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Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are not fully 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,including a dinucleotide-binding motif and a GG motif, was identified in the N-terminus. The results indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6 to 8. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 C, with enzymatic activity deceasing with further increases in temperature due to thermal denaturation of the enzyme at 50 C. 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 the monomeric Th-LAAO (mTh-LAAO) had an antagonistic effect against Rhizoctonia solani. The mTh-LAAO-treated R. solani exhibited hyphal lysis and apoptotic characteristics such as reactive oxygen species (ROS)accumulation. This hyphal lysis was suppressed by a mitochondria-dependent apoptosis inhibitor oligomycin while accompanied by reduction of ROS accumulation. This result suggested that the mitochondria-mediated apoptosis in Rhizoctonia solani was involved in mTh-LAAO-induced growth inhibition, which was supported by the evidence of cytocheome c release and activation of caspase 9 and caspase 3. Furthermore, the data indicated that the mTh-LAAO-induced fungal cell death was also closely interrelated with the interaction of mTh-LAAO with Rhizoctonia solan hyphal cell wall proteins. In further study, we attempted to identify the mechanism behind the antibacterial activity of Th-LAAO against Escherichia coli and Staphylococcus aureus. The results of confocal microscopy and flow cytometry indicate that Th-LAAO interact with bacteria to cause membrane permeabilization, an interaction that may be promoted by the fact that the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs is located at the N-terminus. The findings of increased exogenous H2O2 production and reactive oxidative species (ROS) accumulation in Th-LAAO-treated bacteria indicate that ROS accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. These findings further our understanding of the biological function underlying the antagonistic action of T. harzianum mTh-LAAO against fungal pathogen and provide insight into the mechanism behind the antibacterial activity of Th-LAAO.