Phylogenetic placement of the ectomycorrhizal genus Cenococcum in Gloniaceae (Dothideomycetes)

Cenococcum is a genus of ectomycorrhizal Ascomycota that has a broad host range and geographic distribution. It is not known to produce either meiotic or mitotic spores and is known to exist only in the form of hyphae, sclerotia and host-colonized ectomycorrhizal root tips. Due to its lack of sexual and asexual spores and reproductive structures, it has proven difficult to incorporate into traditional classification within Ascomycota. Molecular phylogenetic studies of ribosomal RNA placed Cenococcum in Dothideomycetes, but the definitive identification of closely related taxa remained elusive. Here we report a phylogenetic analysis of five nuclear loci (SSU, LSU, TEF1, RPB1, RPB2) of Dothideomycetes that placed Cenococcum as a close relative of the genus Glonium of Gloniaceae (Pleosporomycetidae incertae sedis) with strong statistical support. Glonium is a genus of saprobic Dothideomycetes that produces darkly pigmented, carbonaceous, hysteriate apothecia and is not known to be biotrophic. Evolution of ectomycorhizae, Cenococcum and Dothideomycetes is discussed.Keywords: fungi, ectomycorrhizae, Cenococcum, phylogenetics, Dothideomycetes, Gloniaceae, systematic

The Genome of Tolypocladium inflatum: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster

The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology

Phylogenetic placement of the ectomycorrhizal genus Cenococcum in Gloniaceae (Dothideomycetes)

Cenococcum is a genus of ectomycorrhizal Ascomycota that has a broad host range and geographic distribution. It is not known to produce either meiotic or mitotic spores and is known to exist only in the form of hyphae, sclerotia and host-colonized ectomycorrhizal root tips. Due to its lack of sexual and asexual spores and reproductive structures, it has proven difficult to incorporate into traditional classification within Ascomycota. Molecular phylogenetic studies of ribosomal RNA placed Cenococcum in Dothideomycetes, but the definitive identification of closely related taxa remained elusive. Here we report a phylogenetic analysis of five nuclear loci (SSU, LSU, TEF1, RPB1, RPB2) of Dothideomycetes that placed Cenococcum as a close relative of the genus Glonium of Gloniaceae (Pleosporomycetidae incertae sedis) with strong statistical support. Glonium is a genus of saprobic Dothideomycetes that produces darkly pigmented, carbonaceous, hysteriate apothecia and is not known to be biotrophic. Evolution of ectomycorhizae, Cenococcum and Dothideomycetes is discussed. © 2012 by The Mycological Society of America

The Genome of <i>Tolypocladium inflatum</i>: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster

<div><p>The ascomycete fungus <i>Tolypocladium inflatum</i>, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of <i>T. inflatum</i> strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in <i>T. inflatum</i> and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (<i>simA</i>) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. <i>T. inflatum</i> also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The <i>T. inflatum</i> genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.</p></div

Modular domain structure and A-domain specificities of NRPSs grouping within the <i>simA</i> clade.

<p>Color coding of NRPS modules denotes clade assignment of A-domains in phylogeny (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003496#pgen-1003496-g004" target="_blank">Figure 4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003496#pgen.1003496.s003" target="_blank">Figure S3</a>): light blue = groups within cyclosporin (<i>simA</i>) clade, red = groups within enniatin (<i>esyn1</i>) module 1 clade, white = groups with <i>perA</i>-like outside both <i>simA</i> and enniatin module 1 clade (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003496#pgen.1003496.s003" target="_blank">Figure S3</a>). Abbreviations for unusual amino acid substrates: Bmt = (4<i>R</i>)-4-[(<i>E</i>)-2-butenyl]-4-methyl-threonine, Abu = Aminobutyric acid, Hiv = D-2-hydroxyvaleric acid, Hmp = D-Hmp, D-2-hydroxy-3-methylpentanoic acid.</p

Comparison of genome features with closely related ascomycetes.

<p>Comparison of genome features with closely related ascomycetes.</p

Synteny of regions flanking the cyclosporin cluster in other hypocrealean taxa.

