Functional validation of Fusarium wilt and potyvirus resistance genes in melon

Our laboratory has identified melon disease resistance genes by positional cloning. Fom-1 and Prv reside in a head-to head orientation in a single locus, and control resistance to Fusarium races 0 and 2, and to Papaya ring spot virus, respectively. The two genes encode TIR-nucleotide binding-leucine rich repeat (NBL) proteins; in other studies, some paired R genes formed functional units where the two proteins interact. In order to confirm Prv function, we used CRISPR/Cas9 mutagenesis and the Golden Braid cloning system. Transgenic melons from the appropriate resistant genotypes were regenerated, with high frequency of bi-allelic mutations in the target gene. We observed entire deletions of the region between the two targets, and even beyond that area. To our best knowledge, this is a first report of CRISPR mutants in melons. Part of the plants were fertile, and their progeny is being tested for breaking resistance, and for a possible functional interaction between the paired R-genes in the locus. In parallel, we set to mutate the Fom-2 gene that confers resistance to Fusarium races 0 and 1.