Genetic Analysis of Family Transcription Factors in Using New CRISPR-Cas9 Approaches.

Ndt80 family transcription factors are highly conserved in fungi, where they regulate diverse processes. The human fungal pathogen contains three genes (, , and ) that encode proteins with similarity to Ndt80, although the homology is restricted to the DNA binding domain. To better understand their role in virulence functions, we used clustered regularly interspaced short palindromic repeat/CRISPR-associated gene 9 (CRISPR/Cas9) to delete the three -family genes. An Δ mutant showed strong defects in forming hyphae in response to serum or -acetylglucosamine (GlcNAc), which was linked to the ability of Ndt80 to regulate the expression of , an upstream regulator of hyphal signaling. Conversely, the Δ mutant formed hyphal cells on glycerol medium, indicating that Ndt80 is not required for hyphal growth under all conditions. In contrast to our previously published data, a Δ single mutant could grow and form hyphae in response to GlcNAc. However, deleting partially restored the ability of an Δ mutant to form hyphae in response to GlcNAc, indicating a link to GlcNAc signaling. was required for growth on GlcNAc, as expected, but not for GlcNAc or serum to induce hyphae. The mutant was defective in growing under stressful conditions, such as elevated temperature, but not the mutant or mutant. Quantitative assays did not reveal any significant differences in the fluconazole susceptibility of the NDT80-family mutants. Interestingly, double and triple mutant analysis did not identify significant genetic interactions for these family genes, indicating that they mainly function independently, in spite of their conserved DNA binding domain. Transcription factors play key roles in regulating virulence of the human fungal pathogen In addition to regulating the expression of virulence factors, they also control the ability of to switch to filamentous hyphal growth, which facilitates biofilm formation on medical devices and invasion into tissues. We therefore used new CRISPR/Cas9 methods to examine the effects of deleting three genes (, , and ) that encode transcription factors with similar DNA binding domains. Interestingly, double and triple mutant strains mostly showed the combined properties of the single mutants; there was only very limited evidence of synergistic interactions in regulating morphogenesis, stress resistance, and ability to metabolize different sugars. These results demonstrate that , , and have distinct functions in regulating virulence functions.