SUMMARY A hydrophobin-like clone (TasHyd1) was isolated during a PCR differential mRNA display analysis conducted on Trichoderma asperellum mycelia interacting with plant roots. The open reading frame encodes a 145-amino-acid protein showing similarity to Pbhyd1, a Class I hydrophobin from the dimorphic fungus Paracoccidioides brasiliensis. TasHyd1 expression was detected in planta up to 5 days after Trichoderma root inoculation. TasHyd1 is constitutively expressed at low levels in mycelia in young cultures but gene expression is not detected in sporulating hyphae or in non-germinating spores. Carbon limitation stimulates expression of TasHyd1 whereas nitrogen or phosphate starvation down-regulate expression. TasHyd1 fused to an HA tag was over-expressed in Trichoderma and the protein was detected with an anti-HA antibody in the trifluoroacetic-acid-soluble fraction of mycelial cell walls. Over-expressor mutants were not affected in their mycoparasitic activity when tested in vitro against the plant pathogen Rhizoctonia solani and retained root colonization capacity comparable with that of the wild-type. TasHyd1 deletion mutants had no significant reduction in in vitro mycoparasitic activity but were altered in their wettability and were severely impaired in root attachment and colonization. These phenotypes were recovered by complementation of TasHyd1, indicating that the protein is a new hydrophobin that contributes to Trichoderma interaction with the plant.