| Summary: | 碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 96 === Cryptococcus neoformans is a heterothallic basidiomycete and grows vegetatively as yeast. Under nitrogen limitation conditions, strains of opposite mating types, MATa and MATα, produce conjugation tubes and fuse to form sexual dikaryotic filaments with clamp connections. Our previous studies demonstrated that C. neoformans Cwc1 and Cwc2 are two central photoregulators which form complex to inhibit the production of sexual filaments upon light stimulation. To reveal the detailed regulatory mechanisms, Agrobacterium-mediated insertional mutagenesis screen was conducted to identify the components interacting with or functioning downstream of the Cwc1/Cwc2 complex in the pathway. Upon light irradiation, T-DNA mutants restoring different extents of filamentation under the CWC1 overexpression background were identified. In this study, one suppressor mutant, DJ22, was characterized and T-DNA was found to integrate at the C. neoformans CRK1 gene, a homologue of Saccharomyces cerevisiae IME2 and Ustilago maydis crk1. Ime2 is a meiosis-specific gene with the conserved Ser/Thr kinase domain and the significant TXY dual phosphorylation site. Consistent with the findings of other suppressors in our screen, C. neoformans Crk1 played negative role in the mating process. Mating efficiency was increased in the crk1 mutants. Dikaryotic filaments, basidia, and basidiospores produced earlier in the crosses involved the crk1 mutants. Elevation of the CRK1 mRNA level inhibited sexual differentiation. On the other hand, monokaryotic fruiting was defective both in the MATα crk1 mutant and CRK1 overexpression strains. Our study demonstrated that mating process was negatively regulated by the CRK1 gene in C. neoformans.
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