Bioinformatic pipeline for the clustering of secreted protein families and classification and ranking of effector candidates.

<p>The pipeline is composed of six major steps delimited by boxes. Step 1 (Secretome prediction) identifies secreted proteins from the predicted proteomes. A total of 1549 and 1852 secreted proteins were predicted from <i>M. larici-populina</i> and <i>P. graminis</i> f. sp. <i>tritici</i> proteomes, respectively. Step 2 (Markov clustering) groups secreted and non-secreted proteins according to sequence similarities to secreted proteins. A total of 435 secreted protein families (tribes) of at least 3 proteins were defined from the proteomes of the two rust fungi. Step 3 (Functional annotation) implements tribe annotations based on sequence homology searches. Step 4 (<i>De novo</i> motif search) searches for conserved motifs. Step 5 (Effector features annotation) uses the current knowledge of effector features to annotate individual members of secreted protein families. Step 6 (Hierarchical tribe clustering) ranks and classifies the tribes based on their content in proteins with effector features to provide a priority list for functional validation studies. Tools (programs and databases) used are indicated in red. HESP, haustoria expressed secreted protein; AVR, effector protein with defined avirulence activity; NLS, nuclear localization signal; FIR, flanking intergenic region; RCPs, repeat containing proteins; SCRs, small cysteine rich proteins.</p

<i>De novo</i> motif searches in the secretome of <i>M. larici-populina</i> and <i>P. graminis</i> f. sp. <i>tritici</i> reveal conserved cysteine rich motifs.

<p>(A) Amino acid position of 25 motifs in the secretome tribes of rust fungi reported by MEME. Arrows highlight positionally constrained motifs that are abundant in the secreted protein tribes. Position is given after the signal peptide cleavage site when applicable. Grey shading indicates the expected position for putative host-translocation motifs (amino acids 50 to 150). Box plots show median position (bar) first and third quartiles (box), first values outside 1.5 the interquartile range (IQR) (whiskers) and outliers (dots), coloured according to the number of tribes containing at least two proteins harbouring the motif. Motifs are classified by decreasing IQR from top to bottom. (B) Sequence logos of motifs with the highest positional constraint, distribution over the largest number of tribes and greatest number of individual proteins (sites) containing the motif.</p

Non-enzymatic PFAM domains significantly enriched in the secretome of rust fungi.

1<p>Enrichment: Number of PFAM hits in secretome over number of hits in non secreted proteins;</p>2<p>p-value for enrichment in secretome;</p>3<p>number of domains in secretome;</p>4<p>number of domains in haustorial proteins;</p>5<p>tribes containing at least two instances of the domain with number of instances in parenthesis.</p

Comparison between core and lineage-specific secretome tribes of at least three proteins in rust fungi.

<p>(A) Core tribes (containing proteins from both species of rust fungi) represent forty percent (174 out of 435) of the secretome tribes containing three or more proteins. (B) Core secretome tribes of at least three proteins are enriched in proteins annotated by homology searches whereas lineage-specific tribes often remained non-annotated. (C) Size distribution of core secretome tribes of at least three proteins was shifted towards larger tribes compared to lineage-specific tribes. The same conventions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029847#pone-0029847-g002" target="_blank">Figure 2</a> were used in the boxplots.</p

Hierarchical clustering of the secretome reveals clusters of secreted protein families as high priority effector candidates.

<p>A complete hierarchical cluster tree of the 188 secretome tribes with combined score ≥6.342 and secretion signal score >0. The tribe identifiers are indicated at the tip the branches of the boostrap support tree. For each tribe, the number of proteins is indicated on the left of the clustering image and the combined score on the right. When proteins in a tribe show similarity (10e⁻⁵ BlastP e-value threshold) to fungal AVRs and <i>M. lini</i> HESPs, these are indicated along the score bars. We distinguished eight clusters. The number of tribes in a cluster is indicated in parenthesis. AVR, avirulence protein; HESP, haustoria expressed secreted protein; FIR, flanking intergenic region; NLS, nuclear localization signal; RCP, repeat containing protein; SCR, small cysteine rich protein.</p

A selection of 8 tribes of candidate effectors of rust fungi.

1<p>Combined score;</p>2<p>Number of proteins in tribe;</p>3<p>number of secreted proteins in tribe;</p>4<p>number of haustorial proteins in tribe;</p>5<p>number of proteins encoded by <i>in planta</i> induced genes. Values in parenthesis show scores obtained for each property. FIR, Flanking Intergenic Regions; NA; Not Applicable; RCP, Repeat-Containing Protein; SCR, Small Cysteine Rich protein. Tables from Duplessis <i>et al.</i> 2011 cited for gene expression:</p>a<p>Table S11;</p>b<p>Table S14;</p>c<p>Table S15;</p>d<p>Table S10.</p

Distribution of effector features in the <i>M. larici-populina and P. graminis</i> f. sp. <i>tritici</i> secretome proteins and tribes.

