Stability of Tobacco etch virus infectious clones in plasmid vectors.

Tobacco etch virus (TEV) has been traditionally used as a model to research many aspects of the molecular biology of plant RNA virus and, more recently, experimental evolution. However, the only plasmid of this virus species with an infectious clone that has been commonly available to research (pTEV7DA) is rather unstable when propagated in the bacterium Escherichia coli. Here, the TEV infectious clone contained in pTEV7DA is used to construct three new plasmids that allowed infecting the host plants from RNA transcripts synthesized in vitro (pMTEV), directly from plasmid DNA (p35TEV) and by agroinoculation (pGTEV). To increase stability of the three constructed plasmids in E. coli, superfluous vector sequences were removed and the virus expression cassettes were inserted between the plasmid replication origins and antibiotic selection markers in reverse orientation to the latter gene. Although the TEV cDNA in these three new plasmids is not interrupted by any exogenous sequence, they are more stable than the parental pTEV7DA during propagation in E. coli, indicating a major contribution of the plasmid context in virus cDNA stability. Using the different inocula produced from the three new plasmids the TEV infectivity was also compared. The results showed that agroinoculation is the most effective inoculation method and is where symptoms unfold earlier. (c) 2010 Elsevier B.V. All rights reserved.