Partial silencing of a hydroxy-methylglutaryl-CoA reductase-encoding gene in Trichoderma harzianum CECT 2413 results in a lower level of resistance to lovastatin and lower antifungal activity.

In the present article, we describe the cloning and characterization of the Trichoderma harzianum hmgR gene encoding a hydroxymethylglutaryl CoA reductase (HMGR), a key enzyme in the biosynthesis of terpene compounds. In T. harzianum, partial silencing of the hmgR gene gave rise to transformants with a higher level of sensitivity to lovastatin, a competitive inhibitor of the HMGR enzyme. In addition, these hmgR-silenced transformants produced lower levels of ergosterol than the wild-type strain in a minimal medium containing lovastatin. The silenced transformants showed a decrease in hmgR gene expression (up to a 8.4-fold, after 72h of incubation), together with an increase in the expression of erg7 (up to a 15.8-fold, after 72h of incubation), a gene involved in the biosynthesis of triterpenes. Finally, hmgR-silenced transformants showed a reduction in their antifungal activity against the plant-pathogen fungi Rhizoctonia solani and Fusarium oxysporum.

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation.

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.