Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found-a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker

Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found—a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker.The mitochondrial genome sequence is available from the Genbank database (accession number KR704425). NGS sequencing data are available in the SRA database (Bioproject PRJNA283258).Supporting Information: S1 Fig. Secondary structures of predicted tRNA molecules. (TIF)Supporting Information: S2 Fig. Alignment of V. nonalfalfae reads to the V. dahliae mitochondrial genome. (TIF)Supporting Information: S1 File. BLAST results of orf414 analysis. (DOC)Supporting Information: Mitochondrial genome of Verticillium nonalfalfae PLOS ONE | DOI:10.1371/journal.pone.0148525 February 3, 2016 14 / 18 S1 Table. V. nonalfalfae codon usage statistics. (DOC) S2 Table. Collection of Verticillium species included in analysis of the mitochondrial length polymorphism. (XLS) Remove selectedSupporting Information: Mitochondrial genome of Verticillium nonalfalfae PLOS ONE | DOI:10.1371/journal.pone.0148525 February 3, 2016 14 / 18Supporting Information: S2 Table. Collection of Verticillium species included in analysis of the mitochondrial length polymorphism. (XLS)Supporting Information: S1 Table. V. nonalfalfae codon usage statistics. (DOC)VJ received the grant - Javni sklad Republike Slovenije za razvoj kadrov in štipendije - 163. JR (http://www.sklad-kadri.si) BJ received the grant - Javna agencija za raziskovalno dejavnost Republike Slovenije - P4-0077 (www.arrs.gov.si)http://www.plosone.orgam2016Genetic

Species included in the phylogenetic analysis.

<p>Species included in the phylogenetic analysis.</p

Genetic map of the <i>Verticillium nonalfalfae</i> mtDNA.

<p>The concentric circles from the outside inwards represent different tracks. The outermost track represents the coding features of the <i>V</i>. <i>nonalfalfae</i> mitochondrial genome. The direction of the highlights (inward, outward) represents the strand in which the feature is present. The next track represents read counts of RNA-Seq mapping from 2 different pathotypes (mild-blue, lethal-red) of <i>V</i>. <i>nonalfalfa</i>e. The tracks are made of Bowtie2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148525#pone.0148525.ref056" target="_blank">56</a>] mapped RNA-Seq reads and shown as counts per 100bp bins. The counts were normalized with the DESeq method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148525#pone.0148525.ref057" target="_blank">57</a>]. Because of rRNA counts overwhelming the remaining expression profiles, the count number on the graph was capped below 17.5% of the top count profiles (a non-capped graph would show only rRNA expressed), in order to enable visualization of low-expressed regions. Following this track are GC-skew and GC-content tracks, respectively. GC-skew [(G-C)/(G+C)] reflects the relative number of cytosine to guanine and is often used to describe the strand-specific bias of a nucleotide composition. The GC-skew track is shown as a histogram of 250bp sliding windows with calculated gc-skew coefficients. Green regions represent windows for which the coefficient is larger than 0 and red regions windows for which the coefficient is smaller than 0. The neighbouring grayscale heatmap of the GC-content track represents 100bp sliding windows with calculated gc-contents. Regions in the GC content heatmap are shaded in gray, where darker gray represents higher gc-content and lighter gray represents lower gc-content. The two tracks show a similar pattern to other Sordariomycetes and were also used to scan for anomalies in GC content, which could indicate the introduction of heterologous DNA. No anomalies indicating such an event were detected. The cumulative GC skew analysis was also used to try and find the origins of replication and termination of replication loci (data not shown) but we could not determine them with this analysis.</p

RT-qPCR analysis of <i>orf414</i>.

<p>Expression analysis of th<i>e</i> potential long non-coding RN<i>A orf414</i> in two different strains of <i>V</i>. <i>nonalfalfa</i>e (Rec = Slovenian mild strain and T2 = Slovenian lethal strain). Expression values were normalized with <i>ubiquitin (ub</i>q) as a housekeeping reference. Bars indicate standard errors of three biological replicates.</p

tRNA features of the <i>Verticillium nonalfalfae</i> mitochondrial genome.

<p>tRNA features of the <i>Verticillium nonalfalfae</i> mitochondrial genome.</p

Mapping results for mitochondrial sequence for 6 NGS data set used for assembly.

<p>Mapping results for mitochondrial sequence for 6 NGS data set used for assembly.</p

rRNA and <i>orf414</i> features of the <i>Verticillium nonalfalfae</i> mitochondrial genome.

<p>rRNA and <i>orf414</i> features of the <i>Verticillium nonalfalfae</i> mitochondrial genome.</p

Maximum likelihood phylogenetic tree of 20 mitochondrial genomes, based on a conserved set of proteins.

<p>The tree was inferred from an alignment of amino-acid sequences of conserved mitochondrial proteins with 3093 distinct alignment positions and 100 rapid bootstrap inferences. The gamma model of rate heterogeneity and a maximum likelihood estimate of the alpha-parameter were used to prepare the final tree. Numbers above tree nodes represent the bootstrap support values. Next to the tree is a graphical presentation of an alignment of mitochondrial protein-coding genes and their order in the represented species. The conserved set of 14 protein-encoding genes is present in most of the species, with the exception of <i>S</i>. <i>cerevisiae</i> and <i>S</i>. <i>pombe</i> yeasts, which lack the NADH dehydrogenase family of genes, <i>C</i>. <i>lindemuthianum</i>, which lacks the <i>nad2</i> gene, <i>N</i>. <i>crassa</i>, which has two copies of the <i>nad2</i> gene and <i>R</i>. <i>orthosporum</i> and <i>P</i>. <i>anserina</i>, which lack the <i>atp9</i> gene. The phylogenetic tree shows <i>V</i>. <i>nonalfalfae</i> joined with <i>V</i>. <i>dahliae</i> and <i>C</i>. <i>lindemuthianum</i> in a cluster corresponding to the established group of fungi called <i>Glomerellales</i>. <i>C</i>. <i>lindemuthianum</i> can be seen to have a very different gene order compared to the other two members of its group. The <i>Hypocreales</i> and <i>Sordariales</i> groups of the Sordariomycetes can also be seen in the tree. <i>A</i>. <i>chrysogenum</i> of the <i>Hypocreales</i> group contains a translocation of the <i>cox2</i> gene, which distinguishes it from the rest of the members of its group. The <i>Glomerellales</i> and <i>Hypocreales</i> groups show a high degree of synteny within their respective groups and differ only by a translocation of the <i>nad2</i>-<i>nad3</i> gene cluster.</p