HC-Pro中非保守的N436位点突变降低小西葫芦黄花叶病毒的致病力

doi:10.16420/j.issn.0513-353x.2024-0935

园艺学报 ›› 2025, Vol. 52 ›› Issue (11): 3069-3078.doi: 10.16420/j.issn.0513-353x.2024-0935

HC-Pro中非保守的N₄₃₆位点突变降低小西葫芦黄花叶病毒的致病力

任倩¹^,²^,^*, 许泽宇¹^,²^,^*, 张悦¹^,², 吕宜轩¹, 宋丹阳¹, 许小洁¹^,²^,^**(), 栾雅梦³^,^**()

¹ 鲁东大学农林工程研究院，山东烟台 264025
² 烟台市作物高产抗逆分子育种及高效栽培重点实验室，山东烟台 264025
³ 烟台市农业科学研究院，山东烟台 265500

收稿日期:2024-11-26 修回日期:2025-01-18 出版日期:2025-11-26 发布日期:2025-11-26
通讯作者:
^** E-mail：xiaojiexua105@163.com，
18763826098@163.com
作者简介:
*共同第一作者
基金资助:
山东省烟台市高校与地方企业合作项目(2021XDRHXMPT09); 国家自然科学基金项目(32302310); 山东省自然科学基金项目(ZR2023QC094)

Mutation in the Non-Conserved Residue N₄₃₆ of HC-Pro Affected Zucchini Yellow Mosaic Virus Virulence

REN Qian¹^,², XU Zeyu¹^,², ZHANG Yue¹^,², LÜ Yixuan¹, SONG Danyang¹, XU Xiaojie¹^,²^,^**(), LUAN Yameng³^,^**()

¹ The Engineering Research Institute of Agriculture and Forestry， Ludong University，Yantai， Shandong 264025， China
² Yantai Key Laboratory of Molecular Breeding for High-Yield and Stress-Resistant Crops and Efficient Cultivation， Yantai， Shandong 264025， China
³ Yantai Academy of Agricultural Sciences， Yantai， Shandong 265500， China

Received:2024-11-26 Revised:2025-01-18 Published:2025-11-26 Online:2025-11-26

摘要/Abstract

摘要： 通过比对11种马铃薯Y病毒属（Potyvirus）病毒编码的辅助成分——蛋白酶（helper component-proteinase，HC-Pro）氨基酸序列，发现小西葫芦黄花叶病毒（zucchini yellow mosaic virus，ZYMV）HC-Pro中第436位的天冬酰胺（asparagine，N₄₃₆）为非保守位点。为研究N₄₃₆位点在ZYMV致病力中的作用，采用定点突变技术，将HC-Pro中N₄₃₆位点替换为精氨酸（arginine，R），构建了携带绿色荧光蛋白（GFP）报告基因的病毒突变体克隆pZYMV-HC-Pro_N436R-GFP。结果表明，N436R突变显著降低ZYMV在西葫芦（Cucurbita pepo）和甜瓜（Cucumis melo）植株中的积累水平并减轻症状。此外，分析了该突变对ZYMV HC-Pro RNA沉默抑制活性、自身互作和亚细胞定位的影响。与野生型HC-Pro相比，N436R突变显著降低接种区域GFP荧光强度和GFP mRNA积累水平，表明其RNA沉默抑制活性减弱；但该突变未显著影响HC-Pro的亚细胞定位和自身互作强度。综上所述，非保守的N₄₃₆位点是调控ZYMV致病力和HC-Pro RNA沉默抑制活性的关键位点。

关键词: 辅助成分-蛋白酶, 非保守位点, 小西葫芦黄花叶病毒, 致病力

Abstract:

