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Polyclonal antibodies were produced in sheep for the development of a competitive enzyme-linked immunoassay for use in quantifying loline alkaloids in pasture samples. Lolines are aminopyrrolizidine secondary metabolites produced by fungal endophytes present in tall fescue and meadow fescue grasses, and confer increased resistance to a range of grass pests. Immunizing and plate coating antigens were prepared with derivatized loline dihydrochloride. Cross-reactivity studies indicated that the assay developed has broad specificity and antibody binding to loline analogues is affected by structural changes in the side chain of the loline molecule. Results obtained using the optimized immunoassay were compared with those determined by gas chromatography. The assay provides a sensitive and rapid analytical method that detects the loline analogues of interest and has been applied in pasture breeding programmes. The assay limit of quantitation for lolines in pasture is 3 µg/g in dried herbage. [ABSTRACT FROM PUBLISHER]

Endophytes of the genusEpichloë(Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. ManyEpichloëspecies have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterizeEpichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. TheEpichloëspecies so far identified as the closest known relatives of the two progenitors ofE. hybridaareE. festucaevar.loliiandE. typhina. We confirm that the nuclear genome ofE. hybridacontains two homeologs of most protein-coding genes fromE. festucaeandE. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from theE. typhinasubgenome. Mitochondrial DNA is detectable only fromE. festucae, whereas ribosomal DNA is detectable only fromE. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally inE. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers. [ABSTRACT FROM AUTHOR]

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor white M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fnngal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fuoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed. [ABSTRACT FROM AUTHOR]

Bioprotective alkaloids produced by Epichloë and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indolediterpenes. The LTM locus of Neolyphodium Iolii (Lp19) and Epichloë festuce (FI1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloe endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. Iolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement. [ABSTRACT FROM AUTHOR]

While 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. [ABSTRACT FROM AUTHOR]

Deals with a study which focused on the development and application of a multilocus microsatellite-based polymerase chain reaction (PCR) fingerprinting assay for the identification of Epichloe endophytes in culture and in planta. Abundance of microsatellites in Epichloe endophyte genomes; Cloning of microsatellite loci; Primer design and PCR optimization.

Abstract: 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. [Copyright &y& Elsevier]

The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi. [ABSTRACT FROM AUTHOR]

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract: While it is generally accepted that Neotyphodium lolii and related epichloë endophytes are mutualists that provide important benefits to their perennial ryegrass (Lolium perenne) host plants under conditions of biotic stress, relatively little is known about the effect of endophyte on the host under conditions of abiotic stress. Using genetically identical endophyte infected (E+) and uninfected (E−) clones of perennial ryegrass from a natural and a synthetic association grown under conditions of water stress in a controlled environment, we show that N. lolii had minor effects on morphological responses (leaf elongation rate and ground biomass production) but had more pronounced effects on physiological responses (water use efficiency, relative water content and osmotic potential) by the host. The effects were most marked in the natural association. While levels of proline increased in response to water stress, the presence of endophyte had no effect on those levels. The effect of water stress on endophyte bioprotective metabolites was also examined. Ergovaline levels in pseudostem tissue increased in response to increasing water stress for both N. lolii/L. perenne associations but lolitrem B levels only increased in the natural association. No differences in steady state levels of transcripts from genes known to be required for the synthesis of these alkaloids were observed in response to water stress. This study demonstrated that N. lolii can confer protection to perennial ryegrass from water stress and that levels of the bioprotective metabolites, lolitrem B and ergovaline were altered in response to this abiotic stress in a manner that was specific for each symbiotic association. [Copyright &y& Elsevier]

Abstract: Many grasses live in association with asymptomatic fungi (Neotyphodium spp. endophytes), which grow in the intercellular spaces of the grass. These endophytes produce a range of alkaloids that protect the grass against grazing by mammals and insects. One of these alkaloids is an unusual pyrrolopyrazine, peramine. Peramine appears to be continuously produced by the endophyte, but does not progressively accumulate. No mechanism for the removal of peramine by its further metabolism or any other process has been reported. Our aim was to detect peramine or peramine metabolites in plant fluids to determine if peramine is mobilized, metabolized or excreted by the plant. We also wanted to determine if other fungal metabolites are mobilized by the plant, as has been proposed for the loline alkaloids. We developed a highly sensitive method for the analysis of peramine, using a linear ion trap mass spectrometer. We studied the fragmentation pathway of peramine using ESI MS n and ESI FTICRMS. Based on these results we developed a single reaction monitoring method using the fragmentation of the guanidinium moiety. Cut leaf fluid and guttation fluid of different grass endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloë sp.) were analysed. Peramine was detected in the cut leaf fluid of all grass-endophyte associations, but not in the guttation fluid of all associations. In some associations we also detected lolines and ergot peptide alkaloids. This is the first report showing the mobilization of fungal alkaloids into plant fluids by the host plant in grass-endophyte associations. [Copyright &y& Elsevier]

Epoxyjanthitrems I–IV (1–4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichloë festucae var. lolii. Although structures for epoxyjanthitrems I–IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests. [ABSTRACT FROM AUTHOR]