| Summary: | 博士 === 國立東華大學 === 生命科學系 === 101 === Trichoderma harzianum is an important biocontrol agent of several important plant pathogenic fungi. Trichoderma species attacks other fungi by secreting lytic enzymes, including L-amino acid oxidase, β-1,3-glucanase and chitinolytic enzymes. Botrytis cinerea is a common pathogenic fungus which causes blight in most ornamental plants. It may be responsible for serious diseases and postharvest losses in fruits, vegetables and flowers. Previous studies have shown that the extracellular proteins of T. harzianum ETS 323 grown in the presence of deactivated B. cinerea in culture include a putative L-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves 2 steps, that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. In addition, an L-amino acid oxidase (Th-L-AAO) was identified from T. harzianum ETS 323. The secretion of Th-L-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-L-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-L-AAO also showed disease control against the development of B. cinerea on postharvest apple fruits and tobacco leaves. An apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-L-AAO, suggesting that Th-L-AAO triggers programmed cell death in B. cinerea. We show that release of the apoptotic factors cytochrome c, caspase 3, and caspase 9, upon treatment with Trichoderma harzianum-derived L-amino acid oxidase by western blot. Furthermore, we report on a proteomic analysis of secreted proteins from B. cinerea during treatment with T. harzianum ETS 323 L-phenylalanine oxidase (Th-L-AAO) using two-dimensional electrophoresis (2DE). We identified numerous extracellular proteins in the secretions, including translation proteins, ribosomal proteins, and chaperone proteins for folding, metabolism, energy production, and signal transduction. We detected superoxide dismutase (SOD) in un-treated B. cinerea. The results of this study provide important insights into the mechanism of Th-L-AAO action against B. cinerea. In cloning study, we report of the cloning of cDNA encoding a novel T. harzianum ETS 323 L-amino acid oxidase. The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9% and 24%. The molecular mass of the purified protein was 52 kDa, as determined by SDS-PAGE. Th-L-AAO showed detectable optimal activity substrate on phenylalanine. The enzyme optimal pH and temperature for activity were 7 and 40°C, respectively. In Circular Dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% β-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage organisms. A 3D protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.
|
|---|
