Applicants have devised a highly effective, convenient, and expeditious genetic transformation system for filamentous fungi, such as Agaricus bisporus. The preferred method uses an Agrobacterium-mediated transformation protocol. The critical features of this protocol include co-cultivation of the bacterium with fruit body tissue instead of spores. In a preferred embodiment, even higher transformation efficiencies were observed with the use of a homologous promoter in the polynucleotide expression construct in order to drive gene expression.