our lab), GFP specific polyclonal antibody (1/250; Santa Cruz Biotechnology, INC.) and PMMoV-I CP (1/1000; raised in our lab). Detection of antigen-antibody complexes was carried out with peroxidase-conjugated goat anti-rabbit IgG (GARPO) (Nordic) at 1/5000 dilution. The immunoreaction was visualized with ECL chemiluminescence kit (GE Healthcare Amersham) following manufacturer’s instructions. The enzymatic reaction produces a luminescent compound that is detected by visible light sensitive films (Hyperfilm, Pharmacia).
2.9. Protoplast infection assay
Protoplasts were obtained from the four different N. benthamiana transgenic lines as described by Ruiz del Pino et al. (2003) with minor changes. Protoplasts were washed a couple of times with ice-cold solution of 12% mannitol containing 6mM CaCl2. Protoplasts were counted and adjusted to the concentration of 1.3×106 protoplasts/mL using ice-cold electroporation buffer (12% mannitol, 6 mM CaCl2, 80 mM KCl, pH 7.2). For protoplasts infection, 4×105 protoplasts in a final volume of 300µL were taken in a 0.2cm electroporation cuvette along with 20 µg of PMMoV-I RNA and kept on ice. For transformation, a single pulse of 0.12kV and 125 μF was applied with a Gene Pulser (Bio-Rad laboratories, Hercules, CA) immediately after addition of 20 μg of viral RNA. Following the pulse, the protoplasts were kept on ice for 20 min and diluted in CPW-M13. Later, centrifuged at 80×g for 5 min and diluted to 5×105 protoplasts/mL in CPW-M13 and incubated at 25°C under dark. Protoplasts were harvested at 16, 24 and 48 hrs with a short spin of 134g, and a couple of washing in mannitol buffer and the resultant protoplast was re-suspended in 5X Lammeli buffer. The viral infection was detected by Western blot method using PMMoV-I coat protein specific antibody.
2.10. Sub-cellular localization studies of CcGRXS12 protein
For analyzing the sub-cellular localization of CcGRXS12 protein, protoplasts obtained from the different GFP expressing transgenic lines (Nb:GFP, Nb:GRX-GFP and Nb:Δ2MGRX-GFP) were fixed with 4% formaldehyde in 9% mannitol (pH 7.2). To label the nuclei, DAPI staining was performed. In brief, protoplasts (104) were incubated with 100µL of 2µg/mL DAPI solution in PBS-9% mannitol for 5 min. To that 900µl of PBS-9% mannitol was added and kept for another 15 minutes, and then the protoplasts were collected and washed with PBS-9% mannitol for twice and finally suspended in 100 µL PBS-9% mannitol. Around 20µL of this sample was loaded over poly-L-lysine coated glass slides for visualization. For compartmentalization study, autofluorescence from chlorophyll and DAPI staining at nuclei were analyzed. Different fluorescent signals were detected at specific wavelengths [GFP detection at 550nm (excitation 580nm), chlorophyll autofluoresence at 540nm (excitation 600nm) and DAPI at 610nm (excitation at 650nm)] using a TCSP Leica microscope.
2.11. RNA isolation and probe preparation
Total leaf RNA was extracted according to the method prescribed by Chomcynski and Sacchi (1987) by using TRIzol reagent and following the manufacturer’s instruction with a slight modification Briefly, additional centrifugation at 12,000 g for 10 min at 4°C after plant tissue homogenization and an additional final RNA precipitation step with 0.3 M sodium acetate pH 5.2 and 2.5 volumes of 100% ice-cooled ethanol for overnight were performed. The digoxygenin-labelled RNA probes were prepared using the linearized plasmids harbouring various gene products according to the instructions enumerated in MAXIscript kit manual. The cloned gene products, for preparing the riboprobes, were: salicylic acid pathway marker proteins (PR-1, PR-2a and PR-5), the JA/ET pathway marker protein (PR-2d) and the gene for auxin inducible Glutathione-S-Transferase (GST) (pCNT103) (obtained from Carmen Castresana), the clone pT-CPS containing the 593 bp from PMMoV-S CP (Gilardi et al. 1998)
2.12. Northern blot hybridization
For Northern blot analysis, RNA samples (10 μg) were denatured at 65°C for 4 min in MOPS-Acetate-EDTA buffer (20 mM MOPS, 15 mM sodium acetate, 3 mM EDTA pH 7.0) in the presence of 10% formamide, 0.9% formaldehyde and 0.06 mg/mL ethidium bromide. Then the samples were electrophoresed onto 1.5% agarose-formaldehyde gels containing MOPS buffer (50 mM MOPS, 0.4 mM EDTA pH 7) and 6% formaldehyde, using MOPS electrophoresis buffer under a current of 5 V/cm. Once visualized by UV light illumination, the RNAs were transferred to nylon membranes (Hybond-N, Pharmacia), as described in Sambrook et al. (1989), and fixed by irradiation with UV light (120 mJ) in a UV Stratalinker apparatus (Cultek). The membranes with transferred RNA were stored in cold condition for later use.
