Evaluation of the Potential Role of Recombination in Virus-Resistant Transgenic Plants in the Emergence of Novel Viruses

Infections with two or more viruses occur frequently in nature, and when two related viruses replicate simultaneously in the same cells, genetic recombination between them can take place. Recombination can occur also between cellular and viral RNAs. Thus, virus-resistant transgenic plants (VRTPs) that express viral sequences could be a source of novel recombinant viral genomes. From a biosafety point of view, it is therefore important to understand if the recombinants that are generated in VRTPs are novel, and if they could contribute to an increased risk of emergence of recombinant viruses. To enhance the detection of host-messenger/viral RNA recombinants we analyzed them under conditions of high selection pressure in their favor. To do this, we studied transgenic tobacco plants expressing the coat protein and 3’ non-coding region (3’NCR) of R-CMV (Cucumber mosaic virus (CMV) subgroup II). Forty-four plants were inoculated with in vitro transcripts of a mutant I17F-CMV (CMV subgroup I) that had been attenuated by a 6-nt deletion in the 3'NCR of RNA3. Samples were taken from symptomatic plants 8, 10, and 15 days post-inoculation. The plants were screened for recombinant RNA3 by RT-PCR, and the amplicons were analyzed by cloning and sequencing. Many of the recombinants were different from those observed in the same plant line when inoculated with wild-type I17F-CMV under conditions of minimal selection pressure