Geminiviral replicons and cell cycle regulation in sugar beet: investigating key factors for homologous recombination

• Applicant: Dr. Omid Eini, Ph.D.
• Subject Area: Plant Breeding and Plant Pathology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 453640441

SummaryGeminiviruses (Family Geminiviridae) are plant viruses with circular single-stranded DNA genomes that rely on host DNA replication machinery for their genome amplification. They replicate by rolling circle replication (RCR) and recombination-dependent replication (RDR) (Jeske et al. 2001). Therefore, they need to change the host cell cycle into S phase, in which DNA replication and homology-directed DNA recombination (HDR) occur. This scenario supports the high rate of recombination in geminiviruses which resulted in significant diversity among geminiviruses (Martin et al. 2011a). Similarly, HDR is promoted by geminiviral infection in Arabidopsis transgenic plants (Richter et al., 2014), although the viral elements required for this function remain to be determined. In addition, recently geminiviral replicons (GVRs) which contain replication-associated protein (Rep) and intergenic region (IR) have been used for efficient delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) (CRISPR-Cas) and repair DNA into plant cells. Using this system, a higher genome editing (GE) efficiency via HDR for functional gene modification or insertion was reported in tomato, tobacco and potato (Baltes et al. 2014; Čermák et al. 2015; Dahan‐Meir et al. 2018). However, the detailed role of GVRs on HDR pathway was not studied. Therefore, understanding the effect of Rep as well as other viral genes on HDR pathway in plant cells will shed light on both viral homologous recombination and HDR in plants. This may facilitate the way for increasing the efficacy of GE techniques in (crop) plants. This project aims to understand the role of genes (Rep; C2, C3 and C4) from the GVRs derived from Beet curly top virus (BCTV) and Beet curly top Iran virus (BCTIV) in homologous recombination in both viral and plants for generating more efficient GVR in terms of GE. To this end, a model based on a combination of the GVRs and a betasatellite, which replicates within the same cell will be developed for virus recombination assays. For understanding the role of GVR genes for induction of HDR in plant via CRISPR/Cas system, a simple and efficient assay based on the conversion of GFP into YFP in a model plant will be used. To further optimize GVRs for improving HDR in plants, key cell-cycle and recombinant associated genes will be selected from sugar beet transcriptome data from sugar beet and introduced into the GVRs. The efficacy of optimized GVRs for HDR will be tested in the same model plant. The improved and most efficient GVR will be used for GE in sugar beet in the future.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Mark Varrelmann