A symbiosis expressed non‐ribosomal peptide synthetase from a mutualistic fungal endophyte of perennial ryegrass confers protection to the symbiotum from insect herbivory

Abstract

SummaryWhile much is known about the biosynthesis of secondary metabolites by filamentous fungi their biological role is often less clear. The assumption is these pathways have adaptive value to the organism but often the evidence to support this role is lacking. We provide the first genetic evidence that the fungal produced secondary metabolite, peramine, protects a host plant from insect herbivory. Peramine is a potent insect feeding deterrent synthesized by Epichloë/ Neotyphodium mutualistic endophytes in association with their grass hosts. The structure of peramine, a pyrrolopyrazine, suggests that it is the product of a reaction catalysed by a two-module non-ribosomal peptide synthetase (NRPS). Candidate sequences for a peramine synthetase were amplified by reverse transcription polymerase chain reaction. Four unique NRPS products were identified, two of which were preferentially expressed in planta . One of these hybridized to known peramine producing strains. This clone was used to isolate an Epichloë festucae cosmid that contained a two-module NRPS, designated perA . Nine additional genes, which show striking conservation of microsynteny with Fusarium graminearum and other fungal genomes, were identified on the perA -containing cosmid. Associations between perennial ryegrass and an E. festucae mutant deleted for perA lack detectable levels of peramine. A wild-type copy of perA complemented the deletion mutant, confirming that perA is a NRPS required for peramine biosynthesis. In a choice bioassay, plant material containing the perA mutant was as susceptible to Argentine stem weevil (ASW) ( Listronotus bonariensis ) feeding damage as endophyte-free plants confirming that peramine is the E. festucae metabolite responsible for ASW feeding deterrent activity.

Advanced Data-Mining Strategies for the Analysis of Direct-Infusion Ion Trap Mass Spectrometry Data from the Association of Perennial Ryegrass with Its Endophytic Fungus,Neotyphodium lolii

“…Recently, it was shown that peramine is produced by an endophyte-specific two-module nonribosomal peptide synthetase (perA) and that an Epichloë festucae mutant deleted for perA lacks detectable levels of peramine (Tanaka et al, 2005). It was also shown that plant material containing this mutant endophyte was as susceptible to Argentine stem weevil feeding as endophyte-free plants, demonstrating unambiguously that peramine confers resistance to this insect.…”

“…Endophyte-free perennial ryegrass was obtained as described by Tanaka et al (2005). A 2 3 3 factorial design was applied with endophyte-infected (E1) and endophyte-free (E2) plants, and three tissue types, namely, immature leaf, blade, and sheath.…”

Epichloë hybrida, sp. nov., an emerging model system for investigating fungal allopolyploidy

“…-Genes involved in the peramine, indole-diterpene, and ergot alkaloid pathways (Tanaka et al 2005;Young et al 2006;Fleetwood et al 2007) were identified in E. hybrida Lp1 using the BLASTn function of Geneious 8.1.5 (Kearse et al 2012). Alkaloid production by E. hybrida Lp1, E. festucae var.…”

“…Secondary metabolite genes and alkaloid production.-To profile the secondary metabolites of E. hybrida Lp1, genomic reads were mapped against genes previously characterized as being responsible for peramine, indole-diterpene, and ergovaline biosyntheses (Tanaka et al 2005;Young et al 2006;Fleetwood et al 2007;Schardl et al 2013a). Assembled genes have been deposited in GenBank (SUPPLEMENTARY TABLE 1; see also Schardl et al 2013b for additional accessions of these genes).…”

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

“…Of these, the ergot alkaloids and tremorgenic lolitrems cause neurotoxic effects on grazing or granivorous vertebrates (Steyn and Vleggaar 1985;Bacon et al 1986). Peramine is an insect feeding deterrent and protects perennial ryegrass from the Argentine stem weevil, Listronotus bonariensis, a highly destructive insect pest in New Zealand (Rowan and Latch 1994;Tanaka et al 2005). Lolines are potent insecticidal and anti-aphid agents, not being implicated in any mammalian toxicoses (Jackson et al 1996;Wilkinson et al 2000).…”