In this study，the amino acid sequences of potyviral helper component-proteinase（HC-Pro）from 11 potyviruses were compared，and found that asparagine at position 436（N₄₃₆）in zucchini yellow mosaic virus（ZYMV；genus Potyvirus）HC-Pro was not conserved. To analyze the role of this non-conserved amino acid in regulating ZYMV virulence，the residue of N₄₃₆ was substituted with arginine（N436R）usingthe site-directed mutagenesis method and produced plasmid pZYMV-HC-Pro_N436R-GFP carrying the green fluorescent protein（GFP）reporter gene. Results showed that the mutation of N436R reduced the accumulation levels of ZYMV in Cucurbita pepo and Cucumis melo plants，and attenuated viral symptoms. Furthermore，the effects of N436R mutation on the RNA silencing suppression activity，the self-interaction，and the subcellular localization of ZYMV HC-Pro were investigated. Compared with wild-type HC-Pro，N436R mutation significantly reduced the GFP fluorescence intensity and GFP mRNA accumulation levels in leaf patches，indicating that N436R mutation attenuated the RNA silencing suppression activity of HC-Pro. However，N436R mutation did not affect the self-interaction and the subcellular localization of HC-Pro. In conclusion，the non-conserved residue N₄₃₆ played roles in ZYMV virulence and the RNA silencing suppression activity of HC-Pro in plants.

Key words: helper component-proteinase, non-conserved residue, zucchini yellow mosaic virus, virulence

任倩, 许泽宇, 张悦, 吕宜轩, 宋丹阳, 许小洁, 栾雅梦. HC-Pro中非保守的N₄₃₆位点突变降低小西葫芦黄花叶病毒的致病力[J]. 园艺学报, 2025, 52(11): 3069-3078.

REN Qian, XU Zeyu, ZHANG Yue, LÜ Yixuan, SONG Danyang, XU Xiaojie, LUAN Yameng. Mutation in the Non-Conserved Residue N₄₃₆ of HC-Pro Affected Zucchini Yellow Mosaic Virus Virulence[J]. Acta Horticulturae Sinica, 2025, 52(11): 3069-3078.

图/表 5

表1 本试验中用到的引物名称和序列

Table 1 Primer names and sequences used in this study

引物名称Primer name	引物序列（5′-3′）Primer sequences
ZYMV-HC-Pro_N436R-F	GGAACTGTC A GACATTTAATTCAATTTGCCTCAAACGATTTGCAAAG
ZYMV-HC-Pro_N436R-R	GAATTAAATG TCTGACAGTTCCAGCCTTGAGTACATGATACCCAAC
GFP-qRT-F	GTGGAGAGGGTGAAGGTGAT
GFP-qRT-R	CGGATAACGGGAAAAGCATTGA
actin-qRT-F	CTGATGAAGATACTCACAGAAAGAG
actin-qRT-R	CAGGATACGGGGAGCTAATG
GFP-HC-Pro-F	GGAGGTTCTGGCGGTATGTCGTCGCAACCGGAAGTTCAG
GFP-HC-Pro-R	CTCCTCTTAGAATTCTTAGCCGACTCTGTAATGCTTC
AD-HC-Pro-F	GCCATGGAGGCCAGTATGTCGTCGCAACCGGAAGTTCAG
AD-HC-Pro-R	CACCCGGGTGGAATTCGCCGACTCTGTAATGCTTC
BD-HC-Pro-F	ATGGCCATGGAGGCCATGTCGTCGCAACCGGAAGTTCAG
BD-HC-Pro-R	GGATCCCCGGGAATTCGCCGACTCTGTAATGCTTC

图1 11种马铃薯病毒属病毒的部分HC-Pro氨基酸序列比对 ZYMV：小西葫芦黄花叶病毒；WMV：西瓜花叶病毒；TuMV：芜菁花叶病毒；PVY：马铃薯Y病毒；TEV：烟草蚀纹病毒；TVBMV：烟草脉带花叶病毒；BYMV：菜豆黄花叶病毒；LMV：莴苣花叶病毒；SCMV：甘蔗花叶病毒；PenMV：白草花叶病毒；PepMoV：辣椒斑驳病毒。ZYMV HC-Pro中非保守的N436位点由红色虚线框标注

