CRISPR-Cas9 Mutagenesis and Gene Overexpression to Enhance Resistance to Ascochyta medicaginicola in Medicago truncatula

Alfalfa (Medicago sativa), the most widely cultivated forage legume globally, is vulnerable to Ascochyta medicaginicola, the fungus causing spring black stem and leaf spot (SBS) disease, which significantly reduces yield. SBS disease also affects Medicago truncatula, a diploid model legume with extensive genetic resources, including susceptible and resistant accessions. Using comparative genomics, four candidate genes for disease resistance were identified, MtTCAR1, MtPHO2A, MtCPR1-like, and MtPAM16. CRISPR/Cas9 mutagenesis was applied to generate independent mutant plants in the R108 accession, and disease resistance was evaluated by a detached leaf qPCR-based pathogen assay. MtCPR1-like mutant plants exhibited a 34% reduction in pathogen biomass, along with variable constitutive expression of pathogenesis-related genes. Additionally, a fifth candidate gene, MtKCS12, identified through transcriptomic analysis, was overexpressed in transformed plants, resulting in a 71.4 to 80.9% reduction in pathogen biomass compared with wild-type segregants. This study validates gene editing and transgenic approaches for improving SBS disease resistance in M. truncatula, and future research will focus on applying these strategies to enhance resistance in economically important alfalfa.