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• In an attempt to advance the understanding of ectomycorrhizal symbiosis, which improves mineral nutrition and resistance to abiotic and biotic stresses of most woody plants from temperate and boreal regions, we produced a cDNA library of the ectomycorrhizal fungusHebeloma cylindrosporumin a yeast expression vector. The library was used for functional complementation cloning and for sequencing of 4036 expressed sequence tags (ESTs).• Expression of the library in a yeastade2andtrp1mutant strain allowed cloning of twoHebelomagenes,HcADE2andHcTRP1by functional complementation. This experiment highlighted both the efficiency of the complementation strategy and the quality of the library.• All sequence data available onHebeloma(4596) were analysed and contiged. The whole EST resource is accessibleviaa web interface, which allows searching and navigation through clusters and tentative contigs (TCs).• Analysis of the database revealed a low level of counterparts already identified in other ectomycorrhizal fungi. For example, it allowed identification of a large set of membrane transporters, such as phosphate, potassium, sulphate or micronutrient transporters. Thus, this EST resource provides a valuable tool for research on ectomycorrhiza and symbiotic interactions.New Phytologist(2004)doi: 10.1111/j.1469-8137.2004.01185.x© New Phytologist(2004) [ABSTRACT FROM AUTHOR]