Construction Of An Infectious CDNA Clone Of Tobacco Vein Banding Mosaic Virus And Its Use For Cross-protection, Protein Over-expression And VIGS

Infectious cDNA clones are an important tool for investigating the pathogenesis of plantviruses. Using techniques of reverse genetics, an infectious cDNA clone will allow mappingthe viral determinants involved in virus replication, local and systemic movement, symptomdevelopment, and interactions between viral and host factors during virus infection in plants,and obtain the attenuated mutants for cross protection. Infectious cDNA clones of viruses canalso be developed into over-expression and/or VIGS (Virus induced gene silencing) vectorsfor vaccine or antibody production in plants and plant gene function research.Tobacco vein banding mosaic virus (TVBMV) is a distinct species of the largest plantvirus genus Potyvirus and can be transmitted by aphids in a non-persistent manner. In recentyears, the incidence of TVBMV has been increasing in major tobacco-growing areas of Chinaand causing significant yield losses in tobacco leaf production. In this research, weconstructed an introns-containing infectious cDNA of TVBMV, created the mild strains ofTVBMV for cross protection by mutating the conserved amino acids, and generated twovectors for overexpressing and silencing gene in palnts. The results were as following:(1) Determined the full length5′-UTR of TVBMV HN39using classic5′RACE methods. The5′-UTR of TVBMV HN39composed of171nucleotides, which is25nt longer than thatreported previously and contained both 'boxA'(ACAACAU, the 'potybox') and 'boxB'(UCAAGCA) sequences which are highly conserved in the Potyviridae family(2) Constructed infectious cDNA clones, which can be inoculated either by gene gun oragroinfiltration. The complete genome of TVBMV isolate HN39was cloned under theCauliflower mosaic virus35S promoter in pUC19vector. The clone (pTVBMV) wasstabilized by introducing three introns in the P3and CI-encoding regions. All the plants ofNicotiana benthamiana, N. tabacum and N. rustica inoculated with pTVBMV by particlebombardment showed similar symptoms and accumulated similar titers of virus as the plantsinoculated with wild type TVBMV HN39, indicating that pTVBMV is highly infectious. Wealso inserted the virus cassettes of pTVBMV into SalⅠ-SmaⅠ digested pCambia0390vector,and obtained the agroinoculation clone, pCamTVBMV. The green fluorescent protein (GFP)gene was inserted between the NIb and CP encoding regions and expressed stably bypCamTVBMV in the systemically infected N. benthamiana leaves, indicating suitability of pCamTVBMV-GFP for observation of viral movement in plants under UV light.(3) The effects of single amino acid mutations in TVBMV HC-Pro on viral virulence, RNAsilencing suppression and synergism were analyzed in N. benthamiana plants using infectiouscDNA clone pCamTVBMV-GFP. The mutations of IQC to IQI, NRT to ART, FR182NK toFINK, CD198N to CKN and IQN to DEN, alleviated the symptoms of pCamTVBMV-GFP inN. benthamiana plants significantly. The ELISA results for plants inoculated with thesemutants were negative, but real time RT-PCR given a positive results, indicating a role of thefive amino acids of HC-Pro in regulating the virulence of TVBMV. In contrast, the mutantsPAK (PTK to PAK) and APS (NPS to APS) delayed symptom development. Mutants EITC(RITC to EITC), AGS (NGS to AGS) and SQN (IQN to SQN) developed severe symptomsand accumulated virus similar to those of wild type virus. All the mutants were furtheranalyzed for their capacity in suppressing RNA silencing and mediating synergism with PVX.The results showed that five mutants with lower virulence lose both the activity of RNAsilencing suppression and synergism, indicating that these five amino acids were involved inregulation of virulence, RNA silencing suppression and synergism simultenaceously.(4) The cross protection effeciency of mild strain mutants with single, double, or triplemutations was assayed on N. benthamiana. The single mutant DEN caused milder mosaic,double mutant DEN+D198K induced milder vein clearing, while triple mutantDEN+D198K+ART caused inconspicuous symptoms. Cross protection tests indicated thedouble mutant DEN+D198K conferred the best cross proection efficiency and providedcomplete cross protection if inoculated10days before chanllenge inoculation.(5) Constructed over-expression vector and VIGS vector of TVBMV. We inserted multiplecloning sites (MCS-1and-2) exactly before and after coat protein (CP)-encoding sequenceinto TVBMV infectious clone. The gene of elicitin from phytophthora was cloned into MCS-1and the expression of elicitor induced a hypersensitive response in the agroinfiltrated leavesof N. benthamiana, this result showed that the TVBMV over-expression vector can be usedfor foreign proteins expression. We also constructed a VIGS vector, in which we insertedMCS before and after coat protein (CP) encoding sequence into mutant DEN. The GFP genewas cloned into MCS-1, and then inoculated onto N. benthamiana16C line byagroinoculation. Systemic silencing of GFP gene of N. benthamiana16C line was observed10days post inoculation, indicating that the TVBMV VIGS vector can be used for plantfunctional genomics research.