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.

To detect virus deletion variants with much greater sensitivity, Illumina NGS was performed on these five TEV lineages evolved for four 3-week passages. A TEV genome region encompassing the NIaPro, NIb, and CP cistrons (positions 6377-9473 of the wild-type genome) was amplified and sequenced (Materials and Methods). We developed a new approach for identifying deletions, adapting GSNAP (Wu and Nacu 2010) to detect deletions instead of introns (Materials and Methods). As a negative control, the same region was analyzed using NGS data for five lineages of TEV-eGFP evolved for three 9week passages in wild-type N. tabacum plants . No deletions were detected in the entire region, suggesting that our NGS analysis does not generate artifacts. The frequencies of deletion variants (see supplementary table S1, Supplementary Material online) in systemically infected leaves of transgenic plants were significantly higher than the frequencies found in our negative controls (Fisher's exact test, P < 0.001). The NGS results were consistent with RT-PCR data; the deletion variant detected by RT-PCR in Lineage 2 (positions 7621-8484) and Lineage 3 (positions 7046-8500) was the variant found at the highest frequency by NGS, 0.249 and 0.246, respectively. Sixty-four different deletion variants were observed in total, ranging in frequency from 8.44 Â 10 À5 (positions 6538-8478; Lineage 4) to 0.249 (positions 7621-8484; Lineage 2). Interestingly, 6.25% of deletion variants disrupted the reading frame of the coding sequence. Moreover, 43.75% of deletion variants extended into genes flanking NIb, meaning that probably only the remaining 56.25% variants could be viable when transcomplemented by the NIb expressed by the plant.