Fidan Hakan, Calis Ozer, Ari Esin, Atasayar Aydin, Sarikaya Pelin, Tek Mumin Ibrahim, Izmirli Ahmet, Oz Yasemin, Firat Gulsah
Plant Protection Department Faculty of Agriculture Akdeniz University, Antalya, Türkiye.
Agricultural Biotechnology Department, Faculty of Agriculture, Akdeniz University, Antalya, Türkiye.
Front Plant Sci. 2023 Mar 17;14:1143813. doi: 10.3389/fpls.2023.1143813. eCollection 2023.
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. 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 mutations in is to be clarified in -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 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 mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1 or elF4E_3) and double (elF4E_1-3) 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 and elF4E_1 mutants. However, homozygous elF4E_3 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 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.
CRISPR/Cas9是植物育种中最强大的技术之一，能够对基因组进行精确且高效的修饰。该技术正被用于操纵宿主中的目标基因，以培育对植物病原体的抗性。[目标基因名称]是在与马铃薯Y病毒属病毒蛋白基因组连接蛋白（VPg）相互作用期间，在病毒感染中起关键作用的目标基因之一。然而，[目标基因名称]突变的等位基因和位置效应在与VPg的相互作用中尚待阐明。此外，使用CRISPR/Cas9技术大规模生产适合商业生产的抗病品种存在诸多问题。因此，我们针对G27和G247自交系中[目标基因名称]的不同位置，分别使用针对第一外显子和第三外显子的特异性gRNA1和gRNA2，在分离的T1代中筛选出1221株无转基因植株，其中192株G27和79株G247植株在gRNA1或gRNA2的Cas9切割位点处突变最少。进行杂交以观察F1群体中[目标基因名称]突变的等位基因效应，这些群体为纯合和杂合的单突变（elF4E_1或elF4E_3）和双突变（elF4E_1-3）。在未编辑和编辑的F1植株中评估了西瓜花叶病毒（WMV）、番木瓜环斑病毒（PRSV）和西葫芦黄花叶病毒（ZYMV）的病害症状，我们在纯合的elF4E_1-3和elF4E_1突变体中未观察到任何症状。然而，即使接种叶片上没有明显症状，纯合的elF4E_3在逆转录聚合酶链反应（RT-PCR）中呈阳性。酶联免疫吸附测定（ELISA）和定量逆转录聚合酶链反应（qRT-PCR）表明，纯合的elF4E_3中的病毒积累低于杂合和未编辑的植株。还针对这两个基因型全面优化了再生和转化方案。G27和G247每100个外植体的平均芽数分别确定为13.6和18.0。我们在未编辑和编辑的F1植株的产量和形态方面未检测到任何显著差异。我们的结果证明了一种大规模生产对WMV、ZYMV和PRSV具有抗性的黄瓜品种的有效途径。通过这种方式，可以培育出抗病品种，以减少这些病原体在黄瓜生产中造成的损失。