For hybridization with digoxigenin-labelled RNA probes, the membranes were incubated with standard buffer (5xSSC, 0.1% sodium-lauroylsarcosine, 0.02% SDS and 2% blocking reagent, blocking reagent is provided by manufacturer) for 2 hrs at 65°C. Then the membranes were hybridized overnight at 68°C with specific RNA probes in standard buffer containing 100 ng of the corresponding probe. After hybridization, the membranes were washed twice in (2x SSC solution, 0.1% SDS) for 15 min each at room temperature (RT) and twice in (0.1 x SSC, 0.1% SDS) for 15 min at 65°C. Probe detection was performed using the DIG luminescent detection kit (Roche, Penzberg, Germany) according to manufacturer’s protocol. In brief, membranes were incubated in blocking solution of maleic buffer (0.1M Maleic acid, 0.15M NaCl) containing 1% blocking reagent- for 30 min, then in antidigoxigenin alkaline phosphatase conjugated antibody at 1:10,000 for 30 min followed by washing twice in washing buffer. After equilibration in detection buffer, membranes were incubated with chemiluminescent substrate CSPD and exposed to X-ray sensitive films (Hyperfilm, Pharmacia) for 30 min.
2.13. Measurement of pyridine nucleotide (PN) contents
PN contents in the mock and PMMoV-I-infected plants at 28 dpi were analyzed by grinding 30 mg of fresh leaf sample with ball bearings and centrifuged for 12,000 g for 1min. The supernatant obtained was used for selective extraction of NAD(P)H in acid medium and NAD(P)+ in alkaline medium (Hajirezaei et al. 2002). The samples were neutralized to the final pH of 8.0 to 8.5 and made as 100µl aliquots and frozen at -80°C for later analysis. PNs in the neutralized extracts were determined following Gibon et al. (2004). Statistical difference was analysed by SAS 9.1 programme (SAS 9.1 Inc.) using one-way analysis of variance (ANOVA).
2.14. Abiotic stress tolerance assay
Role of CcGRXS12 in abiotic stress tolerance was analyzed by growing different N. benthamiana transgenic plants in one-half MS media in the presence and absence of 3mM H2O2 and 1 µM paraquat. The seedlings were grown horizontally for the first 3 days and then grown in vertical position for another one week. Root length was measured after 10 days. The experiments were performed in triplicates of 30 seedlings each and repeated thrice. The seedlings were grown at 25°C and 16 h of photoperiod. Statistical difference was analysed by SAS 9.1 programme (SAS 9.1 Inc.) using one-way analysis of variance (ANOVA).
2.15. Heterologous expression of CcGRXS12 in S. cerevisae
To clone and express CcGRXS12 mature protein in yeast cells, pGEM-T easy vector (pGEM-GRX), harbouring the full length CcGRXS12 gene was used. Gene specific primers were designed to amplify the full length gene excluding the chloroplast targeting region. Further in order to prevent codon breakage while expression in yeast, we introduced ‘g’ nucleotide (small letter) immediately after the NotI site in the forward primer (5´-ATAAGAATGCGGCCGCgTCGGGTTCATTCGGGTCC-3´) so as to introduce genetic code for alanine at the end of the mitochondrial targeting region. The reverse primer (5´- GAAGATCTGCTTTCTGTTTTTCCAGGATTA-3´) had an overhang of Bgl II site. The amplified fragment was cloned in to pMM221 vector (obtained from Dr.E.Herrero, Universitat de Lleida, Lleida, Spain) which contains the yeast mitochondrial targeting sequence at its 5’ end and 3HA/His6 tag at its 3’ end (Molina et al. 2004). The resultant plasmid containing the CcGRXS12 gene sequence was transformed into yeast Δgrx5 mutants and the selection of the transformants were performed following Rodriguez-Manzaneque et al. (1999).
2.16. Growth conditions of S. cerevisae
The yeast strains employed in this study namely wild type (W303-1B; WT), Δgrx5 mutant (MML100; Δgrx5) and yeast GRX5 expressing in Δgrx5 mutants (MML240; Δgrx5/GRX5) were already reported inRodriguez-Manzaneque et al. (1999) and the CcGRXS12 transformed in yeastΔgrx5mutant strain (Δgrx5/CcGRXS12) (in this work). All yeast strains were grown in YPD, respiratory defective YPG and SC media as mentioned in Rodriguez-Manzaneque et al. (2002). For oxidant sensitive assay, above mentioned yeast strains were grown in the media containing 0.3mM tert-butyl hydroperoxide (t-BOOH) and 1.25mM diamide (Sigma Aldrich, USA) in a serial dilution of 1:10 and incubated for 3 days.
2.17. CcGRXS12 in Fe-S cluster assembly mechanism
Role of CcGRXS12 in Fe-S cluster assembly mechanism was studied by analysing the accumulation level of free iron (Fish 1988), and also by measuring the relative ratio of aconitase to malate dehydrogenase activity (Robinson et al.1987) for the aforementioned yeast strains.