Razzaq Hafiza Arooj, Ijaz Siddra, Haq Imran Ul, Khan Iqrar Ahmad
Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, University Road, Faisalabad, 38000, Pakistan.
Department of Plant Pathology, University of Agriculture, University Road, Faisalabad, 38000, Pakistan.
Mol Biol Rep. 2022 Dec;49(12):11675-11684. doi: 10.1007/s11033-022-07958-1. Epub 2022 Sep 30.
Disease-resistant cultivars are the best solution to get their maximum yield potential and avoid fungicide application. There is no doubt about the contribution, and use of R genes (resistance genes) in resistance development in plants, while S genes (susceptibility genes) also hold a strong position in pathogenesis by resistance repression, and their loss of function contributes to enhanced resistance. Hence, we attempted to knock out the function of the StERF3 gene in potatoes through CRISPR/Cas9-based genome editing and investigated the CRISPR/Cas9 approach as strategic control against late blight disease in potato plants.
The StERF3 gene was edited in late blight susceptible cv. Lady Rosetta. Full allelic edited plants were identified through DnpI, and N1aIV mediated restriction digestion and then further analyzed through Indel Detection by Amplicon Analysis. Sequence analysis of targeted plants for indel identification showed full allelic editing. The detached leaf assay of full allelic edited plants demonstrated the role of the StERF3 gene in susceptibility to late blight in potatoes. In planta disease assay also showed reduced, slowed, and delayed disease progression in StERF3-loss-of-function mutants compared to wild-type (control) plants. Less fungal biomass was quantified in knockouts through Real-time qPCR that supported less susceptibility of edited plants to late blight. Besides, relatively high expression of pathogens-related genes, StPR1, and StNPR1, were also observed in StERF3-loss-of-function mutants compared to the corresponding control.
The results showed the functional inhibition of StERF3 genes using the CRISPR/Cas9 approach. The functional knockouts (StERF3 gene-edited potato plants) revealed enhanced resistance against Phytophthora infestans, thereby demonstrating the best strategic control for late blight disease in potato plants.
抗病品种是实现最大产量潜力并避免使用杀菌剂的最佳解决方案。R基因（抗性基因）在植物抗性发育中的作用毋庸置疑，而S基因（感病基因）在通过抑制抗性来引发病害方面也起着重要作用，其功能丧失有助于增强抗性。因此，我们试图通过基于CRISPR/Cas9的基因组编辑敲除马铃薯中的StERF3基因功能，并研究CRISPR/Cas9方法作为马铃薯晚疫病的战略防控手段。
在晚疫病感病品种罗赛塔夫人（Lady Rosetta）中对StERF3基因进行编辑。通过DnpI和N1aIV介导的限制性消化鉴定出完全等位基因编辑的植株，然后通过扩增子分析进行插入缺失检测进一步分析。用于插入缺失鉴定的靶向植株的序列分析显示了完全等位基因编辑。完全等位基因编辑植株的离体叶片试验证明了StERF3基因在马铃薯对晚疫病的感病性中的作用。与野生型（对照）植株相比，StERF3功能丧失突变体的活体植株病害试验也显示病害进展减少、减缓且延迟。通过实时定量PCR在敲除植株中定量分析发现真菌生物量较少，这支持了编辑植株对晚疫病的易感性较低。此外，与相应对照相比，在StERF3功能丧失突变体中还观察到病程相关基因StPR1和StNPR1的相对高表达。
结果表明利用CRISPR/Cas9方法对StERF3基因具有功能抑制作用。功能敲除（StERF3基因编辑的马铃薯植株）显示出对致病疫霉的抗性增强，从而证明了对马铃薯晚疫病的最佳战略防控效果。
