Tahía Benítez

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.

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.

Increased antifungal and chitinase specific activities of Trichoderma harzianum 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.

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.

Yeast cell viability under conditions of high temperature and ethanol concentrations depends on the mitochondrial genome

SummaryWine yeasts manifest simultaneously a high tolerance to ethanol, thermotolerance, and a high resistance to the mutagenic effects of ethanol on the mitochondrial genome. The transfer of mitochondria from these strains to laboratory yeasts demonstrate that this genome influences the above parameters, since thermotolerance, ethanol-growth tolerance, and the frequency ofrho− mutants were either totally or partially modified in the laboratory recipient strain. When the death rate and the rate of formation ofrho−mutants were measured under extreme conditions of inhibitory ethanol concentrations and high temperature, a perfect correlation was found between these parameters, and both of them were dependent on the strain of mitochondrial genome. Thus, the transfer of wine yeast mitochondria leads to a lower death rate, and a simultaneous increase in thermotolerance and ethanol tolerance in the recipient strain. These results demonstrate the role that viability plays under conditions of high temperatures and high ethanol concentrations. The greater stability of therho+ phenotype shown by the wine yeast mitochondrial genome may be responsible for the increased viability conferred by these mitochondria.

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.

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

Abstract. 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 β-1,6-glucan. The mutant possesses between two and four times more chitinase, β-1,3- and β-1,6-glucanase activities than the wild type, produces about three times more extracellular proteins and secretes higher amounts of a yellow pigment (α-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.

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (μ), fermentation rate (v) or respiration rate (ϱ) of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show Ki (ethanol inhibition constant) values for μ, v and ϱ, higher (Ki>1 M) than those of “petite” mutants or “grande” strains grown in anaerobiosis (Ki=0.7 M). In addition, the relationship between μ or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to “petite” mutants, the resulting strain shows Ki values similar to those of the “grande” strain and the inhibition of μ and v by increasing ethanol concentrations becomes linear.