Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> CECT 2413 displaying nematicidal activity

Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7–4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47–57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.     

Cloning and characterization of bgn16.3, coding for a beta-1,6-glucanase expressed during Trichoderma harzianum mycoparasitism

AIMS: To clone and characterize the gene coding for BGN16.3, a beta-1,6-glucanase putatively implicated in mycoparasitism by Trichoderma harzianum, a biocontrol agent used against plant pathogenic fungi. METHODS AND RESULTS: Using degenerate primed PCR and cDNA library screening, we have cloned the cDNA coding BGN16.3. bgn16.3 showed a significant sequence identity (50%) to bgn16.1; however, they both have low identity to the previously cloned bgn16.2, allowing the identification of amino acid sequences putatively involved in the common catalytic activity of the three proteins. bgn16.3 is a single-copy gene and highly homologous sequences are present in all tested Trichoderma species. bgn16.3 expression pattern is analysed by Northern blot, finding that it is expressed during the interaction of T. harzianum CECT 2413 with Botrytis cinerea, supporting the implication of the enzyme in the mycoparasitic process. CONCLUSIONS: The cloned bgn16.3 completes the knowledge on the beta-1,6-glucanase isozyme system from T. harzianum CECT 2413. A highly homologous gene is present in all analysed Trichoderma strains. bgn16.3 is expressed under few specific conditions, including the mycoparasitic process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the knowledge of beta-1,6-glucanases. It implicates this group of enzymes in the mycoparasitism by some biocontrol agents such as T. harzianum.     

BGN16.3, a novel acidic beta-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413

A new component of the beta-1,6-glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-beta-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic beta-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to beta-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with beta-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement beta-1,6-glucanases in this process is discussed.     

Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum

Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls.     

A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum.

The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls.     

Cloning and heterologous expression of SS10, a subtilisin-like protease displaying antifungal activity from Trichoderma harzianum

Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi. Trichoderma harzianum mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. A gene ( SS10 ) encoding a subtilisin-like protease was cloned from T. harzianum T88, a biocontrol agent effective against soil-borne fungal pathogens. The full-length cDNA was isolated by 5' and 3' rapid amplification of the cDNA ends. The coding region of the gene is 1302 bp long, encoding 433 amino acids of a predicted protein with a molecular mass of 45 kDa and a pI of 6.1. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) (EC 3.4.21.) and had a predicted active site made up of the catalytic residues Asp 187, His 218 and Ser 376. Northern experiments demonstrated that SS10 was induced in response to different fungal cell walls. Subtilisin-like protease gene SS10 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (17.8 U mL⁻¹) 60 h after induction with galactose. The optimal enzyme reaction temperature was 50 °C and the optimal pH was 8. The subtilisin-like protease exerted broad-spectrum antifungal activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Cytospora chrysosperma .     

Increased antifungal and chitinase specific activities of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> CECT 2413 by addition of a cellulose binding domain

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.     

Improved antifungal activity of a mutant of Trichoderma harzianum CECT 2413 which produces more extracellular proteins

Trichoderma harzianum is a well-known biological control agent against fungal plant diseases. In order to select improved biocontrol strains from Trichoderma harzianum CECT 2413, a mutant has been isolated for its ability to produce wider haloes than the wild type, when hydrolysing pustulan, a polymer of beta-1,6-glucan. The mutant possesses between two and four times more chitinase, beta-1,3- and beta-1,6-glucanase activities than the wild type, produces about three times more extracellular proteins and secretes higher amounts of a yellow pigment (alpha-pyrone). This mutant performed better than the wild type during in vitro experiments, overgrowing and sporulating on Rhizoctonia solani earlier, killing this pathogen faster and exerting better protection on grapes against Botrytis cinerea.     

