Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass.

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.

Ryegrass Host Genetic Control of Concentrations of Endophyte-Derived Alkaloids.

Endophytic fungi in pasture grasses produce alkaloids which affect invertebrate and vertebrate herbivores. While the competence to produce an alkaloid is a property of the fungus, the host plant may moderate fungal activity. Host genetic influence on endophyte activity was studied in perennial ryegrass (Lolium perenne L.) infected with a common strain of Neotyphodium lolii (Latch, Christensen & Samuels) Glenn, Bacon & Hanlin. Progeny seedling families of a partial diallel cross and their 12 parent clones were compared in a glasshouse experiment. Peramine and ergovaline concentrations were determined by high pressure liquid chromatography (HPLC), and intensity of endophyte infection was determined by enzyme-linked immunosorbent assay (ELISA). Concentrations of peramine and ergovaline and the amount of endophyte mycelium in plants varied between families, consistently across two glasshouse cells and (for the HPLC data) two harvests. There was no indication of any maternal effects. Host genetic control was evident in significant general combining ability effects and smaller specific combining ability effects. Parent-progeny correlation coefficients were high, and narrow-sense heritability was estimated as 0.70, 0.72, and 0.58 respectively for ergovaline, peramine, and ELISA. Further analysis indicated little interaction between loci, and no directional dominance. The three traits were correlated, indicating that 41 and 65% of the genetically controlled variation in ergovaline and peramine concentrations, respectively, was a function of mycelial mass. However, there were departures from these relationships. Host plant selection may enable development of pastures with controlled low levels of toxic but ecologically beneficial endophyte metabolites.

Identification of Epichloë endophytes in planta by a microsatellite-based PCR fingerprinting assay with automated analysis.

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.

Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response.

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.

Oximes, nitriles and 2-hydroxynitriles as precursors in the biosynthesis of cyanogenic glucosides.

The biosynthesis of the cyanogenic glucosides, linamarin and prunasin, was investigated in linen-flax, peach and cherry-laurel shoots. It was shown that related 2-oximino acids, aldoximes, nitriles and 2-hydroxynitriles were generally good precursors of the aglycone moiety. Studies with double-labelled compounds confirmed the retention of the oximino nitrogen atom from 2-oximinoisovaleric acid and isobutyraldoxime in the biosynthesis of linamarin. A general pathway from amino acids to cyanogenic glucosides involving N-hydroxyamino acids, aldoximes, nitriles and 2-hydroxynitriles is proposed.