瓜类褪绿黄化病毒编码的P6蛋白亚细胞定位及致病特征分析

doi:10.16420/j.issn.0513-353x.2020-0914

摘要/Abstract

摘要：

以瓜类褪绿黄化病毒（cucurbit chlorotic yellows virus,CCYV）山东寿光分离物作为研究对象,对其RNA1编码的P6蛋白进行克隆,发现其基因全长为159 bp,编码1个大约6 kD的蛋白,并含1个跨膜区。进一步构建了带有YFP标签的荧光表达载体P6-YFP,浸润本氏烟叶片后利用激光共聚焦显微镜观察,发现P6-YFP在细胞质和细胞核均有分布。将P6构建到马铃薯X病毒（potato virus X,PVX）异源表达载体获得的PVX-P6接种本氏烟,发现接种PVX-P6的植株新叶伴有明显黄化、皱缩等症状,并沿叶脉出现坏死,而接种PVX空载体的植株仅有轻微的花叶,表明在PVX异源表达系统中,P6诱导寄主植物出现更严重的症状,暗示P6能增强PVX的致病性,可能是参与症状形成的致病相关因子。

关键词: 瓜类褪绿黄化病毒, P6蛋白, 亚细胞定位, 致病性

Abstract:

Cucurbit chlorotic yellows virus（CCYV）is an important whitefly-transmitted Crinivirus,which has become an emerging infectious agent of cucurbits leading to severe disease and significant economic losses. P6 is a small protein encoded by ORF2 of RNA1,one segment of CCYV,and its functions still remains unknown. In this study,P6 was cloned from an isolate of CCYV from Shouguang,Shandong Provience using specific primers by RT-PCR. The results of sequence analysis revealed that P6 gene was 159 bp in length,encoding a protein of approximately 6 kD with a transmembrane domain. To clarify the subcellular localization of P6,a YFP-tagged P6（P6-YFP）was constructed using a 35S:YFP vector and transiently expressed in the Nicotiana benthamiana by agrobacterium infiltration. Fluorescence signal was observed in the cytoplasm and nucleus of the epidermal cells of infiltrated N. benthamiana leaves,which indicated that P6-YFP was distributed in the cytoplasm and nucleus. To test the pathogenicity of P6,a potato virus X（PVX）-based vector was introduced and PVX-P6 was constructed. Chlorotic,crumple and necrosis symptoms were observed in the emerging leaves of N. benthamiana plants inoculated by PVX-P6 and only slight mosaic symptoms was observed in the plant inoculated by PVX, which showed that heterologous expression of P6 in N. benthamiana enhanced the pathogenicity of PVX,indicating that P6 might be an important protein involved in the pathology of CCYV.

Key words: cucurbit chlorotic yellows virus, P6 protein, subcellular localization, pathogenicity

中图分类号:

S642

涂丽琴, 干射香, 吴淑华, 任春梅, 程兆榜, 章松柏, 朱月林, 周益军, 季英华. 瓜类褪绿黄化病毒编码的P6蛋白亚细胞定位及致病特征分析[J]. 园艺学报, 2021, 48(8): 1531-1540.

TU Liqin, GAN Shexiang, WU Shuhua, REN Chunmei, CHENG Zhaobang, ZHANG Songbai, ZHU Yuelin, ZHOU Yijun, JI Yinghua. Characterization of the Subcellular Localization and Pathogenicity of P6 Encoded by Cucurbit Chlorotic Yellows Virus[J]. Acta Horticulturae Sinica, 2021, 48(8): 1531-1540.

图/表 4

表1 引物信息

Table 1 Primers used in this research

引物长度/bp Length	引物名称 Name	序列（5′-3′） Sequence	退火温度/℃ T_m
159	P6-bF	CGggatccATGTATTTTACAACAATATTTTACCTC	48.9
	P6-bR	CGggatccAGCTATTTCTATATTCCTGTGG	49.4
159	P6-ClF	CCatcgatATGTATTTTACAACAATATTTTACCTC	48.9
	P6-SlR	GCgtcgacTCAAGCTATTTCTATATTCCTGT	49.5

图1 CCYV编码的P6氨基酸序列比对（A）和跨膜区分析（B）

Fig. 1 Amino acid sequence alignment of P6 encoded by CCYV（A）and transmembrane domain prediction（B）

图2 P6蛋白亚细胞定位 A：P6-YFP构建示意图;B：P6-YFP亚细胞定位;C：P6-YFP的Western blot及RT-PCR检测。

Fig. 2 Subcellular localization of P6-YFP A：Diagram of P6-YFP construction;B：Subcellular localization of P6-YFP（the distribution of P6 in the nucleus was indicated by H2B in the right small picture）;C：Detection of P6-YFP by RT-PCR and western blot.

