Application of reverse genetic systems to study Beet soil-borne mosaic virus and Beet necrotic yellow vein virus molecular biology, the interaction of species and their use as biotechnological tool

Two members of the Benyviridae family, Beet soil borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organisation, host range and high sequence similarity. To understand functional differences in molecular biology, pathogenicity mechanisms, symptom expression as well as interaction with the host and between viral species, a reverse genetic system represents a prerequisite. Infectious cDNA full length clones of both viruses were constructed by isothermal in vitro recombination. Artificial formation of BNYVV and BSBMV RNA1+2 reassortants were viable and spread long distance in N. benthamiana. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa. Moreover, fluorescence labelled full length clones were achieved by replacing a part of the RNA2 encoded coat protein read through domain. Co infection experiments with labelled BSBMV and BNYVV showed that both viruses remained spatially separated after N. benthamiana agroinoculation. In contrast, a mixture of BSBMV with an unrelated virus resulted in no co infection exclusion. In super infection experiments, BSBMV and BNYVV were unable to establish a secondary infection in plants that were previously infected with BNYVV or BSBMV. In addition, a virus induced gene silencing (VIGS) system was developed. Both viruses were equipped with fragments of the magnesium chelatase subunit H and phytoene desaturase genes. Silencing phenotypes in N. benthamiana induced by both target genes, were comparable to those described in the literature.