Chemotypic diversity of epichloae, fungal symbionts of grasses

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Peramine is an insect-feeding deterrent produced by Epichloë species in symbiotic association with C 3 grasses. The perA gene responsible for peramine synthesis encodes a two-module nonribosomal peptide synthetase. Alleles of perA are found in most Epichloë species; however, peramine is not produced by many perA-containing Epichloë isolates. The genetic basis of these peramine-negative chemotypes is often unknown. Using PCR and DNA sequencing, we analyzed the perA genes from 72 Epichloë isolates and identified causative mutations of perA null alleles. We found nonfunctional perA-⌬R* alleles, which contain a transposon-associated deletion of the perA region encoding the C-terminal reductase domain, are widespread within the Epichloë genus and represent a prevalent mutation found in nonhybrid species. Disparate phylogenies of adjacent A2 and T2 domains indicated that the deletion of the reductase domain (R*) likely occurred once and early in the evolution of the genus, and subsequently there have been several recombinations between those domains. A number of novel point, deletion, and insertion mutations responsible for abolishing peramine production in full-length perA alleles were also identified. The regions encoding the first and second adenylation domains (A1 and A2, respectively) were common sites for such mutations. Using this information, a method was developed to predict peramine chemotypes by combining PCR product size polymorphism analysis with sequencing of the perA adenylation domains. Fungal secondary metabolites are a diverse group of important but often nonessential organic compounds with a wide range of properties that are likely to be advantageous for the producing organism or in some cases essential for pathogenicity or developmental stages (1-3). These low-molecular-weight compounds tend to only be produced under certain environmental or growth conditions. The biosynthetic pathways for production of any particular class of secondary metabolites are common to many fungi, but production of a specific secondary metabolite is often unique to a small phylogenetic group of species (4). Epichloë species are fungal endophytes of C 3 grasses that are known to produce several bioactive alkaloids that provide bioprotective properties to the host plant (5). These secondary metabolites include the indolediterpenes, ergot alkaloids, lolines, and peramine ( Fig. 1) (6, 7). The indole-diterpene lolitrem B and ergot alkaloid ergovaline have significant detrimental effects on the health and production of stock animals that graze infected pastures (7,8). The lolines are insecticidal (9), and peramine is a potent deterrent of feeding by insects, including the agriculturally important invertebrate pest Listronotus bonariensis (Argentine stem weevil) (10-12).Peramine synthesis is catalyzed by the two-module nonribosomal peptide synthetase (NRPS), peramine synthetase (PerA), encoded by the 8.3-kb gene perA (12). The first module of PerA contains an adenylation (A1) domain responsible for selection and activation of t...

Section: Resultsmentioning

confidence: 99%

Disparate Independent Genetic Events Disrupt the Secondary Metabolism Gene perA in Certain Symbiotic Epichloë Species

Berry

Takach

Schardl

et al. 2015

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“…The production of bioactive alkaloids by epichloid endophytes within tall fescue and other cool-season grasses has been demonstrated to provide host protection from herbivory, resulting in substantial ecological and agricultural impacts (3,6,7,43,44,48). Traditionally, the alkaloid chemotype of a given endophyte was tested for the presence of in planta pathway end products, as production in culture (in the absence of the host plant) is most often limited or nonexistent.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, a large part of the success of this germplasm relates to the drought tolerance and antiherbivore properties imparted by the endophyte (2,43,53). All N. coenophialum isolates have been reported to produce peramine and loline alkaloids with known anti-insect properties, but unfortunately several strains also produce ergovaline, which causes fescue toxicosis in mammalian herbivores (12,43,48). Thus, the identification of "livestock-friendly" N. coenophialum isolates lacking ergovaline production is crucial to enhance the agronomic value of elite tall fescue lines without the risk of animal toxicity (6,28,29).…”

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confidence: 99%

Genotypic and Chemotypic Diversity of Neotyphodium Endophytes in Tall Fescue from Greece

Takach

Mittal

Swoboda

et al. 2012

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Epichloid endophytes provide protection from a variety of biotic and abiotic stresses for cool-season grasses, including tall fescue. A collection of 85 tall fescue lines from 15 locations in Greece, including both Continental and Mediterranean germplasm, was screened for the presence of native endophytes. A total of 37 endophyte-infected lines from 10 locations were identified, and the endophytes were classified into five distinct groups (G1 to G5) based on physical characteristics such as colony morphology, growth rate, and conidial morphology. These classifications were supported by phylogenetic analyses of housekeeping genes tefA and tubB, and the endophytes were further categorized as Neotyphodium coenophialum isolates (G1, G4, and G5) or Neotyphodium sp. FaTG-2 (Festuca arundinacea taxonomic group 2 isolates (G2 and G3). Analyses of the tall fescue matK chloroplast genes indicated a population-wide, host-specific association between N. coenophialum and Continental tall fescue and between FaTG-2 and Mediterranean tall fescue that was also reflected by differences in colonization of host tillers by the native endophytes. Genotypic analyses of alkaloid gene loci combined with chemotypic (chemical phenotype) profiles provided insight into the genetic basis of chemotype diversity. Variation in alkaloid gene content, specifically the presence and absence of genes, and copy number of gene clusters explained the alkaloid diversity observed in the endophyte-infected tall fescue, with one exception. The results from this study provide insight into endophyte germplasm diversity present in living tall fescue populations.

“…Agriculturally, ergot alkaloids in forage grasses colonized by endophytic Epichlë spp. (including many fungi recently realigned from the genus Neotyphodium [2]) reduce weight gain and fitness in grazing animals (3,4). Clinically, the structural similarities of ergot alkaloids to neurotransmitters allow ergot alkaloids to treat cognitive and neurological maladies, including dementia, migraines, and Parkinson's disease, in addition to endocrine disorders such as type 2 diabetes and hyperprolactinemia (5)(6)(7)(8).…”

mentioning

confidence: 99%

Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

Robinson

Panaccione

2014

Different lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungus Epichloë festucae var. lolii ؋ Epichloë typhina isolate Lp1 (henceforth referred to as Epichloë sp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate genes easH and cloA were expressed in a mutant strain of the mold Aspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with the Epichloë sp. Lp1 allele of easA, which encodes an enzyme that catalyzed the synthesis of agroclavine from an A. fumigatus intermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressing easA and cloA from Epichloë sp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result of Epichloë sp. Lp1 easA expression in the absence of cloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.