Virus-resistant transgenic plants : benefits, risks and biosafety

Virus-resistant transgenic plants (VRTPs) were first reported two decades ago. However, since the early 1990s, concerns have been raised regarding the potential environmental impact of VRTPs, in particular those which express viral genes. One hypothetical risk is that viral sequences expressed from the transgene could recombine with the genome of a non-target virus, and thus lead to emergence of a novel virus. Recombination between viral RNA and transgene mRNA has already been shown to occur for several viruses under conditions of high selection pressure: when the inoculated virus has been disabled by a genomic deletion. Recent work carried out by the Virology Group at Ca’Tron has focused specifically on cucumoviruses (family Bromoviridae), a genus where viral recombinants have been detected both in the field and laboratory. A sensitive and robust RT-PCR method has been optimized to detect viral recombinants both under conditions of high and low selection pressure, where the virus inoculated is either disabled or wild-type, respectively. Results, under conditions of low selection pressure, showed that those recombinants found in tobacco plants infected with two different cucumoviruses were comparable to those found in tobacco transgenic for part of one cucumovirus genome infected with another cucumovirus. It was therefore concluded that in this specific case novel viral recombinants are not expected to appear. Conditions of high selection pressure for the analysis of recombinants were produced by engineering mutants with six nucleotide deletions at various points along the 3’ non-coding region of Cucumber mosaic virus (CMV) strain I17F. Results so far show an increase in the number of observed recombination sites compared to the low selection pressure study. Detailed studies on and around one of the principal recombination hotspots found (nt 1902) have shown it to be directly involved in the production of a short subgenomic RNA known as RNA5. The RNA structure around hotspot nt 1902 has been determined both in vitro and in silico, with phylogenetic and recombinant analyses identifying its putative role in cucumovirus evolution. It is intended that this study should eventually provide a model for assisting researchers in designing safer viral transgenes, while improving our understanding of the mechanism(s) involved virus RNA recombination, and therefore evolution.