Mutagenesis of the melon Prv gene by CRISPR/Cas9 breaks PRSV resistance and generates an autoimmune allele with constitutive defense responses.

The majority of plant disease resistance (R) genes encode nucleotide binding-leucine rich repeat (NLR) proteins. In melon, two closely linked NLR genes, Fom-1 and Prv, were mapped and identified as candidate genes that control resistance to Fusarium oxysporum f.sp. melonis races 0 and 2, and to papaya ringspot virus (PRSV), respectively. In this study, we validated the function of Prv and showed that it is essential for providing resistance against PRSV infection. We generated CRISPR/Cas9 mutants using Agrobacterium-mediated transformation of a PRSV-resistant melon genotype, and the T1 progeny proved susceptible to PRSV, showing strong disease symptoms and viral spread upon infection. Three alleles having 144, 154 and ~3 kb deletions, respectively, were obtained, all of which caused loss of resistance. Interestingly, one of the Prv mutant alleles, prvΔ154, encoding a truncated product, caused an extreme dwarf phenotype, accompanied by leaf lesions, high salicylic acid levels and defense gene expression. The autoimmune phenotype observed at 25 oC proved to be temperature-dependent, being suppressed at 32 oC. This is a first report on successful application of CRISPR/Cas9 to confirm R-gene function in melon. Such validation opens new opportunities for molecular breeding of disease resistance in this important vegetable crop.