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Rapid screening method of Saccharomyces cerevisiae mutants using calcofluor white and aniline blue

  • Bacterial and Fungal Pathogenesis - Short Communication
  • Published:
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Abstract

Fungal cell walls are composed of polysaccharide scaffold that changes in response to environment. The structure and biosynthesis of the wall are unique to fungi, with plant and mammalian immune systems evolved to recognize wall components. Additionally, the enzymes that assemble fungal cell wall components are excellent targets for antifungal chemotherapies and fungicides. Understanding changes in the cell wall are important for fundamental understanding of cell wall dynamics and for drug development. Here we describe a screening technique to monitor the gross morphological changes of two key cell wall polysaccharides of chitin and β-1,3-glucan combined with polymerase chain reaction (PCR) genotyping. Changes in chitin and β-1,3-glucan were detected microscopically by using the dyes calcofluor white and aniline blue. Combining PCR and fluorescence microscopy, as a quick and easy screening technique, confirmed both the phenotype and genotype of the wild-type, h chitin synthase mutants (chs1Δ and chs3Δ) and one β-1,3-glucan synthase mutant fks2Δ from Saccharomyces cerevisiae knockout library. This combined screening method highlighted that the fks1Δ strain obtained commercially was in fact not FKS1 deletion strain, and instead had both wild-type genotype and phenotype. A new β-1,3-glucan synthase knockout fks1::URA3 strain was created. Fluorescence microscopy confirmed its phenotype revealing that the chitin and the new β-1,3-glucan profiles were elevated in the mother cells and in the emerging buds respectively in the fks1Δ cell walls. This combination of PCR with fluorescence microscopy is a quick and easy screening method to determine and verify morphological changes in the S. cerevisiae cell wall.

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Acknowledgements

Special acknowledgement is given to the BioImaging Platform at La Trobe University and Dr. Peter Lock for the helpful advice and training on various microscopes. The authors would like to thank Dr. Ana Traven, Monash University, who supplied the pPS293 plasmid, containing the URA3 gene, and Dr. Mark Bleackley, La Trobe University, for the guidance and advice for genotyping the mutants. Thank you to Prof. Marilyn Anderson and Dr. Michael Walker for their support, advice, and critical reading of the manuscript.

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Author notes

  1. Francine Perrine-Walker

    Present address: The University of Sydney Institute of Agriculture, 1 Central Avenue, Australian Technology Park, Eveleigh, NSW, 2015, Australia

  2. Francine Perrine-Walker

    Present address: School of Life and Environmental Sciences, The University of Sydney, Life Earth and Environmental Sciences Building (F22), Sydney, NSW, 2006, Australia

  3. Jennifer Payne

    Present address: Department of Biochemistry and Molecular Biology, The Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, 3800, Australia

  4. Jennifer Payne

    Present address: EMBL Australia, Monash University, Clayton, Victoria, 3800, Australia

Authors and Affiliations

  1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia

    Francine Perrine-Walker & Jennifer Payne

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  1. Francine Perrine-Walker
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Contributions

FP-W and JP conceived and designed the research. FP-W and JP conducted the experiments. JP designed the fks1Δ::URA3 mutant and contributed to the PCR/molecular analyses, and FP-W contributed to the microscopy and to image analyses in this study. FP-W wrote the first draft manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Francine Perrine-Walker.

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Perrine-Walker, F., Payne, J. Rapid screening method of Saccharomyces cerevisiae mutants using calcofluor white and aniline blue. Braz J Microbiol 52, 1077–1086 (2021). https://doi.org/10.1007/s42770-021-00515-1

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