Figure 1.
The lifecycle of T. inflatum comprises both an asexual stage (primarily saprotrophic growth in soil) and a sexual stage (only occurs on an insect host). A) Asexual reproductive stage growing on cornmeal agar produces asexual spores on phialides with diagnostic inflated bases. B) Uninfected beetle larva (left) and beetle larva infected with asexual spores (right). C) Sexual reproductive stage growing off of an infected beetle host buried in wood. The sexual stage only occurs when two spores of opposite mating type infect the same insect host.
Table 1.
Comparison of genome features with closely related ascomycetes.
Figure 2.
Phylogenetic relationships and orthologous gene clusters.
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 T. inflatum with each of the major families and associated ecologies within Hypocreales: green = Nectriaceae, primarily plant associated including F. graminearum, F. oxysporum, F. verticillioides, and N. haematococca; blue = Hypocreaceae, primarily fungal associated (Tr. atroviride and Tr. virens) or plant saprobic (Tr. reesei); red = Clavicipitaceae/Cordycipitaceae, primarily animal or insect associated including C. militaris, M. robertsii, M. acridum; and pink = T. inflatum.
Figure 3.
Functional classification of gene content.
A) Profiles of GO Slim (Aspergillus GO Slim) molecular function categories for hypocrealean taxa. Taxa from inside of circle to outside of circle are F. oxysporum, F. verticillioides, F. graminearum, N. haematococca, Tr. atroviride, Tr. reesei, Tr. virens, C. militaris, M. acridum, M. robertsii, and T. inflatum. B) Profiles of GO Slim molecular function categories for genes in species-unique orthologous clusters (Figure 3A) showing percentage of genes in each category out of total annotated genes. Taxa from inside to outside are C. militaris, M. robertsii, M. acridum, and T. inflatum.
Figure 4.
Phylogeny of cyclosporin synthetase (simA).
A) Locations of the cyclosporin (simA) and enniatin synthetase (esyn1) module1 clade showing their disparate locations in the larger NRPS A-domain phylogeny. B) Expanded view of the simA clade, showing modules from six other fungal NRPSs producing cyclic depsipeptides and containing A-domains in the simA clade: module 2 of enniatin synthetase (esyn1), module 2 of beauvericin synthetase (bbBeas), module 2 of bassianolide synthetase (bbBsls), aureobasidin A synthetase (aba1), and modules 5 and 6 of destruxin A synthetase (dtxS1). C) Expanded view of the enniatin synthetase (esyn1) module 1 clade containing module 1 of esyn1, bbBeas, and bbBsls.
Figure 5.
Modular domain structure and A-domain specificities of NRPSs grouping within the simA clade.
Color coding of NRPS modules denotes clade assignment of A-domains in phylogeny (Figure 4, Figure S3): light blue = groups within cyclosporin (simA) clade, red = groups within enniatin (esyn1) module 1 clade, white = groups with perA-like outside both simA and enniatin module 1 clade (Figure S3). Abbreviations for unusual amino acid substrates: Bmt = (4R)-4-[(E)-2-butenyl]-4-methyl-threonine, Abu = Aminobutyric acid, Hiv = D-2-hydroxyvaleric acid, Hmp = D-Hmp, D-2-hydroxy-3-methylpentanoic acid.
Figure 6.
Computational and transcriptional identification of the cyclosporin metabolite cluster.
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 simA 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.
Figure 7.
Organization of the simA biosynthetic cluster.
A) The secondary metabolite gene cluster responsible for cyclosporin biosynthesis as identified by antiSMASH (red), SMURF (blue), and RNA-Seq (green) with predicted protein functions or families listed below. B) The enniatin biosynthetic cluster of F. oxysporum showing genes orthologous to those in the simA cluster in blue [only enniatin synthetase (esyn1)] and genes without orthologs in the simA cluster in grey.
Figure 8.
Cyclophilins in T. inflatum genome.
A) Backbone of maximum likelihood phylogeny of major cyclophilins from T. inflatum, H. sapiens, C. elegans, D. melanogaster, and characterized cyclophilins from other fungi, bacteria, and protists (Figure S7) showing phylogenetic positions of the ten T. inflatum cyclophilins and the simA cluster cyclophilin (red asterisk) within the fungal CypA clade. B) Domain organization of cyclophilins identified in the T. inflatum 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 S. cerevisiae Cpr1 and Cpr3, the simA 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.
Figure 9.
Expression profiling of genes in the simA cluster in relation to insect pathogenesis.
A) Relative expression levels in Reads Per Kilobase per million mapped reads (RPKM) of genes in the simA cluster in SM medium. Significantly (p<.01) upregulated genes are shown with an asterisk below gene. B) Relative expression levels (RPKM) of genes in the simA cluster in Sabouraud Dextrose Broth (SDB), cuticle, and hemolymph media. Genes that are significantly (p<.05) upregulated are shown with either a green star (cuticle media) and/or red star (hemolymph media) below gene. In both A and B, the Y-axis is RPKM, the cluster cyclophilin (TINF00586) is indicated with an asterisk above the histogram bar and the antiSMASH (red), SMURF (blue), and RNA-Seq (green) predicted clusters are indicated by lines below.
Figure 10.
Synteny of regions flanking the cyclosporin cluster in other hypocrealean taxa.
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 T. inflatum 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 simA 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 T. inflatum) which is less than 5 kb in all other hypocrealean taxa except C. militaris and Tr. atroviride. Blue arrows show regions inverted in Tr. atroviride and C. militaris. In Tr. atroviride, an inversion has occurred but the region between adjacent genes is still <5 kb and contains no additional genes. In C. militaris, the inversion has added nearly 500 kb of sequence containing additional genes, none of which were found to have orthologs in the simA cluster or to belong to other secondary metabolite clusters.