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Neotyphodium lolii is a fungal endophyte that lives entirely within the intercellular spaces of its grass host, perennial ryegrass (Lolium perenne, L.). Infection is symptomless and the endophyte relies on the host plant for dissemination via the seed. The association is mutually beneficial since the endophyte confers a number of biotic and abiotic advantages to the host. This paper presents an overview of the functional genomics approaches we are using at AgResearch to dissect the molecular basis of this symbiosis and will broadly describe the fields of genomics, transcriptomics, proteomics and metabolomics, as applied to this system. We have used isogenic ryegrass lines infected or uninfected with endophyte in combination with a suite of molecular biology tools, including Expressed Sequence Tags (ESTs), cDNA and Affymetrix GeneChip® microarray analysis, 2D-gel electrophoresis (to identify novel proteins associated with symbiosis), and metabolic profiling. By using a multidisciplinary approach we aim to identify genes which are important in both the establishment and maintenance of symbiosis, as well as elucidate how endophyte confers enhancements to its host. Keywords: Neotyphodium, Epichloë, symbiosis, functional genomics

Endophytes, belonging to the genus Neotyphodium, live symptomlessly within the intercellular spaces of cool-season grasses, and confer a number of biotic and abiotic advantages to their hosts. We identified a novel endophyte gene (designated Nc25) that is expressed preferentially in planta, is one of the most abundant fungal transcripts in endopyte-infected grasses and which is distributed and highly expressed in a wide range of endophyte/ grass associations. Nc25 is novel and shows no homology to sequence databases or fungal genome initiatives. Characterisation indicates that it encodes a small secreted protein. Re-introduction of a Nc25 deletion strain into perennial ryegrass showed no visible effect on the symbiosis but an unknown oligopeptide, detected only in infected plants, was eliminated. Surprisingly, the oligopeptide is unrelated to the predicted peptide product of Nc25. We hypothesise that Nc25 may regulate the oligopeptide biosynthetic pathway and are investigating this using Affymetrix gene chips to determine how Nc25 affects global gene expression. In addition we are interested in the biological function of this secondary metabolite during symbiosis and in particular whether it has bioactivity that may confer abiotic or biotic advantages to the host plant. Keywords: Neotyphodium, in planta expressed gene, oligopeptide, EGFP

The association between perennial ryegrass (PRG) and fungal endophyte is an important factor in the protection of New Zealand pastures from insect pests, and the genetic background of the host plant significantly affects endophyte performance. Quantitative trait locus (QTL) analysis of phenotypic data from herbage harvested from a mapping population in autumn 2005 identified loci in the PRG genome influencing three traits: (1) endophyte mycelial mass; (2) ergovaline level; (3) peramine level. Three QTL were identified for each trait. For mycelial mass two QTL accounted for more than 75% of the variation (PV) for in the population and have considerable potential for development as pre-inoculation marker-assisted selection (MAS) tools for endophyte colonisation in PRG breeding programmes. The largest-effect QTL for peramine level (18% PV) coincided with a major mycelial mass QTL, implying peramine concentration is partly determined by endophyte colonisation. All other alkaloid QTL, however, were independent of mycelial mass, indicating it may be possible to develop MAS that will facilitate breeding for alkaloid phenotype, independent of the degree of endophyte colonisation. Keywords: DIMS, ELISA, Lolium, map, metabolic profiling, Neotyphodium, SSR

A defensive role for several of the major alkaloids of Neotyphodium spp. endophytes in forage grasses has been confirmed in genetic experiments. However, many aspects of the activity of endophyte-infected grasses towards herbivores remain unaccounted for, suggesting there are further anti-herbivore factors yet to be uncovered. The development of metabolomics has provided new tools for the discovery of novel metabolites without preconceptions of the likely chemical structural class. Prospects for the application of new methods from metabolomics to further elucidate the defensive chemistry of associations of Neotyphodium spp. endophytes with their grass hosts are reviewed. Keywords: Neotyphodium, Epichloë, defensive chemistry, invertebrate, vertebrate, gene deletion

We have studied metabolic and gene expression responses of two white clover genotypes differing in foliar basal flavonol and anthocyanin levels subjected to moderate cold in a controlled climate chamber experiment. The main flavonols and anthocyanins accumulating in white clover leaves are conjugated to galactose and xylose. Concentrations of flavonoids and sugars including starch significantly increased during cold treatment and a strong induction of flavonoid pathway genes was observed in both genotypes. Specific sugars like glucose, fructose, and starch were significantly higher in the genotype with low basal flavonoid levels. By combining metabolic and gene expression analysis we have identified two genes probably involved in the conjugation and deconjugation of flavonoids, a flavonoid galactosyltransferase and a β- galactosidase. The selected flavonoid galactosyltransferase was up-regulated by cold treatment and was also expressed at higher levels in the red leaved genotype. The β- galactosidase studied here was down-regulated by cold and expressed less in the red leaved genotype. These results indicate a tight link between primary and secondary metabolism in clover and demonstrate the value of combined metabolome and transcriptome analysis for the identification of specific gene functions.

