Evaluating the Impact of Viral Markers

To test whether eGFP-based marker genes had an important effect on the biological characteristics of the virus, N. tabacum plants were infected with and equivalent doses of virions of TEV or TEV-eGFP [14] (link). Virions were quantified by means of a quantitative real-time RT-PCR (RT-qPCR), using PrimeScript RT-PCR kit II (TaKaRa), on the coat protein (CP) (primers: 5′-TTGGTCTTGATGGCAACGTG-3′ and 5′-TGTGCCGTTCAGTGTCTTCCT-3′). A Prism 7500 sequence analyzer (Applied Biosystems) was used, as well as Prism 7500 software, version 2.0.4 (Applied Biosystems), to analyze the data. All aerial plant tissue except the inoculated leaf were collected 7 dpi, RNA was extracted, and a second RT-qPCR using CP primers was performed to determine accumulation. There was not a significant difference in accumulation levels between TEV and TEV-eGFP (t-test on log₁₀-transformed data: t₁₄ = 0.754, P = 0.463). Therefore, biological properties of the marked virus are similar to those of the wild-type virus. On the other hand, the insertion of marker proteins does appear to affect viral within-host competitive fitness [21] (link), [37] (link).