The Complete Nucleotide Sequence And Infectious Clones Of A Tomato Aspermy Virus Isolate

Tomato aspermy virus(TAV)is the type species of the genus Cucumovirus ,which includes Peanut stunt viru(sPSV)and Cucumber mosaic virus(CMV),in the family Bromoviridae. TAV is a positive-sense RNA (+ssRNA) virus with a tripartite genome, designated RNAs 1, 2 and 3 in decreasing order of their sizes. Mutation, recombination and reassortment are the three main mechanisms for genetic variation and new strains generation of TAV.TAV is distributed worldwide and has a remarkably host range, and can infects total more than 1000 plant species representing 24 Dicotyledons families and 3 Monocotyledoneae families including Chenopodiaceae and Solanaceae.In this study, a new strain of TAV (designated TAV-BJ) was isolated from natural infected Solamum lycopersicum collected from Beijing suburb, in the year 2009.Host experiment, double strand RNA(dsRNA) extraction, virion extraction, Northern blotting were employed to investigate the characteristic of this strain of TAV.Complete nucleotide sequence was obtain throught TA clone method.The complete sequence analysis results of TAV-BJ revealed that TAV-BJ RNA1 contained 3409 nucleotides (nts), encoding 1a protein of 994 amino acids corresponding to the sequence from 96 to 3077.TAV-BJ RNA2 contained 3032 nucleotides (nts) , encoding 2a protein of 829 amino acids and 2b protein of 78 amino acids, corresponding to the sequences from 86 to 2574 and 2447 to 2680 respectively. RNA3 contained 2218 nts, encoding 3a protein of 247 amino acids and CP of 219 amino acids, corresponding to the sequences from 192 to 932 and 1227to 1883 respectively. RNA1 and RNA2 of TAV-BJ showed approximately 98.6% nucleotide sequence identity with those of KC strain and V strain. The alignment of the TAV-BJ RNA3 nucleotide sequence to V strain(99.6% sequence identity)is higher than KC strain(99.1% sequence identity).The results of phylogenetic analysis of 5 ORFs between TAV-BJ and other 2 strains also revealed that TAV-BJ was high identity with those fully researched TAV strains.The infectious clone of TAV-BJ full-length RNA and TAV-BJ RNA2 2b gene deletion (RNA2△2b) were construced by cloneing into pUC18 vector with one T7 promoter, and their infectious RNA were obtained by in vitro transcription menthod. pT1, pT2, pT3 and pT2△2b were coresspond to the infectious clone of TAV RNA1, RNA2, RNA3 and RNA2△2b.The host experiment was employed to certificate the available of those infectious RNA.CMV-FnyΔ2b, a CMV derivation with 2b gene deletion,show light or even no pathogenicity in host as the absence of the RNA Silencing Suppressor(RSS) 2b protein.The result of co-inoculation of T1, T2 ,T3 and T2△2b respectively with CMV-Fny△2b showed that the recombinant virus with T2 or T3 could cause the pathogenicity in host, so there may be a protein in T3 that palys the some function like the identified RSS TAV 2b protein in T2.The TAV RNA3 chimera infectious clone that replaced with 3a or CP gene fragment of CMV-Fny were constructed by Overlapping PCR, and designated as T3F3a and T3FCP ,respectively.CMV-Fny RNA1 and CMV-Fny RNA2△2b were co-inoculation with T3F3a and T3FCP,respectively.The host experiment show that the recombinant virus with T3FCP ,a chimera virus with TAV 3a gene and CMV-Fny CP gene,still cause the pathogenicity in host,however not the recombinant virus with T3F3a,a chimera virus with CMV-Fny 3a gene and TAV CP gene,and indicated that TAV 3a protein may compensated the function of 2b protein.To further identify the candidated RNA Silencing Suppressor, the transient expression assay-argobacterium co-infiltration experiment was employed.The agrobacteria transient expression vector 35S-GFP (Green Fluorescent Protein) gene could silence the GFP gene in the GFP transgentic Nicotiana benthamiana plant (line 16c) by gene silencing way. In this paper, we constructed agrobacteria transient expression vectors of TAV 2b gene (35S-T2b) and TAV 3a gene (35S-T3a). When agrobacteria with constructed transient expression vector co-infiltrated with 35S-GFP agrobacteria on GFP transgentic N.benthamiana plant (line 16c), the zones co-infiltrated with 35S-GFP and 35S-T2b or 35S-T3a were still bright green(GFP experssed) after 3 days post infiltration under the long UltraViolet light whlie the zones infiltrated with 35S-GFP were red(GFP silenced) .The agrobacteria transient expression vectors of Fny 2b gene(35S-F2b) and Fny 3a gene(35S-F3a) were also constructed as control,and the restlut was that zones co-infiltrated with 35S-F2b and 35S-GFP were still bright green after 3 days post-infiltration under the long UltraViolet light whlie the zones co-infiltrated with35S-F3a and 35S-GFP were red.It seems that TAV 3a protein suppressed local silencing as like 2b protein,and inferred that the 3a protein may be the second RNA Silencing Suppressor in TAV.