CBN-V Video Archives - S8-23
Disecting the Role of Subgenomic Promoter Elements during Homologous Recombination of Brome Mosaic Virus

Wierzchoslawski R., A. Dzianott and J.J. Bujarski

Plant Molecular Biology Center and the Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115 jbujarski@niu.edu

        Previously, we have mapped the intergenic region in the RNA3 segment of a model brome mosaic virus (BMV) to function as an active homologous recombination "hot spot". In this work, the BMV RNA3 constructs, bearing a duplication of the intergenic region downstream to the coat protein ORF, were used to determine the homologous recombination activity within the restriction enzyme marker sites. The engineered RNA3 variants were inoculated pair-wise on the Chenopodium quinoa host plants, and the recombinant RNAs were identified based on the restriction patterns and on sequencing of the RT-PCR cDNA products. The region composed of the subgenomic promoter core, the polyA tract and a downstream hairpin supported nearly 25% of homologous exchanges. Control infections revealed that the crossover frequency in the downstream neighboring region was very low. Also, inactivation of the subgenomic promoter activity inhibited the recombination activity. The in vitro experiments have demonstrated that the observed recombinants were not the RT-PCR artifacts. Our results show that the elements of subgenomic promoter are capable of supporting the efficient exchanges between molecules of the same RNA component during viral infection.

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