COT1作为一种保守的NDR家族蛋白激酶，参与多种真核生物细胞形态分化和极性的调控。我们前期研究结果显示粗糙脉孢菌COT1功能受损会影响麦角甾醇和鞘磷脂的合成，而麦角甾醇的合成受到激酶STK17和转录因子ADS4、CCG8和CSP1的调节。但COT1与真菌细胞膜麦角甾醇、鞘磷脂合成组分（包括调节因子）之间的联系仍不清楚。本研究拟通过中以两个实验的互作合作，采用转录组和蛋白质组分析的方法并辅以细胞膜组分的化学分析，明确COT1途径对麦角甾醇和鞘磷脂合成的影响程度，确定COT1和麦角甾醇、鞘磷脂合成组分（包括调节因子）之间的遗传和物理相互作用，以及COT1和相关信号通路之间的相关作用。本项目将鉴定出COT1影响的下游结构组分，加深人们对NDR信号途径、麦角甾醇/鞘磷脂合成途径及二者之间联系的认识，同时也为利用这些信息改变真菌的生长提供了新的思路。

COT1 is a conserved NDR (nuclear Dbf2-related) protein kinase, involved in determining cell shape and polarity in a variety of eukaryotes. Mutating COT1 results in a pleiotropic effect that includes cell wall abnormalities accompanied by increased sensitivity to drugs targeting synthesis of ergosterols and sphingolipids, critical plasma membrane components. Nonetheless, the link between COT1 function and the fungal plasma membrane has not been thoroughly addressed. Our preliminary data show that regulation/production of ergosterols and sphingolipids are affected by impaired function of COT1 in Neurospora crassa. Furthermore, we have found that ergosterol biosynthesis is regulated by a kinase (STK17) and three transcription factors (ADS4, CCG8 and CSP1). Changes in cot-1 transcription under azole stress and changes in the sphingolipid profile of cot-1 mutants, as well as identification of novel factors regulating ergosterol biosynthesis is the basis of our main objective: Determine the functional link between COT1 and ergosterols/sphingolipids as membrane components in N. crassa. Specifically, we will (i) Determine to what extent ergosterol and sphingolipid biosynthesis are regulated/ affected by the cot-1 pathway and (ii) Establish the mechanistic link between the cot-1 pathway and membrane integrity in N. crassa. We will employ transcriptional and proteomic analyses while utilizing the amenability of N. crassa to classical and molecular genetics. We will determine the genetic and physical (protein-protein) interactions between COT1 and ergosterol/sphingolipid synthesis components (including the above mentioned regulators, but also the structural genes involved, such as the ERG-encoding genes and inositol-phosphorylceramide synthase) and determine the hierarchy and interactions between COT1 and the relevant pathways. .These studies will be complemented by chemical analyses of the membrane components. The significance and innovation of this proposal rests in identification of the downstream structural components (with emphasis on the membrane component of the hyphal cell) affected by cot-1 and elucidation of the mechanistic interaction between a key regulator of hyphal cell development/integrity and the ergosterol/sphingolipid pathways in the fungus. Regardless of the nature of interactions to be revealed, this study will enhance our fundamental understanding of NDR and ERG/sphingolipid pathways, the links between them and will also provide novel ideas for utilizing the information obtained for intervention in fungal growth.

NDR激酶COT-1在真核生物中高度保守，具有调控极性生长等多种细胞学功能。粗糙脉孢菌细胞壁的合成受到COT-1的调控，而与细胞壁紧密相连的细胞膜稳态的调节和COT-1有无联系并不是很清楚。通过遗传分析、基因表达研究、生化分析等手段，我们证明了COT-1和细胞膜麦角甾醇的合成及调控存在着联系。一些重要的元件参与麦角甾醇合成的调控。本项目深入解析了转录激活因子ADS-4和转录抑制子CSP-1在粗糙脉孢菌对唑类药物胁迫响应的调控作用，并证明其调控多个麦角甾醇合成基因的表达。遗传分析表明，COT-1和麦角甾醇合成调控因子CSP-1存在着遗传相互作用，并协同调控唑类药物敏感性和麦角甾醇的合成。RNA-seq分析进一步证实了COT-1和CSP-1之间的协同调控作用，并表明它们协同调控着细胞膜相关的过程。因此，本项目明确了COT-1和细胞膜稳态调节之间的联系，加深了COT-1对细胞生长调控作用的认识。为了深入解析COT-1的调控机制，我们通过蛋白质组和磷酸蛋白质组分析，发现COT-1通过影响信号转导、转录、转录后修饰等过程调控多个生物学功能。通过突变株表型分析，我们找到了三个COT-1下游效应蛋白的候选，为后续的实验提供了便利。