Figure 6
RNA synthesis of LCV RNAs with chimeric YSS.
(a) Schematic diagrams of the infectious cDNA clones of wild type LCV RNA1 (R1; pCM1), wild type LCV RNA2 (R2; pCM2), mutant LCV RNA1 containing the 98-nt of RNA2 (R1-3′R2) and mutant LCV RNA2 containing the 98-nt of RNA1 (R2-3′R1). The locations of the Y-shape structure (YSS) of R1 (R1 YSS; thin uncolored bar) and R2 (R2 YSS; thin gray bar) relative to the 98-nt of both RNAs are as indicated. Numbers (#1–5) represent different combinations of wild type and/or chimeric mutant in vitro transcripts (2 μg each) inoculated into tobacco protoplasts. (b–d) Northern blot analyses of RNA accumulation. Northern blots were performed using total RNA (2 μg each) extracted from tobacco protoplasts harvested at 24, 48 and 96 hours (lanes 24, 48 and 96, respectively) following the inoculation of each of the five combinations of in vitro transcripts (#1–5); and from water (mock)-inoculated tobacco protoplasts harvested at 96 hours post-inoculation (lane W). Hybridizations were conducted using DIG-labeled negative- or positive-sense specific riboprobes II (b,c) and VIII (d) (Fig. 1a). Hybridization signals of minus- and plus-strand genomic RNA1 are indicated as G1(−) and G1(+), respectively and that of minus- and plus-strand genomic RNA2 are indicated as G2(−) and G 2(+), respectively. RNA size estimates and methylene blue-stained 25S rRNA loading controls are as in Fig. 3.
