protein (Fig. 2C) suggests that the C-terminal part of VP3 is not relevant for RNA binding under the experimental conditions used here. Patch 1, Patch 2 and DC mutations were engineered in pNTAP-VP3, and the effect of these modifications on the RNA silencing suppression activity of VP3 was assessed in a dsRNA- triggered silencing assay (Fig. 3). At 6 dpa, leaf patches expressing p35S:GFP, p35S:GF-IR, and either pNTAP-VP3, pNTAP-VP3Patch2, pNTAP-VP3DC or the positive control pNTAP-P1b displayed very high fluorescence levels (Fig. 3A). Northern blot analysis confirmed an efficient protection of GFP mRNA from silencing degradation by the VP3Patch2 and VP3DC mutants. In contrast, leaf patches expressing p35S:GFP, p35S:GF-IR, and either pNTAP-VP3Patch1 or pNTAP- VP3Patch1+2 showed no GFP fluorescence at 6 dpa, like control plants expressing p35S:GFP, p35S:GF-IR and an empty vector, suggesting that Patch1 mutation abolishes the VP3 anti- silencing activity. Northern blot analysis confirmed the fluores- cence data since GFP mRNA was cleared from those tissues expressing VP3Patch1 or VP3Patch1+2 mutants at this time (Fig. 3B). Collectively, these results show a strong correlation between dsRNA interaction and RNA silencing suppression, supporting the idea that VP3 uses its dsRNA binding ability to suppress the RNA silencing. VP3 Proteins Suppress RNA Silencing Homologous VP3 Proteins from Bird-, Fly- and Fish- infecting Viruses Share the Ability to Suppress RNA Silencing The Birnaviridae family consists of viruses able to infect birds, such as IBDV, aquatic organisms and insects. To determine whether homologous proteins to IBDV VP3 from other birna- viruses infecting non-avian hosts are able to suppress RNA silencing, the VP3 coding sequences of IPNV and Drosophila X virus (DXV), which infect fishes and flies, respectively, were synthesized and introduced in the pFastBacHtb and NTAPi vectors to be expressed in insect cells and plants, respectively. IPNV and DXV VP3 proteins purified from insect cells were able to interact with the IBDV genomic dsRNAs, as well as a synthetic 21-nt ds-siRNA, by EMSA (Fig. 4A) in similar manner than their IBDV counterpart. The ability of these VP3 proteins to suppress RNA silencing was assessed in a dsRNA-triggered silencing assay in N. benthamiana plants. At 6 dpa, when leaf patches agroinfiltrated with p35S:GFP plus p35S:GF-IR and an empty vector showed strong silencing of GFP reporter, patches expressing p35S:GFP plus p35S:GF-IR and any of the NTAPi-derived plasmids expressing VP3 from either IBDV, IPNV or DXV displayed strong GFP fluorescence. Concomitantly, high accumulation levels of GFP mRNA were detected in agroinfiltrated leaf patches expressing these birnaviral proteins (Fig. 4B and C), indicating that both IPNV and DXV VP3s also suppress the RNA silencing. Figure 3. Positive correlation between dsRNA binding and RNA silencing suppression activity revealed by a dsRNA-triggered silencing suppression test. GFP fluorescence pictures taken under an epifluorescence microscope (A) and Northern blot analysis of GFP mRNA accumulation (B) of leaves of two N. benthamiana plants infiltrated with agrobacteria carrying the plasmid indicated above each lane (red bars indicate mutated patches). EtBr-stained rRNA is shown as loading control. doi:10.1371/journal.pone.0045957.g003 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45957