Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses

GC proportions in genic and repeat DNA of sequenced genomes.^a

a<p>Abbreviations: CDS = coding sequence, GC = proportion of sequence that is G or C, non-Rpt-IG = nonrepetitive intergenic DNA, Rpt = repetitive DNA.</p>b<p>Statistics for <i>P. ipomoeae</i> are tentative because the assembly was filtered by selecting only contigs containing tBLASTx matches to genome sequences from the other Clavicipitaceae.</p

Alkaloid profiles of sequenced isolates.^a

a<p>Strains are abbreviated as follow: <i>Cpu</i> = <i>Claviceps purpurea</i> 20.1, <i>Cfu</i> = <i>C. fusiformis</i> PRL 1980, Cpa = <i>C. paspali</i> RRC-1481, <i>Eam</i> = <i>Epichloë amarillans</i> E57, <i>Ebe</i> = <i>E. brachyelytri</i> E4804, <i>Eel</i> = <i>E. elymi</i> E56, <i>Ef</i>1 = <i>E. festucae</i> Fl1, <i>Ef</i>2 = <i>E. festucae</i> E2368, <i>Egl</i> = <i>E. glyceriae</i> E2772, <i>Et</i>8 = <i>E. typhina</i> E8, <i>Et</i>5 = <i>E. typhina</i> E5819, <i>Nga</i> = <i>N. gansuense</i> E7080, <i>Ngi</i> = <i>N. gansuense</i> var. <i>inebrians</i> E818, <i>Nun</i> = <i>N. uncinatum</i> E167, <i>Pip</i> = <i>P. ipomoeae</i> IasaF13. Symbols: + = present, (+) = intermediate inferred to be synthesized because downstream product is present, − = not predicted and not detected, (−) = predicted but not detected, nt = predicted but not tested, ERA = ergotamine, ERB = ergobalansine, ERC = ergocryptine, ERV = ergovaline. Blank cells indicate compounds not predicted from genotype, and not tested.</p>b<p>Identification of IDT-436 and terpendoles E, I, J, K, M, M, and A are tentative because authentic standards are unavailable.</p

Summary of loline alkaloid-biosynthesis pathway.

<p>Arrows indicate one or more steps catalyzed by products of the genes indicated. Arrows and genes in blue indicate steps in synthesis of the first fully cyclized intermediate (NANL). Arrows and genes in red indicate steps in modification of NANL to give the variety of lolines found in the epichloae. Asterisks indicate <i>LOL</i> genes that were newly discovered in the genome sequence of <i>E. festucae</i> E2368.</p

Structures of the indole-diterpene biosynthesis loci (<i>IDT/LTM</i>) in sequenced genomes.

<p><i>IDT/LTM</i> genes are indicated by single letters, whereby <i>Q = idtQ</i> or <i>ltmQ</i> (in <i>E. festucae</i>), and so forth. Tracks from top to bottom of each map represent the following: genes, repeats, MITEs, and graphs of AT (red) and GC (blue) contents. Each gene is represented by a filled arrow indicating its direction of transcription. Closed circles indicate telomeres, and distances from the telomere on the <i>E. festucae</i> map are indicated in kilobasepairs (kb). Cyan bars representing repeat sequences are labeled with names or numbers to indicate relationships between repeats in the different species. Vertical bars beneath the repeat maps indicate MITEs. Genes for the first fully cyclized intermediate, paspaline, are indicated in blue, those for subsequent chemical decorations are shown in red, and <i>idt/ltmS</i>, with undetermined function, is in purple. Identifiable genes flanking the clusters are indicated in gray, and unfilled arrows indicate pseudogenes. The major pathway end-product for each strain is listed at the right of its map, abbreviated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, and in bold for those confirmed in this study.</p

Phylogenies of <i>rpbA</i> from sequenced isolates and other Clavicipitaceae.

<p>The phylogenetic tree is based on nucleotide alignment for a portion of the RNA polymerase II largest subunit gene, <i>rpbA</i>. This tree is rooted with <i>Fusarium graminearum</i> as the outgroup. Epichloae are indicated in green, <i>Claviceps</i> species are indicated in blue, <i>Periglandula</i> species are indicated in red, and <i>Aciculosporium take</i> is in black. Species for which genomes were sequenced in this study are shown in bold type, and asterisks indicate plant-associated fungi. Alkaloids listed are the major pathway end-products predicted from the genome sequences, abbreviated as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g002" target="_blank">Figure 2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g004" target="_blank">Figure 4</a>. Other abbreviations: (−) = some genes or remnants present, but not predicted to make alkaloids of this class, – = no genes present for this alkaloid class, EA = ergot alkaloids may be produced; IDT = indole-diterpenes may be produced, (ΔR*) = deletion of terminal reductase domain of <i>perA</i>.</p

Relationships of <i>ltmE</i> and <i>ltmJ</i> with other <i>LTM</i> genes.

<p>Filled boxes indicate coding sequences of exons. Gray polygons indicate closest BLASTp matches to inferred polypeptide sequences for each exon, and are labeled with percent amino-acid identities.</p

Symbiosis of meadow fescue with <i>Epichloë festucae</i>, a heritable symbiont.

<p>Single optical slice confocal micrographs of <i>E. festucae</i> expressing enhanced cyan-fluorescent protein were overlain with DIC bright field images of (A) ovules (bar = 100 µm), (B) embryos (bar = 200 µm), and (C) shoot apical meristem and surrounding new leaves (bar = 200 µm). (D) Asymptomatic (left) and “choked” (right) inflorescences simultaneously produced on a single grass plant infected with a single <i>E. festucae</i> genotype. Vertical (seed) transmission of the symbiont occurs via the asymptomatic inflorescence, whereas the choked inflorescence bears the <i>E. festucae</i> fruiting structure (stroma), which produces sexually derived spores (ascospores) that mediate horizontal transmission.</p