Barley Nepenthesin-Like Aspartic Protease HvNEP-1 Degrades Fusarium Phytase, Impairs Toxin Production, and Suppresses the Fungal Growth

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 ( HvNEP-1 ) protease from barley ( Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum . The quantitative PCR analysis of trichothecene biosynthesis genes ( TRI ) confirmed that rHvNEP-1 strongly repressed the expression of TRI4 , TRI5 , TRI6 , and TRI12 in F . graminearum . The co-incubation of rHvNEP-1 with recombinant F. graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium .