Knockout of elF4E using CRISPR/Cas9 for large-scale production of resistant cucumber cultivar against WMV, ZYMV, and PRSV.

Autor:	Fidan H; Plant Protection Department Faculty of Agriculture Akdeniz University, Antalya, Türkiye., Calis O; Plant Protection Department Faculty of Agriculture Akdeniz University, Antalya, Türkiye., Ari E; Agricultural Biotechnology Department, Faculty of Agriculture, Akdeniz University, Antalya, Türkiye., Atasayar A; Research and Development Department AD ROSSEN Seeds, Antalya, Türkiye., Sarikaya P; Plant Protection Department Faculty of Agriculture Akdeniz University, Antalya, Türkiye.; Research and Development Department AD ROSSEN Seeds, Antalya, Türkiye., Tek MI; Plant Protection Department Faculty of Agriculture Akdeniz University, Antalya, Türkiye., Izmirli A; Agricultural Biotechnology Department, Faculty of Agriculture, Akdeniz University, Antalya, Türkiye., Oz Y; Agricultural Biotechnology Department, Faculty of Agriculture, Akdeniz University, Antalya, Türkiye., Firat G; Research and Development Department AD ROSSEN Seeds, Antalya, Türkiye.
Jazyk:	angličtina
Zdroj:	Frontiers in plant science [Front Plant Sci] 2023 Mar 17; Vol. 14, pp. 1143813. Date of Electronic Publication: 2023 Mar 17 (Print Publication: 2023).
DOI:	10.3389/fpls.2023.1143813
Abstrakt:	CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E -VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1 DEL or elF4E_3 DEL ) and double (elF4E_1-3 DEL ) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3 DEL and elF4E_1 DEL mutants. However, homozygous elF4E_3 DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3 DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2023 Fidan, Calis, Ari, Atasayar, Sarikaya, Tek, Izmirli, Oz and Firat.)
Databáze:	MEDLINE
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