Mutation of gene fragments and strand-specific amplification
Sodium bisulfite-mediated deamination reactions were performed on 120 ng BC1 PCR product using the EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA, USA). Reactions were performed for 5, 10, or 15 min to determine deamination efficiency. Sodium bisulfite-treated SACMV BC1 fragments were PCR-amplified using modified primers [BC1 (mod+XhoI+SpeI) F (5'-GATCCTCGAGACTAGTAAATATTCTACGGACATACG-3') and BC1 (mod +BglII) R (5'-GATCAGATCTTAGTAGCCCAATCTAAGACCTTGT-3')] (Figure 1). These primers were designed to preferentially amplify the positive strand of the sodium bisulfite-treated DNA template. In addition, they result in restriction endonuclease sites that are added on to the 3'- and 5'-ends of the PCR products.Assembly of the IR construct
Strand-specific PCR amplicons were each TA-cloned into the pTZ57R vector (InsT/A Clone PCR Product Cloning kit; Fermentas, Vilnius, Lithuania). Clones containing fragments that had been bisulfite-treated for 10 min [56% (39 of 70) C to T conversion] were screened for inserts in the sense orientation, and clones containing untreated fragments were screened for inserts in the antisense orientation by restriction endonuclease analysis. Selected clones were sequenced using the universal M13/pUC reverse primer. Clones containing the treated [pTZ57R/T-BC1* (-)] and untreated [pTZ57R/T-BC1 (+)] fragments were double-digested with ScaI/BglII and ScaI/BamHI, respectively, and resolved by agarose gel electrophoresis (Figure 1). Bands containing the appropriate restriction fragments were excised and extracted from the gel slice (MinElute Gel Extraction kit; Qiagen GmbH, Hilden, Germany). The inverted repeat was constructed by ligating (T4 DNA ligase; Fermentas) an equal ratio of the restriction fragments containing the untreated fragment to those containing the treated fragment (Figure 1). Four clones (pTZ57R/T-BC1) containing presumptive IR constructs were sequenced using both the universal M13/pUC forward and reverse primers to confirm the presence of the mismatched IR sequence and to determine the number of C to T mutations. A single clone BC1hp7 (i) containing 56% C to T conversion (total of 17% mismatched base pairs) was subsequently used for cloning into pCAMBIA 1303.Generation of IR expression cassettes and transformation of N. benthamiana
The mismatched IR construct was released from BC1hp7 by restriction digestion with XhoI and XbaI and subsequently ligated (T4 DNA ligase; Fermentas) into Xho1/Xba1-restricted pART7 vector to generate an expression cassette. The IR expression cassette was PCR-amplified from pART7 [pART7 F (5'-TTAACGTTTACAATTTCCCATTCGC-3') and pART7 R (5'-GGAATTGTGAGCGGATAAC-3')] and TA-cloned into pTZ57R (InsT/A Clone PCR product cloning kit; Fermentas). The IR expression cassette was released from the pTZ57R clone by digestion with EcoRI and HindIII and ligated to EcoRI and HindIII linearized pCAMBIA1303 (Cambia, Brisbane, Australia) to generate pCambia 1303 SACMV BC1 (pC SACMV BC1) (Figure 1). Agrobacterium tumefaciens strain AglI was transformed with a pCambia 1303 SACMV BC1 clone using the freeze-thaw method (25), and 40 N. benthamiana leaf discs were agro-transformed with AglI-pCambia 1303 SACMV BC1 (26, 27), 20 with pCambia1303 (vector alone control), and 10 with untransformed wild-type AglI. Twenty-one pCambia 1303 SACMV BC1 transformants were regenerated in the presence of the selection antibiotic (hygromycin 50 mg/L) and an ethylene scrubber, potassium permanganate (28), and six vector-only and healthy control plant lines were regenerated.Expression of mismatched BC1 IR cassette
T1 progeny from T0 transformed tobacco plant seeds were screened for expression of the transgene construct, which contains the hygromycin (hptII) marker gene, by real-time RT-PCR since transgene expression of hairpins is often difficult to detect as the hairpin is partially or fully cut by the RNAi machinery. Total RNA extractions were performed using Tri-Reagent method (Sigma-Aldrich, St. Louis, MO, USA), treated with DNase (1 U; Fermentas), resuspended in RNase-free water, and concentrations measured on a Nanodrop spectophotometer (Nanodrop, Thermo Scientific, Wilmington, DE, USA). cDNA was reverse-transcribed using a blend of oligo(dT) random hexamer primers (iScript Reverse Transcriptase; Bio-Rad Laboratories, Hercules, CA, USA) from total RNA isolated from leaf tissue and PCR-amplified [hptII F (5'-CTATTTCTTTGCCCTCGG-3') and hptII R (5'-TTCGATGAT-GCAGCTTGG-3')]. PCR controls included untransformed tobacco leaf tissue (negative) and pCAMBIA 1303 vector (positive hptII). PCR products were run on a 2% agarose gel. Spatial expression of the transgene, in transgenic plants, was also monitored by histochemical staining of β-glucoronidase (GUS). Shoots were placed in staining buffer [1 M sodium phosphate, pH 7.0, 0.5 M EDTA, 10% Triton X-100, 50 mM K3Fe(CN)6, 0.1 M X-Gluc] at 37°C for 24 h, followed by several 50% ethanol washes.