SEARCH · PubMed Health

Results for “Trichoderma”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources

PubMed1994

Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles.

The genetic diversity in Trichoderma harzianum isolates from mushroom compost was assessed using various molecular techniques. Restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) divided the 81 isolates into three major groups, 1, 2 and 3. There was no variation within a group in rDNA, while a low degree of polymorphism was detected in mtDNA. Random amplified polymorphic DNA (RAPD) analysis of 30 randomly chosen isolates, with six primers, in general confirmed the RFLP groups. Nucleotide sequence determination of rDNA internal transcribed spacer (ITS) 1 revealed three distinct ITS types, 1, 2 and 3, possessed by isolates from the respective groups 1, 2 and 3. Based on these molecular data, group 2 isolates, which are aggressive colonizers of mushroom compost, could be clearly distinguished from the isolates belonging to the other two groups.

Base Sequence↗

PubMed2000

Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: cellobiohydrolase I (CBHI) as a model protein.

The authors have developed methodology to study the kinetics of protein synthesis and secretion in filamentous fungi. Production of cellobiohydrolase I (CBHI) by Trichoderma reesei was studied by metabolic labelling of the proteins in vivo with [35S]methionine or [14C]mannose, and subsequent analysis of the labelled proteins using two-dimensional gel electrophoresis. Analysis of the different pl forms of the nascent proteins allowed monitoring of the maturation of CBHI during the transport along the biosynthetic pathway. The maturation of the pi pattern of CBHI as well as secretion into culture medium was prevented by treatment with the reducing agent DTT. The pl forms of CBHI detectable in the presence of DTT corresponded to the early endoplasmic reticulum forms of the protein. Removal of N-glycans by enzymic treatment (endoglycosidase H or peptide-N-glycosidase F), or chemical removal of both N- and O-glycans, changed the pl pattern of CBHI, showing that glycan structures are involved in formation of the different pl forms of the protein. By quantifying the labelled proteins during a time course, parameters describing protein synthesis and secretion were deduced. The mean synthesis time for CBHI under the conditions used was 4 min and the minimum secretion time was 11 min. The methodology developed in this study provides tools to reveal the rate-limiting factors in protein production and to obtain information on the intracellular events involved in the secretion process.

Carbon Radioisotopes↗

PubMed2002

Aspartyl protease from Trichoderma harzianum CECT 2413: cloning and characterization.

A gene that encodes an extracellular aspartyl protease from Trichoderma harzianum CECT 2413, papA, has been isolated and characterized. Based on several conserved regions of other fungal acid proteases, primers were designed to amplify a probe that was used to isolate the papA gene from a genomic library of T. harzianum. papA was an intronless ORF which encoded a polypeptide of 404 aa, including a prepropeptide at the N-terminal region formed by one putative signal peptide, a second peptide which could be cleaved to activate the enzyme and the active protease of calculated 36.7 kDa and pI 4.35. Northern experiments indicated that papA gene was pH regulated, repressed by ammonium, glucose and glycerol, and induced by organic nitrogen sources. The promoter possessed potential AreA, PacC and MYC sites for nitrogen, pH and mycoparasitism regulation respectively, but lacked potential CreA sites for carbon regulation. IEF and zymograms indicated that PAPA was a pepstatin-sensitive aspartyl protease of pI 4.5. Transformants from T. harzianum CECT 2413 cultivated in yeast extract-supplemented medium overexpressed papA and had a fourfold increase in protease activity compared to the wild-type, while transformants that overexpressed the beta-1,6-glucanase gene bgn16.2 and papA had an additional 30% increase in beta-1,6-glucanase activity compared to bgn16.2 single transformants. Overexpression of both genes in ammonium-supplemented medium did not result in higher levels of PAPA and/or BGN16.2 proteins. These results indicated that both PAPA and beta-1,6-glucanase undergo proteolysis in ammonium-supplemented medium but PAPA is not responsible for beta-1,6-glucanase degradation.

Amino Acid Sequence↗

PubMed2006

Intraspecific mitochondrial DNA polymorphism within the emerging filamentous fungal pathogen Trichoderma longibrachiatum.

