BLAST search of the ELI025 amino acid sequence against the genomes, transcriptomes, or proteomes of 18 oomycetes, 10 fungi, 4 algae, 3 diatoms, and one protozoan (the cut-off E-value ≤ 1 x 10–4).

<p>^a Broad institute genome database</p><p>^b Genome portal of the Department of Energy Joint Genome Institute</p><p>BLAST search of the ELI025 amino acid sequence against the genomes, transcriptomes, or proteomes of 18 oomycetes, 10 fungi, 4 algae, 3 diatoms, and one protozoan (the cut-off E-value ≤ 1 x 10–4).</p

<i>P</i>. <i>insidiosum</i>’s transcriptome-derived Exo1 homologous proteins that share the Peptide-A, -B, or -C sequences.

<p>^a Percent length value of a query sequence (i.e., Exo1) that covers or can align with a subject sequence</p><p>^b Percent identity value of query (Exo1) and subject sequences within the Query coverage region</p><p>Predicted structures of these proteins are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#pone.0135239.g002" target="_blank">Fig 2</a>. NCBI accession number, protein length, calculated protein molecular weight (MW), number of 454-derived transcript reads (when <i>P</i>. <i>insidiosum</i> grew at 37°C), and sequence alignment analysis against Exo1 (including: query sequence coverage, <i>E</i>-value, and sequence identity), corresponding to each transcriptome-derived protein, are summarized in the table.</p

Mass spectrometric analyses of the 10- and 15-kDa SDS-PAGE band-derived proteins showing mass-to-charge ratio (m/z), average mass of peptide (Mr; calculated by MASCOT software), peptide sequences (identified by MASCOT software), BLAST search result (against ~15,000 genome-derived predicted proteins of P. insidiosum), and amino acid position of identified peptides.

<p>Mass spectrometric analyses of the 10- and 15-kDa SDS-PAGE band-derived proteins showing mass-to-charge ratio (m/z), average mass of peptide (Mr; calculated by MASCOT software), peptide sequences (identified by MASCOT software), BLAST search result (against ~15,000 genome-derived predicted proteins of P. insidiosum), and amino acid position of identified peptides.</p

Sequence alignment of core promoter regions of the <i>P. insidiosum ELI025</i> gene and various genes from several oomycetes and parasites.

<p>The <i>ELI025</i> sequences (accession number AB971191–3), used for the alignment, are derived from three different <i>P</i>. <i>insidiosum</i> strains. Conserved nucleotides are highlighted in grey. The underlined letters indicate the known transcriptional start site, and is indicated below as "+1". Two putative core promoter components, an initiator element (Inr; 5’-TCATTCC-3’; positions-2 to +5) and a flanking promoter region (FPR; 5’-CAACCTTCC-3’; positions +7 to +15), are found in the upstream region of all genes. (Abbreviation: NCBI, National Center for Biotechnology Information).</p

Immunoreactivity of Exo1 peptides against pythiosis patient sera by ELISA.

<p>ELISA results of serum samples from pythiosis patients (n = 3; PS1-3) and healthy blood donors (n = 3; CS1-3; control) and (<b>A</b>) Peptide-A, (<b>B</b>) Peptide-B, (<b>C</b>) Peptide-C, and (<b>D</b>) a mixture of the peptides (used to coat an ELISA plate). Number in the parenthesis is the mean ELISA signal.</p

Phylogenetic analysis of glucanase genes from oomycetes and fungi.

<p><i>exo1</i> gene sequences from 6 strains of <i>P</i>. <i>insidiosum</i> (accession number: LC033486 to LC033491), and glucanase-encoding genes (top <i>exo1</i>-BLAST hit sequences) from 9 other oomycetes and 26 fungi (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#pone.0135239.t002" target="_blank">Table 2</a>) were included for phylogenetic analysis. Phylogenetic reconstruction was performed using the PhyML program (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#sec002" target="_blank">Methods</a>). Reliability for internal branch was analyzed using the aLRT test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#sec002" target="_blank">Methods</a>).</p

Peptide pre-absorption of the rabbit anti-Exo1 sera for Western blot analysis of <i>P</i>. <i>insidiosum</i> crude protein extracts.

<p>Separated crude proteins (SABH) of <i>P</i>. <i>insidiosum</i> were probed with rabbit pre-immune serum (Lane 1), rabbit post-immune serum (Lane 2), and rabbit post-immune serum pre-absorbed with Peptide-A, -B, and -C (Lane 3), Peptide-A and -B (Lane 4), Peptide-A and -C (Lane 5), and Peptide-B and -C (Lane 6). The arrow and arrow head indicate the 82- and 78-kDa band, respectively. [Abbreviations: SABH, soluble antigen from broken hyphae; Pre-immune, rabbit pre-immune serum; Post-immune, rabbit anti-Exo1 peptide serum; ‘+’, used as probe (pre- and post-immune sera) or used for pre-absorption (Peptide-A, -B, or -C); ‘-’, not used as probe nor used for pre-absorption]</p

Cellular location of ELI025.

<p>Infected arterial tissue from a pythiosis patient was sequentially stained with rabbit anti-rELI025 serum, as the primary antibody, and then mouse anti-rabbit IgG antibody conjugated with horseradish-peroxidase, as the secondary antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118547#sec002" target="_blank">Materials and Methods</a>). Images of the hyphae and location of ELI025 (indicated by arrows) were captured with a bright-field microscope. The scale bar represents 10 μm.</p

Immunoreactivity of the recombinant protein rELI025 and crude protein extracts of <i>P. insidiosum</i>.

<p>Crude proteins (i.e., SABH and CFA) extracted from three different strains of <i>P</i>. <i>insidiosum</i> (Pi-S, MCC18, and P01) and rELI025 are separated in a SDS-PAGE gel <b>(A)</b>. The separated proteins are analyzed by Western blot, using the rabbit anti-rELI025 antibodies <b>(B)</b>, or sera from patients with pythiosis <b>(C)</b>, as probe. The black arrow head indicates the 12.4 kDa band of rELI025. The white arrow heads indicate the 10- and 15-kDa bands of native ELI025. The numbers represent protein molecular weights standards, in kDa. (Abbreviations: SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; CFA, culture filtrate antigen; SABH, soluble antigen from broken hyphae; rELI025, recombinant ELI025).</p

Western blot analysis of <i>P</i>. <i>insidiosum</i>’s crude protein extracts using rabbit anti- Exo1 peptide serum.

<p>Crude proteins (SABH and CFA) extracted from <i>P</i>. <i>insidiosum</i> were separated in a SDS-PAGE gel, transferred to a Western blot membrane, and probed with the rabbit pre-immune or post-immune serum. The arrow and arrow head indicate the 82- and 78-kDa band, respectively. Protein molecular weight markers (7–175) are shown in kDa. (SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CFA, culture filtrate antigen; SABH, soluble antigen from broken hyphae; Pre-immune, rabbit pre-immune serum; Post-immune, rabbit anti-Exo1 peptide serum).</p