图3 PVX-P6的烟草叶片致病特征 A、B：PVX-P6诱导的症状;C：PVX和P6的RT-PCR检测。

Fig. 3 Symptoms induced by PVX / PVX-P6 in Nicotiana benthamiana leaves A,B：Systemic symptoms induced by PVX-P6;C：Detection of PVX and P6 by RT-PCR.

参考文献 34

[1]	Abrahamian P E, Sobh H, Abou-jawdah Y. 2012. First report of cucurbit chlorotic yellows virus on cucumber in Lebanon. Plant Disease, 96 (11):1704-1705.
[2]	Alzhanova D V, Hagiwara Y, Peremyslov V V, Dolja V V. 2000. Genetic analysis of the cell to cell movement of beet yellows closterovirus. Virology, 268:192-200. pmid: 10683341
[3]	Amer M A. 2015. Serological and molecular characterization of cucurbit chlorotic yellows virus affecting cucumber plants in Egypt. International Journal of Virology, 11 (1):1-11. doi: 10.3923/ijv.2015.1.11 URL
[4]	Bananej K, Menzel W, Kianfar N, Vahdat A, Winter S. 2013. First report of cucurbit chlorotic yellows virus infecting cucumber,melon,and squash in Iran. Plant Disease, 97 (7):1005-1005.
[5]	Canizares M C, Navas-Castillo J, Moriones E. 2008. Multiple suppressors of RNA silencing encoded by both genomic RNAs of the Crinivirus,tomato chlorosis virus. Virology, 379 (1):168-174. doi: 10.1016/j.virol.2008.06.020 URL
[6]	Dolja V V, Kreuze J F, Valkonen J P. 2006. Comparative and functional genomics of Closteroviruses. Virus Research, 117 (1):38-51. doi: 10.1016/j.virusres.2006.02.002 URL
[7]	Gan She-xiang, Tu Li-qin, Wu Shu-hua, Fan Xiao-yan, Wu Rui-xue, Zhao Wen-hao, Cheng Zhao-bang, Zhou Yi-jun, Zhu Xiao-ping, Ji Ying-hua, Zhang Song-bai. 2019. Molecular identification of cucurbit chlorotic yellow virus on cucumber in Shouguang,Shandong province. Jiangsu Journal of Agricultural Science, 35 (5):1047-1053. (in Chinese)
	干射香, 涂丽琴, 吴淑华, 范小燕, 吴瑞雪, 赵文浩, 程兆榜, 周益军, 竺晓平, 季英华, 章松柏. 2019. 山东黄瓜上瓜类褪绿黄化病毒的分子鉴定. 江苏农业学报, 35 (5):1047-1053.
[8]	Gu Q S, Liu Y H, Wang Y H, Huangfu W G, Gu H F, Xu L, Song F M, Brown J K. 2011. First report of cucurbit chlorotic yellows virus in cucumber,melon,and watermelon in China. Plant Disease, 95 (1):73.
[9]	Gu Qin-sheng, Peng Bin, Liu Shan-shan, Liu Ying-hua, Wu Hui-jie, Liu Li-feng. 2011. New diseases of cucurbits:cucurbit chlorotic yellows virus. China Cucurbits and Vegetables, 24 (3):32-33. (in Chinese)
	古勤生, 彭斌, 刘珊珊, 刘英华, 吴会杰, 刘丽峰. 2011. 瓜类新病毒病害(一):瓜类褪绿黄化病. 中国瓜菜, 24 (3):32-33.
[10]	Gyoutoku Y, Okazaki S, Furuta A. 2009. Chlorotic yellows disease of melon caused by cucurbit chlorotic yellows virus,a new Crinivirus. Japanese Journal of Phytopathology, 75 (2):109-111. doi: 10.3186/jjphytopath.75.109 URL
[11]	Hamed K, Menzel W, Dafalla G, Gadelseed A M A, Winter S. 2011. First report of cucurbit chlorotic yellows virus infecting muskmelon and cucumber in Sudan. Plant Disease, 95 (10):1321.
[12]	Hong Xue-yu, Chen Jian-ping. 2004. Functions of some proteins encoded by members of family Closteroviridae. Acta Agriculturae Zhejiangensis, 16 (1):47-52. (in Chinese)
	洪雪玉, 陈剑平. 2004. 长线形病毒科成员编码蛋白的功能. 浙江农业学报, 16 (1):47-52.
