Geminivirus-derived replicons: assessment for transient GFP expression in tobacco and tomato

Global food security requires innovative strategies for sustainable crop improvement. Gene editing offers a precise and rapid approach to plant modification, but its success depends on efficient delivery and robust expression systems. Geminivirus-derived replicons (GVRs) enhance transient expression by amplifying introduced DNA within plant cells. In this study, we evaluated three previously deconstructed geminiviral backbones -Bean yellow dwarf virus (BeYDV), Tomato leaf curl virus (ToLCV), and Wheat dwarf virus (WDV)- against a non-replicating T-DNA control for their ability to sustain GFP expression in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). Constructs were delivered via Agrobacterium tumefaciens, and in planta GVR circularization was verified, with accumulation levels dependent on the specific replicon and host species. GFP RNA and protein accumulation was assessed by RT-qPCR, fluorescence imaging, and ELISA; all GVRs prolonged GFP fluorescence relative to control. In tobacco, transcript levels increased significantly by 3 days post infiltration (dpi), reaching up to 221- fold by 6 dpi with BeYDV, while BeYDV and ToLCV produced approximately fivefold higher protein levels. In tomato, ToLCV and WDV showed the strongest enhancement, with transcript and protein levels increasing up to 6.3-fold and 2.4-fold, respectively. These results demonstrate that GVRs markedly enhance and extend transient gene expression in solanaceous hosts, with performance dependent on the replicon and plant species. ToLCV and BeYDV were most effective in tobacco, whereas ToLCV and WDV performed best in tomato. Overall, GVRs represent versatile tools for transient protein production and for improving the delivery and efficiency of genome-editing reagents in plants.