A multidisciplinary approach to dissect the molecular basis of the Neotyphodium lolii/ryegrass symbiosis

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. Introduction Neotyphodium and Epichloë spp (phylum Ascomycota, family Clavicipitaceae) are closely related asexual and sexual endophytic fungi, respectively, that form mutualistic associations with temperate grasses belonging to the family Poaceae. For example, N. lolii and N. coenophialum are fungal endophytes that live entirely within the intercellular spaces of perennial ryegrass (Figure 1) and tall fescue, respectively. Infection is symptomless and the endophyte relies entirely on the host plant for dissemination via the seed or through vegetative structures (Philipson and Christey 1986 reviewed by Schardl, Leuchtmann, and Spiering, 2004). The association is mutually beneficial since the endophyte confers a number of biotic and abiotic advantages to the host, including enhanced plant growth, protection from certain mammalian and insect herbivores, enhanced resistance to nematodes, resistance to some fungal pathogens and in some associations, enhanced drought tolerance (Arechevaleta et al. 1989, Kimmons Gwinn and Bernard 1990, Gwinn and Gavin 1992, Schardl and Phillips 1997, Clay and Holah 1999, Scott 2001, Schardl 2001, Johnson et al. 2003). Endophyte infection has also been implicated in modification of root morphology, osmotic adjustment and mineral uptake (Malinowski and Belesky 1999, Malinowski et al.1999). Some of these benefits are due to the production of fungal secondary metabolites (Figure 2) such as the pyrrolopyrazine (peramine) and aminopyrrolizidine (loline) alkaloids (Bush et al.1997, Blankenship et al. 2001, Spiering et al. 2005; Tanaka et al. 2005). However,