Figure 4
Viral RNA accumulation of LCV RNA2 mutants engineered with deletions in the 3′ terminal region.
(a) Schematic diagram of the 3′ terminal region of LCV RNA2 (WT RNA2) containing the Y-shaped structure. Deletions were made to the nucleotides (nts) at the proximal 3′ or 5′ ends of the 98-nt of RNA2. The number of nts deleted and their proximal locations (marked by bold black lines) are indicated in the names of the mutants: 3′Δ4 (deletion of the last 4 nts), 3′Δ11 (the last 11 nts), 3′Δ24 (the last 24 nts), 3′Δ38 (the last 38 nts), 3′Δ48 (the last 48 nts) and 5′Δ50 (the first 50 nts). Four digit numbers placed next to the sequence represent the nt positions in LCV RNA2. The locations of SL1, SL2 and S3 are as indicated. (b–f) Minus- and plus-strand LCV RNA2 accumulation in tobacco protoplasts inoculated with the in vitro transcripts of LCV RNA1 and that of each of the LCV RNA2 mutants: 3′Δ4 (b), 3′Δ11 (c), 3′Δ24 (d), 3′Δ38 (e), 3′Δ48 (f), 5′Δ50 (f) or wild type (WT) RNA2 (b–f). Total RNA (2 μg each) extracted from the inoculated protoplasts harvested at 24, 48 and 96 hpi (lanes 24, 48 and 96, respectively) and total RNA (2 μg) from water (mock)-inoculated protoplasts harvested at 96 hpi (lane W) were analyzed using DIG-labeled RNA2 negative- or positive-sense specific riboprobe VIII (Fig. 1a). Hybridization signals of minus- and plus-strand genomic RNA2 [G2(−) and G2(+), respectively] are indicated. Estimation of RNA sizes and methylene-blue stained 25s rRNA equal loading controls are as in Fig. 3.
