Identification and Structure Elucidation of Epoxyjanthitrems from Lolium perenne Infected with the Endophytic Fungus Epichloë festucae var. lolii and Determination of the Tremorgenic and Anti-Insect Activity of Epoxyjanthitrem I.

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.

The Role of SreA-Mediated Iron Regulation in Maintaining Epichloë festucae-Lolium perenne Symbioses.

In ascomycetes and basidiomycetes, iron-responsive GATA-type transcriptional repressors are involved in regulating iron homeostasis, notably to prevent iron toxicity through control of iron uptake. To date, it has been unknown whether this iron regulator contributes toward mutualistic endosymbiosis of microbes with plants, a system where the endophyte must function within the constraints of an in-host existence, including a dependency on the host for nutrient acquisition. Functional characterization of one such protein, SreA from Epichloë festucae, a fungal endosymbiont of cool-season grasses, indicates that regulation of iron homeostasis processes is important for symbiotic maintenance. The deletion of the sreA gene (ΔsreA) led to iron-dependent aberrant hyphal growth and the gradual loss of endophyte hyphae from perennial ryegrass. SreA negatively regulates the siderophore biosynthesis and high-affinity iron uptake systems of E. festucae, similar to other fungi, resulting in iron accumulation in mutants. Our evidence suggests that SreA is involved in the processes that moderate Epichloë iron acquisition from the plant apoplast, because overharvesting of iron in ΔsreA mutants was detected as premature chlorosis of the host using a hydroponic plant growth assay. E. festucae appears to have a tightly regulated iron management system, involving SreA that balances endophyte growth with its survival and prevents overcompetition with the host for iron in the intercellular niche, thus promoting mutualistic associations. Mutations that interfere with Epichloë iron management negatively affect iron-dependent fungal growth and destabilize mutualistic Epichloë -ryegrass associations.

Contrasting roles of fungal siderophores in maintaining iron homeostasis in Epichloë festucae.

The symbiosis between Epichloë festucae and its host perennial ryegrass (Lolium perenne) is a model system for mutualistic interactions in which the fungal endophyte grows between plant shoot cells and acquires host nutrients to survive. E. festucae synthesises the siderophore epichloënin A (EA) via SidN, a non-ribosomal peptide synthetase (NRPS). EA is involved in the acquisition of iron, an essential micronutrient, as part of the process of maintaining a stable symbiotic interaction. Here, we mutated a different NRPS gene sidC and showed that it is required for production of a second siderophore ferricrocin (FC). Furthermore mutations in sidA, encoding an l-ornithine N5-monooxygenase, abolished both EA and FC production. Axenic growth phenotypes of the siderophore mutants were altered relative to wild-type (WT) providing insights into the roles of E. festucae siderophores in iron trafficking and consequently in growth and morphogenesis. During iron-limitation, EA is the predominant siderophore and in addition to its role in iron acquisition it appears to play roles in intracellular iron sequestration and oxidative stress tolerance. FC in contrast is exclusively located intracellularly and is the dominant siderophore under conditions of iron sufficiency when it is likely to have roles in iron storage and iron transport. Intriguingly, EA acts to promote but may also moderate E. festucae growth (depending on the amount of available iron). We therefore hypothesise that coordinated cellular iron sequestration through FC and EA may be one of the mechanisms that E. festucae employs to manage and restrain its growth in response to iron fluxes and ultimately persist as a controlled symbiont.

A novel family of cyclic oligopeptides derived from ribosomal peptide synthesis of an in planta-induced gene, gigA, in Epichloë endophytes of grasses.

Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum.

An extracellular siderophore is required to maintain the mutualistic interaction of Epichloë festucae with Lolium perenne.

