Functional validation of a potyvirus resistance gene in melon by CRISPR/Cas9 mutagenesis

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 (PRSV), respectively. The two R genes encode TIR-nucleotide binding-leucine rich repeat (NLR) proteins; a few paired R genes were shown to form functional units where the two proteins interact. In order to confirm Prv function, we used CRISPR/Cas9 mutagenesis and the Golden Braid cloning system. T₀ transgenic melons from the appropriate PRSV-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. Plants were fertile, and their progeny was tested for breaking resistance. Wild type and T₁ seedlings were inoculated with PRSV. Most (83%) of the T₁ progeny showed severe virus disease symptoms compared to the resistant wild type, which provides functional validation of the potyvirus resistance gene. Next we shall test the Prv mutants for Fusarium resistance, in a fusarium resistant cultivar, to check for a possible functional interaction between the two paired R-genes. Interestingly, one of the mutated alleles that we recovered caused a severe dwarf phenotype in the growth chamber, that is alleviated under greenhouse conditions, suggesting that we have generated an autoimmune allele of Prv.