Fluorescent Staining of Arbuscular Mycorrhizal Structures Using Wheat Germ Agglutinin (WGA) and Propidium Iodide

The colonization of a host plant root by arbuscular mycorrhizal (AM) fungi is a progressive process, characterized by asynchronous hyphal growth in intercellular and intracellular spaces, leading to the coexistence of diverse intraradical structures, such as hyphae, coils, arbuscules, and vesicles. In addition, the relative abundance of intercellular and intracellular fungal structures is highly dependent on root anatomy and the combination of plant and fungal species. Lastly, more than one fungal species may colonize the same root, adding a further level of complexity. For all these reasons, detailed imaging of a large number of samples is often necessary to fully assess the developmental processes and functionality of AM symbiosis. To this aim, the use of rapid and efficient staining methods that can be used routinely is crucial. We herein present a simple protocol to obtain high detail images of both overall intraradical fungal colonization pattern and fine morphology, in AM root sections of Lotus japonicus. The procedure is based on tissue clearing, fluorescent staining of fungal cell walls with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), and the combined counterstaining of plant cell walls with propidium iodide (PI). The resulting images can be acquired using traditional or confocal fluorescence microscopes and used for qualitative and quantitative analyses of fungal colonization, of particular interest for the comparison of mycorrhizal phenotypes between different experimental conditions or genetic backgrounds.