Wheat Germ Agglutinin-Alexa Fuor488/Propidium Iodine Costaining and Fluorescence Microscopic Imaging of Ustilago maydis Infected Maize Anthers.

Ustilago maydis infects all aerial maize tissues, forming tumors and altering host development. However, most published methods focus on seedling leaf infections in order to understand the interaction between maize and U. maydis. Cytological analysis, particularly fluorescence microscopy, is vital not only for examining fungal infection progression, but also for understanding the host's response. Thus, a detailed protocol is presented here for dissecting U. maydis-infected maize anthers, followed by costaining with wheat germ agglutinin-Alexa Fluor 488 (WGA-AF488) and propidium iodide (PI). WGA-AF488 specifically labels fungal cell walls, while PI stains all cells. This robust method enables clear cytological analysis of fungal structures and the status of the host cell, overcoming imaging challenges posed by thick maize tissues.