Protein structure of Exo1 and six transcriptome-derived homologous proteins.

<p>Protein domains of Exo1 and homologous proteins (UN05080, UN00475, UN03240, UN01457, UN24957 and UN22794; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#pone.0135239.t003" target="_blank">Table 3</a>) were predicted by SignalP, TMHMM, and NCBI’s conserved domain search programs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#sec002" target="_blank">Methods</a>). The DOG program was used to draw protein structures. Numbers indicate the first and last amino acid positions of each protein. Detailed characteristics of the homologous proteins are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#pone.0135239.t003" target="_blank">Table 3</a>. (Symbols: A, Peptide-A; B, Peptide-B; C/1, Peptide-C (the first half portion); C/2, Peptide-C (the second half portion); SP, signal peptide; BglC, BglC domain; X8, X8 domain; and TM, transmembrane region; The grey regions are sequences that do not match any protein domain defined by the NCBI’s conserved domain search program).</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

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

Expression of <i>exo1</i> in response to temperature, culture duration, and dextrose availability.

<p>Real-time PCR was used to measure <i>exo1</i> mRNA levels in <i>P</i>. <i>insidiosum</i> (strain Pi-S) at 5 culture conditions: (i) 28°C for 7 days (28c-7d-1x); (ii) 37°C for 7 days (37c-7d-1x); (iii) 28°C for 14 days (28c-14d-1x); and 28°C for 7 days with (iv) 2 mg/ml dextrose (28c-7d-0.1x), or (v) no dextrose (28c-7d-0x) supplement. <i>exo1</i> expression in each condition was normalized to the reference actin gene (<i>act1</i>). Asterisk indicates significant up- or down-regulation, relative to 28c-7d-1x.</p

Immunoreactivity of Exo1 peptides against rabbit anti-Exo1 peptide sera.

<p>ELISA result of rabbit pre-immune or anti-Exo1 peptide serum (raised against the combination of Peptide-A, -B, and -C) with the individual peptides (used to coat an ELISA plate).</p

List of primers used in this study.

<p>List of primers used in this study.</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

List of oomycete and fungal microorganisms whose top BLAST-hit DNA sequences vs. the <i>P</i>. <i>insidiosum</i>’s <i>exo1</i> gene were used for phylogenetic analysis.

<p>All sequences were retrieved from the FungiDB database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135239#pone.0135239.ref027" target="_blank">27</a>], and BLAST searched against the NCBI nucleotide database.</p

Measurement of hydrolytic activity of Exo1 by agar plate enzymatic assay:

<p>The pPinsEXO1-harboring <i>E</i>. <i>coli</i> cell suspension [1 x 10⁷ (<b>A</b>) and 1 x 10⁹ (<b>B</b>) cells/ml] and the positive controls [<i>T</i>. <i>harzianum</i>’s lysing enzyme (100 mg/ml; <b>C</b>) and <i>T</i>. <i>reesei</i>’s cellulase (100 mg/ml; <b>D</b>)] gave hydrolytic/clear zones in LB agar supplemented with laminarin. The negative controls [bacteria with the pRSET-C empty plasmid (1 x 10⁹ cells/ml; <b>E</b>) and plain LB broth (<b>F</b>)] did not produce a hydrolytic/clear zone in the laminarin plate. * indicates location where the material (bacteria, enzyme, or broth) was spotted.</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