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

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

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

• Plant Protection • Previous Articles Next Articles

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^,^**(), and 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 Online:2025-11-26 Published:2025-11-26
Contact: XU Xiaojie, and LUAN Yameng

Abstract

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

REN Qian, XU Zeyu, ZHANG Yue, LÜ Yixuan, SONG Danyang, XU Xiaojie, and 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.

Figures/Tables 5

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

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

Fig. 2 Effect of N436R mutation on ZYMV virulence in Cucurbita pepo and Cucumis melo plants

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

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

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