Fungal mating circuity

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The exchange of genetic information through mating is a central biological process. In the ascomycete fungi, mating has been well studied in S. cerevisiae where a receptor/G protein complex activates a MAP kinase module controlling cell cycle arrest, gene expression, and morphology. In the related ascomycete Candida albicans, key elements of the circuitry differ from the S. cerevisiae model. Comprehending the logic of these differences is critical to a full understanding of the fungal mating process. We will investigate two areas where the circuits are different - the requirement for the opaque state for C. albicans mating, and the role of the G alpha subunit. Haploid S. cerevisiae cells are inherently mating competent, while C. albicans cells require a cell-type-dependent epigenetic switch to mating-competence. In the laboratory this switch is rare, and mating competence infrequent. We have identified the ofr1 mutation that allows the cells to bypass normal requirements for mating, and switch to the opaque state and mate even as a/α heterozygotes. We will follow up in two ways – establish the molecular basis for the ofr1 mutation allowing mating, and identify and characterize other mutations that permit a/α heterozygotes to mate. We will first use double mutant analysis to establish which circuits are required for ofr1-triggered mating - mutating the mating signalling pathway (STE4), the classic white-opaque switching machinery (WOR1) and glucosamine uptake (NGT1). To find new elements regulating mating we will identify other mutations in the GRACE collection allowing switching to the opaque form while MTL heterozygous. Strains where gene inactivation stimulates opaque cell formation will be identified as showing the enhanced phloxine staining characteristic of opaque cells. We will also investigate G protein circuitry of C. albicans. Normally the α and βγ subunits play opposing roles in the mating process. In C. albicans we have shown that both α and βγ are required for mating. We will determine if the α and βγ elements perform independent functions required for mating, or coordinate a single function. To distinguish these possibilities we will activate a variety of downstream elements in the pathway, and establish if the activated element suppresses either or both of the α and β mutants. We will also assess other cellular process influenced by the pheromone pathway such as the white-opaque switch to assess whether the roles of α and β are different.