Synchronous Induction and Microscopic Analysis of Hyphal Growth Using Laboratory Strain AB33

In nature, hyphal growth of Ustilago maydis is strictly coupled to plant infection and initiated after mating of two genetically compatible yeast cells on the plant surface. Compatibility is defined by mating type loci: the biallelic a locus mediates recognition and fusion through a pheromone/receptor system, whereas the multiallelic b locus, encoding transcription factor subunits bEast (bE) and bWest (bW), establishes and maintains the dikaryotic hypha controlled via the assembly of compatible subunits into active heterodimers after plasmogamy. The laboratory strain AB33 enables a controlled transition from yeast-like cells to monokaryotic hyphae in liquid culture without mating. This relies on a genetic trick where the expression of the compatible subunits bW2 and bE1 is under the control of a nitrate-inducible promoter, allowing synchronous hyphae induction by switching from ammonium- to nitrate-containing medium. This system allows the study of processes associated with hyphal growth independently of plant infection and permits microscopic and microfluidic observations, providing a powerful tool for addressing fundamental cell biological questions and beyond. Here, a protocol for synchronous induction of hyphal growth in liquid cultures is described, together with practical guidelines for obtaining high-quality microscopic images using formaldehyde fixation, agarose pad preparation, and mitochondrial staining with TMRE.