1887

Abstract

antifungal protein (NFAP) is a β-defensin-like peptide produced by the NRRL 181 isolate. In this study, we investigated the manifestation of the antimicrobial effect of NFAP via heterologous expression of the gene in an NFAP-sensitive fungus, . Heterologous expression of the gene was carried out in CS2902 using a pAMA1-based autonomous replicative vector construct. The effect of the produced NFAP on the germination of conidia was investigated by scanning electron microscopy (SEM), and by DAPI and Calcofluor white (CFW) staining. 2′,7′-Dichlorodihydrofluorescein diacetate staining and an Annexin V–FITC Apoptosis Detection kit were used to reveal the accumulation of reactive oxygen species (ROS) and the possible apoptotic, necrotic effect. The impact of mono- and divalent cations on the antimicrobial activity of NFAP was also examined. Transformants expressing the gene showed reduced hyphal growth compared with the untransformed strain. This effect was absent in the presence of mono- and divalent cations (50 and 100 mM KCl, MgSO, NaSO). Delayed and abnormal germination was observed in the case of transformants. Conidia developed short branching germination tubes with swollen tips. The great majority of germinating conidia were destroyed after 8 h of cultivation, although a few survived and developed into abnormal hyphae. Damage in the organization of the cell wall, the destruction of chitin filaments and the accumulation of nuclei at the broken hyphal tips were detected by SEM, DAPI and CFW staining. The accumulation of ROS and more frequent apoptotic, necrotic events were also observed in the case of the NFAP-producing strain.

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2013-02-01
2019-09-15
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