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园艺学报 ›› 2020, Vol. 47 ›› Issue (8): 1451-1462.doi: 10.16420/j.issn.0513-353x.2019-0847

• 研究论文 • 上一篇    下一篇

柑橘中黄龙病菌效应子SDE70的表达特征及寄主互作蛋白解析

龙俊宏,赵 珂,杜美霞,谢 竹,陈善春,何永睿,邹修平*   

  1. 西南大学/中国农业科学院柑桔研究所,国家柑桔品种改良中心,重庆 400712
  • 出版日期:2020-08-25 发布日期:2020-08-25
  • 基金资助:
    国家自然科学基金项目(31972393);国家重点研发计划专项(2018YFD0201500);国家现代农业产业技术体系建设专项资金项目(CARS-26);广东省科技计划项目(2018B020202009);广西创新驱动发展专项资金课题(桂科AA18118046)

Expression Characteristics and Interacting Proteins of the Effector SDE70 from‘Candidatus Liberibacter asiatics’in Infected Citrus

LONG Junhong,ZHAO Ke,DU Meixia,XIE Zhu,CHEN Shanchun,HE Yongrui,and ZOU Xiuping*   

  1. Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,National Center for Citrus Varieties Improvement,Chongqing 400712,China
  • Online:2020-08-25 Published:2020-08-25

摘要: 柑橘黄龙病菌主要致病种亚洲种(‘Candidatus Liberibacter asiaticus’,Las)具有完整的Sec依赖型分泌系统,可分泌致病因子(效应子)作用于寄主靶标蛋白或基因,调控寄主的抗(感)病反应。为了研究黄龙病菌Sec依赖型效应子在柑橘中的致病机理及其寄主靶标基因,本研究中从感病‘锦橙’中克隆了Las病原菌Sec依赖型效应子基因SDE70(CLIBASIA_02470)。生物信息分析显示,SDE70蛋白全长132个氨基酸,N端20个氨基酸为典型信号肽。大肠杆菌碱性磷酸酶活性分析显示,该信号肽具有胞外分泌功能。亚细胞定位显示,SDE70::GFP融合蛋白在洋葱细胞质和细胞核中积累。定量PCR分析发现,耐黄龙病酸柚显症叶脉中SDE70表达量显著低于未显症叶脉,相反,易感黄龙病‘锦橙’显症叶脉中表达显著高于未显症叶脉;‘锦橙’根中SDE70表达量显著高于叶脉,而幼叶叶脉中显著低于老叶。SDE70在不同品种不同症状发育时期以及不同组织中表达差异显著。利用酵母双杂交试验筛选获得26个与SDE70互作的柑橘蛋白。GO富集分析表明,42.3%的蛋白质与细胞内生物合成过程有关;65.4%的靶标蛋白位于细胞质或质膜上;34.6%的靶标蛋白具有核糖核苷酸结合的分子功能。进一步酵母点对点杂交试验验证3个与SDE70效应子强相互作用的靶标蛋白(CsTRAPP、CsARF、CsRub1),其主要参与植物囊泡转运、类泛素化等过程,暗示SDE70效应子极有可能调控柑橘信号转导和胞质运输途径影响寄主抗(感)病反应。

关键词: 柑橘, 柑橘黄龙病, Sec分泌蛋白, 酵母双杂, 瞬时表达, 定量PCR

Abstract: Citrus Huanglongbing(HLB)is the most devastating disease of citrus. The main pathogenic species is the Asian species(‘Candidatus Liberibacter asiaticus’,Las)in citrus industry. Las has a complete Sec-dependent secretion system which could secrete effectors to target proteins or genes of host to regulate the plant resistance. In order to explore the pathogenesis of Las effectors and their host target genes,a Sec-dependent effector gene SDE70(CLIBASIA_02470)of Las was cloned from infected Jincheng. Informatics analysis showed that the SDE70 protein has a total length of 132 amino acids,and a putative 20 amino acids signal peptide was detected in the N-terminal. The analysis of E. coli alkaline phosphatase activity showed that the signal peptide was functional in transferring protein to outside cell. Subcellular localization analysis showed that the SDE70::GFP fusion protein accumulated in the onion cytoplasm and nucleus. Real-time quantitative PCR analysis found that the expression of SDE70 in the symptomatic veins of tolerant Sour Pomelo was significantly lower than that in the asymptomatic veins,on the contrary,its expression in the symptomatic veins of susceptible Jincheng orange was significantly higher than that in the asymptomatic veins. In Jincheng orange,the expression of SDE70 in roots was significantly higher than that in veins,while its expression in young leaves was significantly lower than that in old leaves. The data showed that SDE70 exhibited significant differential expressions in different disease-resistant varieties,different developmental stages of symptoms,and different tissues. Twenty-six citrus proteins that interact with SDE70 were screened out by yeast two-hybrid assay. Gene Ontology rich analysis indicated that 42.3%,34.6% and 65.4% of these proteins were involved in the cellular biosynthetic process,the molecular function of ribonucleotide binding,and located on the cytoplasmic matrix or membrane,respectively. Further yeast two-hybrid experiments showed that three target proteins interacted strongly with SDE70 and they were involved in plant vesicle transport and ubiquitination-like pathway. These results suggested that SDE70 effector regulate plant response to Las infection through manipulating the signal transduction and/or cytoplasmic transport pathways in citrus.

Key words: citrus, HLB, Sec-dependent secreted protein, Yeast two-hybrid, transient expression, real-time quantitative PCR

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