Biomics

The Arabidopsis EDS1 protein is known to control defense activation and programmed cell death caused by intercellular Toll-like immune receptors that recognize specific pathogen effectors. Unfortunately, the involvement of EDS1 protein in the anti-virus immunity of potato plants has not been studied. During the research, the selection of genomic targets for the introduction of mutations (sequence 20 bp) in the ortholog of the gene LOC102598075 EDS1-like protein was carried out. Design of guide RNA is carry out. Online bioinformatics resources such as CRISPR DESIGN TOOLS, CRISPR GE, CRISPROR, and CHOP-CHOP 2.0 were used to minimize variants of off-target mutations. The Assembly of Cas9/sgRNA expression cassettes based on the pBAtC vector with increased specificity of Cas9 mutation of the ortholog of the EDS1-like protein gene was performed. For better potato transformation arabidopsis promoter in the pBAtC vector was replaced with own pStU6-6 promoter 323 bp. On the basis of the modified vector, an expression vector was obtained with a 20 bp target cloned on the AarI site. Validation of the presence of a 323 bp promoter and a 424 bp Basta gene fragment in the pSt6-6 vector was performed by PCR. The presence of the desired insert is confirmed by sequencing. The created vector will be used for bioballistic transformation of potato plants in vitro.

CRISPR, genome editing, EDS1 protein, pBAtC vector, DNA sequencing

1. Chemeris D.A., Kiryanova O.Yu., Gerashchenkov G.A., Kuluev B.R., Rozhnova N.A., Matniyazov R.T., Baymiev An.Kh., Baymiev Al.Kh., Gubaidullin I.M., Chemeris A.V. Bioinformatic resources for CRISPR/Cas genome editing, Biomics, 2017, vol. 9, no. 3, pp. 203-228. (In Russian)
2. Gerashchenkov G.A., Rozhnova N.A., Kuluev B.R., Kiryanova O.Yu., Gumerova G.R., Knyazev A.V., Vershinina Z.R., Mikhailova E.V., Chemeris D.A., Matniyazov R.T., Baimiev An.Kh., Gubaidullin I.M., Baimiev Al.Kh., Chemeris A.V. Design of guide RNA for CRISPR/Cas plant genome editing. Molecular Biology. 2020. V.54(1).
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. Vol. 31. p. 2456–2474. DOI: https://doi.org/10.1105/tpc.19.00099
4. Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York.
5. 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. 10.1111/jipb.12474
6. Makarova S.S., Makarov V.V., Taliansky M.E., Kalinina N.O. Resistance to viruses of potato: current status and prospects. Vavilov Journal of Genetics and Breeding. 2017. V.21(1). P. 62-73. DOI 10.18699/VJ17.224 (In Russian)
7. 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 Mar;29(5):569-79.
8. Rogers E.E., Ausubel F.M. Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression // Plant Cell. 1997 Mar;9(3):305-16. DOI: 10.1105/tpc.9.3.305
9. Soosaar J.L., Burch-Smith T.M., Dinesh-Kumar S.P. Mechanisms of plant resistance to viruses // Nat Rev Microbiol. 2005 Oct;3(10):789-98. DOI: 10.1038/nrmicro1239