Biomics

Genome-editing strategies have recently emerged as promising tools to impart desired properties to many eukaryotic species, including plants. This technology can CRISPR/Cas9 be used to engineer plant resistance to narrow or wide range of pathogens, reproductive developmental features and other plant properties. It is known that EDS1 arabidopsis protein controls protection activation and programmable cell death due to intercellular Toll-like immune receptors that recognize specific pathogen effectors. Unfortunately, the involvement of EDS1 protein in the antiphytoviral immunity of potato plants has not been studied. Meiosis has a special place in the system of sexual and seeds-without-sex reproduction. Key meiosis genes, and above all the DYAD / SWI1 gene, are a potential candidate in the search for apomixis genes. Binary vectors were obtained on the basis of plasmid pBAtC by the restriction-ligase method. Thus, three expression vectors (p01, p03 and p04) were created for editing the locus EDS1. Two expression vectors (pII-25 and pVIII-29) were created to introduce mutations in the second and eighth exons of the DYAD /  SWI1 arabidopsis gene. In all cases, the presence of cloned inserts was confirmed by DNA sequencing. The created p01, p03, p04 vectors under the pAtU6-6 arabidopsis promoter and the previously obtained p13 vector under the potato pStU6 promoter are used in the work on bioballistic transformation of potato plants in vitro.

CRISPR, genome editing, EDS1 locus, DYAD / SWI1 gene, pBAtC vector, guide RNA, DNA sequencing, arabidopsis, potato

1. Agashe B., Prasad C.K., Siddiqi I. Identification and analysis of DYAD a gene required for meiotic chromosome organisation and female meiotic progression in Arabidopsis. Development. 2002. V.129. P.3935–3943.
2. Boateng K.A., Yang X., Dong F., Owen H.A., Makarof C.A. SWI1 is required for meiotic chromosome remodeling events. Mol Plant. 2008. V.1(4). P.620–633. doi: 10.1093/mp/ssn030
3. Gantner J., Ordon J., Kretschmer C., Guerois R., Stuttmann J. An EDS1-SAG101 Complex is Essential for TNL-mediated Immunity in Nicotiana benthamiana // Plant Cell. 2019. V.31. P.2456–2474. DOI: 10.1105/tpc.19.00099
4. Gerashchenkov G.A., Rozhnova N.A. Kuluev B.R. Design of Guide RNA for CRISPR/Cas Plant Genome Editing. Molecular Biology. 2020. V. 54. P. 24–42. doi: 10.1134/S0026893320010069
5. Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York.
6. Kim H, Kim ST, Ryu J, Choi MK, Kweon J, Kang BC, Ahn HM, Bae S, Kim JS, Kim SG. A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system. J Integr Plant Biol. 2016. V.58(8). P.705-712. doi: 10.1111/jipb.12474
7. Mercier R. SWITCH1 (SWI1): A novel protein required for the establishment of sister chromatid cohesion and for bivalent formation at meiosis. Genes Dev. 2001. V. 15. P.1859–1871.
8. Peart J.R., Cook G., Feys B.J., Parker J.E., Baulcombe D.C. An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus. Plant J. 2002. V.29(5). P.569-579.
9. Ravi M., Marimuthu M.P., Siddiqi I. Gamete formation without meiosis in Arabidopsis. Nature. 2008. V.451(7182). P.1121–1124. doi: 10.1038/nature06557
10. Rozhnova N.A., Gerashchenkov G.A., Chemeris A.V. The creation of an expression vector for genome editing of the EDS 1 gene. Biomics. 2019. V.11(4). P. 422-429. DOI: 10.31301/2221-6197.bmcs.2019-35
11. Yang C., Hamamura Y., Sofroni K., Böwer F., Stolze S.C., Nakagami H., et al. SWITCH 1/DYAD is a WINGS APART-LIKE antagonist that maintains sister chromatid cohesion in meiosis. Nat Commun. 2019. V.10(1):1755. doi: 10.1038/s41467-019-09759-w