ACLSV外壳蛋白与梨两种E3泛素连接酶互作及亚细胞定位
Interaction Between the Coat Protein of Apple Chlorotic Leaf Spot Virus and Two E3 Ubiquitin Ligases of Pear and Their Subcellular Localization
邱辉 1朱德娟 2张永乐 2高玉洁 2李柳 2王国平 1洪霓2
作者信息
- 1. 果蔬园艺作物种质创新与利用全国重点实验室,华中农业大学园艺林学学院,武汉 430070;华中农业大学植物科技学院,湖北省作物病害监测与安全控制重点实验室,武汉 430070
- 2. 华中农业大学植物科技学院,湖北省作物病害监测与安全控制重点实验室,武汉 430070
- 折叠
摘要
为深入了解苹果褪绿叶斑病毒(apple chlorotic leaf spot virus,ACLSV)与寄主植物的互作特性,构建了表达该病毒外壳蛋白(coatprotein,CP)的诱饵载体pGBKT7-CP,采用酵母双杂交技术,从'红香酥'梨cDNA文库中筛选出与ACLSVCP潜在互作的33种寄主因子.酵母双杂交和双分子荧光互补试验验证了CP与来自梨的两种RING型E3泛素连接酶(命名为MIEL和BOI)互作.亚细胞定位分析表明,CP与BOI具有相似的胞间连丝和细胞核分布特点,MIEL主要分布在内质网和细胞核.双分子荧光互补试验结果显示,CP与BOI互作不改变二者的亚细胞分布,而CP与MIEL互作信号分布与MIEL亚细胞定位相似.构建了这3种蛋白的截短突变体,发现含zf-CHY结构域(1～99 aa)的MIEL截短突变体可以与CP互作,而仅全长BOI可以与CP互作,CP截短突变体不能与这两种寄主蛋白互作.
Abstract
Apple chlorotic leaf spot virus(ACLSV)possesses a positive single stranded RNA and widely infects pear and other fruit trees.The coat protein(CP)of the virus functions in the viral pathogenesis.In order to gain a deep understanding of the interaction characteristics between the virus and its host plants,a yeast two hybrid(Y2H)assay was carried out by using pGBKT7-CP(expressing ACLSV CP)as a bait vector to screen pear proteins in a cDNA library of pear'Hongxiangsu',and 33 host factors potentially interacting with ACLSV CP were identified.Furthermore,the interaction relationships between CP and two RING type E3 ubiquitin ligases(named MIEL and BOI)from pear were confirmed by both Y2H and bimolecular fluorescence complementation(BiFC)assays.In BiFC assays,the interaction signal of CP with BOI located at plasmodesma and nucleus,which was similar to the subcellular locations of the two proteins.The interaction signal of CP with MIEL located at endoplasmic reticulum and nucleus,which was similar to the subcellular locations of MIEL.The truncated mutants of the three proteins were constructed.In Y2H assays,only MIEL mutant containing the zf CHY domain(1-99 aa)interacted with CP,and other MIEL mutants and all BOI mutants failed to interact with CP,and all CP mutants cannot interact with the two host proteins.
关键词
苹果褪绿叶斑病毒/外壳蛋白/酵母双杂交/E3泛素连接酶/蛋白互作/亚细胞定位Key words
apple chlorotic leaf spot virus/coat protein/yeast two-hybrid/E3 ubiquitin ligase/protein-protein interaction/subcellular localization引用本文复制引用
基金项目
国家重点研发计划(2019YFD1001800)
出版年
2024
NETL
NSTL
万方数据