Isolation and characterisation of a trichodiene synthase homologous gene in Trichoderma harzianum.

Trichoderma species have long been known as effective biocontrol agents of major plant diseases, and some strains have also been reported to produce toxic metabolites, such as trichothecenes. In order to clarify the molecular aspect of trichothecene biosynthesis the strain ATCC 90237 of Trichoderma harzianum, a recognised producer of the trichothecene harzianum A, was investigated. An amplification fragment of 600 bp obtained by using a degenerate PCR approach showed several degrees of identity with trichodiene synthase genes previously reported from other fungal species. Flanking genomic regions were identified through genomic walking technique. The analysis of resultant sequence allowed the identification of an open reading frame of 1226 nucleotides. The expression of the gene was confirmed by the determination of the corresponding cDNA. The comparison with the genomic sequence revealed the presence of a unique 59 bp intron that presents consensus border and internal sequence typical of filamentous fungi. The deduced putative protein of 388 amino acids displayed an identity of about 60% to the enzyme trichodiene synthase which catalyses the first step in the trichothecene pathway of other mycotoxins producing fungi. The isolation of a putative trichodiene synthase (tri5) gene required for trichothecene biosynthesis was described for the first time in Trichoderma and enabled the design of specific tri5 primers for a PCR assay that demonstrated the presence of the trichodiene synthase gene in the genomes of different Trichoderma species.