Cloning and heterologous expression of aspartic protease SA76 related to biocontrol in Trichoderma harzianum

Trichoderma harzianum is a soil-borne filamentous fungus that exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. The production of hydrolytic enzymes appears to be a key element in the parasitic process. Among the enzymes released by Trichoderma, the aspartic proteases play a major role. A gene (SA76) encoding an aspartic protease was cloned by 3' rapid amplification of cDNA ends from T. harzianum T88. The coding region of the gene is 1,593 bp long, encoding a polypeptide of 530 amino acids with a predicted molecular mass 55 kDa and a pI of 4.5. The catalytic aspartic residues characteristic of aspartic proteases are conserved with an active-site motif (DSG); however, the DSG in the N-terminal lobe is unusual in that Ser replaced Thr. Northern blot analysis indicated that SA76 was induced in response to different fungal cell walls. Aspartic protease SA76 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (10.5 U mL(-1)) 72 h after induction with galactose. The temperature optimum of the enzyme was 45 degrees C and its pH optimum was 3.5. The culture supernatant of the S. cerevisiae strain that expressed the aspartic protease SA76 was able to inhibit the growth of five phytopathogenic fungi. The inhibition of mycelial growth varied between 7% and 38%.     

Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi.

Chitinase, beta-1,3-glucanase, and protease activities were formed when Trichoderma harzianum mycelia, grown on glucose as the sole carbon source, were transferred to fresh medium containing cell walls of Botrytis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by Western blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenously added [U-14C]valine was incorporated in acetone-soluble compounds with an apparent M(r) of < 2,000. These compounds comigrated with the peptaibols trichorzianines A1 and B1 in thin-layer chromatography and released [U-14C]valine after incubation in 6N HCl. Incorporation of radioactive valine into this material was stimulated by the exogenous supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inhibited spore germination as well as hyphal elongation of B. cinerea. Culture supernatants from mycelia placed in fresh medium without cell walls of B. cinerea did not show hydrolase activities, incorporation of [U-14C]valine into peptaibol-like compounds, and inhibition of fungal growth. Purified trichorzianines A1 and B1 as well as purified chitobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than those observed in the culture supernatants. However, when the enzymes and the peptaibols were tested together, an antifungal synergistic interaction was observed and the 50% effective dose values obtained were in the range of those determined in the culture supernatants. Therefore, the parallel formation and synergism of hydrolytic enzymes and antibiotics may have an important role in the antagonistic action of T. harzianum against fungal phytopathogens.     

Detection of peptaibols and partial cloning of a putative peptaibol synthetase gene fromT. harzianum CECT 2413

The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.     

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742)

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-β- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source. This revised version was published online in June 2006 with corrections to the Cover Date.     

Saprotrophic and mycoparasitic components of aggressiveness of Trichoderma harzianum groups toward the commercial mushroom Agaricus bisporus

We examined the mycoparasitic and saprotrophic behavior of isolates representing groups of Trichoderma harzianum to establish a mechanism for the aggressiveness towards Agaricus bisporus in infested commercial compost. Mycoparasitic structures were infrequently observed in interaction zones on various media, including compost, with cryoscanning electron microscopy. T. harzianum grows prolifically in compost in the absence or presence of A. bisporus, and the aggressive European (Th2) and North American (Th4) isolates produced significantly higher biomasses (6.8- and 7.5-fold, respectively) in compost than did nonaggressive, group 1 isolates. All groups secreted depolymerases that could attack the cell walls of A. bisporus and of wheat straw, and some were linked to aggressiveness. Growth on mushroom cell walls in vitro resulted in rapid production of chymoelastase and trypsin-like proteases by only the Th2 and Th4 isolates. These isolates also produced a dominant protease isoform (pI 6.22) and additional chitinase isoforms. On wheat straw, Th4 produced distinct isoforms of cellulase and laminarinase, but there was no consistent association between levels or isoforms of depolymerases and aggressiveness. Th3's distinctive profiles confirmed its reclassification as Trichoderma atroviride. Proteases and glycanases were detected for the first time in sterilized compost colonized by T. harzianum. Xylanase dominated, and some isoforms were unique to compost, as were some laminarinases. We hypothesize that aggressiveness results from competition, antagonism, or parasitism but only as a component of, or following, extensive saprotrophic growth involving degradation of wheat straw cell walls.     

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.     

An antifungal exo-alpha-1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum.

Trichoderma harzianum secretes alpha-1,3-glucanases when it is grown on polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (simulated antagonistic conditions). We have purified and characterized one of these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic. AGN13.1 was an exo-type alpha-1,3-glucanase and showed lytic and antifungal activity against fungal plant pathogens. Northern and Western analyses indicated that AGN13.1 is induced by conditions that simulated antagonism. We propose that AGN13.1 contributes to the antagonistic response of T. harzianum.