FIG. 1.

Expression construct and reversal-of-silencing assay for the determination of suppressor activity by the SARS-CoV 7a ORF. (A) SARS-CoV 7a ORF under 35S promoter of the binary vector pBI121. (B) GFP fluorescence pictures taken under a UV lamp at 8 dpi of agroinfiltrated leaves. Each of the leaves was labeled at the top for the construct with which it was infiltrated. Empty vector was used as a mock control, while MYMIV AC2 and FHV B2 were positive controls in the assay. The tested SARS-CoV 7a protein showed positive reversal of silencing, while SARS virus 3a has been presented as negative for RSS activity. (C) Molecular analysis of reversal of silencing. Western blot analysis for GFP protein and Northern blot analysis for GFP-mRNA and -siRNA are presented. Fifty micrograms of RNA was extracted at 8 dpi from leaf patches infiltrated with Agrobacterium cultures harboring the corresponding plasmids as indicated. The bottom gels show the respective loading controls (Coomassie brilliant blue-stained RuBISCO band for the protein blot, actin-probed band for the Northern mRNA blot, and ethidium bromide [EtBr]-stained tRNA for the siRNA blot).