Role of complementary-sense genes and intergenic region of beet curly top virus in intermolecular recombination frequency upon local infection in plants

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Intermolecular recombination is a critical process in the evolution of plant viruses, including geminiviruses. In this study, we investigated the impact of geminiviral factors on the homologous recombination frequency (HRF) in a model system involving replicons of the beet curly top virus (BCTVRepl) and the cotton leaf curl Multan betasatellite (CLCuMB), which lacks the β C1 gene. BCTVRepl-cGFP and CLCuMB-nGFP replicated together and exhibited intermolecular recombination, which was monitored by reconstituted GFP fluorescence. Mutation of the C2, C3, and C4 genes in BCTVRepl-cGFP led to a significant decrease in HRF. Accumulation of BCTVRepl-cGFP was reduced in the C3 mutant but not in the C2 and C4 mutants. These findings suggest that C4 and, to a greater extent, C2 play a role in homologous recombination (HR) between BCTV and CLCuMB replicons. Furthermore, the BCTV large intergenic region (LIR) sequence to which the C1 (Rep) protein binds to initiate viral replication was introduced into the nGFP construct (referred to as LnGFP). This resulted in a low level of HR (5%) compared to the control nGFP construct, where HR did not occur. Subsequent mutational analysis of the iteron and stem-loop sequences within the LIR of LnGFP revealed the role of these sequences and the C1 (Rep) in the process of DNA recombination. This study presents a new model for assessing HRF in BCTV, which can be applied to other geminiviruses. It also highlights the roles of the C2, C4, C1, and LIR structural sequences in intermolecular recombination and replication in the BCTV replicon. IMPORTANCE Intermolecular recombination is a critical process in the evolution, adaptability, and pathogenicity of plant viruses, including geminiviruses. This study developed a visible and quantifiable system to measure the frequency of homologous recombination between beet curly top virus (BCTV) and cotton leaf curl Multan betasatellite (CLCuMB) replicons based on the reconstitution of GFP fluorescence. Furthermore, mutation analysis of complementary-sense genes in the BCTV replicon indicated that C4 and, to a greater extent, C2 play a role in the recombination between the BCTV and CLCuMB replicons. Subsequent mutational analysis of the iteron and stem-loop sequences within the large intergenic region of BCTV revealed the possible role of these structural sequences in the recombination process.