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.

The occurrence of the alkaloidsN-formyl andN-acetyl loline, peramine, lolitrem B, and ergovaline and the response of aphids to plants containing these compounds were determined in species and cultivars ofFestuca,Lolium, and other grass genera infected with fungal endophytes (Acremonium spp., andEpichloe typhina). Twenty-nine of 34 host-fungus associations produced one or more of the alkaloids, most frequently peramine or ergovaline. Three alkaloids (lolines, peramine, and ergovaline) were found in tall fescue and in perennial ryegrass infected withA. coenophialum, while peramine, lolitrem B, and ergovaline were present in perennial ryegrass and in tall fescue infected withA. lolii and inF. longifolia infected withE. typhina. WhileA. coenophialum andA. lolii produced similar patterns of alkaloids regardless of the species or cultivar of grass they infected, isolates ofE. typhina produced either no alkaloids or only one or two different alkaloids in the grasses tested. Aphid bioassays indicated thatRhopalosiphum padi andSchizaphis graminum did not survive on grasses containing loline alkaloids and thatS. graminum did not survive on peramine-containing grasses. Ergovaline-containing grasses did not affect either aphid.

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Lolitrems are potent tremorgenic mycotoxins that are synthesised by clavicipitaceous fungal endophytes of the Epichloë/Neotyphodium group in association with grasses. These indole-diterpenes confer major ecological benefits on the grass-endophyte symbiotum. A molecular signature for diterpene biosynthesis is the presence of two geranylgeranyl diphosphate (GGPP) synthases. Using degenerate primers for conserved domains of fungal GGPP synthases, we cloned two such genes, ltmG and ggsA, from Neotyphodium lolii. Adjacent to ltmG are two genes, ltmM and ltmK, that are predicted to encode an FAD-dependent monooxygenase and a cytochrome P450 monooxygenase, respectively. The cluster of ltm genes is flanked by AT-rich retrotransposon DNA that appears to have undergone extensive repeat induced point (RIP) mutation. Epichloë festucae, the sexual ancestor of N. lolii, contains an identical ltm gene cluster, but lacks the retrotransposon "platform'' on the right flank. Associations established between perennial ryegrass and an E. festucae mutant deleted for ltmM lack detectable levels of lolitrems. A wild-type copy of ltmM complemented this phenotype, as did paxM from Penicillium paxilli. Northern hybridization and RT-PCR analysis showed that all three genes are weakly expressed in culture but strongly induced in planta. The relative endophyte biomass in these associations was estimated by real-time PCR to be between 0.3 and 1.9%. Taking this difference into account, the steady-state levels of the ltm transcripts are about 100-fold greater than the levels of the endogenous ryegrass beta-tubulin (beta -Tub1) and actin (Act1) RNAs. Based on these results we propose that ltmG, ltmM and ltmK are members of a set of genes required for lolitrem biosynthesis in E. festucae and N. lolii.

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Neotyphodium sp. Lp1, an endophytic fungus from perennial ryegrass (Lolium perenne), produces the mycotoxin ergovaline in infected grasses, whereas a mutant in which a particular peptide synthetase gene is knocked out does not. We examined the impact of this knockout on other constituents of the ergot alkaloid pathway. Two simple lysergic acid amides, ergine and a previously undescribed amide, were eliminated by the knockout. Lysergic acid accumulated in the knockout endophyte, but quantities were only 13% of the total lysergic acid derivatives accumulated in the wild type. Concentrations of several clavines were not substantially affected. However, a novel clavine accumulated to higher concentrations in perennial ryegrass containing the knockout strain. The results indicate that production of simple lysergic acid amides requires the activity or products of the ergovaline-associated peptide synthetase and that the regulation of ergot alkaloid production is modified in response to the relatively late block in the pathway.

Epichloë endophytes are a group of filamentous fungi that include both sexual (Epichloë) and asexual (Neotyphodium) species. As a group they are genetically diverse and form both antagonistic and mutualistic associations with temperate grasses. We report here on the development of a microsatellite-based PCR system for fingerprinting this group of fungi with template isolated from either culture or infected plant material. M13mp19 partial genomic libraries were constructed for size-fractionated genomic DNA from two endophyte strains. These libraries were screened with a mixture of DIG-labeled dinucleotide and trinucleotide repeat probes. Positive clones were sequenced, and nine unique microsatellite loci were identified. An additional microsatellite was serendipitously identified in the 3′ untranscribed region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from N. lolii Lp19. Primers were designed for each locus and a panel of endophytes, from different taxonomic groupings, was screened to determine the degree of polymorphism. On the basis of these results a multiplex assay was developed for strain identification with fluorescently labeled primers for five of these loci. Using this system the size of the products amplified can be precisely determined by automated analysis, and an allele profile for each strain can be readily generated. The assay was shown to resolve endophyte groupings to the level of known isozyme phenotype groupings. In a blind test the assay was used successfully to identify a set of endophytes in planta. A reference database of allele sizes has been established for the panel of endophytes examined, and this will be expanded as new strains are analyzed.

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole‐diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub‐telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole‐diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.