The number of reads generated from the next generation sequence of the amplicons derived from PNRSV RNA1, 2 and 3 and the number of sequence variants from cluster analysis.

<p>The number of reads generated from the next generation sequence of the amplicons derived from PNRSV RNA1, 2 and 3 and the number of sequence variants from cluster analysis.</p

Median-joining haplotype network showing <i>Prunus necrotic ringspot virus</i> (PNRSV) intra-specific diversity of RNA1, RNA2 and RNA3 sequence variants in sample Q15.

<p>Each of the blue, green and orange circles represents a PNRSV methyl transferase (MT), RNA dependent RNA polymerase (RdRp) and coat protein (CP) amplicon sequence variant of RNA1, RNA2 and RNA3 respectively. The yellow circles represent the largest sequence variants (variant made up of highest copy number of reads) of each phylo-group (labeled I, II or III) of RNA1, RNA2 and RNA3 present in sample Q15. The hatch mark indicates the number of mutations separating the haplotypes and the black circles (median vectors) are hypothetical missing intermediates connecting the haplotype groups. Multiple median vectors connecting phylo-groups were collapsed for ease of presentation.</p

The variant phylo-groups identified from phylogenetic and identity analysis of amplicon variant sequences of the 53 <i>Prunus necrotic ringspot virus</i> (PNRSV) samples, the % clusters identity cut-off and the resulting minimum number of variant groups in each amplicon sample RNA.

<p>The variant phylo-groups identified from phylogenetic and identity analysis of amplicon variant sequences of the 53 <i>Prunus necrotic ringspot virus</i> (PNRSV) samples, the % clusters identity cut-off and the resulting minimum number of variant groups in each amplicon sample RNA.</p

Primers used for the RT-PCR amplification of partial methyltransferase (MT), RNA-dependent RNA polymerase (RdRp) and coat protein (CP) gene sequences of RNA1, RNA2 and RNA3 of the <i>Prunus necrotic ringspot virus</i> (PNRSV) genome respectively.

<p>Primers used for the RT-PCR amplification of partial methyltransferase (MT), RNA-dependent RNA polymerase (RdRp) and coat protein (CP) gene sequences of RNA1, RNA2 and RNA3 of the <i>Prunus necrotic ringspot virus</i> (PNRSV) genome respectively.</p

Maximum likelihood relationship phylogeny obtained from alignment of 5,040, 2,083 and 5,486 amplicon sequence variants of methyl transferase (MT), RNA dependent RNA polymerase (RdRp) and coat protein (CP) gene segments on PNRSV RNA1, RNA2 and RNA3 respectively.

<p>The alignments were generated using Muscle version 3.8.31 and the phylogenetic trees constructed with RAxML version 8.0.19 using GTRGAMMA model with 1000 bootstrap replicates. The trees were visualized in FigTree version 1.4.2 with branches having less than 90% bootstrap support collapsed and are indicated by the black circles. Sequence variant labels were removed for ease of presentation and major variant groups colour coded with phylo-group II on RNA3 (shown in green colour), which had the largest number of variants, collapsed for ease of presentation. The number of samples and number of sequence variants forming each major group of each RNA are shown.</p

Circular plot of the pan-genome of <i>E. amylovora.</i>

<p>The CDS of the pan-genome (forward and reverse) are depicted in the two outermost circles (aqua). Moving inwards, the core genome is depicted in yellow and the accessory genome in black. The accessory genome of the individual strains of <i>E. amylovora</i> continue inwards as follows: <i>Rubus</i>-infecting strains MR1 (red), Ea644 (pink) and ATCC BAA-2158 (purple), and Spiraeoideae-infecting strains CFBP 1430 (light blue), ATCC 49946 (royal blue), Ea266 (dark green), CFBP 2585 (tan), 01SFR-BO (sky blue), Ea356 (teal), UPN527 (navy blue), ACW 56400 (orange) and CFBP 1232^T (light green). Variable regions of interest are numbered with a pan-genome locus (PL) of 1 to 32 and are described in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055644#pone-0055644-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055644#pone-0055644-t002" target="_blank">2</a>. Of note are PL 4 (ICE flanking PAI-1), PL 20 (secondary metabolite cluster only found in <i>Rubus</i>-infecting strains), PL 27 (sequence from the <i>Rubus</i>-infecting strains that could not be assembled into contiguous sequence), PL 28 (pEA72), PL 29 (pEA29), PL 30 (pEI70), PL 31 (pEAR5.2 and pEAR4.3) and PL 32 (pEA30).</p

Singleton development plot analysis.

<p>Single development plots defined using 12 strains of <i>E. amylovora</i> including plasmids (A) and excluding plasmids (B), and 9 Spiraeoideae-infecting strains of <i>E. amylovora</i> including plasmids (C) and excluding plasmids (D). All plots indicate that the pan-genome of <i>E. amylovora</i> is ‘open’, predicting that each additional strain sequenced will add 52 (Plot A), 40 (Plot B), 30 (Plot C) and 11 (Plot D) new singletons to their respective pan-genome sets.</p

Percent average amino acid identities (AAI) calculated from the core genome data set using EDGAR and MUMi scores of genomic distance between the 12 <i>E. amylovora</i> strains and closely related <i>Erwinia</i> spp.

<p>MUMi score values vary from 0 for identical genomes to 1 for very distant genomes.</p

Strain metadata and genome sequence statistics for the 12 <i>E. amylovora</i> strains analyzed in this study.

<p>Strain metadata and genome sequence statistics for the 12 <i>E. amylovora</i> strains analyzed in this study.</p

Proteogenomic Analysis of the Venturia pirina (Pear Scab Fungus) Secretome Reveals Potential Effectors

A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors