Schematic diagram of yeast two- and three-hybrid assay design with a representative vector map of pY3HS for ectopic expression of the recombinant antibody.

<p>(<b>A</b>) <b>Yeast two-hybrid selection.</b> The interacting partner AHP protein and recombinant antibody fused to the Gal4 AD and BD domains, respectively, allow recruitment of the transcription machinery which activates the expression of nutritional markers HIS3 and ADE1. (<b>B</b>) <b>Yeast three-hybrid selection.</b> The ectopic expression of recombinant antibody is blocking interaction of the AHP protein and its RD interaction partner. This results in no activation of the expression of nutritional markers HIS3 and ADE1. (<b>C</b>) <b>pY3HS vector map.</b> Schematic representation of the vector for the ectopic expression of recombinant antibodies in Y3H. (AD =  activation domain, BD =  DNA binding domain, HIS3 =  histidine nutritional marker, ADE1 =  adenine nutritional marker, AHP =  ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN, RD =  ARABIDOPSIS HISTIDINE KINASE receiver domain, TEF1 =  strong constitutive promoter, CYC1 =  terminator, Ura3-uracyl nutritional marker, 2μ =  yeast replication origin, AmpR  =  ampicilin resistance, pBS  =  bacterial replication origin).</p

MOESM1 of Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants

Additional file 1. Expression of recombinant proteins in plants. Hemagglutinin derivatives and S·Protein derivatives (30 µg total soluble protein/lane) in plant extracts analyzed by anti-c-myc tag Western blot. S·Protein-pII::H5-S·Tag: co-expression; S·Protein-GCN4::H5-S·Tag: co-expression; S·Protein-pII, S·Protein-GCN4, and H5-S·Tag: single expression; WT: wild-type N. benthamiana

<i>In vitro</i> characterization of scFv hB7A to AHP antigens.

<p>(<b>A</b>) <b>Sequence variability of AHP proteins amino acid sequence.</b> ClustalW tree of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS (AHP1-6). Amino acid sequence identity of AHP proteins (compared to AHP3 in brackets). (<b>B</b>) <b>Far-western blot of recombinant AHP proteins.</b> Recombinant protein expression with incorporated 6x-His-tag detected was confirmed by anti-6x-His-tag antibody immunodetection (top); AHP proteins were detected by recombinant scFv hB7A from the periplasmic extract of the bacterial expression, incorporated c-myc-tag detected by anti-c-myc-tag antibody (bottom). (<b>C</b>) <b>The specificity of scFv hB7A against AHP proteins tested in indirect ELISA.</b> Absorbance values of triplicates (±SD represented with error bars) at 450 nm are displayed for each AHP protein (500 ng/well). (<b>D</b>) <b>The affinity of scFv hB7A to AHP3 tested in competitive ELISA.</b> Absorbance values at 405 nm normalized to 490 nm of quadruplicates from two independent measurements were pooled (±SD represented with error bars). The AHP3 was coated at 20 nM and 2-fold dilutions of 1950 nM AHP3 was equilibrated with 45 nM scFv hB7A prior loading to wells. The data was fitted with DYNAFIT software.</p

Recombinant antibody scFv hB7A is interacting with AHP 3 in <i>A. thaliana</i>.

<p>Confocal images of <i>A. thaliana</i> mesophyll protoplasts co-transformed with nYFP:scFv-hB7A (<b>A</b>) and scFv-hB7A:nYFP (<b>B</b>) interacts irrespective of the fusion orientation with AHP3:cYFP (I. - yellow channel). The signal of the reconstituted split-YFP protein is localized in the cytosol and in the nucleus. Nuclear localized mCherry (II. - red channel) and autofluorescence (>650 nm) of chloroplasts (III. - cyan channel) serve as co-localization markers. The integrity of the cell is visible from the overlay picture together with transmission channel (IV.). Schematic representation of the experiment (V.) Negative control is shown in (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109875#pone.0109875.s008" target="_blank">Figure S8</a></b>). Scale bars: 20 µm.</p

Specific down-regulation of the cytokinin signaling pathway in <i>A. thaliana</i> mesophyll protoplasts after ectopic expression of scFv hB7A.

<p>Levels of the TCS:LUC induced luciferase activity correspond to the activity of the cytokinin signaling pathway activated with 100 nM trans-Zeatin (tZ) and normalized to the transfection control 35S:Renilla Luciferase. The ectopic expression of scFv hB7A is sorted by the amount of total (20 µg) co-transfected DNA (expressed in percent). The Col-0 wild type plants (white) and ahp 1,3,4,5 mutant plants with solitary active AHP2 (gray) show no significant effect. The ahp 1,2,4,5 mutant plants (black) with solitary active AHP3 were severely affected</p

<i>In vivo</i> inhibition of AHP3-CKI1 RD interaction.

<p>CKI1 receiver domain (RD) (Gal4 DNA binding domain BD fusion) interaction with respective AHP interaction partners (Gal4 activation domain AD fusion) upon ectopic expression of scFv hB7A. The visible yeast growth was recorded after incubation for 3 days on a different yeast drop-out media lacking Leu (L), Trp (W), Ura (U) and His (H) or Ade (A) or with added 1 mM 3-Amino-1,2,4-triazole (3AT). Compared to the empty vector pY3HS, scFv hB7A specifically inhibits interactions of AHP3 in the yeast three-hybrid assay as indicated by visible growth inhibition of yeast in different drop-out media. The interaction of AHP5 is naturally very weak and so it is difficult to observe in the yeast three-hybrid assay.</p

Western blot analysis of purified HAs treated/untreated with PNGase F.

<p>Purified HAs from leaves were deglycosylated using the commercial PNGase F enzyme described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#s2" target="_blank">Materials and Methods</a> section. PNGase F-treated and untreated proteins were then separated in 10% SDS-PAGE. Recombinant proteins were detected using an anti-c-myc monoclonal antibody. “−” and “+” indicate PNGase F-untreated and treated samples, respectively.</p

Localization of hemagglutinin-hydrophobin I fusions in tobacco seeds.

<p>A. Fluorescence microscopy. B, C. Electron microscopy. B. Endosperm. C. Embryo. Scarce hydrophobin bodies in the endosperm (arrowheads, A, B). Abundant hydrophobin bodies in the embryo cells (arrowheads, A, C). Hydrophobin bodies show non-uniform electron density (*, C). Endosperm (end), embryo (emb), protein storage vacuole (PSV), ribosomes (arrow). Bars 25 µm (A), 0.5 µm (B, C).</p

Glycosylation profile of recombinant HAs from leaves and seeds.

<p>Man_{6, 7, 8}: Mannose _{6, 7, 8}; Gn: <i>N</i>-acetylglucosamine; X: Xylose; F: Fucose.</p

Immunofluorescence analysis of recombinant HAs in plant leaves.

<p>Leaves were fixed, embedded in PEG and sectioned. Recombinant HAs were immunodecorated with an anti-c-myc monoclonal antibody followed by incubation with secondary antibody (anti-mouse-IgG conjugated with AlexaFluor488) and counterstaining with DAPI. A. H5; B. H5-HFBI; C. H5-ELP; D. wild type. Bars represent 50 µm.</p