<p>Genes within the cyclosporin biosynthetic cluster as delineated by antiSMASH (red), SMURF (blue), and RNA-Seq (green) plus ten genes on the 5′ (green) and 3′ (blue) flanks of the antiSMASH predicted cluster are shown at top and numbered from left (5′) to right (3′) (1–42). Orthologous genes in other hypocrealean taxa identified by best-pairwise BLAST searches are shown below for each species. Grey genes indicate additional genes present in other species while grey shaded areas show regions of synteny between genomes. Genes in <i>T. inflatum</i> in the region between the C2H2 Zn-finger transcription factor (TINF00183) on the 5′end (red line) and the RNA-Seq predicted 3′- end of the <i>simA</i> cluster (at TINF007874) (blue line) mostly lack orthologs in other hypocrealean genomes. The few best-pair orthologs identified in other Hypocreales were found elsewhere in these genomes. Numbers above blue triangles show length of intervening sequence between the 5′ (red line) and 3′ (blue line) flanks of the cluster (approximately 95 kb in <i>T. inflatum</i>) which is less than 5 kb in all other hypocrealean taxa except <i>C. militaris</i> and <i>Tr. atroviride</i>. Blue arrows show regions inverted in <i>Tr. atroviride</i> and <i>C. militaris</i>. In <i>Tr. atroviride</i>, an inversion has occurred but the region between adjacent genes is still <5 kb and contains no additional genes. In <i>C. militaris</i>, the inversion has added nearly 500 kb of sequence containing additional genes, none of which were found to have orthologs in the <i>simA</i> cluster or to belong to other secondary metabolite clusters.</p

Computational and transcriptional identification of the cyclosporin metabolite cluster.

<p>A) Fold changes (log₂ transformed) of gene expression levels from SDB to SM media at time points 1–6 (days 2, 4, 6, 8, 10, 12). Strong upregulation of gene expression occurs after time point 3 (day 6). All genes marked below with an * are upregulated with q-value<0.001 for at least one time point. The boundaries to the cyclosporin <i>simA</i> cluster predicted by antiSMASH (red), SMURF (blue), and RNA-Seq data (green) are indicated by bars below. B) Partial HPLC traces showing the major cyclosporin A peak at 38 min. (marked with a red asterisk) in SM medium for each harvest time point. Trace amounts of cyclosporin A are found at time points 1 and 2, but production spikes at time point 3 (day 6) and peaks at time point 4 (day 8). Additional peaks surrounding the 38 min. major peak are observed after time point 4, consistent with depletion of substrates in the culture media leading to relaxed specificity of NRPS A-domains and production of additional cyclosporin analogs.</p

Cyclophilins in <i>T. inflatum</i> genome.

<p>A) Backbone of maximum likelihood phylogeny of major cyclophilins from <i>T. inflatum</i>, <i>H. sapiens</i>, <i>C. elegans</i>, <i>D. melanogaster</i>, and characterized cyclophilins from other fungi, bacteria, and protists (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003496#pgen.1003496.s007" target="_blank">Figure S7</a>) showing phylogenetic positions of the ten <i>T. inflatum</i> cyclophilins and the <i>simA</i> cluster cyclophilin (red asterisk) within the fungal CypA clade. B) Domain organization of cyclophilins identified in the <i>T. inflatum</i> genome. The cyclosporin cluster cyclophilin (TINF00586) is indicated with a red asterisk and contains a mitochondrial localization signal. C) Expanded view of the fungal CypA clade showing <i>S. cerevisiae</i> Cpr1 and Cpr3, the <i>simA</i> cluster cyclophilin (TINF00586) in red, and TINF04375 in blue. Note that all of the products in blue are likely produced by alternative splicing of the single gene TINF04375.</p

Phylogenetic relationships and orthologous gene clusters.

<p>A) Maximum likelihood phylogeny created from a concatenated alignment of 2769 groups of single copy orthologs identified by the Hal pipeline. Phylogeny constructed using RAxML with best models for each cluster partition identified using ProtTest. Bootstrap values for analyses with the original alignment (top)/the alignment with fast-evolving sites removed (bottom) are shown above nodes. Larger numbers beneath or adjacent to nodes and terminal taxa indicate the number of clusters and genes (in parentheses) within those clusters that map to each node in the phylogeny or are unique to a species. Color coding corresponds to fungal host: green = plant associated, blue = fungal associated, red = animal or insect associated. Hypocreales is delineated at node 1. A major shift from early diverging taxa that have primarily plant-associated hosts to either animal/insect or fungal hosts occurs at node 2. B) The number of both clusters and number of genes (in parentheses) in those clusters that are shared by <i>T. inflatum</i> with each of the major families and associated ecologies within Hypocreales: green = Nectriaceae, primarily plant associated including <i>F. graminearum</i>, <i>F. oxysporum</i>, <i>F. verticillioides</i>, and <i>N. haematococca</i>; blue = Hypocreaceae, primarily fungal associated (<i>Tr. atroviride</i> and <i>Tr. virens</i>) or plant saprobic (<i>Tr. reesei</i>); red = Clavicipitaceae/Cordycipitaceae, primarily animal or insect associated including <i>C. militaris</i>, <i>M. robertsii</i>, <i>M. acridum</i>; and pink = <i>T. inflatum</i>.</p