质膜微区蛋白Remorins调节果实抗病性应答的机理研究

Ripening, aging and deterioration of fruits are always concurrent with structural variations and increase in permeability of plasma membrane, membrane microdomains are basic units for regulating structural compartmentalization and functional specialization of plasma membrane, while functional proteins resided in membrane microdomains are important components for the interaction between cells and pathogenic microorganisms. Remorins are above all the first definitely identified proteins in membrane microdomain, but the underlying molecular and cytological mechanisms for the responses of fruits mediated by Remorins towards pathogenic microorganisms are still largely unclear. The present study is proposed on the basis of our previous work by combining over-expression, CRISPR-Cas9, RNAi, fluorescence label and real-time tracking and other techniques. Emphases will be placed on the studies in three major aspects: (1) to investigate changes in disease-resistant responses of fruits in overexpression lines, knock-out and RNAi lines of REMORINs; (2) to analyze the variations in subcellular localization pattern and constitutive recycling mechanisms of Remorins; (3) to investigate the variations in endocytic events and intracellular sorting of Remorins in response to pathogen infection, finally reveal the spatio-temporal specificity for the responses to pathogen infection, elucidate the mechanisms for signal priming and transduction of Remorins based on protein dynamics in membrane microdomains, and provide new clues for studying the mechanisms for postharvest disease-resistant responses of fruits.

果实衰老和劣变的过程伴随着质膜透性增加和结构变化，质膜微区(membrane microdomain)是调节质膜功能特化的基本单位，而质膜微区蛋白则是细胞与病原菌互作过程中行使相关功能的重要组分。Remorins是植物细胞中最早鉴定的膜微区蛋白，但其在果实抗病性应答过程中的分子细胞学机制还有待研究。本项目拟在前期工作基础上，综合运用基因过表达、CRISPR-Cas9和基因沉默技术、荧光标记和实时追踪等方法，开展如下研究：（1）分析Remorins过表达、基因敲除和RNAi株系中果实抗病性应答反应的变化；（2）阐明Remorins在亚细胞水平上的定位模式、互作因子和细胞内循环途径；（3）探明Remorins响应病原菌侵染后内吞及胞内分选途径的变化，解析其对病原菌侵染响应的时空特异性，揭示质膜微区蛋白Remorins基于膜微区蛋白动态的信号激发机制，为研究果实采后抗病性应答机理提供重要线索。

果实衰老和品质劣变的过程伴随着质膜透性增加和结构变化，质膜微区(membrane microdomain)是调节质膜功能特化的基本单位，而质膜微区蛋白则是细胞与病原菌互作过程中行使相关功能的重要组分。Remorins是植物细胞中最早鉴定的膜微区蛋白，但其调节果实成熟进程和抗性应答过程中的分子细胞学机制还有待研究。本项目在前期工作基础上，运用基因过表达和基因沉默技术、生理指标测定、生物化学分析等方法，取得了如下的实验结果：(1) 转录因子RIN直接调控SlREM1表达；(2) SlREM1蛋白水平在果实发育前期上升，而成熟开始时则快速下降，与转录水平的变化存在一定差异，表明SlREM1可能存在转录后或翻译后修饰；(3) SlREM1定位于细胞质膜；(4) 过表达SlREM1加速番茄果实成熟，促进番茄红素积累和乙烯释放，提高果实抗性；(5) SlREM1通过与乙烯合成途径关键酶SAM、ACS2和ACO1互作调节乙烯合成；(6) 过表达SlREM1诱导烟草叶片细胞死亡，SlREM1 173-186段氨基酸序列对SlREM1诱导细胞死亡是必需的；(7) SlREM1可能通过CRTD、BCPL和NCBP等细胞死亡相关因子，诱导活性氧爆发，最终导致烟草细胞死亡。上述研究结果揭示质膜微区蛋白Remorins基于膜微区蛋白动态的信号激发机制，为研究果实成熟、采后品质保持和抗性应答机理提供重要线索。