Fusarium and PRSV resistance genes in melon: protein interactions and functional validation by CRISPR-Cas9

Fom-1 and Prv genes reside in a head-to head orientation in a single locus, and control melon resistance to Fusarium races 0 and 2, and to Papaya ring spot virus, respectively. They encode TIR-nucleotide binding-leucine rich repeat (NBL) proteins. To confirm Prv function, we used CRISP-Cas9 mutagenesis. Transgenic melons from the appropriate resistant genotypes were regenerated, with high frequency of bi-allelic mutations in the target gene. We observed deletions of the region between 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 is being tested for breaking resistance. Since a few paired R genes reportedly form functional units, we explore the possible interaction between Fom-1 and Prv. In a previous proteomic study, we identified in the xylem a candidate Avr2 for another melon R-gene, Fom-2, and studied their protein interactions. We constructed a yeast-expressed cDNA library from Fusarium infected melon roots and screened it with an Avr2 bait; we also isolated interactors by co-immunoprecipitation. Several putative interactors were identified that could mediate the defense response initiated by Fom-2, and these, as well as plant xylem proteins induced upon infection, serve as a starting point to study Fusarium-melon recognition at the protein level.