Microscopic Observation of Ustilago maydis Mating Interactions

Snetselaar, K. M. 1993. Microscopic observation of Ustilago maydis mating interactions. Experimental Mycology 17, 345-355. Ustilago maydis sporidia grown in liquid medium were concentrated by centrifugation, resuspended in sterile water, and incubated as drop cultures in petri dishes. Haploid and diploid strains with various combinations of mating-type alleles were incubated individually and in mixed cultures for up to 12 h, during which time sporidia were observed and compared with DIC and epifluorescence optics. When paired haploid strains carried unlike a alleles, sporidia formed mating hyphae and conjugated regardless of the b alleles they carded. If the strains also carded unlike b alleles, mating was followed by formation of rapidly growing dikaryotic hyphae. If sporidia with unlike a alleles and identical b alleles were incubated together, multiple fusions were common, and the few hyphae that formed grew slowly and were often multinucleate. When unlike a and disrupted b alleles were involved, more multiple fusions and fewer emergent hyphae resulted. When a diploid strain with compatible alleles at both loci was incubated alone, sporidia formed straight uninucleate hyphae without mating. A diploid strain that was isogenic except for a disrupted b1 locus mated indiscriminately, but straight, dikaryotic hyphae formed only when the mating partner carried an intact b1 allele. These results demonstrated that although the plate-mating assays in common use detect the vigorous filamentous growth associated with pathogenicity, they do not detect all kinds of filaments, nor do they assay mating per se. The correlation of mating cessation with the presence of compatible b alleles in a common cytoplasm implicates the active b product in negative regulation of mating-specific genes. The methods described here could be used to further define the roles of the a and b gene products in controlling mating behavior and subsequent development.