Endoplasmic Reticulum Stress Induced By CMV And Research On The Regulator NbbZIP28 In Nicotiana

The cellular translation machinery is hijacked by large amounts of viral proteins upon virus infection.Then the unfolded proteins accumulated in the lumen of the endoplasmic reticulum(ER)results in ER stress,which triggers the unfolded protein response(UPR)or programmed cell death(PCD).Previous research shows that virus manipulates UPR in favor of its replication and the host initiates PCD of apoptosis to resist virus.But less is known about the signal mechanism and strength of UPR regulated by different viruses.Searching for the key regulator of UPR and PCD is important to antiviral research.So,this article promoted the research on ER stress induced by Cucumber mosaic virus(CMV)and the key regulator NbbZIP28 in Nicotiana.Firstly,resistance source of tobacco strain to CMV was screened through inoculation methods.The results showed that one moderate resistant variety was identified in 3000 species,the others showed susceptible.The purified CMV was used to infect tobacco suspension cell BY-2 by PEG and the cell death was detected by nucleus dying of 4’,6’-diamidino-2-phenylindole.The results showed that PEG could promote CMV to infect BY-2 or protoplast.The ratio of cell death infected by CMV was higher than PBS control at 48-72 h,with 90-100%at 72 h,and more degraded protoplast at 24 h.However,it was difficult to distinguish the nature died cell from CMV-infected cell.The system of inoculation leaves with CMV was used to study ER stress.To investigate if CMV replicated in the tonoplast induced ER stress,the inoculated N.tabacum cv.Samsun NN or N.benthamiana plants by CMV or PBS control were used.At 12-48 h post-inoculation,the transcript level of ER stress genes by RT-PCR was higher in CMV inoculated leaves than PBS control.ER marker(mCherry-HDEL)was transiently expressed in leaf epidermal cells by agroinfiltration in CMV-inoculated leaves.At 5-10 d post-inoculation,under confocal microscopy,ER tubules were rearranged and hyperplasia.Cells ultrastructure by electron microscope showed chloroplast degradation,mitochondria swelling,invagination of vacuole into two double membranous complex structures of microautophagy,and macroautophagic structures in CMV-diseased cells.This revealed CMV infection induces ER stress by up-regulating of ER stress genes,membrane rearrangements of ER tubules.Host may activate microautophagy and macroautophagy to suppress viral infection.Then,the UPR,a highly conserved cellular defense pathway,is activated by ER stress.AtbZIP28 mediates ER stress related UPR when treated by tunicamycin in Arabidopsis.Here,we identified NbbZIP28 induced by virus in N.benthamiana.RACE-PCR and homology-based cloning were conducted.Two GFP-fusion constructes(NbbZIP28-GFP and NbbZIP28Δc-GFP)were generated and transient expressed in N.benthamiana plants.Sequence analyses showed that the NbbZIP28 encodes a protein of 812 amino acids with a putative transmembrane domain(TMD)near its C terminus next to a basic leucine zipper domain.NbbZIP28 protein was located in the ER,and NbbZIP28Δc,a truncated form of NbbZIP28 that lacked the region from the TMD to the carboxy terminus,localized in the nucleus.The transcript levels of NbbZIP28 were significantly up-regulated at 48 h post inoculation with CMV and Tobacco mosaic virus(TMV).NbbZIP28-silenced N.benthamiana plants by VIGS showed no significant phenotypic change,but viruses were able to multiply to higher levels in the silenced plants than in control plants at early stage.The silenced-NbbZIP28 plants showed lower transcript levels of UPR genes including BiP and NF-YC2 at the early stage of virus infection,and the levels were recovered even increased at later stage,indicating that NbbZIP28 may be a key regulator of UPR,but not the only one.NbbZIP28 may play a role in the defense response against viruses at early stage by promoting the UPR pathway activation in N.benthamiana.Last,to further investigate the role of NbbZIP28 in virus induced UPR signals,the mutant of knot-out NbbZIP28 was obtained by Crispr-cas9 and genetic transfection in N.benthamiana.Mutant plants showed no significant difference of germination rate and plant phenotypic.The virus accumulation on mutant plants was higher than wild type plant at early stage of virus inoculation.Mutant seedings showed no significant phenotype difference from wild type upon the stress of salt or drought.In summary,NbbZIP28,a regulator of UPR,plays a negative role in viral infection,inhibiting and delaying virus accumulation at early stage.