Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (4): 771-781.doi: 10.3864/j.issn.0578-1752.2020.04.009

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning of Hsc70-2 and Its Promoting Effect on Potato virus Y Infection in Nicotiana benthamiana

MingYue GONG1,XiaoTian DUAN1,TingTing YU1,Jie WANG2,LiLi SHEN2,Ying LI2,MingHong LIU3,YongLiang LI4,HongKun LÜ5,SongBai ZHANG1(),JinGuang YANG2()   

  1. 1 College of Agronomy, Yangtze University, Jingzhou 434025, Hubei
    2 Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong
    3 Zunyi City Company, Guizhou Tobacco Company, Zunyi 563000, Guizhou
    4 Baoshan City Company, Yunnan Tobacco Company, Baoshan 678000, Yunnan
    5 Hainan Cigar Research Institute, Hainan Provincial Corporation, China National Tobacco Corporation, Haikou 571100
  • Received:2019-08-19 Accepted:2019-09-18 Online:2020-02-16 Published:2020-03-09
  • Contact: SongBai ZHANG,JinGuang YANG E-mail:yangtze2008@126.com;yangjinguang@caas.cn

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Abstract:

【Objective】Potato virus Y (PVY) is an important virus that harms major crops such as tobacco, potato, tomato, pepper and so on, and brings great losses to agricultural production. The objective of this study is to clone NbHsc70-2, analyze the bioinformatics of NbHsc70-2 protein, research the effect of NbHsc70-2 protein on PVY infection in Nicotiana benthamiana, and to provide a theoretical basis for further analyzing the infection mechanism of PVY.【Method】The gene coding sequence (CDS) of Hsc70-2 protein from N. benthamiana was cloned in pEarleyGate100 vector. Multi-sequence alignment and phylogenetic analysis were performed using MEGA 6.0 software. Fluorescence quantitative RT-PCR (qRT-PCR) analysis of NbHsc70-2 expression level was performed in different tissues of N. benthamiana. Bioinformatics analysis using online software BaCeILo and SignalP 4.0.was performed for construction of NbHsc70-2-RFP fusion protein and determination of the subcellular localization of the identified NbHsc70-2 protein. PVY-GFP was used to analyze the effect of PVY infection on NbHsc70-2 in N. benthamiana leaves and localization of this protein. Furthermore, NbHsc70-2 VIGS silencing system and transient expression vector were constructed, and the effect of the protein on PVY expression after silencing/overexpression was analyzed by comparing the results with qRT-PCR assay.【Result】NbHsc70-2 encodes a total of 649 amino acids. The phylogenetic analysis indicated that NbHsc70-2 belongs to the heat shock protein (HSP) family and has the highest similarity with the NaHsc70-2. The C-terminal has the highly conserved motif structure of the HSP family. qRT-PCR analysis showed that NbHsc70-2 had the highest expression level in leaves and low expression in roots and stems. Moreover, BaCeILo prediction and laser confocal microscopy depicted that NbHsc70-2 was localized in the cytoplasm, and after PVY-GFP infection to N. benthamiana, NbHsc70-2-RFP was partially transferred to the nucleus and colocalized with virus in the cytoplasm and nucleus. The induction of NbHsc70-2 gene silencing vector pTRV::NbHsc70-2 into N. benthamiana resulted in stunted growth of infected plants compared to the control at 7 dpi. Whilst, inoculation of silent plants with PVY-GFP didn’t reflect green fluorescence, whereas PVY-GFP inoculation resulted in the green fluorescence production from the leaves of plants kept as control, the number of fluorescent spots in the silent group was about 28% of that in control group. qRT-PCR analysis showed that further inoculation of PVY resulted in accumulation of PVY CP at 1, 3, and 5 d after silencing NbHsc70-2. Moreover, the level of PVY CP decreased after silencing NbHsc70-2, and the difference between 3 and 5 dpi was significant compared to the control, the gene expression level was 14% and 0.004% of that in the control, respectively. Contrarily, high level of PVY CP was observed when N. benthamiana plants were inoculated with NbHsc70-2 overexpression vector along with PVY. The detailed analysis showed that the accumulation of PVY CP was significantly increased at 48 h and 72 h, and the gene expression level was 2.31 and 2.56 times of that in the control group, respectively.【Conclusion】PVY infection causes increased expression of NbHsc70-2. NbHsc70-2 is an important component of PVY infection to N. benthamiana, silencing NbHsc70-2 significantly inhibited PVY expression, and overexpression of NbHsc70-2 significantly increased PVY expression, i.e., the expression level of NbHsc70-2 was positively correlated with PVY replication. NbHsc70-2 protein promoted PVY infection in tobacco.

Key words: Nicotiana benthamiana, NbHsc70-2 protein, Potato virus Y (PVY), gene silencing, overexpression, infection and replication

Table 1

Sequence of the primers used in this study"

引物
Primer		 序列
Sequence (5′ to 3′)		 限制性内切酶
Restriction enzyme
NbHsc70-2 F ATGCGTATTATTAACGAGCCTAC
NbHsc70-2 R TTAATCAACTTCCTCAATCTTAGGAC
NbHsc70-2-XbaI F CTTTAGATCTTCTAGAATGCGTATTATTAACGAGCCTACTG XbaI
NbHsc70-2-KpnI R ATATTAATGTCGACGGTACCATCAACTTCCTCAATCTTAGGACC KpnI
RNAi NbHsc70-2 F TAAGGTTACCGAATTCATTCTTTCAGGTGAGGGTAATGAG EcoRI
RNAi NbHsc70-2 R AGACGCGTGAGCTCGGTACCCAGGAGGAATACCAGAAAGTTC KpnI
NbHsc70-2-AhdI F AGCAGGCTTTGACTTTAGGTCATGCGTATTATTAACGAGCCTACTG AhdI
NbHsc70-2-AhdI R TGGGTCTAGAGACTTTAGGTCTTAATCAACTTCCTCAATCTTAGGA AhdI
NbHsc70-2qRT-F AGGCTCCACTAGGATTCCGAAGG
NbHsc70-2qRT-R AGTGACATCCAACAGCAACAGGTC
PVY-CP-F GATGAATGGGCTTATGGTTTGGTG
PVY-CP-R GATTTGCCTAAGGGTTGGTTTCG
Actin-F CAAGGAAATCACCGCTTTGG
Actin-R AAGGGATGCGAGGATGGA

Fig. 1

Phylogenetic analyses of NbHsc70-2 and its homologues (ML algorithm)"

Fig. 2

Amino acid sequences multiple alignment among NbHsc70-2, NaHsc70-2, NtHsc70-2 and NsHsc70-2"

Fig. 3

Analysis of tissue expression specificity of NbHsc70-2"

Fig. 4

Gene accumulation of NbHsc70-2 after PVY infection in N. benthamiana"

Fig. 5

Subcellular localization of NbHsc70-2 protein by PVY (B) and not by PVY (A)"

Fig. 6

Phenotype analysis of NbHsc70-2 silencing"

Fig. 7

Effects of NbHsc70-2 silencing and overexpression on PVY accumulation"

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