Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (6): 1979-1993.DOI: 10.1016/j.jia.2024.03.049

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尤力克柠檬锌指蛋白ClDOF3.4与柑橘黄化脉明病毒外壳蛋白互作抑制病毒侵染

  

  • 收稿日期:2023-06-30 接受日期:2023-11-07 出版日期:2024-06-20 发布日期:2024-05-30

Eureka lemon zinc finger protein ClDOF3.4 interacts with citrus yellow vein clearing virus coat protein to inhibit viral infection

Ping Liao1, 2, Ting Zeng1, 2, Mengyang Huangfu1, 2, Cairong Zheng1, 2, Jiequn Ren3, Changyong Zhou1, 2, Yan Zhou1, 2#   

  1. 1 Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Citrus Research Institute, Southwest University, Chongqing 400712, China

    2 National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, China

    3 Chongqing Three Gorges Academy of Agricultural Science, Chongqing Three Gorges University, Chongqing 404020, China

  • Received:2023-06-30 Accepted:2023-11-07 Online:2024-06-20 Published:2024-05-30
  • About author:Ping Liao, E-mail: liaoping940324@163.com; #Correspondence Yan Zhou, E-mail: zhouyan@cric.cn
  • Supported by:
    This research was financially supported by the China Agriculture Research System of MOF and MARA (CARS-26-05B), the Innovation Research 2035 Pilot Plan of Southwest University, China (SWU-XDPY22002), the Guangxi Science and Technology Planed Project, China (Gui Ke AD23026090), and the Guangxi Natural Science Foundation, China (2023GXNSFBA026285).

摘要: 柑橘黄化脉明病毒(citrus yellow vein clearing virusCYVCV)是一种新型柑橘病毒,该病毒已对印度、伊朗、土耳其、中国、美国和韩国的柠檬、酸橙和部分宽皮等柑橘品种的种植生产造成了严重的损失。CYVCV外壳蛋白(CP)具有RNA沉默抑制子活性,与病毒致病性和症状密切相关。DOFDNA-binding with one finger)是高等植物特有的锌指蛋白,在植物生长发育和抗病原物侵染的过程中扮演着重要作用。然而,目前尚未开展尤力克柠檬锌指蛋白ClDOFCYVCV的互作研究。因此,本研究通过一系列体内和体外互作验证实验,以及基因瞬时表达、基因瞬时沉默和尤力克柠檬遗传转化等手段,验证了ClDOF3.4CYVCV CP之间的互作关系及其对尤力克柠檬响应CYVCV免疫应答的影响。试验结果表明,ClDOF3.4蛋白在体内和体外均与CP存在相互作用关系,且ClDOF3.4蛋白N端的104-280个氨基酸序列是与CP相互作用的关键区段。在尤力克柠檬叶片中瞬时表达ClDOF3.4可以诱导水杨酸通路相关基因和超敏反应标记基因的上调表达,造成接种部位活性氧爆发和离子渗透坏死、并引起H2O2和水杨酸积累;而ClDOF3.4基因瞬时沉默尤力克柠檬叶片中的水杨酸和超敏反应相关基因下调表达。此外,该研究进一步通过农杆菌接种分别在瞬时表达及沉默ClDOF3.4基因的尤力克柠檬叶片中瞬时表达CP,结果表明ClDOF3.4负向调控CP在尤力克柠檬中的表达和积累。接种CYVCV六个月后,ClDOF3.4转基因尤力克柠檬植株的CYVCV含量约为对照植株的69.4%,与对照植株相比,转基因植株感病后的症状明显减轻,仅出现轻度的叶片斑驳症状。上述研究表明,ClDOF3.4不仅与CYVCV-CP存在相互作用,还可以通过水杨酸信号通路诱导尤力克柠檬对CYVCV的免疫应答。这是首次关于锌指蛋白参与柑橘抗病毒病的报道,该研究结果为进一步研究柑橘抗CYVCV的分子机制奠定了基础。

Abstract: Citrus yellow vein clearing virus (CYVCV) is a new citrus virus that has become an important factor restricting the development of China’s citrus industry, and the CYVCV coat protein (CP) is associated with viral pathogenicity.  In this study, the Eureka lemon zinc finger protein (ZFP) ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.  Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid (SA)-related and hypersensitive response marker genes, and triggered a reactive oxygen species burst, ion leakage necrosis, and the accumulation of free SA.  Furthermore, the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4% that in control plants 6 mon after inoculation, with only mild leaf chlorotic spots observed in those transgenic plants.  Taken together, the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.  This is the first report that ZFP is involved in the immune response of a citrus viral disease, which provides a basis for further study of the molecular mechanism of CYVCV infection.

Key words: citrus yellow vein clearing virus , coat protein ,  ClDOF3.4 ,  plant immune response