Of the 21 independent transgenic lines, four lines (C, D, L, M), showing strong hygromycin and GUS expression, were chosen for further study. Six plants of each line and six nontransgenic control plants were agro-infected using full-length SACMV DNA-A and DNA-B head-to-tail infectious dimers (24). A further six nontransgenic control plants were agro-infected with wild-type Agrobacterium. In order to quantify the levels of viral DNA-B molecules, expressed BC1 from replicating SACMV DNA-B was measured by absolute real-time quantitative PCR (qPCR). Twenty-three days postinfection (dpi) (and 28 dpi for wild-type control plants), total RNA was isolated from 6–8 leaf stage T1 transformants using TRI Reagent (Sigma-Aldrich) and treated with RNAase-free DNase A (Fermentas). cDNA was synthesized from 30 µg DNase-treated total RNA (quantified on a Nanodrop spectrophotometer; Nanodrop) (iScript cDNA synthesis kit; Bio-Rad Laboratories). Absolute quantitative PCR of the BC1 transcript was performed on 2 µL (0.1 ng) cDNA using primers BC1 F (5'-TACGATAACCGACCCAGTTGCGTT-3') and BC1 R (5'-TGCGACTCAA AGGCCGATGTATGA-3') to amplify a 100-bp region situated upstream of the BC1 IR-target region (LightCycler FastStart DNA MasterPLUS SYBR Green I; Roche Applied Science). The number of BC1 transcript molecules present in each cDNA sample was calculated using the equation:
where x is the amount of BC1 cDNA present in grams, calculated from a standard curve. A standard curve was generated from dilutions (10 ng/µL to 1 pg/µL) of SACMV DNA-B in pBluescript spiked into healthy total nucleic acid (TNA) (29) from N. benthamiana. Real-time qPCR on extracted TNA (20 µg/mL RNase-treated; Fermentas) and quantified on a Nanodrop 1000 spectrophotometer (Nanodrop) from challenged and unchallenged transgenic plants was also performed using AC1 primers [AC1 F (5'-GCACAAGCGTCGAT-3') and AC1 R (5'-CTGCCAGTATGCTT-AACGTCA-3')] to quantify the absolute amount of SACMV DNA-A molecules, using the same approach as described above, but no RT-PCR was required.Particle bombardment of N. benthamiana callus tissue with SACMV AC1 mismatched and nonmismatched IR constructs
In order to compare the efficiency of SACMV knockdown between mismatched and nonmismatched IR constructs, a N. benthamiana callus-DNA bombardment system was performed, using a 193-nucleotide AC1 fragment derived from SACMV DNA-A (30). The mismatched AC1 IR construct was designed as described for BC1 above, and the nonmismatched AC1 IR construct was designed using the pHannibal system (19, 30). Both the mismatched and nonmismatched IR inserts in pART7 vector were excised together with the CaMV 35S promoter and ocs 3'-terminator sequences to create minimal gene expression cassettes. The Helios Gene Gun System (Bio-Rad Laboratories) was used to co-bombard either of the minimal gene expression cassettes or pART7 only minimal cassette (CaMV 35S:MCS:ocs 3'; control; 0.25 µg/shot) together with challenging virus DNA [SACMV DNA-A and DNA-B infectious dimers in pBIN19 (24); 0.15 µg DNA-A/shot; 0.15 µg DNA-B/shot] into the callus tissue. All cartridges were prepared according to Helios Gene Gun System instruction manual. Four sets of eight calli each were bombarded once for each minimal gene expression cassette using 200 psi helium pressure under sterile conditions. After bombarding, calli were incubated for 16 h at 25°C in the dark, following which they were transferred to callus medium, pH 5.8 (MS medium plus 1% sucrose and 0.5% agar, supplemented with 50 µM picloram; Sigma-Aldrich), and incubated at 28°C for a 12-h photoperiod in a growth chamber (Binder GmbH, Tuttlingen, Germany) for a further 4 days. DNA was isolated from bombarded callus tissue using a method adapted from Doyle and Doyle (1987) (29), quantified, and DpnI-treated to digest input viral DNA (30).
The absolute amount of replicating viral DNA present in bombarded callus tissue 4 days postbombardment was quantified in duplicate by absolute qPCR of a region of the AV1 ORF of SACMV DNA-A using a LightCycler 1.5 Instrument (Roche Applied Science) with SYBRGreen fluorescent dye (LightCycler FastStart DNA MasterPLUS SYBR Green I kit; Roche Applied Science, Mannheim, Germany).Reactions were set up according to manufacturer's instructions, to which 6 µL purified DNA and 2 µM each PCR primer (AV1F: 5'-GCACAAGCGTCGAT-3'; AV1R: 5'-CTGCCAGTATGCTT-AACGTCA-3') were added. The following PCR conditions were used to amplify the 154-bp fragment: 5 min at 95°C, and 40 cycles of 5 s at 95°C, 10 s at 60°C, and 10 s at 72°C.