FIG. 7.

Evaluation of GFP and ToRSV concentrations in ToRSV-infected plants at various times after induction of GFP silencing. (a) Analysis of GFP mRNA and siRNAs in a subpopulation of ToRSV-infected plants in which silencing of GFP was hindered in systemic leaves. Plants (16c line) were first mock inoculated or inoculated with ToRSV or PVY. Silencing of the GFP transgene was induced after initial development of virus-induced symptoms as described in the legend to Fig. 6c. Plants were tested for the presence of GFP mRNA and siRNA in the agroinfiltrated (i) and systemic (s) leaves at 18 days postagroinfiltration. RNAs from GFP transgenic plants that had not been silenced (lane 7) and from wild-type plants (lane 8) were used as controls. (b) Accumulation of ToRSV in a subpopulation of ToRSV-infected plants in which systemic silencing of GFP was active. Concentrations of GFP protein, GPF mRNA, and GFP-derived siRNAs (lanes 1 to 3) and of ToRSV CP, ToRSV RNA, and ToRSV-derived siRNAs (lanes 6 to 8) were analyzed in three individual plants at 120 dpi. Mock-inoculated silenced (lane 5) or nonsilenced (lane 4) plants were used as controls for the analysis of GFP protein and RNAs. Mock-inoculated (lane 9) or ToRSV-infected (7 dpi) (lane 10) plants were included as controls for the analysis of ToRSV CP and RNAs. Detection of GFP mRNA and ToRSV RNA was done as described in the legend to Fig. 5. Five- and 50-μg samples of total RNA were used for Northern blotting and siRNA detection, respectively. (c) Accumulation of ToRSV in GFP-silenced or -nonsilenced branches late in infection. At 120 dpi, levels of GFP protein and ToRSV CP in red fluorescent (silenced) or green fluorescent (nonsilenced) branches of ToRSV-infected 16c plants were tested by Western blotting.