Rescue of in vitro generated mutants of cloned cauliflower mosaic virus genome in infected plants

Abstract

Cauliflower mosaic virus (CaMV) DNA is an attractive candidate as a vector for introducing foreign DNA into plants because the DNA can be directly introduced into plants by rubbing it on to leaves^1–4. The CaMV genome is a circular double-stranded DNA of about 8 kilobases (kb), and has been entirely sequenced⁵. DNA isolated from the virus has a ‘tangled’ or ‘knotted’ secondary structure⁶ and contains two or three (depending on the isolate) site-specific single-strand interruptions^5,7,8. Cloned viral DNA, when excised from its recombinant plasmid, is infective in linear form. The infected plant can recircularize the DNA ‘knot’ and reintroduce the site-specific single-strand interruptions¹. For CaMV to be a useful vector, sites in the genome are required into which foreign DNA can be inserted without destroying infectivity, and ideally the inserted DNA would be expressed under viral control. Here we report the generation in vitro of small insertion and deletion mutations in cloned CaMV DNA. Most mutations destroy the infectivity of the viral DNA except for one, a linker insertion in the large intergenic region. Co-infection of plants with non-overlapping mutants usually leads to the production of virus particles, which seem to be produced by recombination.