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One-step targeted gene deletion in Candida albicans haploids

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Abstract

The recent discovery of haploids in Candida albicans and the construction of tool strains carrying multiple auxotrophic markers have enabled, for the first time, performing one-step gene deletions in this fungal human pathogen. This breakthrough promises to greatly facilitate the molecular and genetic study of C. albicans biology and pathogenicity. However, the construction of gene-deletion mutants in C. albicans haploids involves many technical difficulties, particularly low transformation efficiency and autodiploidization. Here we describe a highly effective protocol for designing and performing one-step gene deletion in C. albicans haploids, which takes ∼11 d to complete (not including plasmid construction, which may take ∼2 weeks). A gene deletion cassette is constructed on a plasmid and subsequently released for transformation by lithium acetate incubation or electroporation. Desired gene-deletion mutants are identified and their ploidy is assessed simultaneously by colony PCR before final confirmation by flow cytometry.

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Figure 1
Figure 2: Schematic diagram of the gene deletion construct.
Figure 3: Visualization of the gene deletion cassette.
Figure 4: Examples of transformants on plates.
Figure 5: Characterization of transformants by colony PCR.
Figure 6: Ploidy analysis of transformants by flow cytometry.

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References

  1. Brown, G.D. et al. Hidden killers: human fungal infections. Sci. Transl. Med. 4, 165rv113 (2012).

    Article  Google Scholar 

  2. Homann, O.R., Dea, J., Noble, S.M. & Johnson, A.D. A phenotypic profile of the Candida albicans regulatory network. PLoS Genet. 5, e1000783 (2009).

    Article  Google Scholar 

  3. Noble, S.M., French, S., Kohn, L.A., Chen, V. & Johnson, A.D. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat. Genet. 42, 590–598 (2010).

    Article  CAS  Google Scholar 

  4. Blankenship, J.R., Fanning, S., Hamaker, J.J. & Mitchell, A.P. An extensive circuitry for cell wall regulation in Candida albicans. PLoS Pathog. 6, e1000752 (2010).

    Article  Google Scholar 

  5. Ryan, O. et al. Global gene deletion analysis exploring yeast filamentous growth. Science 337, 1353–1356 (2012).

    Article  CAS  Google Scholar 

  6. Noble, S.M. & Johnson, A.D. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot. Cell 4, 298–309 (2005).

    Article  CAS  Google Scholar 

  7. Fonzi, W.A. & Irwin, M.Y. Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Berman, J. & Sudbery, P.E. Candida albicans: a molecular revolution built on lessons from budding yeast. Nat. Rev. Genet. 3, 918–930 (2002).

    Article  CAS  Google Scholar 

  9. De Backer, M.D., Magee, P.T. & Pla, J. Recent developments in molecular genetics of Candida albicans. Annu. Rev. Microbiol. 54, 463–498 (2000).

    Article  CAS  Google Scholar 

  10. Wilson, R.B., Davis, D. & Mitchell, A.P. Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J. Bacteriol. 181, 1868–1874 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Morschhauser, J., Michel, S. & Staib, P. Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination. Mol. Microbiol. 32, 547–556 (1999).

    Article  CAS  Google Scholar 

  12. Alani, E., Cao, L. & Kleckner, N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116, 541–545 (1987).

    Article  CAS  Google Scholar 

  13. Wilson, R.B., Davis, D., Enloe, B.M. & Mitchell, A.P. A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast 16, 65–70 (2000).

    Article  CAS  Google Scholar 

  14. Gola, S., Martin, R., Walther, A., Dunkler, A. & Wendland, J. New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region. Yeast 20, 1339–1347 (2003).

    Article  CAS  Google Scholar 

  15. Walther, A. & Wendland, J. PCR-based gene targeting in Candida albicans. Nat. Protoc. 3, 1414–1421 (2008).

    Article  CAS  Google Scholar 

  16. Gietz, R.D., Schiestl, R.H., Willems, A.R. & Woods, R.A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360 (1995).

    Article  CAS  Google Scholar 

  17. Walther, A. & Wendland, J. An improved transformation protocol for the human fungal pathogen Candida albicans. Curr. Genet. 42, 339–343 (2003).

    Article  CAS  Google Scholar 

  18. Gietz, R.D. & Schiestl, R.H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).

    Article  CAS  Google Scholar 

  19. De Backer, M.D. et al. Transformation of Candida albicans by electroporation. Yeast 15, 1609–1618 (1999).

    Article  CAS  Google Scholar 

  20. Thompson, J.R., Register, E., Curotto, J., Kurtz, M. & Kelly, R. An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast 14, 565–571 (1998).

    Article  CAS  Google Scholar 

  21. Hickman, M.A. et al. The 'obligate diploid' Candida albicans forms mating-competent haploids. Nature 494, 55–59 (2013).

    Article  CAS  Google Scholar 

  22. Gomez-Raja, J., Andaluz, E., Magee, B., Calderone, R. & Larriba, G. A single SNP, G929T (Gly310Val), determines the presence of a functional and a non-functional allele of HIS4 in Candida albicans SC5314: detection of the non-functional allele in laboratory strains. Fungal Genet. Biol. 45, 527–541 (2008).

    Article  CAS  Google Scholar 

  23. Chibana, H., Uno, J., Cho, T. & Mikami, Y. Mutation in IRO1 tightly linked with URA3 gene reduces virulence of Candida albicans. Microbiol. Immunol. 49, 937–939 (2005).

    Article  CAS  Google Scholar 

  24. Brand, A., MacCallum, D.M., Brown, A.J., Gow, N.A. & Odds, F.C. Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus. Eukaryot. Cell 3, 900–909 (2004).

    Article  CAS  Google Scholar 

  25. Reuss, O., Vik, A., Kolter, R. & Morschhauser, J. The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341, 119–127 (2004).

    Article  CAS  Google Scholar 

  26. Huxley, C., Green, E.D. & Dunham, I. Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet. 6, 236 (1990).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Berman and members of the Wang lab for critical reading of the manuscript. This work was funded by the Agency for Sciences, Technology and Research of Singapore.

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Authors and Affiliations

  1. Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore

    Guisheng Zeng, Yan-Ming Wang, Fong Yee Chan & Yue Wang

  2. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Yue Wang

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  1. Guisheng Zeng
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  2. Yan-Ming Wang
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  3. Fong Yee Chan
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  4. Yue Wang
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Contributions

G.Z. designed and performed the experiments and wrote the first draft of the manuscript. F.Y.C. contributed to plasmid construction, and Y.-M.W. helped with flow cytometry analysis. Y.W. discussed and commented on the results at all stages and revised the manuscript.

Corresponding author

Correspondence to Yue Wang.

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The authors declare no competing financial interests.

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Zeng, G., Wang, YM., Chan, F. et al. One-step targeted gene deletion in Candida albicans haploids. Nat Protoc 9, 464–473 (2014). https://doi.org/10.1038/nprot.2014.029

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