Regulation of the expression of the nag1 gene (N-acetyl-ß-D-glucosaminidase) in Trichoderma harzianum

Mycoparasitism defines a process by which certain (mostly imperfect) fungi attack others and use them as a nutrient source. Several fungi of the genus Trichoderma are active mycoparasites, a property with both positive (antagonism of plant pathogens) as well as negative (attack on mushroom farms) impacts on agriculture. Since most fungi of this genus do not attack plants, some of them have been extensively studied as potent biocontrol agents. Particularly T. harzianum is known to be capable of attacking a great variety of phytopathogenic fungi. Mycoparasitism has been described as a complex, multi-step interaction process between the host hyphae and the hyphae of T. harzianum. The mycoparasite hyphae grow towards the target mycelium, and - upon contact with the host - coil around or grows along the host hyphae and form hook-like structures, presumably appressoria, that aid in penetration of the host`s hyphal cell wall. Subsequent penetration into the target hyphae is then aided by secretion of hydrolyzing enzymes. Chitinases have been shown to be important factors in the ability of Trichoderma harzianum to biocontrol plant pathogens, and we thus consider an understanding of their formation essential. In this proposal we offer a strategy which should lead to the understanding of regulation of expression of one of the chitinases, i.e. the nag1 -gene product, which is based on the following experiments: (a) the use of in vivo (promoter deletions) and in vitro (EMSA-footprinting) experiments to identify the nucleotide motif involved in induction of nag1 expression; (b) the use of the determined nucleotide motif to clone the gene encoding the respective DANN-binding protein (e.g. by screening of an expression gene library using the novel One-Hybrid- System; (c) The regulatory gene so obtained shall be characterized in Vivo and in vitro by standard techniques; (d) A new system shall be established using REMI mutagenesis to obtain regulatory mutants of nag1 expression, that will enable us to isolate further regulatory genes involved in the induction of nag1.

In this research project we identified the DNA region that is responsible for the regulation of the expression of a gene encoding a chitinolytic enzyme in the mycoparasitic biocontrol fungus Trichoderma atroviride (=T. harzianum P1). We also isolated a gene whose gene product can bind to this regulatory DNA region, but is not involved in the control of the chitinase gene. Instead, it is most likely involved in the protection against certain environmental stress factors. The chitinase gene (nag1, encoding an N-Acetyl-ß-D-glucosaminidase) is strongly expressed when the fungus is grown on medium containing N-Acetyl-ß-D-glucosamine as carbon source, even in very low concentrations. N- Acetyl-ß-D-glucosamine is the basic building block of chitin, a biopolymer which is present in the cell wall of. Chitin degrading enzymes such as N-Acetyl-ß-D-glucosaminidase are implied to play a major role in mycoparasitism, i.e. the killing and utilization of a fungus by another fungus. The goal was to identify and characterize certain motifs in the regulatory regions of the gene, which are responsible for governing this pattern of expression. First a region of 90 nucleotides, located 200 - 290 bases before the actual coding region of the gene, was identified as being absolutely necessary for the increase of nag1 expression in the presence of N-Acetyl-ß-D-glucosamine. Using experimental procedures that can visualize interactions of proteins with DNA sequences, we could identify even shorter regions of nucleotides that interact with proteins and are presumably essential for the regulation. Position and nucleotide sequence of these short motifs had a strong similarity to the situation in the gene promoter of a cutinase gene from the plant pathogenic fungus Haematonectria haematococca (Fusarium solani). The function of nag1 and the encoded protein is believed to be comparable to this gene, and the regulation of enzyme formation is similar. We therefore isolated a gene from T. atroviride based on the sequence similarity with the gene from H. haematococca. The isolated gene was shown to code for a protein that can bind one of the identified motifs. A fungal strain was then constructed where the gene, named seb1, was destroyed and which therefore did not contain a functional Seb1 protein. This strain, however, did not show any changes in the expression of nag1, and the interactions of the nag1 regulatory region with regulatory proteins was intact. We therefore conclude that the isolated gene does not play a role in the expression of nag1. In order to identify a function for this gene we compared growth, biomass formation and morphology of the original strain and the one lacking the seb1 gene. We found a strongly reduced growth in the presence of high concentrations of salt in the growth medium in the constructed strain as compared to an only slight reduction in the original strain. We therefore conclude that the gene is somehow involved in the protection against certain environmental stress factors. It does not, however, fulfill the same function as a certain stress regulator gene from the model organism bakers yeast.