Bioluminescence-Driven Optimization of Geminivirus-Based Vectors as Tools for Plant Biotechnology

Viral replicons are valuable tools in plant biotechnology, widely utilized to increase recombinant protein production. Their ability to amplify gene dosage in a trigger-dependent manner also opens doors to regulatory applications. This work focuses on optimizing geminivirus-based vectors for Synthetic Biology applications in plants, using autobioluminescence as a sensitive, real-time reporter to characterize gene expression. Specifically, geminivirus-based synthetic replicons derived from bean yellow dwarf virus (BeYDV), tomato yellow leaf curl virus (TYLCV), and beet curly top virus (BCTV) were engineered and assessed for basal expression, inducibility, and recombinant protein coexpression potential. Our study provided insights into the strengths and limitations of each geminiviral replicon. BeYDV replicon displayed a robust activation profile suitable for complex tasks such as multigene expression, while TYLCV showed high expression levels despite moderate basal leakage. In contrast, BCTV demonstrated less favorable control and expression levels. Through a bioluminescence-based screening, the TYLCV system was further optimized to improve regulatory precision. These findings highlight the versatility of geminivirus replicons, paving the way for future engineering of synthetic gene circuits in plants.

This research was supported by the Ministerio de Ciencia e Innovación (Spain) through Agencia Estatal de Investigación (grant PID2022-141438OB-I00 to D.O. and grant PID2022-139376OB-C31 to E.R.B. and A.G.C.), the European Commission─NextGenerationEU through the Ministerio de Ciencia e Innovación (Spain) and Agencia Estatal de Investigación (grant TED2021-131171B–I00). Work in R.L-D.’s laboratory is partially funded by the Excellence Strategy of the German Federal and State Governments. E.G.-P. is recipient of ACIF-2020 fellowship (Generalitat Valenciana). KSS’ lab is funded by the MRC Laboratory of Medical Sciences (UKRI MC-A658-5QEA0) and UKRI Biotechnology and Biological Sciences Research Council through the International Science Partnerships Fund (ISPF) grant number UKRI249

Peer reviewed