TMV/CMV Induces Tobacco Endoplasmic Reticulum Stress And Functional Characterization Of Regulator NbNAC089

Endoplasmic reticulum(ER)is an important site for protein synthesis,and it is easy to be influenced by the environment in the process of protein synthesis,which causes accumulation of unfolded proteins and misfolded proteins in the ER reticulum and triggers the unfolded protein response(UPR).When ER stress is unmitigated or prolonged,it can promote programmed cell death(PCD).At present there are a lot of studies on the ER stress caused by abiotic factors.This paper focuses on the relationship between the virus and ER stress and the function of the key membrane-associated transcription factor NbNAC089.This study confirmed that Tobacco mosaic virus(TMV)or Cucumber mosaic virus(CMV)infection could induce ER stress and the change of the ER morphology.Fluorescent protein–tagged ER and quantitative real-time PCR were used to investigate the ER ultrastructure and the expression of ER stress-related genes in TMV-inoculated or CMV-inoculated leaves,including BiP?PDI?CAM?SKP1?bZIP60.Nicotiana benthamiana leaf inoculated with TMV showed fluorescent spots on the ER and ER tubules were rearranged and clustered together.In CMV-infected leaf,the continuous network of ER membranes was hyperplastic.In addition,TMV or CMV infection could induce the up-regulation of ER stress-related genes,these results indicate that virus infection could trigger ER stress.The results of RACE and homology-based cloning showed that NbNAC089,encodes 409 amino acids,a typical NAC transcription factor,was predicted to have a conserved NAC domain in the N-terminal region and a putative TMD in the C-terminal region.The subcellular localization of NbNAC089 showed that it is a typical ER-localized protein,and relocated to the nucleus after removing the transmembrane domain(TMD)and the C-terminus sequence.NbNAC089 has a high homology to Arabidopsis ANAC089.ANAC089 is a membrane-associated transcription factor,which controls the PCD pathway induced by ER stress.The results of qRT-PCR and western blot showed that NbNAC089 was up-regulated and was cleaved releasing truncated form relocated to the nucleus upon the sensing of TMV or CMV infection.NbNAC089-silenced plants by VIGS showed upregulation of the UPR genes,reduced expression of the PCD genes and allowed higher TMV or CMV accumulation compared with the control.Thus,NbNAC089 plays a role in suppressing viral infection by inhibiting the UPR signal and promoting the PCD pathway.