Establishment Of Virus Induced Gene Silencing Using Geminivirus DNA1 Molecule And Its Application

Virus induced gene silencing(VIGS)is currently recognized as a powerful reverse genetics tool for application in functional genomics.VIGS is induced by infecting a plant with a plant virus that has had its genome modified to include a sequence identical to that in RNA transcribed from the host gene to be silenced. DNA1,a single-stranded DNA molecule associated with begomoviruses(Family Geminiviridae),was shown to replicate autonomously but required the helper virus for its dissemination.In this paper,we developed a VIGS vector based on the DNA1 component of Tobacco curly shoot virus(TbCSV),a monopartite begomovirus,by inserting a multiple cloning site(MCS)between the Rep ORF and the A-rich region for subsequent insertion of DNA fragments of genes targeted for silencing.When the modified DNA1 vector was inserted with host gene(sulfur,Su)or transgene(green fluorescent protein,GFP)and co-agroinoculated with TbCSV,efficient silencing of the cognate gene was observed in Nicotiana spp.plants.More interestingly,we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic TV.benthamiana or endogenous Su in tobacco plants with Tomato yellow leaf curl China virus(TYLCCNV),another monopartite begomovirus that does not induce any viral symptoms in plants.A gene-silencing system in Nicotiana spp.,Solarium lycopersicum and Petunia hybrida plants was then established by using TYLCCNV and the modified DNA1 vector.The system can be used to silence genes involved in meristem and flower development,and to silence two genes simultaneously.We also developmented the TbCSV and 2mDNA1 silencing system in N.tabacum.The system could successfully silence theβ-glucuronidase(GUS)gene in the transgene plants and Su(sulphur desaturase)gene in five N.tabacum cultivars.Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants,and expressed in a variety of tissues and organs including leaf,shoot,stem,root and flower.The system works well in N.tabacum plants between 18-32°C.We used the 2mDNA1 vector to study gene functions in plants.The modified DNA1 vector was used to silencing the AtTOM homologue genes NbTOM1 and NbT0M3 in N.benthamiana,silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to lower level and silencing of the both genes simultaneously can completely inhibit tobamovirus multiplication.We investigated the role of NtEDS1 in the N-mediated resistance to Tobacco mosaic virus(TMV).Our VIGS analysis suggested that resistance against TMV mediated by tobacco N was NtEDS1-dependent.The 2mDNA1 silencing vector was also used to study the function of NbPHAN,an AS1/PHAN orthologue gene,in leaf development.We found leaf lose adaxial/abaxial polarity when NbPHAN was silenced.In the absence of NbPHAN,dowmwards curly and crimple leaf and de novo reinitiation of ectopic leaf blades along the midvein were observed.Using yeast two hybridization approach,we found NbPHAN can interact with AtAS2,and deletion analysis indicated that the middle domain has important role for this interaction.We also found a conserved C-terminal domain(CTD)mediate homodimerization of NbPHAN.Subcelluar localization of NbPHAN using GFP-NbPHAN fusion protein showed it was exclusively localized in nucleolus.Using deletion mutant,we found the MYB domain of NbPHAN determined this localization.The role of NbPHAN in pathogenicity of geminivirus infection will be further investigated.