Genetic Analysis And Efficient Construction Of Infectious Clone Of Papaya Leaf Distortion Mosaic Virus(PLDMV)

At present,the main control object of papaya virus disease in Hainan papaya is papaya ringspot virus,but in recent years,it has been found that PLDMV Potyvirus can infect not only non-transgenic papaya,but also transgenic papaya resistant to PRSV.And it may accelerate the spread of PLDMV,so PLDMV is a new threat to papaya production,which is as destructive as PRSV.In this paper,we construct the viral expression vector of full-length cDNA of PLDMV-DF and insert foreign GFP reporter gene to study PLDMV.It should provide more theoretical basis for the new strategy of prevention and treatment of PLDMV in the future.In this paper,agroinfection-compatible PLDMV full-length and fluorescent protein-tagged infectious cDNA clones driven by the Cauliflower mosaic virus 35S promoter were successfully constructed using one-step Gibson assembly.The clones were directly transformed into Agrobacterium tumefaciens to prevent potential problems such as plasmid instability during propagation in Escherichia coli.95%of papaya seedlings infected with PLDMV-GFP or PLDMV-mCherry developed systemic symptoms typical of those caused by wild-type PLDMV GFP green and mCherry red fluorescence was observed in leaves,stems,and roots of infected papaya plants.The full-length and fluorescent protein-tagged agroinfectious PLDMV cDNA clones were stable in papaya for more than 90 days and during six serial passages at 30-day intervals.The availability of these infectious clones will contribute to research on PLDMV-host interactions and can be applied in the papaya breeding program for PLDMV resistance.This is the first report of this new cloning strategy based on one-step In-Fusion method and direct A.tumefaciens transformation.This method is simpler,more efficient,and faster than traditional methods to construct agroinfection-compatible full-length infectious cDNA clones of plant viruses.In this study,we constructed the viral expression vector of PLDMV-DF full-length cDNA infection and inserted foreign GFP,mCherry reporter gene,which not only laid a theoretical and experimental basis for studying the pathology and molecular mechanism of PLDMV,but also for the prevention and treatment of PLDMV.It also provides a new method and building methods for the construction of infectious clone of Potato virus Y.