An efficient transformation system for Trichoderma atroviride using the pyr4 gene as a selectable marker

The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride . To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5 mM uracil restores their growth and conidia production, and 1 mg ml −1 is the lethal dose of 5-fluoroorotic acid in T. atroviride . Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura − 1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆ tmk3 and ∆ pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆ blr1 and ∆ blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1 , grg2 , phr1 , and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride .