Geminiviral replicons and cell cycle regulation in sugar beet: investigating key factors for homologous recombination
Final Report Abstract
Geminiviruses (Family Geminiviridae) rely on host DNA replication machinery for their genome amplification. They need to change the host cell cycle into S phase, in which DNA replication and homology-directed DNA recombination (HDR) occur. In addition, HDR is promoted by geminiviral infection in Arabidopsis transgenic plants, although the viral elements required for this function remain to be determined. Recently, geminiviral replicons (GVRs) including the Beet curly top virus (BCTV) replicon have been used for efficient delivery of CRISPR/Cas components for targeted genome editing in plant cells. However, the detailed role of GVRs on HDR pathway was not studied. Further, understanding the effect of GVRs on the regulation of HDR and cell cycle genes will shed light on both viral homologous recombination and HDR in plants. This project aimed to understand the role of genes (Rep, C2, C3 and C4) from the GVRs derived from BCTV in homologous recombination in both viral and plants for generating more efficient GVRs in terms of GE. For this, first, a model based on a combination of the BCTV replicon and Cotton leaf curl betasatellie (CLCB), which replicates only in the presence of the helper virus was developed to test virus recombination. Then, the role of BCTV replicon genes on the virus HDR was tested after mutation of each gene. This showed that C4 and more significantly C2 have clear effects on the rate of HDR, while C3 enhanced HDR events because of higher virus replication. Transcriptome data showed that both BCTV and BCTIV replicons induce expression of HDR and cell cycle genes in locally infected B. vulgaris plants. Initial results showed that coinfiltration of some of these genes including RAD51, BLM, XRCC4 and Histone H4 with BCTVRepl-cGFP and CLCB-nGFP improved the number of GFP cells following the HDR event. Further, the effect of selected HDR and cell cycle genes was tested in gfp mutant Nicotiana benthamiana plants for HDR events using CRISPR/Cas system. The results showed that BCTV replicon improves HDR rate in gfp mutant plants significantly compared with T- DNA. However, the effect of the selected HDR and cell cycle genes on such HDR events remained inconclusive. In conclusion, we developed a model that phenotypically evaluates the rate of HDR between two geminiviruses in a short time (3 days). This model can be used to investigate host or viral factors that control BCTV recombination. Transcriptome data highlighted signalling pathways, defence response and cell cycle regulators in B. vulgaris in response to local and systemic BCTV and BCTIV infection, which can be used for improving resistance to the virus infection. Outstanding HDR and cell cycle genes for improving HDR can be considered for improving the efficacy of genome editing in B. vulgaris in the future.
Publications
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Regulation of cell cycle genes by geminiviral replicons in sugar beet. International Advances in Plant Virology. Poster presentation3 (P3). Online meeting
Omid Eini & Mark Varrelmann
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Beet curly top virus factors for intermolecular recombination frequency upon local infection in plants. International Advances in Plant Virology. Ljubljana, Slovenia. Poster presentation (P48)
Omid Eini & Mark Varrelmann
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Differential response of sugar beet genotypes to the local and systemic Beet curly top Iran virus infection: virus accumulation and transcriptome assay. 12th International Congress of Plant Pathology. Lyon, France, Poster presentation.
Omid Eini & Mark Varrelmann
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Evidence for the splicing of complementary -sense transcripts of Beet curly top Iran virus (BCTIV). 55. Tagung des DPG- Arbeitskreises “Viruskrankheiten der Pflanzen“. March 20th and March 21th 2023, Julius- Kühn-Institute, Berlin. Platform presentation.
Omid Eini , Saedeh Ebrahimi & Mark Varrelmann
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Response of sugar beet cultivars to Beet curly top virus infection: virus accumulation and transcriptome assay. ASSBT Biennial Meeting, Savannah, Georgia, USA, Platform presentation.
Mark Varrelmann & Omid Eini
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Insights into the molecular basis of beet curly top resistance in sugar beet through a transcriptomic approach at the early stage of symptom development. Journal of General Virology, 105(9).
Eini, Omid; Benjes, Kristin; Dietrich, Katrin; Reichelt, Michael; Schumann, Nadine & Varrelmann, Mark
