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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

Two controlled environment experiments were performed to test the effects of nitrogen, phosphorus and carbohydrates on endophyte (Neotyphodium lolii) and alkaloid concentrations in ryegrass (Lolium perenne). Three perennial ryegrass cultivars ('high sugar grasses' AberDove and AberDart; control Fennema) that differ in carbohydrate content were infected with three strains of N. lolii (common strain, CS; AR1; AR37). Infected and uninfected plants were grown under high (9 mM) and low (2.25 mM) nitrogen (AberDove, Fennema; CS, AR1, AR37) or under high (2 mM KH2PO4) and low (0.05 mM KH2PO4) phosphorus (AberDart, Fennema; CS, AR1). Quantitative realtime Polymerase Chain Reaction (qPCR) was used to estimate endophyte concentrations in harvested leaf tissues. High N and P supply as well as high carbohydrate content of the host grass reduced endophyte concentrations. Alkaloid production was also reduced under both increased N supply and in the high sugar cultivar, and was linearly related to endophyte concentration (except ergovaline). The results stress the need for wider quantification of fungal endophytes in the grassland/ foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes, or increasing nutrient inputs, affect the role of endophytes in grassland ecosystems. Keywords: Neotyphodium lolii, foliar endophyte, Lolium perenne, perennial ryegrass, qPCR, high sugar ryegrass, nitrogen, phosphate, carbohydrate, AR1, AR37, alkaloids

Grass associations with Epichloë/Neotyphodium endophytes display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. To gain a comprehensive understanding of the complex biological interactions that occur between a plant host and fungal symbiont, a transcriptomics approach using custom designed Affymetrix GeneChip®s was employed. We are currently comparing and analysing symbiotic interactions of perennial ryegrass (Lolium perenne) with endophytes N. lolii and E. festucae as well as comparing it against endophyte-free perennial ryegrass. Both N. lolii and E. festucae have been grown in culture in order to compare in planta versus in vitro gene expression. Additionally, targeted gene replacements in E. festucae have been performed and a comparative analysis of the knock-outs with wild-type E. festucae infected plants is in progress. These comparative analyses have revealed changes in gene expression which may lead to the identification of gene pathways/networks and the roles of these genes in symbiosis. Keywords: Neotyphodium, Epichloë, transcriptomics, symbiosis

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.

Symbiotic grass associations with fungal endophytes (genera Neotyphodium and Epichloë) display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. Perennial ryegrass, an important agronomic grass, is typically associated with the N. lolii endophyte. The endophyte lives within the intercellular spaces without inducing any symptoms in the plant. The aim of this study is to elucidate the biosynthetic function of fungal secondary metabolite gene clusters. Non-ribosomal peptide synthetase genes (NRPSs) of unknown function were targeted, as these genes are commonly associated with the production of bioactive peptides some of which are ecologically important. Some novel endophyte NRPS genes have been identified using a degenerate PCR screen; one of these, NRPS5 will be discussed here. Clones were obtained by screening a fosmid Epichloë festucae genomic DNA library and we are currently determining gene function by using targeted gene replacement followed by an assessment in vitro and in planta using metabolomics and appropriate bioassay screens. Keywords: endophyte, NRPS, secondary metabolism

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

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

Pastoral Genomics is engaged in applying molecular mapping and functional genomics to characterise and manipulate key phenotypic traits associated with improved productivity in the commercially important forage crop white clover (Trifolium repens L.). We report on the development of a framework linkage map for white clover and the application of this map to the identification of quantitative trait loci (QTL) associated with vegetative and seed yield traits. Homologues of genes responsible for the traits of interest have been identified and cloned in order to identify 'perfect markers' for marker assisted breeding and to develop a cisgenic® clover cultivar. We introduce western clover (T. occidentale), a diploid perennial clover species closely related to white clover, as a stoloniferous clover model to dissect low heritability traits initially intractable in white clover. We have identified extensive genetic variation in this species, which previously has been reported to be remarkably uniform. We have developed a framework map for T. occidentale and demonstrated conservation of marker order and spacing with white clover. An ethyl methane sulfonate (EMS) mutant population has been used to isolate three distinct groups of condensed tannin (CT) mutants. We have also investigated our model's transformation potential, achieving frequencies 2-3 fold higher than for white clover and suggest that this model is potentially useful for genomic analysis of white clover and other clonal species