Screening Of Metal Ions Inhibiting Tobacco Mosaic Virus Replication And Preliminary Study On Its Mechanism

Metal ions are vital for sustaining the activity upto 30%of enzymes,whcih are indispensable for life activities.It was well documented,the metal ions present distinct functions in the processes of animal viral replication and infection,nevertheless,few studies have been conducted in plant viruses.Tobacco Mosaic virus(TMV)belongs to genus Tobamovirus,family Vigaviridae,which is a positive single-stranded RNA virus transmitted by mechanical damage and friction.TMV resulted in host symptoms including mosaic and shrinking in leaves,which seriously reduces the yield and quality of commercial crops.The study would conduct the effect of metal ions on TMV replication in host,and the resulsts would scientifically understand the role of metal ions in plant viruses replication,and also is helpful to develop novel prevention technology against TMV.The 14 metal ions including monovalence of Na⁺,K⁺,divalence of Mg²⁺,Mn²⁺,Zn²⁺,Ca²⁺,Cu²⁺,Cd²⁺,Ni²⁺,Co²⁺,Cs²⁺,Fe²⁺,and tridivanlence of Fe³⁺,Al³⁺were selected to test the activity on TMV replication by leaf disc floating method.The TMV genomic RNA level was quantified by the specific RT-q PCR.The results showed that6 metal ions of Na⁺,Zn²⁺,Cu²⁺,Cd²⁺,Fe²⁺and Fe³⁺relatively inhibited the TMV genomic RNA accumulation.P54 gene was transcribed by the subgenome of TMV,which inset in the P183 gene,including partial RNA dependent RNA replication(Rd Rp)structure.The P54 gene of TMV was prokaryotic expressed,the recombinent P54 protein(with 6×His tag)was purified by affinity chromatography.The insoluble substances existed at co-incubating the P54 protein with metal ions of Na⁺,Zn²⁺,Cd²⁺,Fe²⁺and Fe³⁺.Exogenous P54protein inhibiting the TMV genomic RNA accumulation.Add exogenous P54 protein could release the inhibiting of TMV genomic RNA accumulation by metal ions of Zn²⁺,Fe²⁺,Fe³⁺,probably hinted P54 protein direct binding with metal ions of Zn²⁺,Fe²⁺,Fe³⁺.The P54 gene was transient overexpressed in the leaves of Nicotiana tabacum var Samsun NN.The subcellular localization of P54 protein was observed and photoed by the laser confocal microscopy.The results revealed the the P54 protein localized at cell membrane and the nucleus,which hinted multiple regulating functions in TMV replication by P54 protein.