FIG. 1.

TCV CP binds long dsRNAs. (A) Long-dsRNA-binding activity of plant viral proteins was studied in gel mobility shift assays. Long dsRNAs were incubated with crude viral extracts prepared from symptomatic N. benthamiana (PoLV, CymRSV, BYV, PCV, PVY, PVX, CMV, TMV, and TAV), Arabidopsis (TCV), tobacco (TEV), or wheat leaves (BSMV). Extracts prepared from mock-inoculated plants (−) and extracts isolated from plants that were infected with PΔ14, a mutant PoLV that was unable to express P14 suppressor protein, were used as negative controls. (B) Plant-expressed TCV CP binds long ds-sRNA. Extracts were prepared from nontreated (−) N. benthamiana leaves and from leaves which were infiltrated with PoLV P14 (P14), TCV CP (CP), BYV P21 (P21), PCV P15 (P15), TEV HC-Pro (Hc-TEV), BSMV γB (γB), or reovirus Sigma3 (Reo). P14 and Reo were used as positive controls. Reo strongly binds long dsRNA but does not bind ds-sRNA. (C) High- and low-molecular-weight RNA gel blot analyses were carried out to study the effect of TCV CP and BYV P21 on accumulation of hairpin transcripts, GFP mRNAs, and sRNAs. N. benthamiana leaves were infiltrated (GFP-IR+35SGFP) with 35SGFP and with agrobacteria expressing hairpin GFP transcripts (−), or the leaves were coinfiltrated with GFP-IR+35SGFP and with TCV CP (CP) or with BYV P21 (P21). RNA samples were isolated at 3 d.p.i. GFP-ir indicates hairpin transcripts derived from GFP-IR, while GFP refers to GFP mRNA transcribed from 35SGFP (upper panel). Note that the probe we used for sRNA hybridization (bottom panel) detected both hairpin transcript- and GFP mRNA-derived sRNAs (GFP sRNA). (D) Effect of TCV CP and BYV P21 on accumulation of hairpin transcripts and hairpin-derived sRNAs. N. benthamiana leaves were infiltrated with GFP-IR (−) or coinfiltrated with GFP-IR and with TCV CP (CP) or with P21 (P21). Samples were taken at 3 d.p.i.