We have identified from the mutualistic grass endophyte Epichloë festucae a non-ribosomal peptide synthetase gene (sidN) encoding a siderophore synthetase. The enzymatic product of SidN is shown to be a novel extracellular siderophore designated as epichloënin A, related to ferrirubin from the ferrichrome family. Targeted gene disruption of sidN eliminated biosynthesis of epichloënin A in vitro and in planta. During iron-depleted axenic growth, ΔsidN mutants accumulated the pathway intermediate N(5)-trans-anhydromevalonyl-N(5)-hydroxyornithine (trans-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of Lolium perenne (perennial ryegrass) with ΔsidN mutants resulted in perturbations of the endophyte-grass symbioses. Deviations from the characteristic tightly regulated synchronous growth of the fungus with its plant partner were observed and infected plants were stunted. Analysis of these plants by light and transmission electron microscopy revealed abnormalities in the distribution and localization of ΔsidN mutant hyphae as well as deformities in hyphal ultrastructure. We hypothesize that lack of epichloënin A alters iron homeostasis of the symbiotum, changing it from mutually beneficial to antagonistic. Iron itself or epichloënin A may serve as an important molecular/cellular signal for controlling fungal growth and hence the symbiotic interaction.

The use of genomics and metabolomics methods to quantify fungal endosymbionts and alkaloids in grasses.

The association of plants with endosymbiotic micro-organisms poses a particular challenge to metabolomics studies. The presence of endosymbionts can alter metabolic profiles of plant tissues by introducing non-plant metabolites such as fungal specific alkaloids, and by metabolic interactions between the two organisms. An accurate quantification of the endosymbiont and its metabolites is therefore critical for studies of interactions between the two symbionts and the environment.Here, we describe methods that allow the quantification of the ryegrass Neotyphodium lolii fungal endosymbiont and major alkaloids in its host plant Lolium perenne. Fungal concentrations were quantified in total genomic DNA (gDNA) isolated from infected plant tissues by quantitative PCR (qPCR) using primers specific for chitinase A from N. lolii. To quantify the fungal alkaloids, we describe LC-MS based methods which provide coverage of a wide range of alkaloids of the indolediterpene and ergot alkaloid classes, together with peramine.

Alkylation or Silylation for Analysis of Amino and Non-Amino Organic Acids by GC-MS?

Gas chromatography-mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies.

Structure of a eukaryotic nonribosomal peptide synthetase adenylation domain that activates a large hydroxamate amino acid in siderophore biosynthesis.

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.

A reverse-phase liquid chromatography/mass spectrometry method for the analysis of high-molecular-weight fructooligosaccharides.

Many important crop and forage plants accumulate polymeric water-soluble carbohydrates as fructooligosaccharides (or fructans). We have developed an improved method for the analysis of the full fructan complement in plant extracts based on porous graphitized carbon chromatography coupled to negative electrospray ionization mass spectrometry. By the use of profile data collection and multiple charge state ions, the effective mass range of the ion trap was extended to allow for the analysis of very high-molecular-weight oligosaccharides. This method allows the separation and quantification of isomeric fructan oligomers ranging from degree of polymerization (DP) 3 to DP 49.

From differentiating metabolites to biomarkers.

The current developments in metabolomics and metabolic profiling technologies have led to the discovery of several new metabolic biomarkers. Finding metabolites present in significantly different levels between sample sets, however, does not necessarily make these metabolites useful biomarkers. The route to valid and applicable biomarkers (biomarker qualification) is long and demands a significant amount of work. In this overview, we critically discuss the current state-of-the-art of metabolic biomarker discovery, with highlights and shortcomings, and suggest a pathway to clinical usefulness.

E/Z-Thesinine-O-4'-alpha-rhamnoside, pyrrolizidine conjugates produced by grasses (Poaceae).

Based on direct infusion mass spectrometry we identified a novel alkaloid as a major component of perennial ryegrass (Lolium perenne). Initial mass spectral data suggested it to be a pyrrolizidine conjugate. As this class of alkaloids has not been described before from grasses, we isolated it to elucidate its structure. The isolated alkaloid proved to be a mixture of two stereoisomers. The structures of the two compounds as determined by 1D and 2D NMR spectroscopy, were E-thesinine-O-4'-alpha-rhamnoside (1) and Z-thesinine-O-4'-alpha-rhamnoside (2). These identifications were supported by the characterisation by GC-MS and optical rotation of (+)-isoretronecanol as the necine base released on alkaline hydrolysis of these alkaloids. 1 and 2 together with the aglycone and a hexoside were also detected in tall fescue (Festuca arundinacea). This is the first report of pyrrolizidine alkaloids produced by grasses (Poaceae).

