Visualization of Growth and Morphology of Fungal Hyphae in planta Using WGA-AF488 and Propidium Iodide Co-staining

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).

Microscopy

Fungus

Propodium iodide

Confocal

Maize