The genetic diversity of the emerging fungal pathogen Trichoderma longibrachiatum was examined at the level of mitochondrial DNA. The 17 investigated strains, comprising nine clinical and eight non-clinical isolates, exhibited seven and ten different mitochondrial DNA profiles by using the restriction enzymes BsuRI and Hin6I, respectively. The sizes of mitochondrial DNAs varied from 34.9 to 39.5 kb. The discriminatory power of the method was higher than that of internal transcribed spacer sequence analysis and therefore should be more suitable for identification and epidemiological investigations. However, clinical and non-clinical isolates did not form separate clusters on the resulting dendrogram and thus there was no indication of a correlation between genetic structure and pathogenicity of the isolates.

DNA, Mitochondrial↗

PubMed2003

Intron requirement for AFP gene expression in Trichoderma viride.

The 430 bp ORF of the Aspergillus giganteus antifungal protein (AFP) gene, containing two small introns, was fused between the promoter and the terminator of the Aspergillus nidulans trpC gene. The AFP gene in this vector produced detectable levels of spliced mRNA in Trichoderma viride. In contrast, in the same vector configuration, its 285 bp intronless derivative showed no accumulation of mRNA when transformed into T. viride. Such expression results were confirmed at the protein level. This fact demonstrated that the introns were required for AFP gene expression in T. viride. This is thought to be a novel phenomenon found in filamentous fungi. Although the mechanism of splicing in filamentous fungi might be similar to that in other eukaryotes, little is known of how it affects expression. This study suggests that the small introns in filamentous fungal genes may not only act as intervening elements, but may also play crucial roles in gene expression by affecting mRNA accumulation. Furthermore, it may provide new evidence for intron-dependent evolution.

Fungal Proteins↗

PubMed2004

Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme.

Previous studies on Melanocarpus albomyces laccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungus Trichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin-laccase fusion protein. Both proteins were expressed in T. reesei under the cbh1 promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l(-1)). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin-laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l(-1). The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed2004

BLR-1 and BLR-2, key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride.

In fungi, phototropism, the induction of carotenogenesis and reproductive structures, and resetting of the circadian rhythm are controlled by blue light. Trichoderma atroviride, a fungus used in biological control, sporulates in a synchronized manner following a brief pulse of blue light. Due to its apparent simplicity, this response was chosen for pursuing photoreceptor isolation. Two genes were cloned, blue-light regulators 1 and 2 (blr-1 and blr-2), similar to the Neurospora crassa white-collar 1 and 2, respectively. The BLR-1 protein has all the characteristics of a blue-light photoreceptor, whereas the structure of the deduced BLR-2 protein suggests that it interacts with BLR-1 through PAS domains to form a complex. Disruption of the corresponding genes demonstrated that they are essential for blue-light-induced conidiation. blr-1 and blr-2 were also shown to be essential for the light-induced expression of the photolyase-encoding gene (phr-1). Mechanical injury of mycelia was found to trigger conidiation of T. atroviride, a response not described previously. This response was not altered in the mutants. A novel effect of both red and blue light on mycelial growth was found involving another light receptor, which is compensated by the BLR proteins.

Amino Acid Sequence↗

PubMed2005

The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei.

Trichoderma reesei was cultivated in chemostat cultures on lactose-containing medium. The cultures were characterized for growth, consumption of the carbon source and protein production. Secreted proteins were produced most efficiently at low specific growth rates, 0.022-0.033 h(-1), the highest specific rate of total protein production being 4.1 mg g(-1) h(-1) at the specific growth rate 0.031 h(-1). At low specific growth rates, up to 29 % of the proteins produced were extracellular, in comparison to only 6-8 % at high specific growth rates, 0.045-0.066 h(-1). To analyse protein synthesis and secretion in more detail, metabolic labelling of proteins was applied to analyse production of the major secreted protein, cellobiohydrolase I (CBHI, Cel7A). Intracellular and extracellular labelled CBHI was quantified and analysed for pI isoforms in two-dimensional gels, and the synthesis and secretion rates of the molecule were determined. Both the specific rates of CBHI synthesis and secretion were highest at low specific growth rates, the optimum being at 0.031 h(-1). However, at low specific growth rates the secretion rate/synthesis rate ratio was significantly lower than that at high specific growth rates, indicating that at low growth rates the capacity of cells to transport the protein becomes limiting. In accordance with the high level of protein production and limitation in the secretory capacity, the transcript levels of the unfolded protein response (UPR) target genes pdi1 and bip1 as well as the gene encoding the UPR transcription factor hac1 were induced.

Biological Transport↗

PubMed2006

ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum.

