Data_Sheet_8_CRISPR-Cas9 Targeting of the eIF4E1 Gene Extends the Potato Virus Y Resistance Spectrum of the Solanum tuberosum L. cv. Desirée.pdf

Translation initiation factors and, in particular, the eIF4E family are the primary source of recessive resistance to potyviruses in many plant species. However, no eIF4E-mediated resistance to this virus genus has been identified in potato (Solanum tuberosum L.) germplasm. As in tomato, the potato eIF4E gene family consists of eIF4E1, its paralog eIF4E2, eIF(iso)4E, and nCBP. In tomato, eIF4E1 knockout (KO) confers resistance to a subset of potyviruses, while the eIF4E1/2 double KO, although conferring a broader spectrum of resistance, leads to plant developmental defects. Here, the tetraploid potato cv. Desirée owning the dominant Ny gene conferring resistance to potato virus Y (PVY) strain O but not NTN was used to evaluate the possibility to expand its PVY resistance spectrum by CRISPR-Cas9-mediated KO of the eIF4E1 susceptibility gene. After a double process of plant protoplast transfection-regeneration, eIF4E1 KO potatoes were obtained. The knockout was specific for the eIF4E1, and no mutations were identified in its eIF4E2 paralog. Expression analysis of the eIF4E family shows that the disruption of the eIF4E1 does not alter the RNA steady-state level of the other family members. The eIF4E1 KO lines challenged with a PVYNTN isolate showed a reduced viral accumulation and amelioration of virus-induced symptoms suggesting that the eIF4E1 gene was required but not essential for its multiplication. Our data show that eIF4E1 editing can be usefully exploited to broaden the PVY resistance spectrum of elite potato cultivars, such as Desirée, by pyramiding eIF4E-mediated recessive resistance.

翻译起始因子，尤其是真核翻译起始因子4E家族（eIF4E family），是众多植物物种抵御马铃薯Y病毒属（potyviruses）病毒的隐性抗病性的主要遗传基础。然而，在普通栽培马铃薯（Solanum tuberosum L.）种质资源中，尚未发现由eIF4E介导的针对该病毒属的抗病性。与番茄类似，马铃薯的eIF4E基因家族包含eIF4E1、其旁系同源基因eIF4E2、eIF(iso)4E以及核帽结合蛋白（nCBP）。在番茄中，敲除（knockout, KO）eIF4E1可使植株对部分马铃薯Y病毒属病毒产生抗性；而同时敲除eIF4E1与eIF4E2的双敲除株系虽能拓宽抗病谱，但会导致植株出现发育缺陷。本研究以携带显性Ny基因的四倍体马铃薯栽培品种‘Desirée’为材料，该基因可使植株抵御马铃薯Y病毒（potato virus Y, PVY）O株系，但无法抵御NTN株系；研究旨在通过CRISPR-Cas9介导敲除感病基因eIF4E1，以此拓展该品种对PVY的抗病谱。经过植物原生质体转染-再生两步流程，成功获得了eIF4E1敲除的马铃薯植株。该敲除仅特异性作用于eIF4E1，其旁系同源基因eIF4E2未检测到任何突变。对eIF4E家族的表达分析结果显示，敲除eIF4E1并不会改变该家族其他成员的RNA稳态水平。用PVYNTN分离物接种eIF4E1敲除株系后，植株体内的病毒积累量显著降低，病毒诱导的症状也得到缓解，这表明eIF4E1基因是病毒增殖所必需的，但并非不可或缺。本研究结果表明，可通过聚合eIF4E介导的隐性抗病性，利用eIF4E1基因编辑技术有效拓宽‘Desirée’这类优良马铃薯栽培品种对PVY的抗病谱。