Soybean proteins identified from the xylem saps of both <i>F. virguliforme</i>-infected and -uninfected, healthy soybean plants.

a<p><i>Glycine max</i> protein identification number from Phytozome database (<a href="http://www.phytozome.net/search.php" target="_blank">http://www.phytozome.net/search.php</a>).</p>b<p>Exponentially modified protein abundance index.</p

Soybean proteins differentially accumulated in the <i>F. virguliforme</i>-infected (A) or <i>F. virguliforme</i>-uninfected (B) soybean plants.

a<p><i>Glycine max</i> protein identification number from Phytozome database.</p>b<p>Number of times the peptides were identified among five biological replications.</p>c<p>Exponentially modified protein abundance index.</p

Classification of the 112 most abundant proteins identified from xylem saps of both <i>F. virguliforme-</i>infected and <i>F. virguliforme-</i>uninfected soybean plants based on molecular function.

<p>(A) Percentage of proteins in different functional categories at ontology level 2, with a cutoff of 5. (B) Secondary functional categories based on KEGG pathway. Only the prominent pathways with a sequence cutoff of 3 are reported here.</p

Analyses of the Xylem Sap Proteomes Identified Candidate <i>Fusarium virguliforme</i> Proteinacious Toxins

<div><p>Background</p><p>Sudden death syndrome (SDS) caused by the ascomycete fungus, <i>Fusarium virguliforme</i>, exhibits root necrosis and leaf scorch or foliar SDS. The pathogen has never been identified from the above ground diseased foliar tissues. Foliar SDS is believed to be caused by host selective toxins, including FvTox1, secreted by the fungus. This study investigated if the xylem sap of <i>F. virguliforme-</i>infected soybean plants contains secreted <i>F. virguliforme-</i>proteins, some of which could cause foliar SDS development.</p><p>Results</p><p>Xylem sap samples were collected from five biological replications of <i>F. virguliforme</i>-infected and uninfected soybean plants under controlled conditions. We identified five <i>F. virguliforme</i> proteins from the xylem sap of the <i>F. virguliforme-</i>infected soybean plants by conducting LC-ESI-MS/MS analysis. These five proteins were also present in the excreted proteome of the pathogen in culture filtrates. One of these proteins showed high sequence identity to cerato-platanin, a phytotoxin produced by <i>Ceratocystis fimbriata</i> f. sp. <i>platani</i> to cause canker stain disease in the plane tree. Of over 500 soybean proteins identified in this study, 112 were present in at least 80% of the sap samples collected from <i>F. virguliforme</i>-infected and -uninfected control plants. We have identified four soybean defense proteins from the xylem sap of <i>F. virguliforme</i>-infected soybean plants. The data have been deposited to the ProteomeXchange with identifier PXD000873.</p><p>Conclusion</p><p>This study confirms that a few <i>F. virguliforme</i> proteins travel through the xylem, some of which could be involved in foliar SDS development. We have identified five candidate proteinaceous toxins, one of which showed high similarity to a previously characterized phytotoxin. We have also shown the presence of four soybean defense proteins in the xylem sap of <i>F. virguliforme</i>-infected soybean plants. This study laid the foundation for studying the molecular basis of foliar SDS development in soybean and possible defense mechanisms that may be involved in conferring immunity against <i>F. virguliforme</i> and other soybean pathogens.</p></div

<i>F. virguliforme</i> peptides identified from the xylem sap of <i>F. virguliforme-</i>infected soybean plants.

a<p>Protein identification numbers are same as the gene IDs of the <i>F. virguliforme</i> genome database (<a href="http://fvgbrowse.agron.iastate.edu" target="_blank">http://fvgbrowse.agron.iastate.edu</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093667#pone.0093667-Srivastava1" target="_blank">[56]</a>.</p>b<p>Number of times the peptides were identified from five biological replicates of xylem saps collected from <i>F. virguliforme</i>-infected soybean plants. The total number of times a peptide(s) was identified is presented in parentheses.</p>C<p>Exponentially modified protein abundance index. This equals to 10^PAI-1, which is proportional to the protein content in a protein mixture.</p

Collection of xylem sap from 14 to 21-day old <i>F. virguliforme-</i>infected or -uninfected soybean plants.

<p>The free end of a rubber tube attached to a 1 mL syringe was securely fasten to the cut soybean hypocotyl and sealed with Vaseline. Low pressure was created by pulling the plunger of the syringe to facilitate xylem sap accumulation.</p

Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.

<p>Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.</p

Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.

<p>Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.</p

Physical map of NBS-LRR like genes present on the <i>Rps12</i> region.

<p>(A) Genetic map of the <i>Rps12</i> region (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169950#pone.0169950.g004" target="_blank">Fig 4B</a>). (B) Physical map of eight NBS-LRR-like genes identified from the <i>Rps12</i> region of the Williams 82 genome.</p

Analysis of <i>NBSRps4/6</i>-specific molecular markers linked to a novel <i>Phytophthora</i> resistance gene.

<p>(A) The <i>NBSRps4/6</i> specific sequence (GenBank accession no. AY258630 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169950#pone.0169950.ref050" target="_blank">50</a>]) used for developing molecular markers. Primer sequences used for PCR are underlined and marked with half arrows. The PCR primers for amplified targets, NBSRps4/6-1272, NBSRps4/6-869, NBSRps4/6-533 and NBSRps4/6-130, are shown along the primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169950#pone.0169950.s003" target="_blank">S2 Table</a>). (B) The <i>NBSRps4/6</i> specific molecular markers linked to the novel <i>Rps</i> gene. L, 100 bp DNA Ladder (New England Biolabs, USA); SP, susceptible parent AR2; RP, resistant parent PI399036; RB, bulk of 10 resistant homozygous RILs; SB, bulk of 10 susceptible RILs. NBSRps4/6-533, <i>NBSRps4/6</i> specific NBSRps4/6-533 marker; NBSRps4/6-130, <i>NBSRps4/6</i> specific NBSRps4/6-130 marker.</p