棉花质外体防御蛋白GbCRR1的功能研究

Apoplast, the compartment of plant cell outside the plasma membrane, serves as the first line of defense against microbe invasion, and plays a vital role in plant immunity response. In previous experiments, the root apoplast secretome of the Verticillium wilt-resistant island cotton (Gossprium barbadense cv Hai) was analyzed through Two-dimensional differential gel electrophoresis (2D-DIGE) and Mass spectrometry (MALDI-TOF/TOF). In total, 49 differentially expressed proteins were identified from Verticillium dahliae-infected cotton root. Among them, GbCRR1, encoding a cysteine-rich protein, preferentially expressed in cotton roots, and increased in abundance in response to V. dahilae challenge. Subcellular localization examinations suggested GbCRR1-EGFP florescence was localized to apoplast and cell membrane, indicating that it could be secreted into apoplastic space upon V. dahilae infection. Ectopic expression of GbCRR1 enhanced the tolerance to P. parasitica in transgenic tobacco plants, which suggests that GbCRR1 overexpression could confer broad-spectrum disease resistance. When GbCRR1 was silenced by VIGS (Virus-induced Gene Silence) method, the deteriorated symptoms of V. dahilae infection were observed in cotton plants, which supports that GbCRR1 might be an important regulator in the cotton resistance to Verticillium wilt, however its molecular mechanism remains intangible. To decipher the mystery of GbCRR1-meidated defense response to V. dahilae, biochemistry, molecular and cellular biology, and transgenic approaches will be employed on the cotton-Verticillium system, and ultimately the GbCRR1’s working model in adaption to V. dahilae infection could be postulated based on the identification of its physiological roles. Our results is expecting to, on one hand, contribute the better understanding on the apoplastic immunity in cotton roots, and to bring more evidence on cotton-Verticillium interaction; on the other hand, GbCRR1, as promising candidate gene, could benefit the molecular breeding of new cotton cultivar with verticillium-resistance.

质外体是植物抵御病原菌侵染的第一道屏障，在免疫反应中发挥着重要作用。前期根分泌蛋白组研究分离到一个半胱氨酸富集的棉花蛋白GbCRR1，在棉花根部特异表达，且受大丽轮枝菌侵染的诱导。抑制GbCRR1表达可显著降低棉花对黄萎病的耐受性，而烟草异源表达GbCRR1可提高烟草对黑胫病的抗性，推测GbCRR1可能是个广谱性的抗病因子，但其详细的分子机制尚不清楚。本研究拟以棉花-大丽轮枝菌为研究系统，通过生物化学、分子生物学和细胞生物学以及转基因等方法，深入分析GbCRR1介导的防卫反应在棉花应答大丽轮枝菌侵染过程中的生理功能及作用机制。相关研究结果不仅可为解析质外体组分在棉花防御黄萎病菌侵染的过程中所发挥的作用提供理论依据，充实棉花-黄萎病菌互作机制的研究，同时也可为棉花抗黄萎病分子育种提供候选目标基因。

本研究以棉花-大丽轮枝菌为研究系统，通过生物化学、分子生物学和细胞生物学以及转基因等方法，深入分析GbCRR1介导的防卫反应在棉花应答大丽轮枝菌侵染过程中的生理功能及作用机制。. 质外体作为植物宿主和入侵微生物之间的第一屏障，在抵御外源病害侵入过程中具有重要的作用。因此，植物与病原体相互作用的研究越来越集中在质外体免疫上。本研究以棉花(gossypium sp)根组织细胞质外体为材料，在植物与病原真菌相互作用的过程中，发现了3种蛋白质的大丽轮枝菌。在这些蛋白质中，包含棉花寄主细胞分泌的几丁质酶28(Chi28)和富含胱氨酸的重复蛋白1(Cys-rich repeat protein 1，CRR1)，以及病原菌则释放的蛋白酶VdSSEP1。生化分析表明，大丽轮枝菌分泌的Ser蛋白酶1（VdSSEP1）能水解Chi28，但是CRR1能保护 Chi28不被VdSSEP1分解。根据体外实验结果，CRR1与Chi28在酵母和植物细胞中可以相互作用，减弱了植物细胞质外体受病原菌侵染时Chi28水平的下降。棉花中对CRR1和Chi28进行基因敲低（Knockdown），转基因棉花表现出对大丽轮枝菌侵染的敏感性增加，而过表达CRR1基因的植株增强了对大丽轮枝菌的抗性、对真菌葡萄灰霉病菌和卵菌烟草黑胫病菌的抗性。相对应的，大丽轮枝菌VdSSEP1基因敲除菌株缺失了真菌的致病性。. 项目提供了棉花质外体免疫中多因子互作的证据，为解析质外体组分在棉花防御黄萎病菌侵染的过程中所发挥的作用提供了理论依据，也可为棉花抗黄萎病分子育种提供候选目标基因。