Balasubramanian Muthusamy, Shahar Nizan, Amalia Bar‐Ziv, Rafael Perl‐Treves
ABSTRACT Functional validation of NLR genes is critical for confirming their specific roles and developing durable disease‐resistant crops. The Fom‐1 gene of Cucumis melo , controlling resistance to races 0 and 2 of F. oxysporum f. sp. melonis (FOM), had been identified by map‐based cloning as MELO3C022146, which encodes a TIR‐NBS‐LRR (TNL) protein. It resides in a head‐to‐head orientation adjacent to another TNL gene, Prv , controlling resistance to papaya ring spot virus (PRSV). In this study we validated the function of Fom‐1 in mediating FOM resistance by applying CRISPR/Cas9 editing to the resistant cultivar, Védrantais. Two gRNAs were designed to target exons 1 and 2, respectively, and mutations were introduced at both target sites, resulting in truncated open reading frames in both alleles. Inoculation assays of T 1 plants with FOM races 2 and 0 revealed a breakdown of resistance, manifested by leaf necrosis and wilting, and susceptibility was stably inherited in the T 2 generation. This proved that MELO3C022146, the candidate gene for Fom‐1 , is responsible for resistance in melon cultivar Védrantais. Future studies will address the molecular functions of this gene as well as possible interactions between Fom‐1 and its neighbour R ‐gene, Prv .