Identification of efficient geminivirus-derived LIR-elements for exogenous protein expression

Viral vectors have emerged as powerful platforms for producing medical and metabolite products. To explore how diverse regulatory components can enhance gene expression, and leveraging the abundant diversity of long intergenic regions (LIRs) in geminiviruses, we systematically screened the bidirectional promoter activities of 209 LIR fragments from 8 genera within the Geminiviridae family. This screening was performed using a dual-reporter vector (GFP/Firefly luciferase) via transient expression in Nicotiana benthamiana, leading to the identification of four highly active LIR elements (LIR-2, 37, 51, and 62) derived from three distinct genera. Corresponding geminivirus expression vectors were then constructed based on these LIRs. Among them, the GM2, GM37, and GM62 vectors significantly enhanced GFP/GUS expression levels, showing 1.27 ± 0.20- to 1.82 ± 0.17-fold and 1.16 ± 0.10- to 1.64 ± 0.33-fold increases at 3 dpi, respectively, compared to the cauliflower mosaic virus 35S promoter in the pCAMBIA1300 plant vector. Furthermore, these vectors successfully expressed the medically relevant protein human papillomavirus 16 L1 (HPV16 L1), which is typically difficult to express using the pCAMBIA1300 vector. The GM2 vector exhibited the highest expression level, reaching 1.70 ± 0.38 times that of GM37, underscoring its potential as a superior expression platform. Collectively, the LIR elements identified in this study enrich the toolbox for developing virus-based expression vectors in plants, and are particularly suitable for applications in plant bioreactors requiring simultaneous expression of multiple target molecules.