The role of calcineurin and protein phosphatase 2A in morphology, mating, pathogenicity and cell viability in ustilago maydis
Egan, John Duick
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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 have elucidated signal transduction pathways involving cAMP-dependent protein kinase A (PKA) and mitogen-activated protein (MAP) kinases as being important for normal cellular function in U. maydis. Although most of the target substrates of these pathways are unidentified, it appears that the phosphorylation states of these target substrates determine mating, filamentous growth and pathogenicity. The purpose of this study was to determine the roles of two protein phosphatases that could be involved in the PKA and MAPK pathways in U. maydis; a reverse genetics approach was taken to identify the catalytic subunits of calcineurin (ucn1) and protein phosphatase 2A (upa2). Strains that are mutant in ucn1 have a multiple budding phenotype with their cell cluster size inversely dependent on the inoculum density. Mating between ucn1 mutants and wild-type strains was reduced, and mating between two ucn1 mutants was severely reduced. This reduction in mating can be attributed to ucn1 mutants’ inability to upregulate mfa1 during mating induction. Through pathogenicity assays, calcineurin was identified as a critical virulence factor within maize plants. A synergistic phenotype was observed in the uac1 ucn1 double mutant; both filamentous and multiple budding. The ucn1 multiple budding mutant phenotype was determined to be epistatic to the filamentous solopathogenic SG200 phenotype. Cyclosporine A was successful in generating the ucn1 mutant phenotype in wild-type strains and a distinct pattern of bud emergence was observed during calcineurin inhibition. A gene disruption in the U. maydis PP2A (upa2) catalytic subunit was not obtained, even after multiple attempts. Consistent with this result, experiments with okadaic acid, a potent inhibitor of PP2A, stopped the growth of U. maydis cells at 2nM concentration, suggesting the probable lethality of a PP2A null mutation. Protein phosphorylation patterns between U. maydis wild-type and mutant strains could not be determined using a hexahistidine-tagged Prf1 transcription factor, or through western blotting of total protein lysates using antiphosphoserine/ threonine antibodies.