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

Abstract

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...

Orthologous peramine and pyrrolopyrazine‐producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi

“…Degenerate PCR primers were designed using perA sequences from Epichloë spp. that were known to encode functional peramine synthetases (Berry et al, 2015b;Schardl et al, 2013) and the MR_perA sequence obtained from the M. rileyi RCEF 4871 genome . Primers were designed to highly conserved perA target regions, incorporating the minimum number of degenerate base codes necessary to achieve reliable amplification from all aligned sequences.…”

“…The known distribution of perA was previously limited to Epichloë spp., though the presence of a perA pseudogene in the closely related species Periglandula ipomoeae suggested that the evolutionary origins of perA extend back to at least the common ancestor of these genera (Berry et al ., ; Schardl et al ., ). Our results demonstrate that MR_perA encodes a functional peramine synthetase, establishing a precedent for distribution of perA orthologs beyond genus Epichloë .…”

“…Furthermore, in this study we describe a new method for qualitative and quantitative assessment of peramine using hydrophilic interaction chromatography (HILIC). Due to the different interaction between the highly polar peramine and the HILIC stationary phase, an improved peak shape and assay sensitivity is achieved compared with previous methodologies that used C18 reversed-phase high performance liquid chromatography (Berry et al, 2015b;Rasmussen et al, 2012).…”

Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis

“…lolii (LpTG-1; Lolium perenne taxonomic group) and LpTG-2 possess intact perA genes, while non-peramineproducing strains (belonging to LpTG-3 and LpTG-4) are characterised by partial deletions and premature stop codons (Hettiarachchige et al 2015). The complete perA gene encodes two adenylation (A1 and A2) domains, two thiolation (T1 and T2) domains, a methylation (M) domain, a condensation (C) domain, and a reductase (R) domain, which may be sufficient to produce peramine (Schardl et al 2013b;Berry et al 2015). Several variants of perA with variability of the number of functional domains have been identified in different Epichloë species.…”

“…Several variants of perA with variability of the number of functional domains have been identified in different Epichloë species. Expression of a perA variant which lacks the reductase domain may produce a metabolite related to peramine (Schardl et al 2013b;Berry et al 2015).…”