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.

Concentrated saps of TEV and TEV-DNIb were obtained by grinding 500 mg of 7 days postinoculation (dpi) infected tissue in a mortar with 500 ml of grinding buffer. Nicotiana tabacum 35S::NIb plants were inoculated with a 1:1 mixture of homogenates of both viruses. Twenty plants were inoculated by abrasion of the third true leaf with 30 ml mixed homogenate, and 20 plants were taken as a mock-inoculated control. After 7, 21, and 63 dpi, total RNA was extracted from the youngest leaves of five plants. The region flanking the NIb cistron was reverse transcribed using Moloney Murine leukemia virus (M-MLV) reverse transcriptase (RT) (Fermentas) and primer 97-101-R (supplementary table S1, Supplementary Material online). PCR amplification was performed using the high-fidelity Phusion DNA polymerase (Finnzymes) and the 73-80-F/ 92-96-R primers (supplementary table S1, Supplementary Material online). PCR products were resolved on a 1% agarose gel to verify the presence of both genotypes. RT-qPCR was performed to compare viral accumulation in case of direct competition between the two genotypes. RT-qPCR for the CP cistron was used to determine the total level of viral accumulation (i.e., accumulation of both genotypes), using the specific primers TEV-CP-qPCR-F and TEV-CP-qPCR-R (supplementary table S1, Supplementary Material online). RT-qPCRs were performed using One-Step SYBR PrimeScript RT-PCR kit II (Takara) following the instructions provided by the manufacturer. CP was chosen because it locates in the 3 0 end of TEV genome and hence would only quantify complete genomes but not partial incomplete amplicons. Each RNA sample was quantified three times in independent experiments. Amplifications were done using the ABI PRISM Sequence Analyzer 7000 (Applied Biosystems). The thermal profile was as follows: RT phase consisted of 5 min at 42 C followed by 10 s at 95 C; and PCR phase of 40 cycles of 5 s at 95 C and 31 s at 60 C. Quantification results were examined using SDS7000 software v. 1.2.3 (Applied Biosystems). RT-qPCR for NIaPro/NIb cistrons was then performed to determine viral accumulation of the TEV genotype only, using specific primers TEV-NIaPro/NIb-F and TEV-NIaPro/NIb-R (supplementary table S1, Supplementary Material online) and otherwise identical conditions when compared with the quantification of the CP. The ratio of TEV-DNIb to TEV (R) is then R ¼ (n CP Àn NIaPro/NIb )/n NIaPro/NIb , where n CP and n NIaPro/NIb are the copy numbers of the CP and NIaPro/NIb, respectively, as measured by RT-qPCR. To estimate the replicative advantage W (Carrasco et al. 2007 ) of TEV-DNIb, we performed linear regression on the infection time in days and the log-transformed ratio data. The antilog-transformed slope is W.