柑橘黄化斑驳相关病毒侵染性克隆构建及致病性分析

doi:10.1016/j.jia.2023.08.014

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (10): 3034-3041.DOI: 10.1016/j.jia.2023.08.014

柑橘黄化斑驳相关病毒侵染性克隆构建及致病性分析

收稿日期:2023-01-18 接受日期:2023-08-03 出版日期:2023-10-20 发布日期:2023-10-07

Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants

WU Jia-xing^1*,ZHANG Song^2*, LIANG Xiao-fei¹, XING Fei¹, Sagheer ATTA³, WANG Xue-feng^1#, CAO Meng-ji^1#

¹National Citrus Engineering and Technology Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Citrus Research Institute, Southwest University, Chongqing 400712, P.R.China
² Guangxi Citrus Breeding and Cultivation Technology Innovation Center, Guangxi Academy of Specialty Crops, Guilin 541004, P.R.China
³ Department of Plant Protection, Faculty of Agricultural Sciences, Ghazi University, Dera Ghazi Khan 32200, Pakistan

Received:2023-01-18 Accepted:2023-08-03 Online:2023-10-20 Published:2023-10-07
About author:#Correspondence CAO Meng-ji, E-mail: caomengji@cric.cn; WANG Xue-feng, E-mail: wangxuefeng@cric.cn * These authors contributed equally to this study.
Supported by:
This study was supported by the Chongqing Science Funds for Distinguished Young Scientists, China (CSTB2022NSCQ-JQX0027), the Fundamental Research Funds for the Central Universities, China (SWU-XDPY22002), the National Natural Science Foundation of China (32072389, 32370005) and the Chongqing Talents of Exceptional Young Talents Project, China (cstc2022ycjh-bgzxm0143).

摘要/Abstract

摘要：

柑橘黄化斑驳相关病毒（Citrus yellow mottle-associated virus，CiYMaV）属马铃薯X病毒属（Potexvirus）、印度柑橘病毒亚属（Mandarivirus），最早于2018年在巴基斯坦被发现，目前该病毒在巴基斯坦多地柑橘产区有发生。本研究首先借助植物双元表达载体pCass4-RZ构建了3个CiYMaV的全长cDNA克隆（pCiYMaV-FL-1、pCiYMaV-FL-18和pCiYMaV-FL-22），随后通过农杆菌真空浸润接种强德勒柚（Citrus grandis）验证其侵染活性。结果表明，在接种40-60天，3个克隆均引起严重的叶脉黄化、叶片斑驳和植株矮化的症状。直接组织点免疫（direct tissue blot immunoassay，DTBIA）和反转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT-PCR）的检测结果表明3个克隆的侵染率为94.7-100%。尽管3个克隆引发的症状相似，pCiYMaV-FL-22在接种60天时，其在植株内的RNA和蛋白积累水平最高。因此，选择pCiYMaV-FL-22将其接种到7种柑橘和3种草本寄主上，结果表明pCiYMaV-FL-22能成功侵染7种柑橘寄主，并且在5个柑橘品种上引起明显症状。通过透射电镜，在接种pCiYMaV-FL-22的柑橘植株组织中观察到长线型、弯曲的病毒颗粒。综上所述，本研究构建了CiYMaV的全长感染性cDNA克隆，完成了该病毒的柯赫氏法则验证，为进一步研究该病毒侵染的分子机制和开发能应用于柑橘上的病毒载体奠定了基础。

Abstract:

Citrus yellow mottle-associated virus (CiYMaV) belonging to the subgenus Mandarivirus within the genus Potexvirus, was first identified in 2018 from Pakistan (CiYMaV-PK), where it is endemic in several regions. Here, three full-length cDNA clones (pCiYMaV-FL-1, pCiYMaV-FL-18, and pCiYMaV-FL-22) corresponding to the genomic RNA of CiYMaV were constructed and then agroinfiltrated on Chandler pummelo (Citrus grandis) seedlings using the vacuum infiltration method. All the inoculated plants developed severe vein yellowing, leaf mottling, and dwarfing symptoms by 40 days post-infiltration (dpi). The results of a direct tissue blot immunoassay and reverse transcription polymerase chain reaction detection showed 94.7–100% infection rates of pCiYMaV-FL at 60 dpi. Despite there being no observed difference among the three clones in the severity of symptom, pCiYMaV-FL-22 showed the highest accumulation levels of viral RNA and coat proteins. Moreover, pCiYMaV-FL-22 successfully infected seven other citrus varieties and induced symptoms in five of them. Transmission electron microscopy identified the presence of filamentous virus particles in extracts from systemic leaves of the plants infected with pCiYMaV-FL-22 at 6-months post-infiltration. Taken together, the results indicate that Koch’s postulates were fulfilled for CiYMaV in citrus plants. This is the first report of full-length infectious cDNA clones of CiYMaV, and thus, the data provide a basis for further study of the molecular mechanisms of virus infection and the development of a viral vector to express foreign genes in citrus plants.

Key words: Mandarivirus , vein yellow , vacuum infiltration , direct tissue blot immunoassay , virus morphology

WU Jia-xing, ZHANG Song, LIANG Xiao-fei, XING Fei, Sagheer ATTA, WANG Xue-feng, CAO Meng-ji. 柑橘黄化斑驳相关病毒侵染性克隆构建及致病性分析[J]. Journal of Integrative Agriculture, 2023, 22(10): 3034-3041.

WU Jia-xing, ZHANG Song, LIANG Xiao-fei, XING Fei, Sagheer ATTA, WANG Xue-feng, CAO Meng-ji. Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants[J]. Journal of Integrative Agriculture, 2023, 22(10): 3034-3041.

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柑橘黄化斑驳相关病毒侵染性克隆构建及致病性分析

Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants

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