<p>(A) Percentage of known avirulence effectors (AVRs) from <i>M. lini</i>, <i>P. infestans</i>, <i>L. maculans</i> and <i>C. fulvum</i> showing each effector property. A red cross indicates no match; N.A., not available. (B) Number of proteins grouped in secretome tribes showing each one of eight effector properties. Numbers on charts refers to total number of proteins. (C) The distribution of eight effector features in core and lineage-specific secretome tribes of rust fungi. *Five PFAM domains associated with pathogenicity and enriched in secretome tribes of rust fungi (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029847#pone-0029847-t001" target="_blank">Table 1</a>) and PFAM-B domains were permitted.</p

Illustration of floral mimicry produced by the pseudoflower-forming rust fungus <i>Pucciniamonoica</i>.

<p>(<b>A</b>) Picture of uninfected flowering <i>Boechera</i><i>stricta</i> plant (left) and a close up picture of its light pink flowers (right). (<b>B</b>) Pictures of vegetative tissues of <i>B</i><i>. stricta</i> plants that produce pseudoflowers upon infection with <i>Pucciniamonoica</i> (left) and a close up of a yellow <i>P</i><i>. monoica</i> pseudoflower (right). Samples from <i>B</i><i>. stricta</i> (<b>A</b>) and pseudoflowers (<b>B</b>) were collected near Gunnison, Colorado, United States of America.</p

Differentially expressed genes in pseudoflowers and uninfected <i>Boechera</i><i>stricta</i> flowers using rank products (RP) analysis.

<p>(<b>A</b>) Volcano plots showing changes in gene expression in <i>Pucciniamonoica</i>-induced pseudoflowers (‘Pf’) vs. uninfected <i>Boechera</i><i>stricta</i> plant stems and leaves (‘SL’). (<b>B</b>) Volcano plots showing changes in gene expression in uninfected <i>B</i><i>. stricta</i> flowers (‘F’) vs. uninfected <i>B</i><i>. stricta</i> stems and leaves (‘SL’). Each point in the volcano plot represents changes in gene expression from a single <i>Arabidopsis thaliana</i> gene. Red points indicate genes that are significantly up or down-regulated with a RP FDR value < 0.05. X-axis correspond the log₂ ratio (‘Pf’/’SL’ or ‘F’/’SL’ comparison) and the y-axis correspond to the –log₁₀ of RP FDR value. (<b>C</b>) Venn diagram showing number of genes that are differentially regulated specifically in ‘Pf’ vs. ‘SL’ and ‘F’ vs. ‘SL’ comparisons.</p

qRT-PCR validation of differentially expressed genes in pseudoflowers.

<p>Quantitative Real Time PCR (qRT-PCR) on a panel of seven genes was used to verify the transcriptional changes observed by microarray analysis. Consistent with the microarray results, expression of <i>TEOSINTE </i><i>BRANCHED1, CYCLOIDEA, and </i><i>PCF </i><i>TRANSCRIPTION </i><i>FACTOR3</i> (<i>TCP3</i>, At1g53230), <i>SUGAR </i><i>TRANSPORTER1</i> (<i>SWEET1</i>, At1g21460), <i>SUGAR </i><i>TRANSPORTER15</i> (<i>SWEET15</i>, At5g13170) and <i>TYROSINE </i><i>TRANSAMINASE</i> enzyme encoding gene (<i>TT</i>, At4g23590) genes was up-regulated in <i>Pucciniamonoica</i>-induced pseudoflowers (‘Pf’) compared to <i>B</i><i>. stricta</i> stems and leaves (‘SL’), while <i>ALTERED </i><i>MERISTEM </i><i>PROGRAMMING1</i> (<i>AMP1</i>, At3g54720), <i>KNOTTED-LIKE1</i> (<i>KNAT1</i>, At4g08150) and <i>FLOWERING </i><i>LOCUS </i><i>T</i> (<i>FT</i>, At1g65480) genes was down-regulated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075293#pone-0075293-t001" target="_blank">Table 1</a>). In addition, <i>SWEET15</i> and <i>FT</i> genes were confirmed to be down-regulated in uninfected <i>B</i><i>. stricta</i> flowers (‘F’) compared to ‘SL’ as shown by microarray analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075293#pone.0075293.s002" target="_blank">Table S2</a>). To indicate the mode of regulation we used two symbols: ‘*’ for significant up-regulation and ‘^#’ for significant down-regulation. The number of symbols indicates level of significance: one for <i>P</i> < 0.05, two for <i>P</i> < 0.01 and three for <i>P</i> < 0.001. The error bars represents standard error of the mean.</p