Comparative Analysis Of The Subcellular Localization And The Impact On Tobacco Resistance Of Six Kinds Of Viruses Coat Protein

Plant viruses are the second important pathogen just after the fungus, caused serious lost of the important economic crops and food crops, and the degree of lost increased year by year. With the rapid development of genetic engineering, disease resistance breeding has become one of the effective means of prevention and treatment of plant viral diseases. Viral coat protein gene has been widely used for pathogen-derived resistance. Transgenic plants expressing virus genes or sequences have shown good resistance against plant virus, which is associated with the genome’s structure, function and subcellular localization. CPs in various different viruses have different functions, and subcellular localization of the viral coat protein. CPs of different viruses have different functions. So their subcellular localization in plant cells may also be different. Therefore, we present a comparative analysis of CPs of six viruses in our laboratory. They are the Tobacco mosaic virus(TMV), Potato virus Y(PVY), Turnip mosaic virus(TuMV), Potato virus X(PVX), Tobacco curly shoot virus(TbCSV), Tobacco rattle virus(TRV).Specific primer pairs were designed according to the published nucleotide sequence of five viruses, respectively. With the primer pairs, the coat protein(CP) gene of each virus was cloned by RT-PCR and inserted into the vector pSAT1-EGFP-C1 or pSAT6-EGFP-N1. Then each expression cassette was digested by PI-PSP I or ASC I and inserted into the plant expression vector pPZP-RCS1. The positive clone confirmed by PCR and digestion was used for transformation. The recombinant plasmids were introduced into Agrobacterium tumefaciens EHA105 by freezing and thawing method, respectively.Transient expression was performed via agroinfection assay in Nicotiana Benthamiana and tobacco(NC87). After three days, the fluorescent of the fused protein transient expressed in the leaves were observed by laser cofocol scanning microscopy. The results showed that the fusion protein CP-GFP of PVX, PVY, TuMV, TMV diffused in the cytoplasm and cell membrane(or cell wall), respectively. CP-GFP of TuMV, TMV and TRV could diffuse in the nucleus, respectively. CP-GFP of PVY, TuMV, TMV and TRV formed network structures and attached on cytoskeletons. CP-GFP of TRV also could be observed in some vacuoles or the mitochondria of the leaves. While TbCSV CP-GFP diffused in N. Benthamiana epidermal cells and located at the edge of the cell walls, forming discontinuous green fluorescent spots at the plasmodesmata. In addition, only CP-GFP of PVX and TbCSV CP were present in the chloroplast and stomata. The subcellular localizations of CP-GFP of PVX, TRV, TuMV and TMV were slightly different in tobacco(NC87). The expression levels of CP-GFP of PVX and TRV CP were reduced, and CP-GFP of PVX mainly distributed in the cell membrane, forming small aggregates. There are less network structures but more aggregates in the leaves expressed CP-GFP of TuMV.Our previous work showed that ToMV CP and IP-L could interact with each other. In order to learn more about the specificity of the interaction between ToMV CP and IP-L, transient expression was performed via agroinfection assay in N. Benthamiana. The results showed the CPs of PVX, TRV, TuMV and TbCSV co-localizated with IP-L, suggesting there may be interactions between each of them. But there was no co-localization observed between PVY CP and IP-L.In order to compare the resistance differencesinduced by CPs of different viruses, we measured the activity of defense enzymes and the content of chlorophyll of the N. Benthamiana ransient expressed each CP of 6 viruses to clear the physiological and biochemical changes of plants. Our results showed that there were different degrees of reduction of chlorophyll a, total chlorophyll and chlorophyll a/b in the leaves. Chlorophyll b also decreased except TMV CP treatment group. Activities of superoxide dismutase(SOD), peroxidase(POD), catalase(CAT), and polyphenol oxidase(PPO) all increased at various levels in 5 days after inoculation. To further evaluated the effect of the virus CP on the plant resistance, we inoculated TMV-GFP with TMV-GFP infectious clone via agroinfection assay in N. Benthamiana, in which CP-GFP of six kinds of viruses. The movement of TMV-GFP in the leaves was observed by UV light on 3 days after inoculaton. The results showed the movement of TMV-GFP was inhibited in the N. Benthamiana expressed each of the virus CP.