A Dku70 mutant of Trichoderma virens as a tool for efficient gene targeting

The antagonistic abilities of the genus Trichoderma make them ideal candidates for biocontrol against phytopathogenic fungi in integrated pest management. Strains of Trichoderma spp. (including the T.virens I10 isolate) have proven to be effective mycoparasites of important plant-pathogenic fungi. An I10 GFP transformant has been exploited for monitoring the sclerotia colonisation in Sclerotinia spp. Gene targeting by homologous recombination (HR) is a powerful technique that can be applied to study gene functions and to alter interesting properties of fungal strains. However, in filamentous fungi this is often hampered by very low frequencies of HR and it is therefore not trivial to target a gene of interest to a specific genomic locus or to delete an endogenous gene. To increase the recombination rate in T.virens transformation the orthologue of the human KU70, which is required for the nonhomologous and joining (NHEJ) pathway and responsible for ectopic DNA integration, has been identified and deleted. The effect on gene targeting of the absent ku70 in T.virens was tested by deleting a laccase gene. Efficiency of gene targeting was 90% in the I10 ku70 strain, which is a significant increase compared to the parental strain (non-ku70 deleted) where only a 10-15% gene knock-out frequency was observed. The generated T.virens ku70 mutant can now be exploited for functional gene studies. The double mutant T.virens ku70/lac will be used to study the physiological role of the laccase gene product including a potential function in mycoparasitism against sclerotial fungi such as B. cinerea or S. sclerotiorum.