Targeted editing of the Cspmr4 gene via CRISPR/Cas9 to enhance tolerance to Phytophthora cinnamomi in Castanea sativa

ABSTRACT Castanea sativa Mill. is a highly versatile species of great ecological, economic, and cultural importance throughout Europe. However, it faces serious threats such as ink disease and chestnut blight, caused respectively by Phytophthora cinnamomi (Pc) and Cryphonectria parasitica, whose severity has increased in recent years due to climate change. A promising gene editing strategy to confer pathogen tolerance involves inactivating susceptibility genes (S genes) in plants. Here, the susceptibility gene Cspmr4, which encodes a callose synthase, was knocked-out for the first time in a forest species using CRISPR/Cas9. To achieve this, somatic embryos from two embryogenic chestnut lines (CI-9 and CI-3) were transformed using Agrobacterium tumefaciens strain EHA105 carrying a CRISPR/Cas9 construct targeting two sites in Cspmr4 . Only line CI-9 produced kanamycin resistant transgenic embryos, with an average transformation efficiency of 5.9%. To assess editing efficiency and identify mutation types, target sites were amplified using PCR, followed by Sanger sequencing and mutation analysis using TIDE and ICE tools. Editing was confirmed in five of seven embryogenic lines. All five lines have at least 97% editing efficiency in at least one gRNA. Edited somatic embryos showed the highest survival rates, exceeding 50% in three of the analyzed lines. In addition, regenerated plants from line CI-9- pmr4 -1 exhibited a marked reduction in root necrosis, decreasing from 57% in wild-type explants to only 8% in the edited line.