[13]	Huang L H, Tseng H H, Li J T, Chen T C. 2010. First report of cucurbit chlorotic yellows virus infecting cucurbits in Taiwan. Plant Disease, 94 (9):1168.
[14]	Kiss Z A, Medina V, Falk B W. 2013. Crinivirus replication and host interaction. Frontiers in Microbiology, 4:99.
[15]	Landeo-Rios Y, Navas-Castillo J, Moriones E, Canizares M C. 2017. The heterologous expression of the p 22 RNA silencing suppressor of the Crinivirus tomato chlorosis virus from tobacco rattle virus and potato virus X enhances disease severity but does not complement suppressor-defective mutant viruses. Viruses, 9 (12):358. doi: 10.3390/v9120358 URL
[16]	Liu Shan-shan, Peng Bin, Wu Hui-jie, Liu Tang-jing, Kong Xiang-yi, Shi Yan, Gu Qin-sheng. 2013. Molecular identification of cucurbit chlorotic yellows virus in Hainan and Henan provinces. Journal of Fruit Science, 30 (2):291-293. (in Chinese)
	刘珊珊, 彭斌, 吴会杰, 柳唐镜, 孔祥义, 施艳, 古勤生. 2013. 海南省和河南省发生甜瓜褪绿黄化病的分子鉴定. 果树学报, 30 (2):291-293.
[17]	Lu Shao-hua, Li Jing-jing, Liu Ming-yang, Wang Xue-li, Yan Feng-ming. 2015. Effects of cucurbit chlorotic yellows virus on feeding behavior of Bemisia tabaci. Entomological Research in Central China, 11 (2):310-311. (in Chinese)
	卢少华, 李静静, 刘明杨, 王雪丽, 闫凤鸣. 2015. 瓜类褪绿黄化病毒对烟粉虱取食行为的影响. 华中昆虫研究, 11 (2):310-311.
[18]	Navas-Castillo J, Lopez-Moya J J, Aranda M A. 2014. Whitefly-transmitted RNA viruses that affect intensive vegetable production. Annals of Applied Biology. 165:155-171. doi: 10.1111/aab.2014.165.issue-2 URL
[19]	Okuda M, Okazaki S, Yamasaki S, Okuda S, Sugiyama M. 2010. Host range and complete genome sequence of cucurbit chlorotic yellows virus,a new member of the genus Crinivirus. Phytopathology, 100 (6):560-566. doi: 10.1094/PHYTO-100-6-0560 URL
[20]	Orfanidou C G, Maliogka V I, Katis N I. 2014. First report of cucurbit chlorotic yellows virus in cucumber,melon,and watermelon in Greece. Plant Disease, 98 (10):1446-1447.
[21]	Orfanidou C G, Maliogka V I, Katis N I, Kontosfyris G, Smith T, Caglayan K. 2017. First report of cucurbit chlorotic yellows virus in cucumber in Turkey. Plant Disease, 99 (2):533.
[22]	Pan Wei-ping, Zhang Yi-he, Ji Yan-ling. 2017. The first report of cucurbit chlorotic yellows virus infecting melon in Turpan. Vegetables,(2):60-61. (in Chinese)
	潘卫萍, 张以和, 吉艳玲. 2017. 吐鲁番首次发生甜瓜褪绿黄化病毒病. 蔬菜,(2):60-61.
[23]	Peremyslov V V, Pan Y W, Dolja V V. 2004. Movement protein of a Closterovirus is a type Ⅲ integral transmembrane protein localized to the endoplasmic reticulum. Journal of Virology, 78 (7):3704-3709. pmid: 15016890
[24]	Qiao W, Helpio E L, Falk B W. 2018. Two Crinivirus-conserved small proteins,P5 and P9,are indispensable for efficient lettuce infectious yellows virus infectivity in plants. Viruses, 10 (9):459. doi: 10.3390/v10090459 URL
[25]	Qiao W, Medina V, Falk B W. 2017. Inspirations on virus replication and cell-to-cell movement from studies examining the cytopathology induced by lettuce infectious yellows virus in plant cells. Frontiers in Plant Science, 8:1672. doi: 10.3389/fpls.2017.01672 URL
