Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.

Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis 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 two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. 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 fruit and tobacco leaves. Furthermore, 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. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.

Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40°C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50°C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H 2 O 2 production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H 2 O 2 production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.

The monomeric L-amino acid oxidase (mTh-LAAO) of Trichoderma harzianum ETS 323 has been suggested to antagonize Rhizoctonia solani by an unknown mechanism. Here, the mTh-LAAO-treated R. solani exhibited hyphal lysis and apoptotic characteristics such as DNA fragmentation, reactive oxygen species (ROS) accumulation, lipid peroxidation, and mitochondrial membrane potential depolarization. This hyphal lysis was suppressed by the mitochondria-dependent apoptosis inhibitor oligomycin while accompanied by reduction of ROS accumulation. This result suggested that mitochondria-mediated apoptosis in R. solani was involved in mTh-LAAO-induced growth inhibition, which was supported by the evidence of cytocheome c release and activation of caspases 9 and 3. Furthermore, the data indicated that the mTh-LAAO-induced fungal cell death was also closely interrelated with the interaction of mTh-LAAO with R. solani hyphal cell wall proteins. These results illuminate the biological function and mechanism underlying the antagonistic action of T. harzianum mTh-LAAO against fungal pathogens.

L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. 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 microorganisms. Furthermore, a three-dimensional 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.