隱球菌CRK1基因功能特性之分析研究

Abstract

隱球菌(Cryptococcus neoformans)，為一伺機性人類病原真菌，在分類地位上屬於擔子菌，具有酵母菌與菌絲體兩種形態。隱球菌存在MATa 與 MATα兩種交配型細胞，在氮素源缺乏的環境下，MATa 與 MATα 細胞產生接合管進行細胞融合，並產生雙核生殖菌絲，擔子柄及擔孢子完成有性生活史。本實驗室過去的研究發現，隱球菌藉由Cwc1與Cwc2兩蛋白質形成之複合體，在光照環境下，感應藍光抑制雙核生殖菌絲的生長。為了進一步瞭解藍光抑制雙核菌絲生長的機制，我們利用農桿菌轉殖系統(Agrobacterium-mediated insertional mutagenesis)，針對CWC1過度表現株進行逢機突變，並篩選於持續光照環境下，表現型由雙核生殖菌絲完全被抑制回復生長雙核生殖菌絲的突變株，藉以找出與藍光調控機制有關的下游基因。其中一株回覆突變株DJ22，進一步分析發現T-DNA插入破壞隱球菌CRK1基因。啤酒酵母菌(Saccharomyces cerevisiae) IME2基因及玉米黑穗病菌(Ustilago maydis) crk1基因為隱球菌CRK1之同源基因。Ime2 是一個減數分裂專一性蛋白質，具有高度保守性之Ser/Thr激酶區與TXY雙重磷酸化位置。在我們研究中發現，crk1突變株細胞融合效率提高，產生之雙核生殖菌絲多於野生株，且其雙核菌絲、擔子柄(basidia)、擔孢子(basidiaspore)的發生皆早於野生株。過度表現CRK1則會抑制雙核生殖菌絲的形成。另一方面，crk1突變株與CRK1過度表現株皆會抑制monokaryotic fruiting菌絲的發生。由本研究結果得知，CRK1在C. neoformans有性生殖過程中，扮演一負調控因子的角色。

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.