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