Antonio Llobell

Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.

Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum

A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized. Partial amino-acid sequences from the purified 33-kDa chitinase were obtained. The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA. The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids. All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase. A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases. However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase. The chit33 gene appears as a single copy in the T. harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls. The 33-kDa chitinase seems to be very stable except under starvation conditions. The independent regulation of each of the chitinases in T. harzianum indicates different specific roles.

An Antifungal Exo-α-1,3-Glucanase (AGN13.1) from the Biocontrol Fungus Trichoderma harzianum

ABSTRACT Trichoderma harzianum secretes α-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 α-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.

Carbon source control on β-glucanases, chitobiase and chitinase from Trichoderma harzianum

The cell-wall degrading enzymes β-glucanase and chitinase have been suggested to be essential for the mycoparasitic action of Trichoderma species against plant fungal pathogens. For this reason, the production in different carbon sources of extracellular β-1,3-glucanase, β-1,6-glucanase, chitobiase and chitinase was studied in a mycoparasitic strain of Trichoderma harzianum. Maximal β-glucanase specific activities were detected in media supplemented with either pustulan (β-1,6-glucan), nigeran (α-1,3-glucan alternating with α-1,4-glucan), chitin or Saccharomyces cerevisiae or Botrytis cinerea purified cell walls, whereas the highest chitinase specific activity was obtained in medium supplemented with chitin. Furthermore, β-glucanase, chitobiase and chitinase activities showed an increase parallel to increasing concentrations of either pustulan or chitin added to the cultures, although the extent of this increase varied with the different enzymes. The culture filtrates of T. harzianum grown in these carbon sources also showed lytic activity on purified cell walls of S. cerevisiae and B. cinerea. The enzyme synthesis seemed to be repressed by glucose, 8-hydroxyquinoline, which inhibits transcription, or cycloheximide, an inhibitor of protein synthesis.

Microscopic and transcriptome analyses of early colonization of tomato roots by "Trichoderma harzianum"

The capacity of the fungus Trichoderma harzianum CECT 2413 to colonize roots and stimulate plant growth was analyzed. Tobacco seedlings (Nicotiana benthamiana) transferred to Petri dishes inoculated with T. harzianum conidia showed increased plant fresh weight (140%) and foliar area (300%), as well as the proliferation of secondary roots (300%) and true leaves (140%). The interaction between strain CECT 2413 and the tomato-root system was also studied during the early stages of root colonization by the fungus. When T. harzianum conidia were inoculated into the liquid medium of hydroponically grown tomato plants (Lycopersicum esculentum), profuse adhesion of hyphae to the plant roots as well as colonization of the root epidermis and cortex were observed. Confocal microscopy of a T. harzianum transformant that expressed the green fluorescent protein (GFP) revealed intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips. Analysis of the T. harzianum-tomato interaction in soil indicated that the contact between T. harzianum and the roots persisted over a long period of time. This interaction was characterized by the presence of yeast-like cells, a novel and previously undescribed developmental change. To study the molecular mechanism underlying fungal ability to colonize the tomato-root system, the T. harzianum transcriptome was analyzed during the early stages of the plant-fungus interaction. The expression of fungal genes related to redox reactions, lipid metabolism, detoxification, and sugar or amino-acid transport increased when T. harzianum colonized tomato roots. These observations are similar to those regarding the interactions of mycorrhiza and pathogenic fungi with plants.

Molecular characterization and heterologous expression of an endo-β-1,6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum

Hydrolytic enzymes from the filamentous fungus Trichoderma harzianum have been described as critical elements of the mycoparasitic action of Trichoderma against fungal plant pathogens. In this report we describe the first genomic and cE)NA clones encoding a β-1,6-endoglucanase gene. The deduced protein sequence has limited homology with other β-glucanases. Northern experiments show a marked repression of mRNA accumulation by glucose. The protein has been successfully produced in Saccharomyces cerevisiae upon construction of a transcriptional fusion of the cDNA with a yeast promoter. This S. cerevisiae recombinant strain shows a strong lytic action on agar plates containing β-1,6-glucan.

Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413 using the TrichoEST functional genomics approach.

