The aim of this project was to undertake large scale transcript profiling of endophyte and plant genes during symbiosis, and to determine the impact of targeted endophyte gene deletions on expression of plant and endophyte genes. We have designed and developed an Affymetrix NimbleExpress™ GeneChip® representing expressed sequence tags (ESTs) of the fungal endophyte Neotyphodium lolii Lp19 and its ryegrass host, Lolium perenne. In total, 8511 genes were represented on the microarrays with approximately eleven 25 base pair oligonucleotides per gene. Experiments were conducted to analyse differential expression of genes from endophyte-infected and endophyte-free plant material, and from endophytes grown in culture. In some symbioses, endophytes had targeted mutations in genes involved in signalling, synthesis of secondary metabolites or in genes of unknown function. Here we describe the processes which guided design of the GeneChip®, the results of quality control assessments of hybridised arrays and considerations concerning statistical analyses of gene expression. Keywords: Affymetrix, GeneChip®, NimbleExpress, Neotyphodium lolii, Epichloë festucae, ryegrass, Lolium perenne, endophyte, symbiosis.

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