1887

Abstract

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte (anamorph: ), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the gene adapted to the pathogenic yeast () or a synthetic codon-optimized () gene with neomycin resistance () cassette for one-step marker excision. Both genes were under control of the copper-repressible promoter (). Molecular analyses of resultant transformants showed that only the -harbouring module was functional in . Applying this system, we successfully produced the recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) () or serine protease () genes, producing mutant strains with double or triple gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

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2014-10-01
2019-11-22
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