Robust Cre recombinase activity in the biotrophic smut fungus Ustilago maydis enables efficient conditional null mutants in planta

Abstract

Abstract Site-specific recombinases have been used in higher eukaryotes, especially in animals, for a broad range of applications, including chromosomal translocations, large deletions, site-specific integration, and tissue-specific as well as conditional knock-outs. The application of site-specific recombination has also been demonstrated in simple eukaryotes like fungi and protozoa. However, its use in fungal research, especially in phytopathogenic fungi, has often been limited to “recycle” the marker genes used in transformation experiments. We show that Cre recombinase can be used for conditional gene deletions in the phytopathogenic fungus Ustilago maydis. Conditional gene knock-outs can be generated via the transcriptional control of the recombinase by U. maydis promoters specifically activated during the biotrophic phase of fungal growth, enabling gene deletions at defined developmental stages inside the plant tissue. Also, we show that a tamoxifen-activated Cre-recombinase allows the tight control necessary for the induced deletion of essential genes by the addition of tamoxifen. These tools will be helpful to address the function of genes under both axenic and in planta conditions for the U. maydis-maize pathosystem and should pave the way for similar approaches in other plant pathosystems.

Abstract Site-specific recombinases have been used in higher eukaryotes, especially in animals, for a broad range of applications, including chromosomal translocations, large deletions, site-specific integration, and tissue-specific as well as conditional knock-outs. The application of site-specific recombination has also been demonstrated in simple eukaryotes like fungi and protozoa. However, its use in fungal research, especially in phytopathogenic fungi, has often been limited to “recycle” the marker genes used in transformation experiments. We show that Cre recombinase can be used for conditional gene deletions in the phytopathogenic fungus Ustilago maydis. Conditional gene knock-outs can be generated via the transcriptional control of the recombinase by U. maydis promoters specifically activated during the biotrophic phase of fungal growth, enabling gene deletions at defined developmental stages inside the plant tissue. Also, we show that a tamoxifen-activated Cre-recombinase allows the tight control necessary for the induced deletion of essential genes by the addition of tamoxifen. These tools will be helpful to address the function of genes under both axenic and in planta conditions for the U. maydis-maize pathosystem and should pave the way for similar approaches in other plant pathosystems.