Research The Mechanism Of Tobacco Curly Shoot Virus V2 Protein Regulating Pathogenicity

Tobacco curly shoot virus(TbCSV)is a monopartite geminiviruses belongs to the begomovirus of geminiviridae.The DNA-A component of the virus is considered to be an evolutionary intermediate type of monopartite geminiviruses,which can be independently infected the host or accompanied by complex infection with tobacco curly shoot betasatellite(TbCSB).TbSCV can be spread by Bemisita tabaci,has been widely occurring in the southwest of China.It is an important pathogen of tomato,pepper and tobacco,causing huge economic losses to a variety of crops and cash crops.It has been reported that the V2 protein of begomovirus is a multifunctional protein,which can participate in a series of processes such as the virus pathogenesis,movement and inhibition of host gene silencing,and plays an important role in the interaction between the virus and host.At present,the function of TbCSV V2 protein in the process of virus infection has not been reported.In this study,we cloned the TbCSV V2 coding gene sequence,analyzed its effect on virus infection and host growth through over-expression and viral gene deletion mutation,and analyzed the basic of biological characteristics of V2 such as subcellular localization,cell necrosis induction and gene silencing inhibitory activity,the correlation between several biological characteristics of V2 was studied by amino acid point mutation.In order to know whether V2 in TbCSV is involved in the pathogenic process of the virus and the molecular mechanism of its regulation,to provide the basis for studying the pathogenic mechanism of virus infection and the effective prevention of disease.In order to clarify the pathogenicity of TbCSV V2,a PVX over-expression vector of V2 and a functionally deficient mutant of the TbCSV V2 were constructed to infect N.b,and the results showed that PVX mediated over-expression of V2 can cause systemic necrosis of N.b leaves.By RT-q PCR detection found that the accumulation of PVX in the group of PVX-V2 was significantly higher than PVX-GUS.The results showed that the infectious clone of TbCSV V2 mutation showed no obvious symptoms after infecting the N.b,it's co-infection with TbCSB can lead to significantly reduced the leaf curl and shrinkage,the incidence and the virus accumulation,which showed that TbCSV V2 was an important protein required for virus pathogenesis.Then,the silencing inhibitory activity of TbCSV V2 was investigated.The results showed that the leaves of the plants inoculated with p GD-GFP + p GD-V2 had obvious green fluorescence,which was slightly less than the group of p GD-GFP +p GD-P19,and the negative control group p GD-GFP + p GD had no green fluorescence.Western blot experiments confirmed that the accumulation of GFP protein in the co-infiltrated leaves of p GD-GFP and p GD-V2 was similar to the positive control.These results showed that TbCSV V2,like P19,is an RNA silencing suppressor that suppresses post-transcriptional gene silencing(PTGS).In order to explore the biological function of V2 and analyze the expression pattern of V2 during the TbCSV infection,the invasive clone of Y35-FLAG was constructed.The results showed that,the expression of TbCSV V2 protein was highest at about 10 dpi after inoculation with N.b,15 dpi took the second place,and the lowest at 5dpi.The accumulation of TbCSV virus increased from 5dpi to the highest at 15 dpi,and then decreased after 15 dpi.V2 protein is composed of 115 amino acids.In order to clarify the key regions and amino acid positions where V2 to suppress RNA silencing,a series of truncated mutants was constructed and identified their silencing inhibitory activity through bioinformatics analysis of V2 functional domains.The results showed that the amino acids in position 6 to 110 of V2 protein had gene silencing inhibitory activity.In order to screen the key sites of V2 inhibiting RNA silencing,10 basic amino acids and 5 cyclic side-chain amino acids in V2 sequence that may be involved in RNA binding were mutated by alanine scanning through the screening results of multiple alignment and binding of amino acid sequences to key amino acid regions.The results showed that the conserved basic amino acids such as histidine,arginine and lysine or phenylalanine and tyrosine in the cyclic side chain amino acids in the mutant sites lost the silencing inhibitory activity of V2.It has been reported that co-expression of PVX with certain heterologous viral silencing suppressors will induce HR response,according to these results,the 9th phenylalanine and 105 th lysine that affect the silencing inhibitory activity of the V2 gene and the 105 th lysine that did not affect its silencing inhibitory activity were inserted into the PVX vector for systemic overexpression to clarify V2 correlation between silencing inhibitory activity and induced cell necrosis.The results showed that the mutation of phenylalanine at position 9 of V2 had no significant effect on its induced cell necrosis,while the position at 105 of lysine was significantly reduced.And V2 was 9th and 105 th amino acid mutations can lead to a significant reduction in large aggregates in the cytoplasm.