Fungal mating circuity

通过交配交换遗传信息是一个重要的生物学过程。在子囊菌真菌中，已经对酿酒酵母的交配进行了很好的研究，其中受体/G蛋白复合物激活MAP激酶模块，控制细胞周期阻滞、基因表达和形态。在相关的子囊菌白色念珠菌中，电路的关键元件与酿酒酵母模型不同。理解这些差异的逻辑是充分理解真菌交配过程的关键。我们将研究两个不同回路的区域——对白色念珠菌交配的不透明状态的要求，以及G α亚基的作用。单倍体酿酒酵母细胞天生具有交配能力，而白色念珠菌细胞需要依赖于细胞类型的表观遗传开关来获得交配能力。在实验室里，这种转换很少见，交配能力也不常见。我们已经确定了ofr1突变，该突变允许细胞绕过正常的交配需求，并切换到不透明状态，甚至作为a/α杂合子进行交配。我们将以两种方式进行后续研究——建立允许交配的ofr1突变的分子基础，以及识别和表征允许a/α杂合子交配的其他突变。我们将首先使用双突变分析来确定哪些电路是由ofr1触发的交配所必需的——突变交配信号通路（STE4）、经典的白色不透明开关机制（WOR1）和氨基葡萄糖摄取（NGT1）。为了找到调节交配的新元素，我们将在GRACE集合中识别其他突变，这些突变允许MTL杂合时切换到不透明形式。基因失活刺激不透明细胞形成的菌株将被鉴定为显示不透明细胞增强的苯氧辛染色特征。我们还将研究白色念珠菌的G蛋白回路。通常α和βγ亚基在交配过程中起相反的作用。在白色念珠菌中，我们已经证明α和βγ都是交配所必需的。我们将确定α和βγ元素是否执行交配所需的独立功能，还是协调单一功能。为了区分这些可能性，我们将激活途径中的各种下游元件，并确定激活元件是否抑制α和β突变体中的一个或两个。我们还将评估受信息素途径影响的其他细胞过程，如白色不透明开关，以评估α和β的作用是否不同。

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.