Proteolytic enzymes (EC 3.4) secreted by Trichoderma strains are receiving increasing attention because of their potential implication in the Trichoderma biocontrol abilities. We have used an expressed sequence tag (EST) approach to identify genes encoding extracellular peptidases in T. harzianum CECT 2413 grown under several biocontrol-related conditions. Based on BlastX results and Gene Ontology annotation, a total of 61 (among 3478) unique sequences (unisequences) were predicted to encode enzymes with peptidase activity, three corresponding to secreted peptidases already known from this Trichoderma strain (PAPA, PRA1 and P6281). Further manual screening based on the functional identity and cellular location of the best matches revealed ten unisequences encoding novel extracellular peptidases. We report the characterization of the corresponding genes as well as a potential orthologous gene of the intracellular peptidase PAPB from T. asperellum. In each case, full-length coding sequences were obtained, and deduced proteins were compared at phylogenetic level with peptidases from other organisms. T. harzianum CECT 2413 novel peptidases included six serine endopeptidases (EC 3.4.21) belonging to the families S1, S8 and S53, three aspartic endopeptidases (EC 3.4.23) of the family A1, one metallo-endopeptidase (EC 3.4.24) of the family M35, and one aminopeptidase (EC 3.4.11) of the family M28. Results obtained by Northern blot analyses demonstrated that the genes within a family are differentially regulated in response to different culture conditions, suggesting that they have diverse functional roles.

Expression of an α‐1,3‐glucanase during mycoparasitic interaction of Trichoderma asperellum

Trichoderma species have been investigated as biological control agents for over 70 years owing to their ability to antagonize plant pathogenic fungi. Mycoparasitism, one of the main mechanisms involved in the antagonistic activity of Trichoderma strains, depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. The antifungal activity of an α‐1,3‐glucanase (EC 3.2.1.59, enzymes able to degrade α‐1,3‐glucans and also named mutanases) has been described in T. harzianum and its role in mycoparasitic processes has been suggested. In this study, we report on the purification, characterization and cloning of an exo‐α‐1,3‐glucanase, namely AGN13.2, from the antagonistic fungus T. asperellum T32. Expression at the transcription level in confrontation assays against the strawberry pathogen Botrytis cinerea strongly supports the role of AGN13.2 during the antagonistic action of T. asperellum.

Cell wall-degrading isoenzyme profiles of Trichoderma biocontrol strains show correlation with rDNA taxonomic species

Trichoderma is known for being the most frequently used biocontrol agent in agriculture. A fundamental part of the Trichoderma antifungal system relies on a series of genes coding for a variety of extracellular lytic enzymes. Characterization of the polymorphism between five putative isoenzymatic activities [β-1,3-glucanase (EC 3.2.1.39, EC 3.2.1.58), β-1,6-glucanase (EC 3.2.1.75), cellulase (EC 3.2.1.4; EC 3.2.1.21, EC 3.2.1.91), chitinase (EC 3.2.1.30, EC 3.2.1.52), protease (EC 3.4.11; EC 3.4.13–19; EC 3.4.21–24, EC 3.4.99)] was carried out using 18 strains from three sections of Trichoderma. Of these, seven strains were from T. sect. Pachybasium, nine from T. sect. Trichoderma and two from T. sect. Longibrachiatum. Thirty-seven different alleles in total were identified: 13 for β-1,3-glucanase, four for β-1,6-glucanase, three for cellulase, eight for chitinase and nine for protease activity. A dendrogram (constructed by the unweighted pair group method with arithmetic averages) based on isoenzymatic data separated the 18 strains into three main enzymatic groups: T. harzianum, T. atroviride/T. viride/T. koningii and T. asperellum/T. hamatum/T. longibrachiatum. Isoenzymatic groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location, based on their sequence of internal transcribed spacer 1 in ribosomal DNA and their antifungal activities.

A putative catabolite-repressed cell wall protein from the mycoparasitic fungus Trichoderma harzianum.

A cDNA clone encoding a putative cell wall protein (Qid3) was isolated from a library prepared from chitin-induced mRNA in cultures of the mycoparasitic fungus Trichoderma harzianum. The predicted 14 kDa protein shows a potential signal peptide, several hydrophobic domains and certain motifs that are structurally similar to proline-rich and glycine-rich plant cell wall proteins. Expression of the qid3 gene is derepressed in the absence of glucose. When introduced in yeast, qid3 expression causes cell division arrest into cytokinesis and cell separation, probably due to its cell wall localization.