Maintenance of Sex-Related Genes and the Co-Occurrence of Both Mating Types in <i>Verticillium dahliae</i>

<div><p><i>Verticillium dahliae</i> is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of <i>V. dahliae</i> suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of <i>V. dahliae</i>. Only 1% of isolates in a global collection of 1120 phytopathogenic <i>V. dahliae</i> isolates contained the <i>MAT1-1</i> idiomorph, whereas 99% contained <i>MAT1-2</i>. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of <i>V</i>. <i>dahliae</i>, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including <i>MAT1-1-1</i> and <i>MAT1-2-1</i>, were constitutively expressed in <i>V</i>. <i>dahliae</i> cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in <i>V</i>. <i>dahliae MAT1-1-1</i> and <i>MAT1-2-1</i> sequences were indistinguishable from the ratios observed in the <i>MAT</i> genes of sexual fungi in the <i>Pezizomycotina</i>. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen <i>V</i>. <i>dahliae</i> sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in <i>V. dahliae</i>.</p></div

Reverse-transcriptase PCR results of 10 <i>Verticillium dahliae</i> orthologs of genes associated with the sexual cycle in model fungal systems; gene names are provided for each lane; A) RT-PCR results from <i>V. dahliae</i> strain 58 (<i>MAT1-1)</i>.

<p>B) RT-PCR results from <i>V. dahliae</i> strain Ls 17 (<i>MAT1-2</i>);</p

Comparison of codons under positive (relaxed) and purifying selection in 18 sex-related genes in <i>Verticillium dahliae</i> using the MEC model.

1<p>Fungal taxa and gene accessions used to estimate selective pressures in <i>V. dahliae</i> genes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112145#pone.0112145.s003" target="_blank">Table S2</a>. Color-coded SELECTON results for each gene are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112145#pone.0112145.s001" target="_blank">Figure S1</a>.</p><p>Comparison of codons under positive (relaxed) and purifying selection in 18 sex-related genes in <i>Verticillium dahliae</i> using the MEC model.</p

Comparison of codons under positive (relaxed) and purifying selection in <i>MAT1-1-1</i>, in a variety of fungi in the subphylum <i>Pezizomycotina</i> using the MEC model.

1<p>SELECTON results for the putatively asexual fungus <i>V. dahliae</i> were calculated by analyzing a <i>MAT1-1-1</i> codon sequence alignment including sequences from all other fungi listed except <i>P. fulva</i>. Results for the ten species <i>A. fumigatus – S. sclerotiorum</i> were calculated using a codon alignment of only these ten species.</p>2<p>Homothallic fungus.</p>3<p>Results for the putatively asexual fungus <i>P. fulva</i> were calculated by analyzing a <i>MAT1-2-1</i> codon sequence alignment including sequences from all other fungi listed except <i>V. dahliae</i>.</p><p>Comparison of codons under positive (relaxed) and purifying selection in <i>MAT1-1-1</i>, in a variety of fungi in the subphylum <i>Pezizomycotina</i> using the MEC model.</p

New Fungus-Insect Symbiosis: Culturing, Molecular, and Histological Methods Determine Saprophytic Polyporales Mutualists of Ambrosiodmus Ambrosia Beetles

Ambrosia symbiosis is an obligate, farming-like mutualism between wood-boring beetles and fungi. It evolved at least 11 times and includes many notorious invasive pests. All ambrosia beetles studied to date cultivate ascomycotan fungi: early colonizers of recently killed trees with poor wood digestion. Beetles in the widespread genus Ambrosiodmus, however, colonize decayed wood. We characterized the mycosymbionts of three Ambrosiodmus species using quantitative culturing, high-throughput metabarcoding, and histology. We determined the fungi to be within the Polyporales, closely related to Flavodon flavus. Culture-independent sequencing of Ambrosiodmus minor mycangia revealed a single operational taxonomic unit identical to the sequences from the cultured Flavodon. Histological sectioning confirmed that Ambrosiodmus possessed preoral mycangia containing dimitic hyphae similar to cultured F. cf. flavus. The Ambrosiodmus-Flavodon symbiosis is unique in several aspects: it is the first reported association between an ambrosia beetle and a basidiomycotan fungus; the mycosymbiont grows as hyphae in the mycangia, not as budding pseudo-mycelium; and the mycosymbiont is a white-rot saprophyte rather than an early colonizer: a previously undocumented wood borer niche. Few fungi are capable of turning rotten wood into complete animal nutrition. Several thousand beetle-fungus symbioses remain unstudied and promise unknown and unexpected mycological diversity and enzymatic innovations

Ecological characteristics of multilocus microsatellite types that comprised isolates of both mating types.

a<p>“0” indicates no amplification at locus; alleles are presented in the order: VD2.VD1.VD9.VD11.VD92.VD97.VD69.VD12.VD27.VD73.VD8.VD10.VD3. ^{b, c} Total number of <i>MAT1-1</i>, <i>MAT1-2</i> isolates for each microsatellite type.</p><p>Ecological characteristics of multilocus microsatellite types that comprised isolates of both mating types.</p

Color-coded results of SELECTON analyses of <i>Verticillium dahliae MAT1-2-1</i>, compared to sequences from nine different sexual fungi in the <i>Pezizomycotina</i>.

<p>Shades of yellow (colors 1 and 2) indicate a Ka/Ks ratio>1 (positive selection) and shades of purple (colors 3 through 7) indicate a Ka/Ks ratio<1 (purifying selection); A) results from the M8 model; B) results of the MEC model; amino acid sequence of the HMG domain is indicated by black border.</p

Primers used to amplify <i>V. dahliae</i> sex-related genes with RT-PCR.

1<p>The genome of <i>V. dahliae</i> strain Ls 17 contains only <i>MAT1-2-1. MAT1-1-1</i> sequence obtained from NCBI GenBank.</p><p>Primers used to amplify <i>V. dahliae</i> sex-related genes with RT-PCR.</p