Neubauer, M.; Vollen, K.; Ascencio-Ibanez, J. T.; Hanley-Bowdoin, L.; Stepanova, A. N.; Alonso, J. M.

Viral vectors can be useful tools for expressing recombinant proteins as well as delivering gene-editing machinery. Despite their utility, the development and subsequent optimization of these tools is often a difficult and tedious process. Thus, although considerable work has been done to create useful viral vectors for gene editing and protein expression, there is a lack of understanding of how best to design these vectors for specific applications. For instance, it is often unclear whether the inclusion of heterologous promoter sequences or different viral components will improve cargo expression or replicon accumulation. To address some of these hurdles, we designed a GoldenBraid (GB) compatible viral vector system based on the geminivirus Beet curly top virus (BCTV). This system allows for simple, modular cloning of a variety of reporter constructs. Making use of this modular cloning strategy, we compared a variety of alternative viral vector architectures. Interestingly, native BCTV promoters outperformed the constitutive 35S promoter, while the removal of the BCTV virion-sense genes promoted reporter expression. Intriguingly, these modifications had no effect on total replicon accumulation. These results show the utility of the new modular BCTV-based viral vectors for protein expression and gene targeting applications, as well as uncover design principles that may inform future geminivirus-based viral vector architectures. We anticipate that the availability of this new modular system will spark the broad adoption of replicon-based strategies in protein expression and gene editing experiments in plants.