Development of convenient potyvirus-based vectors for simultaneous expression of two heterologous proteins in solanaceous plants

Plant virus-based vectors can deliver foreign nucleic acid sequences into host plants for expression, serving as powerful biotechnological tools in agricultural applications, such as plant functional genomic study, agronomic trait improvement, pest management, genome editing. Viruses in the genus Potyvirus represent the largest group of plant-infecting RNA viruses, employing polyprotein processing as the genome expression strategy. A variety of potyvirus-based vectors have been documented, whereas the ones with a simple cloning strategy for simultaneous expression of two heterologous proteins await to be exploited. In this study, we developed an infectious cDNA clone of a potato virus Y (PVY) isolate PVY-N, termed pPVY. Next, we engineered Golden Gate-compatible derivatives of pPVY and pPVMV (an infectious cDNA clone of an attenuated strain of pepper veinal mottle virus) by incorporating Type IIS restriction enzyme sites into the P1/HCPro and NIb/CP junctions of the viral genomes. The resulting recombinant clones, pPVY-2×GoldG and pPVMV-2×GoldG, are highly infectious in their natural Solanaceae hosts. Both vectors allow simultaneous insertion of two reporter genes (mCherry and GFP) for robust expression in Nicotiana benthamiana and N. tabacum (for PVY) and N. benthamiana and pepper plants (for PVMV). Serial passage revealed that the recombinant viruses harboring two heterologous genes were genetically stable. Considered the fact that both viruses have a wide host range in Solanaceae plants, the PVY- and PVMV-based vectors would serve as valuable biotechnological tools and have a promising application prospect.