Analysis of recombination between viral RNAs and transgene mRNA under conditions of high selection pressure in favour of recombinants

Abstract

One possible environmental risk related to the utilization of virus-resistant transgenic plants expressing viral sequences is the emergence of new viruses generated by recombination between the viral transgene mRNA and the RNA of an infecting virus. This hypothesis has been tested recently for cucumber mosaic virus (CMV) by comparing the recombinant populations in transgenic and non-transgenic plants under conditions of minimal selection pressure in favour of the recombinants. Equivalent populations were observed in transgenic and non-transgenic plants but, in both, there was a strongly dominant hotspot recombinant which was shown recently to be nonviable alone in planta, suggesting that its predominance could be reduced by applying an increased selection pressure in favour of viable recombinants. Partially disabled I17F-CMV mutants were created by engineering 6 nt deletions in five sites in the RNA3 39-non-coding region (39-NCR). One mutant was used to inoculate transgenic tobacco plants expressing the coat protein and 39-NCR of R-CMV. A total of 22 different recombinant types were identified, of which 12 were, as expected, between the transgene mRNA and the mutated I17F-CMV RNA3, while 10 resulted from recombination between the mutated RNA3 and I17F-CMV RNA1. Twenty recombinants were of the aberrant type, while two, including the dominant one detected previously under conditions of minimal selection pressure, were homologous recombinants. All recombinants detected were very similar to ones observed in nature, suggesting that the deployment of transgenic lines similar to the one studied here would not lead to the emergence of new viruses.

Shrinkage of Genome Size in a Plant RNA Virus upon Transfer of an Essential Viral Gene into the Host Genome

“…Our study has important implications for the development and agronomic use of transgenic plants resistant to viruses generated by inserting complete viral genes into the plant genome, in particular by inserting viral replicase genes (reviewed in Kundu and Mandal [2001]). From a biosafety standpoint, it has been known for several years that recombination between viral sequences transgenically expressed by plants and virus genomes may occur (Turturo et al 2008;Morroni et al 2009), raising concerns about whether this transgenic strategy would favor the emergence of new viruses (Tepfer 2002;Thompson and Tepfer 2010). Here, we have illustrated a different evolutionary scenario: the expression by the plant of functional viral proteins creates the conditions in which viruses with long or even full deletions of the complemented protein may dominate the population, thus precluding their spread in nontransgenic plants.…”

Fixation of Emerging Interviral Recombinants in Cucumber Mosaic Virus Populations

“…We were not surprised to see interviral recombinants in the populations of reassortant viruses, as these have been described previously (12)(13)(14)(15)(16)23 Based on the sizes of the inserted fragments and depending on the insertion sites, we could distinguish several different recombinants ( Fig. 1), designated Rec-1 to Rec-5.…”

“…Several such recombinants described in the literature involved the 3= nontranslated region (3= NTR) of RNA 3 of CMV (13)(14)(15)(16)(17). In this work, we describe recombinants in the progenies of a virus obtained by reassortment between CMV strains Fny-CMV (subgroup IA) and LS-CMV (subgroup II) and we studied their fixation process in viral populations developed in competition experiments in five host plants.…”

The Search for Resistance to Cassava Mosaic Geminiviruses: How Much We Have Accomplished, and What Lies Ahead

“…Furthermore, because whole viral genes or long genome segments had been used in most hpRNA studies, there was the risk of recombination between the transgenes and the infecting virus genomes, leading to emergence of new viruses (Morroni et al, 2009; Tepfer et al, 2015). Indeed, Frischmuth and Stanley (1998) reported a recombination between an ACMV AV1 transgene in N. benthamiana and an agroinoculated ACMV DNA with a deleted AV1 gene, resulting in ACMV with a functional CP.…”