Functional inhibition of the StERF3 gene by dual targeting through CRISPR/Cas9 enhances resistance to the late blight disease in Solanum tuberosum L.

Abstract

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.