Abstract
Fungal pathogens colonizing plants show a varying degree of symptoms. Microscopy techniques have been used to study the infection and proliferation of fungal hyphae inside the host. One of the best optimized and commonly used method is the co-staining with Wheat Germ Agglutinin- Alexa Fluor 488 conjugate (WGA-AF488) and propidium iodide (PI), which stains fungal hyphae and the plant cell wall in contrasting shades. This technique is widely used to characterize the various behaviors of fungal hyphae, e.g., in fungal knockout mutants being attenuated during differential stages of host colonization. We describe the protocol for sample preparation of WGA-AF488– PI staining of infected plant tissue. Here, we have used an infected sample with the basidiomycetous smut fungus Ustilago maydis that infects its host plant maize (Zea mays L.) and Ustilago hordei that infects barley (Hordeum vulgare L.). This protocol helps to understand growth, biomass and morphology of fungus in planta by confocal laser scanning microscopy (Doehlemann et al., 2011; Redkar et al., 2015).
Keywords: Microscopy, Fungus, Propodium iodide, Confocal, Maize
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Data processing can be done by using the Leica Image Analysis Software in the SP8 confocal microscopy or with the freely available ImageJ.
Recipes
Acknowledgments
Our work is supported by the Cluster of Excellence on Plant Science (CEPLAS), the German Research foundation (DFG) and the European Research Council (ERC). AR acknowledges support by EMBO Long Term Fellowship (ALTF-842-2015), co-funded by the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Curie Actions. The protocol is adapted from Doehlemann et al. (2011) and Redkar et al. (2015). The authors declare no conflicts of interest exists.
References
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