Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi

<div><p>Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus <i>Fusarium</i> are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some <i>Fusarium</i> species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (<i>TRI</i>) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in <i>TRI</i> gene duplication and number of <i>TRI</i> loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the <i>TRI</i> cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of <i>TRI</i> cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi.</p></div

Biosynthetic pathways for the trichothecene analogs deoxynivalenol and T-2 toxin in <i>Fusarium graminearum</i> and <i>F</i>. <i>sporotrichioides</i>, respectively.

<p>The inset at the top right shows the structure and numbering systems for trichodiene and 12,13-epoxytrichothec-9-ene (EPT).</p

Representative diversity of trichothecene structures in fungi other than <i>Fusarium</i>.

<p>See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.g001" target="_blank">Fig 1</a> for examples of <i>Fusarium</i> trichothecenes.</p

Functional analysis of <i>TRI3</i> in <i>Trichoderma arundinaceum</i>.

<p>(<b>A-C</b>) High performance liquid chromatograms showing harzianum A (HA) production by: (<b>A</b>) wild-type progenitor strain Ta37; (<b>B</b>) <i>tri3</i> mutant strain tri3.1; and (<b>C</b>) strain tri3.1.C1, <i>tri3</i> mutant strain tri3.1 complemented with a wild-type copy of <i>TRI3</i>. (<b>D</b>) Quantification of HA production in the wild type and <i>tri3</i> deletion mutant strains tri3.1, tri3.30, tri3.33, and tri3.48. (<b>E</b>) Quantification of HA production in the wild type, <i>tri3</i> mutant strain tri3.1, and three tri3.1-derived strains that were complemented with a wild-type copy of <i>TRI3</i> (strains tri3.1.C1, tri3.1.C4 and tri3.1.C5). (<b>F</b> and <b>G</b>) Total ion chromatograms from gas chromatography-mass spectrometry analysis of culture extracts of the (<b>F</b>) wild-type strain and (<b>G</b>) <i>tri3</i> mutant strain tri3.1. The peaks labeled 4OH and ISD are for trichodermol (4-hydroxy EPT) and isotrichodiol, respectively.</p

Functions of trichothecene biosynthetic genes.

<p>Functions of trichothecene biosynthetic genes.</p

Gene content and arrangement at <i>TRI</i> loci in fungi examined in this study.

<p>The phylogenetic tree to the left was inferred by maximum likelihood analysis of concatenated nucleotide sequences of <i>TRI3</i>, <i>TRI5</i>, and <i>TRI14</i>, the only three <i>TRI</i> genes common to all the fungi. Numbers near branch nodes are bootstrap values based on 1000 pseudoreplicates. The four colored blocks (labeled A, B, C and D) indicate four lineages of <i>TRI</i> genes. The diagrams to the right show the content and arrangement of genes at <i>TRI</i> loci. Green arrows represent homologs of previously described <i>TRI</i> genes, and numbers within arrows indicate <i>TRI</i> gene designations (e.g., <i>14</i> indicates <i>TRI14</i>). <i>TRI22</i> was originally described as <i>TRI11</i> in <i>Trichoderma</i>, but here we consider <i>TRI11</i> and <i>TRI22</i> to be functionally and phylogenetically distinct genes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.s004" target="_blank">S1 Fig</a>). For the purposes of this study, paralogs are indicated by the lowercase letters <i>a</i>, <i>b</i>, and <i>c</i>; e.g., <i>TRI6</i> paralogs are indicated as <i>6a</i>, <i>6b</i>, and <i>6c</i>. Gray arrows represent genes present in the <i>TRI</i> cluster of only one genus; orange arrows represent genes unique to <i>Beauveria</i> and <i>Cordyceps</i>; and purple arrows represent genes unique to <i>Stachybotrys</i> and <i>Myrothecium</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.s005" target="_blank">S2 Fig</a>). Arrows overlaid on the same line indicate genes on the same contig, whereas arrows overlaid on different lines indicate genes on different contigs. For <i>Stachybotrys</i>, the numbers 7711 and 40293 above <i>TRI</i> paralogs indicate strains in which the paralogs occur. <i>TRI3b</i> in <i>S</i>. <i>chartarum</i> strain 40293 is truncated relative to other <i>TRI3</i> homologs and, as a result, is likely nonfunctional.</p

Functional analysis of <i>TRI17</i> in <i>Trichoderma arundinaceum</i>.

<p>(<b>A</b>) Quantitation of harzianum A production in the wild-type strain Ta37, <i>tri17</i> mutant strain tri17.139, and three tri17.139-derived strains (tri17.C1, tri17.C4 and tri17.C10) that were complemented with a wild-type copy of <i>T</i>. <i>arundinaceum TRI17</i>. (<b>B</b> and <b>C</b>) Total ion chromatograms of culture extracts of (<b>B</b>) wild-type progenitor strain Ta37 and (<b>C</b>) <i>tri17</i> deletion mutant tri17.139. The trichothecene biosynthetic intermediate trichodermol (4-hydroxy EPT) is indicated at 5.268 min. 4OH indicates trichodermol (4-hydroxy EPT).</p

Comparison phylogenetic tree inferred from concatenated sequences of <i>TRI3</i>, <i>TRI5</i> and <i>TRI14</i> (left) and a species phylogeny inferred from concatenated sequences of 20 housekeeping genes (right).

<p>Numbers near branch nodes are bootstrap values from 1000 pseudoreplicates. Only bootstrap values greater than 70% are shown. The housekeeping genes used in this analysis are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.s003" target="_blank">S3 File</a>.</p

Scenarios for changes in trichothecene biosynthesis during evolutionary divergence of <i>TRI</i> cluster homologs.

<p>The scenarios presume the ancestral <i>TRI</i> cluster and trichothecene pathway presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.g012" target="_blank">Fig 12</a>. In each scenario, the tree is a simplified version of the tree inferred from concatenated sequences of <i>TRI3</i>, <i>TRI5</i>, and <i>TRI14</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006946#ppat.1006946.g003" target="_blank">Fig 3</a>). A change in biosynthesis from the ancestral state is indicated by a change in color of a branch from black to blue. <b>A.</b> acquisition of C8 oxygen by gain of Tri1 in <i>Fusarium</i> and an unknown enzyme(s) in <i>Microcyclospora</i> and <i>Trichothecium</i>; <b>B.</b> acquisition of C15 oxygen by gain of Tri11 in <i>Fusarium</i>, <i>Beauveria</i> and <i>Cordyceps</i>, and an unknown enzyme in <i>Myrothecium</i> and <i>Stachybotrys</i>; <b>C.</b> acquisition of macrolide ring by gain of unknown enzymes in <i>Myrothecium</i> and <i>Stachybotrys</i>; <b>D.</b> change in C4 hydroxylation enzyme from Tri22 to Tri13 during divergence of <i>Fusarium TRI</i> cluster; <b>E.</b> change in Tri3 function from C4 acylation to C15 acetylation during divergence of <i>Fusarium TRI</i> cluster; and <b>F.</b> change in Tri4 function from 3 to 4 oxygenations during divergence of <i>TRI</i> cluster lineage in <i>Fusarium</i>, <i>Beauveria</i>, and <i>Cordyceps</i>.</p

<i>TRI</i> gene content of fungi examined in the current study.

<p>A gray box indicates that a <i>TRI</i> gene is present in the genome of a fungus, while a white box indicates the gene was not detected. Numbers within boxes indicate the number of paralogs. The Greek letter ψ indicates that a large portion of the gene is present but that it is a pseudogene.</p