VP3 Proteins Suppress RNA Silencing Figure 1. VP3 protein from IBDV suppresses the RNA silencing. ssRNA-triggered (A) and dsRNA-triggered (B) silencing assays in N. benthamiana plants. The figure shows GFP fluorescence pictures taken under an epifluorescence microscope using the same exposure time (left panels), and Northern blot analyses of GFP mRNA and GFP-derived siRNAs (right panels) in agroinfiltrated leaf patches from two plants expressing the indicated constructs, and collected at 6 days post agroinfiltration. Two different probes were used for detection of GFP siRNAs: the GF probe (for primary plus secondary siRNAs) corresponds to the GFP fragment included in the inverted repeat RNA expressed from the silencing trigger plasmid p35S:GF-IR; and the P probe (specific for secondary siRNAs) corresponds to the 39 terminal region of the GFP gene, which is not included in p35S:GF- IR. EtBr-stained rRNA and 5S+tRNA are shown as loading controls. doi:10.1371/journal.pone.0045957.g001 resolved by gel electrophoresis (Fig. 2B). As expected from previous reports, purified IBDV VP3 was able to bind IBDV genomic dsRNA molecules producing a typical smear-like gel shifting of the dsRNA probe akin to that observed in isolated IBDV ribonucleoprotein complexes (Fig. 2B and [55]). Impor- tantly, this protein was also able to interact with siRNA duplexes of both 21- and 26-nt of length, without apparent size specificity (Fig. 2B). dsRNA Binding is Crucial for VP3-mediated RNA Silencing Suppression The relevance of interactions between VSRs and diverse RNA molecules in silencing pathways has been previously reported (reviewed in [15,18]). A mutagenic approach was followed to assess the importance of VP3-dsRNA interactions in the VP3 silencing suppression activity. Positively charged amino acids of the IBDV VP3 protein, predicted from crystal structure [56] to be from the region involved in dsRNA binding, were replaced by negatively charged Asp residues (Patch 1 and Patch 2, Fig. 2A). In addition, a deletion of the highly hydrophilic C- terminal region of IBDV VP3 (position 222–258), which was previously proposed as potentially relevant in both nucleic acid and protein-protein interactions [56], was engineered (DC, Fig. 2A). Mutant proteins were produced and purified from insect cells, and tested for their dsRNA binding capacity against the IBDV genomic RNAs, as well as a 32P-labelled 21-nt siRNA duplex, by EMSA (Fig. 2C). None of the multiple substitutions of positively charged amino acids affected the capacity of IBDV VP3 to form dimers (Fig. S1), suggesting that these mutations do not cause a drastic disturbance PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45957