Scanning electron microscopy of <i>Oncopeltus fasciatus</i> salivary glands infected with <i>Phytomonas serpens</i>.

<p>The parasites were injected laterally into the thorax of the insects and the salivary glands were explanted at different time points after injection. (<b>A</b>) Outer surface of the salivary gland showing a high number of parasites between two salivary gland lobes and a few parasites attached to the gland, 48 h post-infection. Scale bar: 10 µm. (<b>B–C</b>) Large numbers of parasites bound to the salivary gland, 72 h post-infection. Scale bars: 100 µm (<b>B</b>) and 50 µm (<b>C</b>). SG: salivary gland; P: parasite; SGL1, SGL2 and SGL3: salivary gland lobes. The asterisk (*) indicates the salivary duct. For further details see the Methods.</p

Ligand blotting showing the salivary gland proteins of <i>Oncopeltus fasciatus</i> recognized by biotin-tagged membrane proteins and live biotinylated parasites.

<p>(<b>Lane a</b>) Coomassie Blue staining of total salivary gland proteins separated by 12% SDS-PAGE. The numbers on the left indicate the relative position of molecular mass markers expressed in kilodaltons. (<b>Lane b</b>) Ligand blotting that shows the presence of a 130 kDa protein recognized by biontinylated live parasites. Prior to the incubation with the membrane, the parasite surface proteins were tagged with Sulfo-NHS-Lc-Biotin. The arrow indicates the position of the 130 kDa protein. For further details see the Methods.</p

Immunoblotting of the 130 kDa salivary gland protein probed with anti-human laminin-5 β3 chain antibodies.

<p>Total salivary gland proteins were extracted and separated by 10% SDS-PAGE (<b>a</b>). The 130 kDa band was cut and purified from the gel and the purity of the product was evaluated by 10% SDS-PAGE stained with silver nitrate (<b>b</b>). The purified 130 kDa protein was probed with anti-human laminin-5 β3 chain antibodies (<b>c</b>). The arrow shows the position of the 130 kDa protein on SDS-PAGE and the stained band by immunoblotting.</p

Matching of the amino acid sequences of the 130 kDa protein of <i>Oncopeltus fasciatus</i> salivary glands with the human laminin-5 β3 chain amino acid sequence.

<p>(<b>A</b>). Protein spots cut from the gel were treated with porcine trypsin and the peptides were spotted onto a MALDI-TOF sample plate (Voyager- DE, Applied Biosystem, CA, USA). Peptide mass fingerprints were analyzed using Protein Prospector MS-Fit interface (<a href="http://prospector.ucsf.edu" target="_blank">http://prospector.ucsf.edu</a>). (<b>B</b>). Underlined letters represent the amino acid sequences of the salivary gland protein that matched the mass spectrometry data to protein sequences in the NCBI database. The underlined sequences indicate the peptides of the salivary gland protein that were matched with the amino acid sequence of the precursor of the human laminin-5 β3 chain.</p

2D SDS-PAGE and corresponding ligand blotting that show the binding of live parasites to the 130 kDa polypeptide spot present in the total protein extract of <i>Oncopeltus faciatus</i> salivary glands.

<p>(<b>A</b>) 2D SDS-PAGE of total protein extract from salivary glands stained with Coomassie Blue. The arrow indicates spots at the 130 kDa region. The numbers on the horizontal axis refer to the pH gradient (pI) and the numbers on the vertical axis refer to the molecular mass standard expressed in kilodaltons. (<b>B</b>) Ligand blotting showing one spot at the 130 kDa region. The spot on the PVDF membrane indicates the binding of biotinylated live parasites to the 130 kDa protein. For further details see the Methods.</p

Comparison of laminin subunit γ through pairwise alignment of γ1 of <i>Homo sapiens</i> (LamG1Hs, gi|145309326), γ1-like of the hemipteran <i>Acyrtosiphon pisum</i> (LamG1Ap, gi|328717115) and γ1-like of <i>O. fasciatus</i> (LamG1Of).

<p>(<b>A</b>) The alignment shows two regions with highly conserved amino acids, consistent with the patterns found in EGF-like domains of γ1 subunits (highlighted in the red rectangles). (<b>B</b>) The alignment of the three γ1 subunits also reveals a sequence similar to the domain VI of γ1 of <i>O. fasciatus</i> transcriptome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-EwenCampen1" target="_blank">[28]</a>. The region similar to domain VI is highlighted in the red rectangles in the alignment. Both in (<b>A</b>) and (<b>B</b>) black shaded residues are identical or similar amino acids present in all three sequences. Grey shaded residues are identical or similar amino acids present in two of the three sequences. The consensus sequence is represented under alignment lines. The alignments were performed using GENEDOC software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-Nicholas1" target="_blank">[90]</a>.</p

Comparison of laminin subunit β through pairwise alignment of β3 laminin of <i>Homo sapiens</i> (LamB3Hs, gi|119613854), β1-like of the turkey <i>Meleagris gallopavo</i> (LamB1Mg, gi|326911240) and β1-like of <i>Oncopeltus fasciatus</i> (LamB1Of).

<p>The alignment of β3 laminin of <i>H. sapiens</i> and β1-like of <i>M. gallopavo</i> with the partial sequence of β1-like identified in the transcriptome of <i>O. fasciatus</i> embryo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-EwenCampen1" target="_blank">[28]</a> shows a highly conserved region at the domain VI among these molecules. Black shaded residues are identical or similar amino acids present in all three sequences. Grey shaded residues are identical or similar amino acids present in two of the three sequences. The consensus sequence is represented under alignment lines. In the red rectangles are highlighted the regions with higher similarity between all three sequences. The alignments were performed using GENEDOC software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-Nicholas1" target="_blank">[90]</a>.</p

Inhibition of the <i>in vitro</i> interaction between <i>Phytomonas serpens</i> and salivary glands explanted from <i>Oncopeltus fasciatus</i>.

<p>The parasites were pre-incubated for 30 min in the presence of human laminin-5 (Lam-5) (<b>A</b>) or in the presence of the purified 130 kDa salivary gland protein (p130) (<b>B</b>). In a parallel system, the salivary glands were pre-incubated in the presence of anti-human laminin-5 β3 chain antibodies (anti-β3 antibodies) (<b>C</b>). In the control systems, the parasites and salivary glands were pre-incubated in the absence of the proteins and antibodies, respectively. The proteins and antibodies were used at the indicated concentrations or dilutions. Each bar represents the mean ± standard error of at least three independent experiments. The <i>P</i> values are indicated on the panels.</p