Complementation of a REM mutant in Ustilago maydis

Ustilago maydis is a fungal pathogen of corn (Zea mays). Its complex life cycle consists of three cell types: (1) non-pathogenic, yeast-like haploids, (2) mycelial, pathogenicdikaryons, and (3) diploid cells produced upon karyogamy within the plant. Mating and pathogenicity in U. maydis are under the control of two loci, a and b. Heterozygousity at both loci is required for fusion of cells, pathogenesis, and subsequent development of the fungus within the plant. The aim of this project was to identify genes, other than a and b, that are involved in the pathogenesis of U. maydis. To this end, mycelial haploid mutants were isolated by UV mutagenesis of a yeast-like strain. A single mutant (designated 87-18) was selected for further study due to the strength and stability of the mycelial phenotype. This mutation was named rem (repressor of mycelial phenotype). The phenotype of mutant 87-18 was remarkable because the strain grows with a filamentous morphology in both solid and liquid media. This phenotype is not demonstrated by any of the cell types of U. maydis, regardless of the condition of the a and b loci. Pathogenicity assays indicate that 87-18, which was derived from an auxotrophic strain, was not solopathogenic, but could cause disease when co-inoculated with a compatible wild-type strain. Prototrophic, mutantprogeny of this cross were not solopathogenic. To clone the gene(s) responsible for the rem phenotype,a wild-type genomic library was constructed in the cosmid vector, pJW42. Transformation of the mutant 87-18 with the cosmid library yielded six yeast-like colonies. Cosmid DNA was isolated from three of these transformants and restriction digest patterns indicated that the insert DNA in the three cosmids was highly similar. Cosmid DNA gave 100%yeast-like transformants when reintroduced into strain 87-18.Thus, these results indicate the isolation of a DNA sequence that complements the mutation of strain 87-18. This complementing DNA provides the starting material for a molecular analysis of the determination of cell morphology in U. maydis.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat