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Trichoderma guizhouense NJAU 4742 (Harzianum clade) can suppress the causative agent of banana wild disease Fusarium oxysporum f. sp. cubense 4 (Foc4). To identify genes involved in this trait, we used T-DNA insertional mutagenesis and isolated one mutant that was unable to overgrow Foc4 and had reduced antifungal ability. Using the high-efficiency thermal asymmetric interlaced-PCR, the T-DNA was located in the terminator of a neutral metalloprotease gene (encoding a MEROPS family M35 protease), which was named nmp1. The antifungal activity of the mutant was recovered by retransformation with wild-type nmp1 gene. The purified NMP1 (overexpressed in Pichia pastoris) did not inhibit the growth and germination of other fungi in vitro. Its addition, however, partly recovered the antifungal activity of the mutant strain against some fungi. The expression of nmp1 is induced by the presence of fungi and by dead fungal biomass, but the time-course of transcript accumulation following the physical contact depends on mode of interaction: it increases in cases of long-lasting parasitism and decreases if the prey fungus is dead shortly after or even before the contact (predation). We thus conclude that NMP1 protein of T. guizhouense has major importance for mycotrophic interactions and defence against other fungi., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)