Shrinkage of Genome Size in a Plant RNA Virus upon Transfer of an Essential Viral Gene into the Host Genome

Tromas, Nicolas; Zwart, Mark P.; Forment, Javier; Elena, Santiago F.

Recombination between TEV-ÁNIb and the Transcripts from the 35::NIb Transgene Borja et al. (1999) reported that inoculation of transgenic N. benthamiana plants expressing the CP protein of tomato bushy stunt tombusvirus (TBSV) with TBSV mutants carrying defective CP resulted in the restoration of wild-type virus throughout a double-recombination event between the viral genome and the mRNA transcribed from the transgene. In sharp contrast, several authors have failed to detect recombination events between transgenes and potyviruses (Chung et al. 2007; Dietrich et al. 2007) . To test for possible recombination events in our system, we performed an evolution experiment consisting of five independent lineages of TEV-ÁNIb serially passaged four times in N. tabacum 35S::NIb transgenic plants, with each passage being 3 weeks long. At the end of the evolution experiment, we analyzed the evolved lineages by RT-PCR using a combination of primers that will specifically amplify full-genome viruses (supplementary table S1, Supplementary Material online). We found no evidence for recombination between TEV-ÁNIb and host-derived NIb transcripts. Therefore, we conclude that, in our experimental set up, regeneration of the wild-type genome by recombination is an unlikely phenomenon. This outcome is congruent with the estimates of within-host fitness for TEV and TEV-ÁNIb; recombinants analogous to the wild-type virus would be displaced by TEV-ÁNIb, and the process could occur rapidly as de novo variation will initially be present at low frequencies in the virus population. On the other hand, this result may reflect the possibility that recombination between TEV-ÁNIb and the transgene transcripts is very rare, as shown for other potyviruses (Chung et al. 2007; Dietrich et al. 2007 ).