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

Purification of ELPylated hemagglutinin (H5-ELP) from transgenic leaves and seeds by membrane-based ITC.

<p>ELPylated hemagglutinins from leaves (A) and seeds (B) were purified by the standard or improved mITC methods (C) 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. Proteins in the raw plant extract (RE), in the supernatant after passage through a 0.2 µm cellulose acetate membrane (Sm) and in the eluent (Pm) were collected during the mITC purification process and separated by 10% SDS-PAGE. Recombinant proteins were then detected using Coomassie Brilliant Blue staining (left) or an anti-c-myc monoclonal antibody (right).</p

Expression cassettes for hemagglutinin (HA) in plants.

<p>HAs were stably expressed in both leaves and seeds under the control of the CaMV 35S and the seed-specific promoters as the naked form (H5), hydrophobin I fusion protein (H5-HFBI) and ELPylated H5 (H5-ELP). All recombinant HAs contained His and c-myc tags for affinity chromatography purification and Western blotting, respectively. The LeB4 signal peptide and KDEL motif were used to ensure ER retention.</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

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

Influence of hydrophobin and elastin-like polypeptide on hemagglutinin accumulation in transgenic plants under the control of either the CaMV 35S promoter (A) or the seed-specific USP promoter (B).

<p>Each column shows the mean value of expression level of the transgenic plants, and the error bars indicate the standard deviation. TSP: total soluble protein.</p

Localization of hemagglutinin-ELP fusions in tobacco seeds.

<p>A, B. Fluorescence microscopy.C, D. Electron microscopy.A, C. Endosperm. B, D. Embryo. Note the ELP bodies (arrowheads, A, B) and those that are loosely packed (arrowheads, C, D). Cell wall (cw), oil bodies (ob), protein storage vacuole (arrows), nucleus (n). Bars 50 µm (A, B), 1 µm (C, D).</p

Expression of influenza HAs in transgenic tobacco plants.

<p>The extracted proteins from transgenic tobacco leaves (A) or seeds (B) were separated by 10% SDS-PAGE, blotted and detected with anti-c-myc monoclonal antibody followed by horseradish peroxidase-linked sheep anti-mouse IgG as a secondary antibody. “+”: anti-hTNFα-VHH-ELP was used as a Western blot standard <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#pone.0099347-Conrad1" target="_blank">[19]</a>; Wt: wild type; TSP: total soluble protein. The numbers refer to independent primary transgenic plants.</p

Transgenic tobacco plants expressing recombinant hemagglutinins.

<p>Transgenic plants were screened by Western blot using an anti-c-myc monoclonal antibody. H5: hemagglutinin; H5-HFBI: hemagglutinin fusion with hydrophobin I; H5-ELP: ELPylated hemagglutinin.</p><p>*Plants expressing H5 and H5-ELP under the control of the CaMV 35S promoter were described by Phan and co-workers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#pone.0099347-Phan2" target="_blank">[25]</a>.</p