Trichoderma harzianum is a widespread mycoparasitic fungus, able to successfully colonize a wide range of substrates under different environmental conditions. Transcript profiling revealed a subset of genes induced in T. harzianum under hyperosmotic shock. The hog1 gene, a homologue of the MAPK HOG1 gene that controls the hyperosmotic stress response in Saccharomyces cerevisiae, was characterized. T. harzianum hog1 complemented the hog1Delta mutation in S. cerevisiae, but showed different features to yeast alleles: improved osmoresistance by expression of the hog1 allele and a lack of lethality when the hog1(F315S) allele was overexpressed. ThHog1 protein was phosphorylated in T. harzianum under different stress conditions such as hyperosmotic or oxidative stress, among others. By using a ThHog1-GFP fusion, the protein was shown to be localized in nuclei under these stress conditions. Two mutant strains of T. harzianum were constructed: one carrying the hog1(F315S) allele, and a knockdown hog1-silenced strain. The silenced strain was highly sensitive to osmotic stress, and showed intermediate levels of resistance against oxidative stress, indicating that the main role of ThHog1 protein is in the hyperosmotic stress response. Stress cross-resistance experiments showed evidences of a secondary role of ThHog1 in oxidative stress. The strain carrying the hog1(F315S) allele was highly resistant to the calcineurin inhibitor cyclosporin A, which suggests the existence of links between the two pathways. The two mutant strains showed a strongly reduced antagonistic activity against the plant pathogens Phoma betae and Colletotrichum acutatum, which points to a role of ThHog1 protein in fungus-fungus interactions.

Antibiosis↗

PubMed2006

Novel light-regulated genes in Trichoderma atroviride: a dissection by cDNA microarrays.

The influence of light on living organisms is critical, not only because of its importance as the main source of energy for the biosphere, but also due to its capacity to induce changes in the behaviour and morphology of nearly all forms of life. The common soil fungus Trichoderma atroviride responds to blue light in a synchronized manner, in time and space, by forming a ring of green conidia at what had been the colony perimeter at the time of exposure (photoconidiation). A putative complex formed by the BLR-1 and BLR-2 proteins in T. atroviride appears to play an essential role as a sensor and transcriptional regulator in photoconidiation. Expression analyses using microarrays containing 1438 unigenes were carried out in order to identify early light response genes. It was found that 2.8 % of the genes were light responsive: 2 % induced and 0.8 % repressed. Expression analysis in blr deletion mutants allowed the demonstration of the occurrence of two types of light responses, a blr-independent response in addition to the expected blr-dependent one, as well as a new role of the BLR proteins in repression of transcription. Exposure of T. atroviride to continuous light helped to establish that the light-responsive genes are subject to photoadaptation. Finally, evidence is provided of red-light-regulated gene expression and a possible crosstalk between the blue and red light signalling pathways.

Blotting, Northern↗

PubMed1994

Induction of ethylene biosynthesis in Nicotiana tabacum by a Trichoderma viride xylanase is correlated to the accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase transcripts.

Xylanase (EIX) from the fungus Trichoderma viride elicits ethylene biosynthesis in leaf tissues of Nicotiana tabacum cv Xanthi but not in cv Hicks. The increase in ethylene biosynthesis is accompanied by an accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC), an increase in extractable ACC synthase activity, and increases in ACC synthase and ACC oxidase transcripts. Priming of increases in ACC synthase and ACC oxidase transcripts. Priming of leaves with ethylene (120 microL/L, 14 h) sensitizes the tissue, resulting in an enhanced response to EIX and increases in both the in vivo ACC oxidase activity and ACC oxidase transcript level. EIX and ethylene independently induce ACC oxidase. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine is not accompanied by a reduction in ACC oxidase transcript level, indicating that ethylene biosynthesis is not required. In contrast to the differential induction of ethylene biosynthesis by EIX in Xanthi versus Hicks cultivars, both cultivars respond to a chemical stress (induced by CuSO4) by enhancing ethylene production. This induction is accompanied by an increase in ACC synthase transcript but not in that of ACC oxidase.

Amino Acid Oxidoreductases↗

PubMed2001

Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum.

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.

Adaptation, Physiological↗

PubMed2000

Purification, crystallization and preliminary X-ray study of beta-xylosidase from Trichoderma reesei.

An extracellular multifunctional beta-xylosidase was purified from a culture of the fungus Trichoderma reesei. The active 95 +/- 5 kDa enzyme has been crystallized from sodium acetate buffer using PEG as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.75, b = 98.54, c = 227.25 A, and diffract beyond 2.7 A resolution. X-ray data were collected from frozen crystals on a synchrotron source.

