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.

As we just described, serial passages of TEV in N. tabacum 35S::NIb led to the rapid occurrence of a whole spectrum of deletion variants. How would these deletion mutants further evolve in transgenic plants, and would their evolution be different from that of a full-length TEV? To address this question and gain insight into the long-term evolution of viruses harboring NIRV-induced deletions, we performed a second evolution experiment for both TEV and TEV-ÁNIb passaged in N. tabacum 35S::NIb, with five independent lineages for each virus. Li and Carrington (1995) showed that beta-glucuronidase (GUS) activity in leaves systemically infected by the TEV-ÁNIb-GUS mutant was comparable to that of the parental TEV-GUS. We found a similar result for the accumulation of TEV and TEV-ÁNIb in N. tabacum 35S::NIb using RT-qPCR ( fig. 3A) : there was not a significant difference in virus accumulation at 7 dpi (t 8 ¼ 1.394, P ¼ 0.201). The infection dynamics of the two viruses should therefore be similar, allowing us to compare the evolutionary outcomes. However, to compare the evolution of the full-length TEV and TEV-ÁNIb, we had to devise a setup in which the integrity of the NIb gene is ensured for TEV. Two previous studies have found that marker genes were highly stable in the viral genome when many 1-week passages were performed, although the markers were quickly lost in longer passages (Dolja et al. 1993; Zwart et al. 2014) . By performing 30 one-week passages with TEV and TEV-ÁNIb in N. tabacum 35S::NIb, we therefore hoped to avoid the occurrence of large genomic deletions in TEV. On the other hand, we realize that selection may not operate as effectively as during longer passages, due to short passage duration and frequent bottleneck associated to the mechanical transmissions among plants.