Figure 5
Engineered mutations targeting stem-loops 1 and 2 that form the Y-shape structure in LCV RNA2.
(a) Schematic diagrams of the secondary structure of SL mutants: ΔSL1 (with SL1 deleted), ΔSL2 (with SL2 deleted), SLD1-1 (with a 6-nucleotide substitution in the right arm of the lower stem of SL1 engineered to disrupt SL1), SLD1-2 (with a 6-nucleotide substitution in the left arm of the lower stem of SL1 engineered to disrupt SL1) and SLR1 (with a compensatory 6-nucleotide substitution in the left arm of the lower stem of SLD1-1 engineered to restore SL1). Light gray letters in ΔSL1 and ΔSL2 represent the nucleotides deleted from SL1 and SL2, respectively. Bold letters in SLD1-1, SLD1-2 and SLR1 represent non-viral nucleotides engineered to substitute the viral nucleotides. The locations of SL1, SL2 and S3 are as indicated. (b–f) Tobacco protoplasts were inoculated with the in vitro transcripts of wild type LCV RNA1 along with those of LCV RNA2 mutants ΔSL1 (b), ΔSL2 (c), SLD1-1 (d), SLD1-2 (e), SLR1 (f), or wild type LCV RNA2 (WT). Total RNA (2 μg each) extracted from transcript-inoculated protoplasts 24, 48 and 96 hpi (lanes 24, 48 and 96, respectively) and total RNA (2 μg) extracted from water (mock)-inoculated protoplasts (lane W; harvested at 96 hpi) were analyzed using DIG-labeled negative- or positive-sense specific riboprobe VIII (Fig. 1a). Hybridization signals of minus- and plus-strand genomic RNA2 are indicated as G2(−) and G2(+), respectively. Estimation of RNA sizes and methylene-blue stained 25s rRNA equal loading controls are as in Fig. 3.
