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

Abstract

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.

The melon Fom‐1–Prv resistance gene pair: Correlated spatial expression and interaction with a viral protein

“…To directly demonstrate functional cooperation between Prv and Fom‐1 , we will need to mutate each of them in a suitable genetic background, and see whether mutation in one gene will affect resistance to both FOM and PRSV; this represents a notable challenge in melon. Recently, we have been able to knock‐out Prv by CRISPR‐Cas9 in a PRSV resistant genotype and prove that it is indeed required for PRSV resistance (Nizan et al, 2023 ); however, functional proof of Fom‐1 function and its possible interaction with Prv is still lacking. Another important objective would be to identify the F. oxysporum Avr factor recognized by Fom‐1/Prv.…”

“…The Fom‐1 gene, MELO3C022146, was found in close proximity to Prv , MELO3C022145, in a head‐to‐head orientation, their respective start codons being separated by 1.3 kb. In a recent study (Nizan et al, 2023 ), Prv was knocked out and proved to be essential for PRSV resistance. We still do not know whether Prv and Fom‐1 act cooperatively, but interestingly , Prv has an extra NBS domain at its carboxy‐terminus, which could function as an integrated decoy.…”

The Hypersensitive Response to Plant Viruses

“…Antiviral NLRs with supplemental domains that could fit the integrated decoy model are scarce, although this type of NLR is widespread in plant genomes [ 128 ]. They include PvVTT1 which contains a second TIR domain [ 90 ], Sw5-b with an extended N-terminal domain referred to as the Solanaceae domain (SD) shown to contribute to Avr recognition [ 124 ], and the melon Prv containing an additional NBS domain in its C-terminus [ 97 ]. Many ID-containing NLRs (ID-NLRs) act in a pair with a second NLR [ 127 ]; the ID-NLR is the sensor, whereas the second, a canonical NLR, is the executor acting as a signal transducer.…”

“…The paired NLR genes are in close proximity, in a head-to-head orientation, and they share promoter sequences [ 132 , 133 , 134 ]. Such a head-to-head orientation has been described for the Prv and the Fom-1 genes of Cucumis melo conferring resistance to the papaya ring spot virus and to some races of the Fusarium oxysporum fungus [ 97 ]. Whether this pair of TNLs encoding genes and other resistance genes to viruses fully fits the paired integrated decoy model will need further investigations.…”

Optimization of an Efficient Direct Shoot Induction System and Histological Evaluation of Shoot Formation in Cucurbita maxima Duch

“…With the development of genome sequencing and genome editing technologies, the precise improvement of plant traits through genetic transformation has become a new approach for many species of the Cucurbitaceae. According to previous reports, cucumber [5][6][7][8], melon [9][10][11][12][13], and watermelon [14][15][16][17] have successfully undergone gene transformation and genome editing.…”