We have identified a novel siderophore from the mutualistic grass symbiont Epichloë festucae. Targeted gene replacement of a non-ribosomal peptide synthetase (termed sidF) from E. festucae eliminated biosynthesis of this extracellular siderophore. Structural characterisation, based on extensive mass spectrometry showed similarities to fusarinine-type siderophores. Plants inoculated with strains carrying a targeted deletion in sidF have a dramatic phenotype. Siderophore loss changed a mutualistic symbiont into an antagonistic fungus, causing de-regulation and proliferation of fungal hyphae, plant stunting and sometimes even tiller death. We hypothesise that loss of the biosynthesis of the siderophore disrupts iron homeostasis within the whole symbiotum, which is a critical factor for maintenance of mutualistic endophyte-grass symbioses. Keywords: siderophore, iron homeostasis, NRPS

Many grasses live in association with fungi (Neotyphodium spp. endophytes) growing in the intercellular spaces of the grass. The endophytes produce a range of alkaloids that protect the grass against grazing animals and insects. There has been speculation about the mobilisation and translocation of different alkaloids. We aimed to determine if metabolites produced by endophytes are mobilised and translocated by the plant. We developed a versatile method for the analysis plant fluids, using a linear ion trap mass spectrometer. Cut leaf fluid and guttation fluid of different grass endophyte associations in ryegrass and tall fescue were analysed. Peramine was detected in the cut leaf and guttation fluid of all ryegrass-endophyte associations that are able to produce peramine, but were found only in the cut leaf fluid of the tall fescue association. Lolines and ergot peptides were also detected in some associations. Keywords: Neotyphodium, ryegrass, tall fescue, guttation, peramine, ergovaline, loline

In fungal pathogenesis the cAMP signalling cascade is usually essential for virulence. Deletion of the adenylate cyclase gene, the enzyme that synthesises cAMP, often results in an attenuated or avirulent phenotype. Our aim was to identify the signalling mechanisms regulating colonisation of perennial ryegrass (Lolium perenne) by the fungal symbiont Epichloë festucae Fl1. We have identified genes from several signalling networks, and here report on the outcomes of targeted disruption of the E. festucae Fl1 adenylate cyclase gene (acyA). A dual genome (endophyte/ ryegrass) Affymetrix GeneChip® has been synthesised and we are undertaking large scale transcript profiling of the L. perenne/ E. festucae ΔacyA symbiotum to identify target genes regulated by the endophyte cAMP signalling network. Keywords: cAMP, adenylate cyclase, acyA, Neotyphodium lolii, Epichloë festucae, symbiosis, Affymetrix GeneChip

Neotyphodium lolii is a fungal endophyte that lives entirely within the intercellular spaces of its grass host, perennial ryegrass (Lolium perenne L.). Infection is symptomless and the endophyte relies on the host plant for dissemination via the seed. The association is mutually beneficial since the endophyte confers a number of biotic and abiotic advantages to the host. This paper will present an overview of the multidisciplinary approach we are using at AgResearch to dissect the molecular basis of this symbiosis. Topics covered will include how we intend to link the knowledge gained from basic biology and cytology with the fields of Genomics, Transcriptomics, Proteomics and Metabolomics. To achieve this we are using isogenic ryegrass lines infected or uninfected with endophyte in combination with a suite of molecular biology tools, including Expressed Sequence Tags (ESTs), Microarray analysis, 2D-gel electrophoresis (to identify novel proteins associated with symbiosis), and metabolic profiling. To aid our proteomics approach, we have also generated both fungal genomic and fungal EST resources. By linking these approaches we hope to identify genes which are important in both the establishment and maintenance of symbiosis. In addition, by combining transcriptomic with metabolomic approaches, we intend to elucidate how endophyte infection influences host secondary metabolism. We hypothesise the latter is correlated with many of the endophyte-conferred enhancements to its host

Endophytic fungi of temperate grasses form stable symbiotic associations that provide mutual benefits for both species.