Analysis of recombination between viral RNAs and transgene mRNA under conditions of high selection pressure in favour of recombinants

One possible environmental risk related to the utilization of virus-resistant transgenic plants expressing viral sequences is the emergence of new viruses generated by recombination between the viral transgene mRNA and the RNA of an infecting virus. This hypothesis has been tested recently for cucumber mosaic virus (CMV) by comparing the recombinant populations in transgenic and non-transgenic plants under conditions of minimal selection pressure in favour of the recombinants. Equivalent populations were observed in transgenic and non-transgenic plants but, in both, there was a strongly dominant hotspot recombinant which was shown recently to be nonviable alone in planta, suggesting that its predominance could be reduced by applying an increased selection pressure in favour of viable recombinants. Partially disabled I17F-CMV mutants were created by engineering 6 nt deletions in five sites in the RNA3 3'-non-coding region (3'-NCR). One mutant was used to inoculate transgenic tobacco plants expressing the coat protein and 3'-NCR of R-CMV. A total of 22 different recombinant types were identified, of which 12 were, as expected, between the transgene mRNA and the mutated I17F-CMV RNA3, while 10 resulted from recombination between the mutated RNA3 and I17F-CMV RNA1. Twenty recombinants were of the aberrant type, while two, including the dominant one detected previously under conditions of minimal selection pressure, were homologous recombinants. All recombinants detected were very similar to ones observed in nature, suggesting that the deployment of transgenic lines similar to the one studied here would not lead to the emergence of new viruses.