Generation of stable infectious clones of plant viruses by using Rhizobium radiobacter for both cloning and inoculation

A novel Rhizobium radiobacter (synonym Agrobacterium tumefaciens)-mediated approach was developed to generate stable infectious clones of plant viruses. This method uses R. radiobacter for both cloning and inoculation of infectious clones, bypassing the requirement of cloning in E. coli to avoid the instability. Only three steps are included in this method: (i) construct viral genome-encoding plasmids in vitro by one-step Gibson assembly; (ii) transform the assembled DNA products into R. radiobacter; (iii) inoculate plants with the R. radiobacter clones containing the viral genome. Stable infectious clones were obtained from two potyviruses papaya ringspot virus (PRSV) and papaya leaf distortion mosaic virus (PLDMV) using this method, whereas attempts utilizing "classical" E. coli cloning system failed repeatedly. This method is simple and efficient, and is promising for a wide application in generation of infectious clones of plant virus, especially for those which are instable in E. coli.