Proteomic analysis of secreted proteins from Trichoderma harzianum. Identification of a fungal cell wall-induced aspartic protease

Trichoderma mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. We have analysed the extracellular proteome secreted by T. harzianum CECT 2413 in the presence of different fungal cell walls. Significant differences were detected in 2DE maps, depending on the use of specific cell walls or chitin. A combination of MALDI-TOF and liquid chromatography mass spectrometry allowed the identification of a novel aspartic protease (P6281: MW 33 and pI 4.3) highly induced by fungal cell walls. A broad EST library from T. harzianum CECT 2413 was used to obtain the full-length sequence. The protein showed 44% identity with the polyporopepsin (EC 3.4.23.29) from the basidiomycete Irpex lacteus. Lower identity percentages were found with other pepsin-like proteases from filamentous fungi (<31%) and animals (<29%). Northern blot and promoter sequence analyses support the implication of the protease P6281 in mycoparasitism.