Functional Validation of the Melon Fom‐1 Gene, Controlling Resistance to Fusarium oxysporum Races 0 and 2, by CRISPR/ Cas9 Mutagenesis

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 .