Phenotypic characterization of transposon-inserted mutants of Clostridium proteoclasticum B316T using extracellular metabolomics.

The development of high-throughput DNA sequencing techniques has enabled the sequencing of several hundred bacterial genomes. However, the major step towards understanding the molecular basis of an organism will be the determination of all gene functions in its genome. Current gene assignments by sequence homology generate numerous hints to putative or unknown functions. Even hits with good homology are often not specific enough to describe the in vivo biochemical functions and the underlying biological roles. In this work we applied metabolic footprinting analysis to characterize Tn916-inserted mutants of a hemicellulose-degrading rumen bacterium grown on complex culture medium. Interestingly, the most distinctive phenotypic difference was observed in a mutant with a transposon insertion in a non-coding region of the genome, while disruption of a gene with high homology to a known alpha-glucosidase/xylosidase showed no distinctive phenotypic effect. Our results demonstrate that extracellular metabolomics data coupled to genome information is a powerful and low-cost approach to rapidly screen and characterize microbial mutants with single gene deletions. However, metabolomics as a stand-alone technique is unlikely to give a complete answer to define gene functions, and, therefore, is an approach to be used to generate hypotheses and direct new experiments to confirm gene function.

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.

Direct-infusion mass spectrometry (MS) was applied to study the metabolic effects of the symbiosis between the endophytic fungus Neotyphodium lolii and its host perennial ryegrass (Lolium perenne) in three different tissues (immature leaf, blade, and sheath). Unbiased direct-infusion MS using a linear ion trap mass spectrometer allowed metabolic effects to be determined free of any preconceptions and in a high-throughput fashion. Not only the full MS(1) mass spectra (range 150-1,000 mass-to-charge ratio) were obtained but also MS(2) and MS(3) product ion spectra were collected on the most intense MS(1) ions as described previously (Koulman et al., 2007b). We developed a novel computational methodology to take advantage of the MS(2) product ion spectra collected. Several heterogeneous MS(1) bins (different MS(2) spectra from the same nominal MS(1)) were identified with this method. Exploratory data analysis approaches were also developed to investigate how the metabolome differs in perennial ryegrass infected with N. lolii in comparison to uninfected perennial ryegrass. As well as some known fungal metabolites like peramine and mannitol, several novel metabolites involved in the symbiosis, including putative cyclic oligopeptides, were identified. Correlation network analysis revealed a group of structurally related oligosaccharides, which differed significantly in concentration in perennial ryegrass sheaths due to endophyte infection. This study demonstrates the potential of the combination of unbiased metabolite profiling using ion trap MS and advanced data-mining strategies for discovering unexpected perturbations of the metabolome, and generating new scientific questions for more detailed investigations in the future.

Isolation and characterisation of procyanidins from Rumex obtusifolius.

An acetone:water (7:3) extract obtained from the leaves of Rumex obtusifolius was fractionated into procyanidin oligomer and polymer fractions using a linear gradient and a simple step method on Sephadex LH-20. The chemical characteristics of the procyanidin fractions were studied by 13C-NMR spectroscopy, acid-catalysed degradation in the presence of benzyl mercaptan, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS and electrospray ionisation (ESI) MS. The 13C-NMR showed that the polymer fraction consisted predominantly of procyanidin polymers, some with galloyl groups attached. The thiolysis reaction products indicated a mean degree of polymerisation (DP) of 4.3 for the step method, and a range of 2.3-8.2 mean DP for the gradient fractionation, with epicatechin as the most abundant flavan-3-ol extension unit, while the terminal units consisted of equal proportions of catechin, epicatechin and epicatechin gallate. Singly charged ions observed in MALDI-TOF/MS showed a range of oligomeric procyanidins and their polygalloyl derivatives. These species (in the range DP 2-7) were also observed by ESI/MS but the spectra were more complex due to overlapping multiply charged ions. Isolation of oligomers from the Sephadex LH-20 fraction by chromatography on polyamide and C18 yielded B1, B2, B3 and B7 dimers, an A-type trimer and a B2 3,3'-O-digallate.

