Development of powdery mildew (erysiphe necator) resistant plants in grapevine (Vitis vinifera l.) using CRISPR/Cas9 genome editing technology

The economic losses in grape cultivation worldwide are largely attributed to the powdery mildew disease caused by Erysiphe necator. It is generally accepted that almost all varieties of Vitis vinifera L., which represent approximately 90% of the total grape production worldwide, are susceptible to this disease. The global spread of the pathogen necessitates frequent pesticide applications in viticulture. The chemicals used for this purpose not only result in additional production costs for growers but also adversely affect the health of workers in the vineyard and beneficial organisms present in the vineyard. In recent years, numerous studies have indicated the potential use of genes known as S (susceptible) genes, which enable a harmonious interaction between the plant and pathogen, in breeding for resistance. Various research has explored the possibility of imparting powdery mildew resistance to plants by knocking out S-genes located in loci called MLO. Successful outcomes of such applications in plants like A. thaliana, barley, and tomatoes have provided hope for gene knock-out studies targeting MLO genes in grapevine. In this study, the application of CRISPR technology aims to create mutations in MLO and ALS (acetolactate synthase) genes through the combined use of gene knock-out and base editing. Agrobacterium tumefaciens and the CRISPR/Cas9 system are employed to select both transient and stable transfers of guide RNAs prepared for grapevines. In this context, in addition to the vector design prepared for knocking-out MLO genes, an additional design targeting ALS (acetolactate synthase) genes has been integrated into the plant using Agrobacterium tumefaciens EHA105 strain. For this purpose, the transformation vector prepared for ALS genes is designed to perform a base substitution (C→U(T)) in the relevant genes, converting Proline (Pro 74 for ALS1 gene and Pro 175 for ALS2 gene) amino acid to Serine. Numerous studies in the literature have demonstrated that this modification imparts resistance to sulfonylurea herbicides in plants. The embryogenic calli used in the transformation studies have been exposed to a sulfonylurea herbicide to monitor the events of transient transformation. At the end of the study, it was determined that 28 out of 132 plants regenerated in Black Khismish variety transformed in a stable manner. Among them, single-base mutations were observed in the Gly177 residues in 2 plants. No mutations were observed in the remaining 104 plants evaluated for transient transformation. The results obtained from the study are expected to be effectively used in gene transfer studies to be followed in the Black Khismish variety, and they are anticipated to guide future studies for obtaining non-transgenic and mutant plants in terms of MLO genes.