[26]	Shakeel M T, Amer M A, Al-Saleh M A, Al-Shahwan I M, Kamran A, Orfanidou C G, Katis N I. 2018. Molecular characterization of Crinivirues involved in the etiology of cucumber yellowing disease in Riyadh region,Saudi Arabia. European Journal of Plant Pathology, 150 (9):39-47. doi: 10.1007/s10658-017-1250-5 URL
[27]	Tang Xin, Zhang De-yong, Li Fan, Yan Fei, Zhou Xu-guo, Zhang You-jun, Shi Xiao-bin, Liu Yong. 2017. The first report and the epidemical dynamic of the cucurbit chlorotic yellows virus(CCYV)in Hunan province. Acta Phytopathologica Sinica, 47 (4):573-576. (in Chinese)
	唐鑫, 张德咏, 李凡, 燕飞, 周序国, 张友军, 史晓斌, 刘勇. 2017. 瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus)在湖南省的首次报道及其流行动态研究. 植物病理学报, 47 (4):573-576.
[28]	Tu Li-qin, Wu Shu-hua, Zhao Wen-hao, Fan Xiao-yan, Gan She-xiang, Cui Xiao-yan, Cheng Zhao-bang, Chen Xin, Zhu Yue-lin, Zhou Yi-jun, Ji Ying-hua. 2020. Preliminary Characterization of the subcellular localization of NIa-Pro protein encoded by clover yellow vein virus from broad Bean. Acta Horticulturae Sinica, 47 (1):153-160. (in Chinese)
	涂丽琴, 吴淑华, 赵文浩, 范小燕, 干射香, 崔晓艳, 程兆榜, 陈新, 朱月林, 周益军, 季英华. 2020. 蚕豆中三叶草黄脉病毒编码的NIa-Pro蛋白亚细胞定位特征初步研究. 园艺学报, 47 (1):153-160.
[29]	Tzanetakis I E, Martin R R, Wintermantel W M. 2013. Epidemiology of Criniviruses:an emerging problem in world agriculture. Frontiers in Microbiology, 4:119. doi: 10.3389/fmicb.2013.00119 pmid: 23730300
[30]	Yao Yu-rong, Chen Guo-hua, Feng Lan-xiang, Xie Bing-yan. 2013. Molecular detection of pepper viruses in southern vegetable production bases. China Vegetables,(10):84-89. (in Chinese)
	姚玉荣, 陈国华, 冯兰香, 谢丙炎. 2013. 北运蔬菜基地辣椒病毒病病原种类的分子检测. 中国蔬菜,(10):84-89.
[31]	Wu Shu-hua, Zhao Wen-hao, Li Ting-fang, Cheng Zhao-bang, Pan Bao-gui, Guo Qing-yun, Wang Shu-bin, Ji Ying-hua, Zhou Yi-jun. 2015. Molecular identification of a virus causing mottle symptoms in pepper leaves in Nanjing. Jiangsu Journal of Agricultural Science, 31 (6):1284-1290. (in Chinese)
	吴淑华, 赵文浩, 李廷芳, 程兆榜, 潘宝贵, 郭青云, 王述彬, 季英华, 周益军. 2015. 南京辣椒上一种斑驳类型病毒病的分子鉴定. 江苏农业学报, 31 (6):1284-1290.
[32]	Wei Y, Shi Y J, Han X Y, Chen S Y, Li H L, Chen L L, Sun B J, Shi Y. 2019. Identification of cucurbit chlorotic yellows virus P4.9 as a possible movement protein. Virology Journal, 16:82. doi: 10.1186/s12985-019-1192-y URL
[33]	Yang Shi-an, Li Zhan-biao, Qin Bi-xia, Xie Hui-ting, Cui Li-xian, Su Qin, Deng Tie-jun, Cai Jian-he. 2017. Molecular detection and identification of three viruses isolated from melons in Guangxi. Plant Protection, 43 (3):83-89. (in Chinese)
	杨世安, 李战彪, 秦碧霞, 谢慧婷, 崔丽贤, 苏琴, 邓铁军, 蔡健和. 2017. 广西三种甜瓜病毒分离物的分子检测与鉴定. 植物保护, 43 (3):83-89.
[34]	Zhang Rü-nan, Li Yuan-yuan, Li Da-wei, Yu Jia-lin, Han Cheng-gui. 2012. Characterization of the genome sequence of cucurbit chlorotic yellow virus isolate from Beijing. Proceedings of the annual meeting of Chinese Society of Plant Pathology,(1):628. (in Chinese)
	张汝楠, 李源源, 李大伟, 于嘉林, 韩成贵. 2012. 瓜类褪绿黄化病毒(CCYV)北京分离物基因组序列分析. 中国植物病理学会学术年会论文集,(1):628.