植物类受体激酶FLS2内吞途径的研究

The endocytosis in plant cells maintains the uptake of molecules into cells and the turn over of intracellular protein trafficking, therefore it is essential for the plant development. However, the endocytic pathways and regulatory mechanism remain to be further investigated. The receptor-like kinase FLAGELLIN -SENSITIVE2 (FLS2), which internalizes specifically only under the ligand induction, is one of ideal proteins for analysis of plant endocytosis. In the present study, we will investigate the endocytic pathways of receptor-like kinase FLS2 in Arabidopsis as well as its molecular mechanism of internalization by means of dynamic analysis in the living cells together with genetic approaches. First, we will analyze the distribution patterns and dynamic characteristics of FLS2 before and after the ligand induction in plasma membrane. Then, we will determine the co-localization between FLS2 and endocytosis-associated proteins clathrin or flotillin in plasma membrane. Finally, we will further explore the function of endocytosis-associated proteins in the internalization of FLS2 into cytoplasm. In the course of the investigation, some new methods and techniques including variable-angle total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy will be used in this study with the aim of uncovering the function of clathrin and lipid raft protein flotillin in the endocytosis of FLS2 receptor and revealing the mode of FLS2 endocytosis under the ligand induction. Surely, the results of this study will lay foundation for the further investigation of molecular mechanism for plant endocytosis.

植物内吞作用不仅维持着胞外物质吸收和胞内蛋白运输的平衡，而且在植株的生长发育中起重要作用。然而，有关植物细胞的内吞方式和分子机制尚缺乏深入的研究。植物类受体激酶FLS2经配体刺激特异性地进行内吞，是研究植物细胞内吞作用理想的目标蛋白。本项目针对前人工作的不足，我们将以拟南芥为材料，着重展开三方面的研究：(1)观察类受体激酶FLS2经配体刺激前后在质膜上的分布模式并测定相关动力学参数；(2)研究类受体激酶FLS2与内吞相关蛋白clathrin、flotillin在质膜上的共定位；(3)揭示内吞相关蛋白在类受体激酶FLS2内吞进入胞质中的作用。在本项目研究中，拟采用包括可变角度的全内反射荧光显微术、荧光相关光谱、荧光交叉相关光谱等一些新方法、新技术，借助这些新技术和新方法，旨在揭示笼型蛋白和脂筏在FLS2内吞过程中的作用，从而阐明FLS2的内吞模式，为进一步研究植物内吞作用的分子机理奠定基础。

植物虽不像动物那样具有免疫细胞，但在与病原菌长期共生过程中，进化出了一套极其复杂精细的感受信号的体系，来特异地识别进入宿主细胞的各种信号，并启动相应的防御反应。植物利用细胞表面模式识别受体（Pattern-recognition receptors, PRRs）来感知病原相关分子模式（PAMPs，Pathogen-associated molecular patterns），启动PAMPs触发的免疫（PAMPs-triggered immunity, PTI），是植物感知病原菌入侵最常见防御反应之一。FLS2蛋白是一个富含亮氨酸重复序列的受体激酶，通过识别细菌鞭毛蛋白激活植物的防御反应，在PAMPs引起的免疫反应中发挥至关重要的作用。. 本研究首先克隆了拟南芥FLS2基因，构建了内源启动子驱动的GFP标记的FLS2融合表达载体，并获得FLS2-GFP转基因拟南芥植株。通过功能回补实验证明，FLS2-GFP融合蛋白具有生物学功能。在此基础上，本论文利用可变角度全内反射荧光显微术（Variable angle-total internal reflection fluorescence microscopy，VA-TIRFM）、荧光互相关光谱（fluorescence correlation spectroscopy，FCS）等技术，并结合生物化学的方法，对拟南芥FLS2-GFP在质膜上的动力学特征、胞吞途径及胞吞调控机制进行了比较深入的研究。研究发现：(1) FLS2在细胞质膜上的寡聚状态和定位是高度动态的；(2) FLS2蛋白存在多种胞吞途径，其中clathrin介导的胞吞途径在FLS2-GFP的胞吞过程中起到重要作用，并且膜微区也参与了质膜上FLS2-GFP的胞吞；(3) 磷酸化修饰参与调控FLS2的胞吞转运。本研究在单分子水平上探讨了FLS2蛋白的分布、运动、聚合和胞吞之间的联系，并揭示了磷酸化修饰参与调控FLS2的动态和胞吞，为今后开展FLS2胞吞调控机制的研究提供了新思路。