FIG. 2.

High steady-state levels of ToRSV RNA are found in time course experiments conducted on several plant species. (a) Time course analysis of ToRSV RNA concentration in infected N. benthamiana plants. Leaves from three individual plants were analyzed for each time point. Lane M, mock-inoculated plants; lanes i, inoculated leaf; lanes 3, third leaf from the top; lanes 1, apical leaf. (b) Detection of negative- and positive-strand ToRSV RNA in symptomatic (5 dpi) or recovered (30 dpi) leaves. (c and d) Time course analyses of viral RNA levels in N. clevelandii (c) and cucumber (d) plants. Fragments of ToRSV RNA corresponding to the CP and MP open reading frames (amplified with primers p47F [5′-GTCCCATGGGGTCTTCTCTAGGAACTCCTGGT] and p51R [5′-GTCCTCCAGGCCACGCCCGAAAGGAT]) were used for hybridization of blots in panels a, c, and d. The strand-specific probes used for panel b consisted of a 139-nt fragment from the 3′-nontranslatable region of ToRSV (67), which is identical in RNA 1 and RNA 2. Sense and antisense radiolabeled transcripts were generated in vitro using the T7 and SP6 polymerases and the cDNA fragment inserted between the corresponding promoters. Because the probes were produced using different polymerases, the ratio of positive- to negative-strand RNA could not be inferred directly.