Fig. 1 Alignment of eleven potyviruses HC-Pro amino acid sequences ZYMV：Zucchini yellow mosaic virus；WMV：Watermelon mosaic virus；TuMV：Turnip mosaic virus；PVY：Potato virus Y；TEV：Tobacco etch virus；TVBMV：Tobacco vein banding mosaic virus；BYMV：Bean yellow mosaic virus；LMV：Lettuce mosaic virus；SCMV：Sugarcane mosaic virus；PenMV：Pennisetum mosaic virus；PepMoV：Pepper mottle virus（PepMoV）. The corresponding amino acids for N436 in ZYMV HC-Pro were indicated in the red box

图2 N436R突变显著降低ZYMV在西葫芦和甜瓜植株中的积累水平 A：野生型ZYMV及其突变体在接种西葫芦（Cucurbita pepo）和甜瓜（Cucumis melo）植株后8 d引起的症状；B：Western blot检测野生型ZYMV及其突变体接种后8 d的西葫芦和甜瓜上部非接种叶片中ZYMV CP积累量。以丽春红染色的条带（Ponceau S staining，PSS）作为内参，使用ImageJ软件测量条带强度。数字表示PSS归一化后ZYMV CP的积累水平

Fig. 2 Effect of N436R mutation on ZYMV virulence in Cucurbita pepo and Cucumis melo plants A：Symptoms caused by wild-type and mutant ZYMV in Cucurbita pepo and Cucumis melo plants at 8 days post agroinfiltration；B：Western blot analysis of the accumulation levels of ZYMV CP in upper non-inoculated Cucurbita pepo and Cucumis melo leaves at 8 days post agroinfiltration. Ponceau S staining（PSS）as a loading control. Band intensities were measured using the ImageJ software. Numbers indicate the accumulation levels of GFP normalized to PSS staining

图3 N436R突变降低ZYMV HC-Pro RNA沉默抑制活性 A：野生型ZYMV HC-Pro及突变体在农杆菌共接种试验中的RNA沉默抑制活性；B：RT-qPCR检测16C本氏烟叶片不同接种区域的GFP mRNA积累水平。组间Tukey多重检验确定均数间的统计学差异，不同字母表示差异显著（P < 0.05）；C：Western blot检测16C本氏烟叶片不同接种区域的GFP蛋白积累水平

Fig. 3 Effect of N436R mutation on ZYMV HC-Pro RNA silencing suppression activity A：The RNA silencing suppression activity of wild-type and mutant ZYMV HC-Pro in Agrobacterium co-infiltration assay. B：RT-qPCR analysis of GFP mRNA accumulation levels in agroinfiltrated N. benthamiana line 16C leaf patches. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicate significant differences（P < 0.05）. C：Western blot analysis of GFP accumulation levels in agroinfiltrated leaf patches of N. benthamiana line 16C

图4 N436R突变对ZYMV HC-Pro自身互作和亚细胞定位的影响 A：酵母双杂交实验分析野生型ZYMV HC-Pro及其突变体的自身互作情况；B：野生型ZYMV HC-Pro及其突变体的亚细胞定位情况。白色箭头指示ZYMV HC-Pro通过自身互作形成的聚集体

Fig. 4 Effect of N436R mutation on ZYMV HC-Pro self-interaction and subcellular localization A：Yeast two-hybrid assay analysis of the self-interaction of wild-type ZYMV HC-Pro and its mutant. B：Subcellular localization of wild-type ZYMV HC-Pro and its mutant. White arrows indicated ZYMV HC-Pro formed aggregates via its self-interaction

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HC-Pro中非保守的N₄₃₆位点突变降低小西葫芦黄花叶病毒的致病力

Mutation in the Non-Conserved Residue N₄₃₆ of HC-Pro Affected Zucchini Yellow Mosaic Virus Virulence

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