Abstract
Among various defence strategies used to protect potato plants from a viral infection, a deficiency of the eukaryotic translation initiation factors (eIF) provided by the host-mediated translational suppression has been characterized as a promising approach in modern crop breeding. The strategy used in the present study relies on knocking down the genes of the eIF4E-encoding family in a susceptible potato cultivar by the RNAi silencing mechanism. Taking into account the high concern regarding the potential effects of biotech crops on product safety and public acceptance, no bacterial/viral sequences and selectable marker genes were used. A hairpin construct carrying the fragment of potato eIF4E1 gene was created under control of potato Lhca3 (photosystem I 24 kDa light-harvesting protein) gene promoter and terminator. As a result of marker-free Agrobacterium-mediated transformation, two independent events were identified; one was confirmed to be clean from vector-backbone DNA sequences. Due to high homology between the genes encoding eIF4E1 and eIF4E2 potato factors, the silencing of both genes was achieved. During the two seasonable experiments, the intragenic line with higher production of small RNAs due to the expression of hpRNAi cassette showed a strong level of resistance after the mechanical inoculation with agriculturally significant PVYNTN strain and produced tubers phenotypically similar to non-infected control. Overall, the results obtained here indicate that RNAi-mediated transcriptional regulation of targeted eIF4E gene family using plant tissue-specific promoters in marker-free intragenic potato plants is a promising strategy for improving the viral resistance in clonally propagated crops without affecting plant phenotype and productivity.
Key message
This study is a proof-of-concept for successful generation of viral resistance in potato by the RNAi-mediated transcriptional regulation of targeted eIF4E gene family using intragenic approach.
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Acknowledgements
The authors thank Alexander Chuenko (Doka-Gene Technology Ltd) for kindly providing the potato cv. ‘Pirol NN’ and the local PVYNTN strain, as well as for supporting the idea of research. The work is done for the assignments # 0101-2019-0037 from the Russian Federal Agency for Science and Education.
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DM, VT, SD conceived and designed the experiments; SD contributed reagents/materials and coordinate the research; VT designed and engineered the vector for RNAi interference and performed qRT-PCR analysis; DM and AO carried out the Agrobacterium-mediated transformation and selected intragenic plants; DM and AK made the inoculation with virus; AK, TS and AP performed ELISA test; AK and AP carried out the PCR, RT-PCR and Southern blot; TS and AP extracted sRNAs and performed Northern blot; DM, VT, AP, SD analyzed the data; DM wrote the paper with assistance from all authors.
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Miroshnichenko, D., Timerbaev, V., Okuneva, A. et al. Enhancement of resistance to PVY in intragenic marker-free potato plants by RNAi-mediated silencing of eIF4E translation initiation factors. Plant Cell Tiss Organ Cult 140, 691–705 (2020). https://doi.org/10.1007/s11240-019-01746-9
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DOI: https://doi.org/10.1007/s11240-019-01746-9