Crystallization↗

PubMed2003

Crystallization and preliminary X-ray characterization of Trichoderma reesei hydrophobin HFBII.

Hydrophobins are small proteins found in filamentous fungi and characterized by their ability to change the character of a surface by spontaneous self-assembly on a hydrophobic-hydrophilic interface. Hydrophobin HFBII from Trichoderma reesei was crystallized by the hanging-drop vapour-diffusion method at 293 K. Two crystal forms were obtained: a native form and a form crystallized in the presence of manganese chloride. The native crystals were of high symmetry, cubic I23, but only diffracted to 3.25 A. The crystals grown in the presence of manganese were monoclinic and diffracted to 1.0 A with a synchrotron-radiation source. The anomalous difference Patterson map calculated from the home laboratory data showed a strong single peak, possibly caused by manganese present in the crystallization solution.

Crystallization↗

PubMed2004

Crystallization and preliminary X-ray analysis of a novel Trichoderma reesei xylanase IV belonging to glycoside hydrolase family 5.

Xylanase IV (XYN IV) is a new recently characterized xylanase from Trichoderma reesei. It is able to degrade several different xylans, mainly producing xylose. XYN IV has been crystallized by the hanging-drop vapour-diffusion method, using PEG 6000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 86.3, b = 137.5, c = 196.1 A, alpha = beta = gamma = 90 degrees. Assuming a molecular weight of 50.3 kDa, the V(M) values indicate there to be four XYN IV monomers in an asymmetric unit and the solvent content of the crystals to be 57%. Based on dynamic light-scattering measurements, XYN IV is a dimer in solution. A native data set to 2.8 A resolution has been collected at a home laboratory and a data set to 2.2 A resolution has been collected using synchrotron radiation.

Crystallization↗

PubMed2004

Crystals of family 11 xylanase II from Trichoderma longibrachiatum that diffract to atomic resolution.

Xylanases catalyse the cleavage of various forms of xylan. A new crystal form of xylanase II from the fungus Trichoderma longibrachiatum that diffracts to better than 1 A resolution was grown from 12% PEG 8K, 0.1 M Tris pH 8.5, 0.2 M CaCl(2) with the addition of 2% glycerol to overcome crystal twinning. The crystals grow in a body-centered orthorhombic Bravais lattice, with unit-cell parameters a = 66.78, b = 67.94, c = 79.18 A. The solvent content is 42% with one molecule per asymmetric unit. Molecular-replacement analysis reveals the space group to be I222. This atomic resolution structure will provide important insights that will lead to a better understanding of the enzymatic mechanism of the family 11 xylanases.

Crystallization↗

PubMed2004

Purification, crystallization and preliminary X-ray diffraction analysis of the Trichoderma reesei hydrophobin HFBI.

Hydrophobins are fungal proteins that are capable of altering the hydrophobicity of surfaces by self-assembly at hydrophilic-hydrophobic interfaces. Here, the growth of hydrophobin crystals suitable for X-ray crystallography is reported. The hydrophobin HFBI from Trichoderma reesei was crystallized by vapour diffusion in hanging drops in 30% PEG 4000, 0.1 M sodium citrate pH 4.3 buffer containing 0.2 M ammonium acetate and CYMAL-5 detergent (initial concentration of 2.4 mM). HFBI crystals are hexagonal and belong to space group P6(1) (or P6(5)), with unit-cell parameters a = b = 45.9, c = 307.2 A. The HFBI used in the crystallization experiments was purified from fungal cell walls.

Crystallization↗

PubMed2006

Structure of an orthorhombic form of xylanase II from Trichoderma reesei and analysis of thermal displacement.

An orthorhombic crystal of xylanase II from Trichoderma reesei was grown in the presence of sodium iodide. Crystal structures at atomic resolution were determined at 100 and 293 K. Protein molecules were aligned along a crystallographic twofold screw axis, forming a helically extended polymer-like chain mediated by an iodide ion. The iodide ion connected main-chain peptide groups between two adjacent molecules by an N-H...I-...H-N hydrogen-bond bridge, thus contributing to regulation of the molecular arrangement and suppression of the rigid-body motion in the crystal with high diffraction quality. The structure at 293 K showed considerable thermal motion in the loop regions connecting the beta-strands that form the active-site cleft. TLS model analysis of the thermal motion and a comparison between this structure and that at 100 K suggest that the fluctuation of these loop regions is attributable to the hinge-like movement of the beta-strands.

Binding Sites↗