De novo Analysis of the Epiphytic Transcriptome of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii and Identification of Candidate Secreted Effector Proteins.

The cucurbit powdery mildew fungus Podosphaera xanthii is a major limiting factor for cucurbit production worldwide. Despite the fungus's agronomic and economic importance, very little is known about fundamental aspects of P. xanthii biology, such as obligate biotrophy or pathogenesis. To design more durable control strategies, genomic information about P. xanthii is needed. Powdery mildews are fungal pathogens with large genomes compared with those of other fungi, which contain vast amounts of repetitive DNA sequences, much of which is composed of retrotransposons. To reduce genome complexity, in this work we aimed to obtain and analyse the epiphytic transcriptome of P. xanthii as a starting point for genomic research. Total RNA was isolated from epiphytic fungal material, and the corresponding cDNA library was sequenced using a 454 GS FLX platform. Over 676,562 reads were obtained and assembled into 37,241 contigs. Annotation data identified 8,798 putative genes with different orthologues. As described for other powdery mildew fungi, a similar set of missing core ascomycete genes was found, which may explain obligate biotrophy. To gain insight into the plant-pathogen relationships, special attention was focused on the analysis of the secretome. After this analysis, 137 putative secreted proteins were identified, including 53 candidate secreted effector proteins (CSEPs). Consistent with a putative role in pathogenesis, the expression profile observed for some of these CSEPs showed expression maxima at the beginning of the infection process at 24 h after inoculation, when the primary appressoria are mostly formed. Our data mark the onset of genomics research into this very important pathogen of cucurbits and shed some light on the intimate relationship between this pathogen and its host plant

De novo Analysis of the Epiphytic Transcriptome of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii and Identification of Candidate Secreted Effector Proteins.

The cucurbit powdery mildew fungus Podosphaera xanthii is a major limiting factor for cucurbit production worldwide. Despite the fungus's agronomic and economic importance, very little is known about fundamental aspects of P. xanthii biology, such as obligate biotrophy or pathogenesis. To design more durable control strategies, genomic information about P. xanthii is needed. Powdery mildews are fungal pathogens with large genomes compared with those of other fungi, which contain vast amounts of repetitive DNA sequences, much of which is composed of retrotransposons. To reduce genome complexity, in this work we aimed to obtain and analyse the epiphytic transcriptome of P. xanthii as a starting point for genomic research. Total RNA was isolated from epiphytic fungal material, and the corresponding cDNA library was sequenced using a 454 GS FLX platform. Over 676,562 reads were obtained and assembled into 37,241 contigs. Annotation data identified 8,798 putative genes with different orthologues. As described for other powdery mildew fungi, a similar set of missing core ascomycete genes was found, which may explain obligate biotrophy. To gain insight into the plant-pathogen relationships, special attention was focused on the analysis of the secretome. After this analysis, 137 putative secreted proteins were identified, including 53 candidate secreted effector proteins (CSEPs). Consistent with a putative role in pathogenesis, the expression profile observed for some of these CSEPs showed expression maxima at the beginning of the infection process at 24 h after inoculation, when the primary appressoria are mostly formed. Our data mark the onset of genomics research into this very important pathogen of cucurbits and shed some light on the intimate relationship between this pathogen and its host plant

The most expressed GO categories in the epiphytic transcriptome of <i>P</i>. <i>xanthii</i>.

<p>Representation of the number of average reads within the most expressed GO categories in the <i>P</i>. <i>xanthii</i> epiphytic cDNA library. For each category, the number of genes within the group of Top50 most expressed transcripts is shown on the right side of the bars. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163379#pone.0163379.s004" target="_blank">S2 Table</a> for details on Top50 most expressed genes.</p

N-terminal amino acid Y/F/WxC motif and natural selection in <i>P</i>. <i>xanthii</i> CSEPs.

<p>The N-terminal amino acid motif Y/F/WxC was screened manually within the pool of CSEPs, and only the motif WxC was observed following the secretion signal. Alignments were performed with ClustalW2, and the sequence logo was represented using the Seq2Logo online server (<a href="http://www.cbs.dtu.dk/biotools/Seq2Logo/" target="_blank">www.cbs.dtu.dk/biotools/Seq2Logo/</a>).</p

Summary statistics of the annotation process of the <i>P</i>. <i>xanthii</i> epiphytic transcriptome.

<p>Summary statistics of the annotation process of the <i>P</i>. <i>xanthii</i> epiphytic transcriptome.</p

Expression of selected <i>P</i>. <i>xanthii</i> CSEP genes in a time-course experiment.

<p>Total RNA was isolated from zucchini cotyledons infected with <i>P</i>. <i>xanthii</i> at different time points, and the relative expression of five putative effector-genes (<i>CSEP01</i>, <i>CSEP02</i>, <i>CSEP05</i>, <i>CSEP021</i> and a homologue to <i>Scp160</i>) was analysed by quantitative RT-PCR. Transcript abundance was normalised to the endogenous control β-tubulin gene <i>PxTUB</i>2 (KC333362). Relative expression of each gene was calibrated to the sample 8 h post inoculation. Points represent the mean ± standard deviation of three technical replicates from two cDNA samples obtained from two different zucchini cotyledons.</p

Pie chart representing the functional annotation of the <i>P</i>. <i>xanthii</i> secretome deduced from the predicted proteins from the epiphytic transcriptome.

<p>The diagram displays the relative abundance of GO categories among the total of 137 putatively secreted proteins identified; 84 secreted proteins were grouped by biological functions, and 53 had no GO terms and, consequently, were catalogued as candidate secreted effector proteins (CSEPs). The number of proteins within each functional category is presented in brackets. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163379#pone.0163379.t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163379#pone.0163379.s006" target="_blank">S4 Table</a> for details.</p

Phylogenetic analysis of <i>P</i>. <i>xanthii</i> CSEPs.

<p>The unrooted phylogenetic tree is based on NJ analysis. The confidence of groupings was estimated by using 1,000 bootstrap replicates, and the percentage of replicate trees supporting each branch is shown next to the branching point. The analysis showed the existence of potential families within the <i>P</i>. <i>xanthii</i> CSEPs.</p

List of CSEPs identified in the <i>P</i>. <i>xanthii</i> epiphytic secretome and orthologues present in the genome of <i>B</i>. <i>graminis</i> f. sp. <i>hordei</i>.

<p>List of CSEPs identified in the <i>P</i>. <i>xanthii</i> epiphytic secretome and orthologues present in the genome of <i>B</i>. <i>graminis</i> f. sp. <i>hordei</i>.</p

Pie chart representing the functional annotation of predicted proteins from the epiphytic transcriptome of <i>P</i>. <i>xanthii</i>.

<p>The diagram displays the relative abundance of <i>C</i>. <i>albicans</i> GOSlim categories among the 6,645 annotated predicted proteins. All data were submitted to the NCBI database under the accession GEUO00000000.</p