Characterization of the Subcellular Localization and Pathogenicity of P6 Encoded by Cucurbit Chlorotic Yellows Virus

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (8): 1531-1540.doi: 10.16420/j.issn.0513-353x.2020-0914

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Characterization of the Subcellular Localization and Pathogenicity of P6 Encoded by Cucurbit Chlorotic Yellows Virus

TU Liqin¹^,², GAN Shexiang¹^,³, WU Shuhua¹, REN Chunmei¹, CHENG Zhaobang¹, ZHANG Songbai³, ZHU Yuelin², ZHOU Yijun¹, JI Yinghua¹^,^**()

¹Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China
²College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China
³College of Agriculture,Yangtze University,Jingzhou,Hubei 434025,China

Received:2020-12-17 Revised:2021-05-06 Online:2021-08-25 Published:2021-09-06
Contact: JI Yinghua E-mail:jiyinghua@jaas.ac.cn

Abstract

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

CLC Number:

S642

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.

Figures/Tables 4

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

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

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.

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.

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Characterization of the Subcellular Localization and Pathogenicity of P6 Encoded by Cucurbit Chlorotic Yellows Virus

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