Abstract:

【Objective】 Potato Y virus (PVY) is one of the most important viruses that endanger the tobacco production in China. NAC transcription factors are closely related to plant disease resistance and stress resistance. The objective of this study is to clone NbNAC062, analyze its bioinformatics and research its role in the process of PVY infection, and to provide a target for the development of tobacco antiviral agents. 【Method】 Nicotiana benthamiana was used as the material to clone NbNAC062, and MEGA, UniProt, SMART, TMHMM Server 2.0, Sol Genomics Network, PlantCARE and other technologies were used for bioinformatics analysis. Laser confocal microscope and quantitative real-time PCR (qRT-PCR) were used to clarify the localization of NbNAC062 protein and the change of NbNAC062 mRNA expression before and after PVY infection. Based on virus-induced gene silencing (VIGS) technology and over-expression technology, the pTRV::NbNAC062 silencing vector and the pEarleyGate100::RFP::NbNAC062 over-expression vector were constructed. qRT-PCR and Western blot were used to detect the changes of PVY accumulation and the expression of unfolded protein response (UPR) related gene BiP after silencing and over-expression in N. benthamiana.【Result】NbNAC062 encodes 646 amino acids, the N-terminal 28-179 aa is the NAC domain, 129-185 aa is the DNA binding region, and the C-terminal 621-643 aa is a hydrophobic transmembrane structure. Phylogenetic tree and protein sequence analysis show that N. benthamiana NbNAC062 is closely related to N. attenuata NaNAC062. The NbNAC062 promoter contains a variety of cis-acting elements related to abscisic acid, methyl jasmonate, salicylic acid and stress response. PVY infection activates NbNAC062 to transfer from cell membrane to nucleus and induces NbNAC062 up-regulation of expression. For 5 and 7 days after PVY infection, the NbNAC062 mRNA level in the treatment group was 2.52 and 1.95 times of that of the control group, respectively. For 3 days after PVY infection, the BiP mRNA expression was 2.39 times of that of the control group, and for 7 days after PVY infection, the expression of BiP was significantly lower than that of the control group, which was down-regulated by 56.77%. NbNAC062 was silenced and PVY was inoculated, compared with the control group, the expression of PVY CP mRNA was up-regulated in the silence group at 3, 5, and 7 days after inoculation, which was 2.12, 2.41, and 1.38 times of that of the control group, respectively. However, the expression of BiP mRNA was down-regulated by 28.19%, 58.11%, and 10.77%, respectively. The PVY CP protein content of the silence group was also significantly higher than that of the control group at 5 and 7 days after vaccination. NbNAC062 was over-expressed and PVY was inoculated, compared with the control group, the expression of PVY CP mRNA in the over-expression group at 24, 48, 72 hours after inoculation was down-regulated by 22.60%, 34.51%, and 36.21%, respectively, and BiP mRNA was up-regulated at 48 and 72 hours after inoculation, which was 1.56 and 1.35 times of that of the control group, respectively. The content of PVY CP in the over-expression group was also lower than that of the control group.【Conclusion】NbNAC062 belongs to the NAC class of membrane-bound transcription factors, which can be activated by PVY infection and transferred to the nucleus. It may regulate the expression of the UPR-related gene BiP to promote cell survival and inhibit early PVY infection.

Key words: NbNAC062, potato virus Y (PVY), gene silencing, transient over-expression