(rBV hisVP3Patch2) that carries four point mutations (R159D, R168D, H198D and R200D) was done using the approach described above. The construction of the rBV expressing a 6 6 His-tagged version of VP3 Patch1+2 mutant (rBV hisVP3- Patch1+2) was performed by replacing the BamHI/XbaI fragment from the hisVP3Patch2 ORF by that of its hisVP3Patch1 counterpart. The construction of rBVs expressing the VP3 from DXV and IPNV was performed as follows. DNA fragments coding for the DXV VP3 and the IPNV VP flanked by BamHI and EcoRI were generated by gene synthesis (Genscript) and inserted into the EcoRV site of pUC57. Cloning of these ORFs into the baculovirus transfer vector pFastBacHtb to generate pFBhisVP3dxv and pFBhisVP3ipnv, and the generation of the corresponding rBVs, were carried out as described above. To construct plasmids expressing the different versions of VP3 (wild type and mutants) and Influenza A NS1 in plants, GATEWAY technology (Invitrogen) was applied. Hence, pDONR-207 (Invitrogen) was used as donor vector, whereas pMDC32 (provided by Mark Curtis, University of Zurich) [98] and pNTAPi (provided by Michael Fromm, University of Nebraska) [99] were used as destination vectors. Primers and templates used for PCR amplifications to generate the different entry vectors are listed in Tables S1 and S2 in the supplemental material. Expression vectors producing untagged CVYV P1b (pMDC32-P1b), untagged and TAP-tagged Influenza NS1 (pMDC32-NS1 and p35S-NTAP-NS1, respectively), untagged and TAP-tagged IBDV VP3 (pMDC32-VP3 and p35S-NTAP- VP3, respectively), TAP-tagged mutant versions of IBDV VP3 (p35S-NTAP-VP3patch1, p35S-NTAP-VP3patch2, p35S-NTAP- VP3patch1+2 and p35S-NTAP-VP3DC), TAP-tagged IPNV VP3 (p35S-NTAP-VP3ipnv) and TAP-tagged DXV VP3 (p35S-NTAP-VP3dxv) were constructed by LR clonase reactions between the corresponding pDONR entry vectors and either the destination vectors pMDC32 or pNTAPi (Table S1). Plasmid pDONR-P1b and p35S-NTAP-P1b were previously described [63]. Cloning of the IBDV VP3 ORF in a PPV full-length cDNA yielding pICPPV-VP3 made use of an intermediate clone, pGEMT-p1p3, and the full-length cDNA clone pICPPV-NK- GFPn, according to the procedure described by Maliogka et al. (manuscript submitted for publication). Primers and templates used for IBDV VP3 amplification are listed in Tables S1 and S2. pICPPV-NK-GFPn (P. Sa´enz, M.R. Ferna´ndez-Ferna´ndez and J.A. Garc´ıa, unpublished results), a PPV full-length cDNA clone derived from pICPPV-NK-GFP (Ferna´ndez-Ferna´ndez, 2001 #3289}, and pICPPV-DHC [80] were used as control. The accuracy of all the constructs was verified by restriction digestion analysis, as well as DNA sequencing of all regions derived from PCR amplifications. A. tumefaciens C58C1 strains carrying p35S:GFP [100] plus pCH32 [101], and p35S:GF-IR [102] were kindly provided by David Baulcombe (University of Cambridge, United Kingdom). Agroinfiltration and Fluorescence Imaging N. benthamiana plants were infiltrated with A. tumefaciens C58C1 strain carrying the indicated plasmids as previously described [103]. GFP fluorescence was observed under a Leica MZ FLIII fluorescence microscope with excitation and barrier filters of 480/ 40 and 510 nm, respectively. Pictures of GFP were caught with an Olympus DP 70 camera and the software DP Controller and DP manager. VP3 Proteins Suppress RNA Silencing RNA Extraction and Northern Blot Analysis Samples of large and small RNAs were prepared from agroinfiltrated leaf patches and subjected to Northern blot analysis as previously described [103]. GFP siRNA were detected with 32P- labeled GF and P riboprobes, which were prepared by transcrip- tion with SP6 RNA polymerase from SacII-linearized pGEMT-GF and pGEMT-P, respectively. These plasmids contain the nt 4 to 403 (GF) and 404 to 717 (P) of the GFP gene cloned in pGEM-T. Expression and Purification of Different VP3 Proteins from Insect Cells Procedures to express recombinant 6xHis-tagged VP3s in insect cells by using rBVs as well as to purify the polypeptides by metal- affinity chromatography were previously described [52]. Gel Filtration Analysis Affinity-purified 6xHis-tagged VP3s were analyzed by gel filtration using a fast protein liquid chromatography system ( A¨KTA FPLC, Pharmacia) with a Superdex-200 5/150 column (Pharmacia) previously calibrated with catalase (158 kDa), serum albumin (68 kDa), ovoalbumine (50 kDa) and chymotrypsinogen A (21 kDa). Column equilibration and chromatography were performed at a flow rate of 0.5 ml/min in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl at 4uC. Electrophoretic Mobility Shift Assay (EMSA) Synthetic ds-siRNA with 2-deoxinucleotides 39 overhands (59CUUACGCGAGUCUUCGATT39/59UCGAAGUACU- CAGCGUAAGTT39) (Sigma) were labelled with c-32P-ATP using T4 PNK (Promega). IBDV genomic double-stranded RNAs were prepared accordingly to Luque et al [55]. Either labelled siRNA (2 nM final concentration) or genomic IBDV RNAs (7.5 nM final concentration) were incubated with different amounts of the indicated affinity chromatography- purified proteins for 30 min at room temperature in a reaction mixture (20 ml) containing small RNA-binding buffer (10 mM TrisHCl pH8, 10 mM Glycine, 2 mM DTT) or long dsRNA- binding buffer (50 mM TrisHCl pH8, 150 mM NaCl, 0.5mg/ul BSA), respectively. After incubation, protein-small RNA and protein-long dsRNA complexes were resolved on 5% poly- acrylamide-containing 0.56Tris-borate-EDTA gels and 1% aga- rose-containing 0.56Tris-borate-EDTA gels, respectively. Poly- acrylamide gels were dried and exposed to X-ray sensitive films. Agarose gels were stained with ethidium bromide and exposed under UV light. Biolistic Inoculation The Helios Gene Gun System (Bio-Rad, Hercules, CA, U.S.A.) was used for biolistic inoculation. Microcarrier cartridges were prepared from 2 different clones per construct, with 1.0 mm gold particles coated with the different plasmids at a DNA loading ratio of 2 mg/mg of gold and a microcarrier loading of 0.5 mg/ shooting. Helium pressure of 7 bars was used for shooting plants. Each cartridge was shot twice onto two leaves of each plant. Western Blot of Infected Plants Tissue samples of inoculated leaves were harvested under UV light from GFP expressing foci, whereas tissue of upper non- inoculated leaves were harvested from the indicated whole leaves. Control samples corresponding to non-infected leaves were taken from equivalent areas. Preparation of protein samples, SDS-PAGE electrophoresis, and electroblotting were done as previously described [103]. Specific proteins were detected using anti-IBDV PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45957