Fungal Mutant Construction Strategies.

Strains used in this study along with their genotypes are listed in SI Appendix, Table S2. The parental strains C. tropicalis CAY3764, C. parapsilosis CPL2H1, C. glabrata HTL, and C. auris {"type":"entrez-nucleotide","attrs":{"text":"B11804","term_id":"2092924","term_text":"B11804"}}B11804 (wild-type Colombian isolate of the South American clade IV) were used to generate homozygous deletion mutants using available auxotrophic and drug resistance marker–based strategies (79–81). Gene-replacement cassettes were prepared using a PCR-assisted gene splicing by overlap extension DNA assembly procedure (82). At least two independent mutants were created for each gene of interest. Gene deletion complementation with a single wild-type gene copy and an antibiotic-resistance marker was done either with nourseothricin in case of C. tropicalis and C. parapsilosis or with hygromycin B in case of C. glabrata and C. auris, respectively. Correct integration sites during gene deletion and complementation procedures were confirmed by routine PCR. The primers utilized for strain construction and genetic manipulations are listed in SI Appendix, Table S3.