A complex ergovaline gene cluster in epichloe endophytes of grasses.

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.

High-throughput direct-infusion ion trap mass spectrometry: a new method for metabolomics.

A fast method was developed to directly infuse raw plant extracts into a linear ion trap mass spectrometer, using the ion trap to isolate and fragment as many ions as possible from the extract. The full mass spectra can be analysed by multivariate statistics to determine discriminating ions, and the fragmentation data allows rapid classification or identification of these ions. The methodology was used to screen a wide range of strains of endophytic fungi in perennial ryegrass seeds for differences in metabolic profiles. The results show that this newly developed methodology is able to determine discriminating ions that can be present in very low concentrations. It also yielded sufficient fragmentation data to classify or identify the discriminating ions.

Peramine and other fungal alkaloids are exuded in the guttation fluid of endophyte-infected grasses.

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 MSn 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.

Transformation of the ryegrass endophyte Neotyphodium lolii can alter its in planta mycelial morphology.

The fungus Neotyphodium lolii grows in the intercellular spaces of perennial ryegrass as a mutualistic endosymbiont. One of the benefits it conveys to the plant is the production of alkaloids toxic to herbivores. We wanted to determine in planta expression patterns of the N. lolii 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) gene, believed to be involved in the synthesis of two of these alkaloid toxins, lolitrem B and ergovaline. We transformed the N. lolii strain Lp19 with plasmids, in which DNA fragments upstream of the open reading frame of the N. lolii HMG CoA reductase gene controlled expression of the GUS (gusA; Escherichia coli beta-glucuronidase) reporter gene. In exponentially growing cultures, the GUS gene was not expressed if the length of upstream sequence was less than 400 bp, and >1100 bp were required for maximum expression. When reintroduced into ryegrass plants, transformants often showed highly increased hyphal branching compared to the wild-type parent strain, although in culture their growth kinetics and morphology were indistinguishable from that of the wild-type. Deterioration of hyphae and the hypha-plant interface occurred and in one transformant reduced tillering (formation of new plants, referred to in agronomy as tillers) and death of infected plants. We found no evidence that these abnormalities were caused by interference of the construct with the function of the native gene, as judged by analysis of the site of integration of the promoter-GUS cassette, expression of the native gene and lolitrem B and ergovaline levels in infected plants. However, there was some correlation between GUS expression and the degree of hyphal branching, suggesting that high levels of beta-glucuronidase may disturb the symbiotic interaction. Levels of another alkaloid, peramine, were also not significantly affected by transformation. In previous studies increased in planta branching of the endophyte has been shown to be associated with a severe reduction of alkaloid production. Our results show that a plant-endophyte association in which increased branching occurs is still able to produce alkaloids.

Extracellular metabolomics: a metabolic footprinting approach to assess fiber degradation in complex media.

This work reports the implementation and optimization of a method for high-throughput analysis of metabolites produced by the breakdown of natural polysaccharides by microorganisms. Our simple protocol enables simultaneous separation and quantification of more than 40 different sugars and sugar derivatives, in addition to several organic acids in complex media, using 50-mul samples and a standard gas chromatography-mass spectrometry platform that was fully optimized for this purpose. As an implementation proof-of-concept, we assayed extracellular metabolite levels of three bacterial strains cultivated on complex medium rich in polysaccharides and under identical growth conditions. We demonstrate that the metabolic footprinting profile data distinguish among sample types such as typical metabolomics data. Moreover, we demonstrate that the differential metabolite-level data provide insight on specific fibrolytic activity of the different microbial strains and lay the groundwork for integrated proteome-metabolome studies of fiber-degrading microorganisms.