Figure 2. SPMMV P1 suppresses RNA silencing by mechanisms that diverge from other viral suppressors.

(A) P1 and HC-Pro proteins of SPMMV were agroinfiltrated into N.benthamiana leaves (according to the schematic drawing shown at the left) along with a reporter 35S-GFP construct to visualize (central panels) and to analyze by northern blot (right panel) their effects on RNA silencing. The P1b suppressor of CVYV was used as positive control, while an empty pBin61 served as negative control. (B) ³²P-labeled ds 21nt siRNA was incubated with extracts of leaves infiltrated with constructs expressing SPMMV P1, SPMMV HC-Pro, both P1 and HC-Pro of SPMMV in cis (P1HC-Pro), TEV HC-Pro and GFP. Bound complexes were electrophoretically separated on a native gel. (C) ³²P-labeled ds 49 nt RNA was incubated with extracts of leaves infiltrated with P1, P1HC-Pro, and HC-Pro of SPMMV, Reovirus sigma3 and GFP. Bound complexes were electrophoretically separated on a native gel. (D) RNA preparations from leaves of 16c transgenic lane of N. benthamiana plants expressing GFP infiltrated with SPMMV P1, SPMMV HC-Pro and TEV HC-Pro were analyzed by northern blot with GFP-specific probes for the presence of mRNA and siRNA (upper and central panels); loading charge is shown at the lower panel. (E) Analysis of 3′ methylation of miR168 in the presence of the indicated viral suppressors. RNA samples with or without oxidation and ß-elimination were separated in 12% polyacryamide gel and then blotted. Small RNA blot was hybridized with LNA oligonucleotide probes detecting miR168 star (miR168*) and miR168 mature strands. Faster miRNA bands indicate the non-methylated miRNAs. (F) Detail of a leaf from a N. benthamiana 16c plant under UV light at 8 days after co-agroinfiltration with the reporter 35S-GFP and pBin empty vector (left) or SPMMV P1 (right). The development of the red halo around the agroinfiltrated patch indicated that SPMMV P1 did not abolish movement of the signal.