Kou-Cheng Peng

Induced proteome of Trichoderma harzianum by Botrytis cinerea.

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including beta-1,3-glucanases, beta-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.

Proteomic study of biocontrol mechanisms of Trichoderma harzianum ETS 323 in response to Rhizoctonia solani.

To elucidate the entire range of proteins that are secreted by Trichoderma harzianum ETS 323 in its antagonism with Rhizoctonia solani, an in vivo interaction between them was mimicked and not only the secreted cell wall-degrading enzymes (CWDEs) but also all of the proteome were investigated. Seven CWDEs, chitinase, cellulase, xylanase, beta-1,3-glucanase, beta-1,6-glucanase, mannanase, and protease,were revealed by activity assay, in-gel activity stain, 2-DE, and LC-MS/MS analysis. Extracellular protein extracts from media that contained R. solani exhibited much higher CWDE activities than media that did not contain R. solani. Cellulase and mannanase activity, however, were insignificant. Activity stain also revealed that beta-1,3-glucanase, beta-1,6-glucanase, and xylanase activity occurred exclusively in media that contained R. solani. Furthermore, 35 of the 43 excised spots on the 2-DE gel were successfully analyzed by LC-MS/MS, and eight proteins were identified. They were two glycoside hydrolases, two proteases, two beta-glucosidases, one endochitinase and, interestingly, one amino acid oxidase. Additionally, a possible mechanism was proposed to elucidate how the cell walls of R. solani are systematically enveloped and disintegrated.

A Novel l-Amino Acid Oxidase from Trichoderma harzianum ETS 323 Associated with Antagonism of Rhizoctonia solani

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.

Identification of antibacterial mechanism of l‐amino acid oxidase derived from Trichoderma harzianum ETS 323

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 Trichodermau2003harzianum 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 40u2003°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 50u2003°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 H2O2 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 H2O2 production are involved in the mechanism responsible for the antibacterial activity of Th‐LAAO.

Purification and characterization of novel glucanases from Trichoderma harzianum ETS 323.

Trichoderma harzianum ETS 323 secretes two glucanases, a 23.5 kDa endoglucanase (EG Th1) and a 61 kDa exoglucanase (ExG Th1). They were identified by their hydrolysis products and were purified to homogeneity. The optimal temperature and pH for both EG Th1 (7.3-fold purification, 5.0% yield) and ExG Th1 (33.7-fold purification, 0.15% yield) were 50 °C and pH 4.5, respectively. The kinetic parameters of EG Th1 (K(m) = 23 mg mL(-1), V(max) = 294 μM min(-1), specific activity = 7.4 U mg(-1)) and ExG Th1 (K(m) = 85 mg mL(-1), V(max) = 385 μM min(-1), specific activity = 24.6 U mg(-1)) toward carboxymethyl cellulose were determined. Both enzymes favored CMC and maintained 100% activity for 10 days at 38 °C. KCl, MgCl(2), HgCl(2), and FeCl(3) showed approximately 30% inhibition against EG Th1 but not ExG Th1. They catalyzed transglycosylation of glucose in the presence of cellobiose, but ExG Th1 exhibited better activity and higher product diversity.

Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea mycelium in culture conditions.

ABSTRACT 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.

Study on the anthraquinones separated from the cultivation of Trichoderma harzianum strain Th-R16 and their biological activity.

The biocontrol fungal species of Trichoderma, which colonizes plant roots, are well-known for their potential to control plant pathogens. Six anthraquinones, of which four have been identified for the first time from Trichoderma and two have already been reported in other strains, were purified from Trichoderma harzianum strain Th-R16 to evaluate their biological activities. The structures of the compounds were determined by one- and two-dimensional NMR. The compounds were shown to exhibit stronger antifungal activity than antibacterial activity. Low yield compounds, like 1,5-dihydroxy-3-hydroxymethyl-9,10-anthraquinone, were found to be more active against fungal pathogens than pachybasin and crysophanol, which were found to be the major extracellular metabolites. Test anthraquinones with higher oxidation numbers had better antifungal activity, and their activities were concentration-dependent.

Monomeric L-amino acid oxidase-induced mitochondrial dysfunction in Rhizoctonia solani Reveals a novel antagonistic mechanism of Trichoderma harzianum ETS 323.

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.

Cloning of a Novel l-Amino Acid Oxidase from Trichoderma harzianum ETS 323 and Bioactivity Analysis of Overexpressed l-Amino Acid Oxidase

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.

Trichoderma harzianum ETS 323-mediated resistance in Brassica oleracea var. capitata to Rhizoctonia solani involves the novel expression of a glutathione S-transferase and a deoxycytidine deaminase.

Plant interactions with microbial biocontrol agents are used as experimental models to understand resistance-related molecular adaptations of plants. In a hydroponic three-way interaction study, a novel Trichoderma harzianum ETS 323 mediated mechanism was found to induce resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets. The R. solani challenge on leaves initiate an increase in lipoxygenase activity and associated hypersensitive tissue damage with characteristic programmed cell death that facilitate the infection. However, B. oleracea plantlets whose roots were briefly (6 h) colonized by T. harzianum ETS 323 developed resistance to R. solani infection through a significant reduction of the host hypersensitive tissue damage. The resistance developed in the distal leaf tissue was associated with the expression of a H(2)O(2)-inducible glutathione S-transferase (BoGST), which scavenges cytotoxic reactive electrophiles, and of a deoxycytidine deaminase (BoDCD), which modulates the host molecular expression and potentially neutralizes the DNA adducts and maintains DNA integrity. The cDNAs of BoGST and BoDCD were cloned and sequenced; their expressions were verified by reverse-transcription polymerase chain reaction analysis and were found to be transcriptionally activated during the three-way interaction.