Abstract
In this protocol we describe a cell wall stress assay for the fungal pathogen F. oxysporum, based on exposure to the two anionic dyes Calcofluor White (CFW) and Congo Red (CR). Both compounds have been used to exert stress upon the fungal cell wall in vitro (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Leach et al., 2012; Heilmann et al., 2013; Garcia et al., 2015). CFW perturbs chitin assembly, whereas CR interferes with β-glucan synthesis, resulting in cell wall-weakening and activation of the cell wall stress response (Ram and Klis, 2006; Kopecka and Gabriel, 1992; Roncero and Duran, 1985). Presumably, the signaling pathways and cell wall changes associated with this response reflect cell wall homeostasis during normal growth as well as cell wall remodeling events in response to stresses encountered during the fungus-host interactions. The conditions for preparation of CFW and CR culture medium specified in this protocol are based on the paper by Ram and Klis entitled “Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red”, published in Nature protocols (Ram and Klis, 2006). This paper established the optimum conditions for preparation of CFW and CR stock solutions and suggested maintaining the culture medium at a constant pH to avoid acidification, protonation and precipitation of these dyes. This cell wall stress assay has been widely used in our group for the characterization of F. oxysporum mutants in mitogen activated protein kinase (MAPK) signaling pathway genes involved in cell wall integrity (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Turra et al., 2014).
Materials and Reagents
Note: CFW and CR are hazardous and potentially carcinogenic so caution must be taken to avoid skin contact or inhalation of these compounds.
Equipment
Note: No other special equipment is required.
Software
Procedure
A major requirement for this protocol is to experimentally establish the optimum concentration of CFW or CR to be used. This may be influenced by several parameters, including size of the inoculum, genetic background of the strains and composition of the growth medium. The wild type strain used in our laboratory was Fusarium oxysporum f.sp. lycopersici (strain 4287/FGSC 9935). Appropriate CFW and CR concentration should be sublethal for the wild-type or reference strain used in the experiment. We typically define this parameter by inoculating serial dilutions of conidia in the form of spots on plates containing CFW and CR, with concentrations in the range of 10-200 μg/ml of CFW and 5-600 μg/ml of CR.
Representative data
Figure 1 shows the sensitivity of several F. oxysporum MAPK pathway mutants to CFW and CR (Perez-Nadales and Di Pietro, 2011). In F. oxysporum, the Fmk1 MAPK pathway is essential for plant infection (Di Pietro et al., 2001). This experiment revealed that Msb2, a signaling mucin receptor functioning upstream of Fmk1 and also the Fmk1 MAPK are required for cell wall integrity in F. oxysporum under these conditions. Figure 1. Cell wall stress assay for F. oxysporum. A. Plates containing YPD medium or YPD medium supplemented with CR or CFW were spot-inoculated with the indicated strains and amounts of microconidia, incubated for three days at 28 °C and photographed. B. Colony diameter of the indicated media was measured at day 3 post-inoculation and plotted relative to the wild-type strain (100%). Error bars represent standard deviations calculated from five independent plates. Values with the same letter are not significantly different according to Mann–Whitney test (P ≤ 0.05).
Notes
Recipes
Acknowledgments
This research was supported by the SIGNALPATH Marie Curie Research Training Network (MRTN-CT-2005-019277) and by grants BIO2008-04479-E, EUI2009-03942, and BIO2010-15505 from the Spanish Ministerio de Ciencia e Innovación.
References
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