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Ustilago maydis is a dimorphic basidiomycete and the causal agent of corn smut disease. It serves as a genetic model for understanding dimorphism, pathogenicity, and mating response in filamentous fungi. Previous studies indicated the importance of regulated cAMP-dependent protein kinase A (PKA) for filamentous growth and pathogenicity in U. maydis. The roles of two protein phosphatases that potentially act antagonistically to PKA were assessed. A reverse genetics approach to mutate the catalytic subunits of calcineurin (CN, protein phosphatase [PP]2B) and PP2A in U. maydis was employed. A mutation in the CN catalytic subunit ucn1 caused a dramatic multiple-budding phenotype and mating between two ucn1 mutants was severely reduced. The pathogenicity of ucn1 mutant strains was also severely reduced, even in a solopathogenic haploid strain. Importantly, mutations disrupting protein phosphorylation by PKA were epistatic to ucn1 mutation, indicating a major role of ucn1 as a PKA antagonistic phosphatase. Genetic and inhibitor studies indicated that the U. maydis PP2A catalytic subunit gene (upa2) was essential.