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Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red

Nature Protocols volume 1, pages 2253–2256 (2006)Cite this article

Abstract

The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the fungal cell wall. Here we present a simple approach for detecting these genes. The method is based on the observation that cell wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the cell wall. CFW-based and CR-based susceptibility assays identify cell wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4–6 h (excluding the incubation time required for fungal growth).

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Figure 1: The chemical structures of CFW (a) and CR (b).
Figure 2: Susceptibility of A.niger cell wall mutants to CFW (200 μg ml−1) or CR (150 μg ml−1).

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Acknowledgements

We thank A. Franken and M. Arentshorst for their excellent technical assistance, and S. Langeveld for her help with preparing the figures. F.K. acknowledges financial support from the European Union Specific Targeted Research or Innovation Projects (STREP) FungWall program (1540260).

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Authors and Affiliations

  1. Institute of Biology Leiden, Leiden University, Molecular Microbiology, Wassenaarseweg 64, AL Leiden, 2333, The Netherlands

    Arthur F J Ram

  2. Swammerdam Institute for Life Sciences, University of Amsterdam, BioCentrum Amsterdam, Nieuwe Achtergracht 166, WV Amsterdam, 1018, The Netherlands

    Frans M Klis

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Correspondence to Arthur F J Ram.

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Ram, A., Klis, F. Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat Protoc 1, 2253–2256 (2006). https://doi.org/10.1038/nprot.2006.397

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