Fungi are heterotrophic microorganisms with devastating impact on agriculture and human health. Agricultural production worth billions of € is destroyed annually by fungal diseases. Human fungal infections have severe consequences on the growing number of immuno-compromised patients, with high mortality rates associated with Aspergillus and Candida infections. There is a critical need to further the development of new antifungals, which requires a comprehensive molecular knowledge on fungal pathogenesis and signal transduction. ARIADNE uses a comparative biological approach at the systems biology level to identify and validate signalling target genes that play an essential causative role in fungal virulence on both plant and human hosts. To establish a comprehensive view of fungal pathogenesis seven pathogenic species will be studied, including the two major fungal pathogens of humans Aspergillus fumigatus and Candida albicans, the devastating plant pathogens Fusarium oxysporum, Magnaporthe grisea and Mycospherella graminicola, as well as two established model pathogens of filamentous and dimorphic fungi, Ashbya gossypii and Ustilago maydis. This array of fungal species cuts across diverse taxonomic groups, covering several hundred million years of evolution. The central scientific and technological challenges for ARIADNE are to 1) integrate parallel transcriptomics, proteomics, and chemical genetics for a comprehensive set of major fungal pathogens, 2) identify evolutionarily conserved and species specific signalling effectors which act as key players in fungal virulence, 3) validate these proteins as candidate targets for small molecule inhibitors. This groundbreaking scientific and teaching approach will open up new avenues on how to look at fungal pathogenicity and will generate an unprecedented knowledge platform, which will facilitate the discovery and validation of antifungal targets in both the agricultural and the pharmaceutical sectors.