Abstract
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.
Highlights
Nepenthesins are the first group of proteases reported from the nepenthesin-like plant aspartic proteases (PAPs)
We characterized recombinant HvNEP1 after expression in Pichia pastoris, we studied its effect on Fusarium growth and mycotoxin synthesis, and we uncovered that rHvNEP-1 mediates the proteolysis of F. graminearum and F. culmorum phytases
The Mass Spectrometry (MS) of the most inhibitory fractions identified several proteins of which HvNEP-1 appeared a likely candidate inhibitor (Supplementary Data Set), The result corresponds to the finding that phytase inactivation in the crude protein extracts of barley grains is ascribed to aspartic protease activity in the seed extract (Bekalu et al, 2017)
Summary
Nepenthesins are the first group of proteases reported from the nepenthesin-like plant aspartic proteases (PAPs). As described for the nepenthesin-like PAPs, nepenthesins are characterized by diverse N-terminal sequence and nepenthesin-type PAP insertion (NAP-I) sequence (Simoes and Faro, 2004; Soares et al, 2019; Bekalu et al, 2020a). They were initially described from the pitcher fluid of the carnivorous plant, Nepenthes (Athauda et al, 2004). Several genes coding for protein homologs to nepenthesins have been identified in Arabidopsis thaliana and Oryza sativa (Takahashi et al, 2008; Chen et al, 2009) They display a high diversity and widespread tissue expression, suggesting their participation in various physiological processes in plants. In addition to their extended biological roles, nepenthesins have recently been implicated for various industrial applications, for example, as a tool for Digestion in Hydrogen/Deuterium Exchange Mass Spectrometry (Yang et al, 2015), and treatment for celiac disease (Rey et al, 2016)
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