Grapevine (Vitis vinifera) is a valuable crop in Europe for both economical and cultural reasons, but highly susceptible to Downy mildew (DM). The generation of resistant vines is of critical importance for a sustainable viticulture and can be achieved either by introgression of resistance genes in susceptible varieties or by mutation of Susceptibility (S) genes, e.g., by gene editing. This second approach offers several advantages: it maintains the genetic identity of cultivars otherwise disrupted by crossing and generally results in a broad-spectrum and durable resistance, but it is hindered by the poor knowledge about S genes in grapevines. Candidate S genes are Downy mildew Resistance 6 (DMR6) and DMR6-Like Oxygenases (DLOs), whose mutations confer resistance to DM in Arabidopsis. In this work, we show that grapevine VviDMR6-1 complements the Arabidopsis dmr6-1 resistant mutant. We studied the expression of grapevine VviDMR6 and VviDLO genes in different organs and in response to the DM causative agent Plasmopara viticola. Through an automated evaluation of causal relationships among genes, we show that VviDMR6-1, VviDMR6-2, and VviDLO1 group into different co-regulatory networks, suggesting distinct functions, and that mostly VviDMR6-1 is connected with pathogenesis-responsive genes. Therefore, VviDMR6-1 represents a good candidate to produce resistant cultivars with a gene-editing approach.

Several pathogens continuously threaten viticulture worldwide. Until now, the investigation on resistance loci has been the main trend to understand the interaction between grapevine and the mildew causal agents. Dominantly inherited gene-based resistance has shown to be race-specific in some cases, to confer partial immunity, and to be potentially overcome within a few years since its introgression. Recently, on the footprint of research conducted in Arabidopsis, putative genes associated with downy mildew susceptibility have been discovered also in the grapevine genome. In this work, we deep-sequenced four putative susceptibility genes—namely VvDMR6.1, VvDMR6.2, VvDLO1, VvDLO2—in 190 genetically diverse grapevine genotypes to discover new sources of broad-spectrum and recessively inherited resistance. Identified Single Nucleotide Polymorphisms were screened in a bottleneck analysis from the genetic sequence to their impact on protein structure. Fifty-five genotypes showed at least one impacting mutation in one or more of the scouted genes. Haplotypes were inferred for each gene and two of them at the VvDMR6.2 gene were found significantly more represented in downy mildew resistant genotypes. The current results provide a resource for grapevine and plant genetics and could corroborate genomic-assisted breeding programs as well as tailored gene editing approaches for resistance to biotic stresses.

The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JA-responsive gene expression by the bioactive form JA-isoleucine (JA-Ile) have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. As they are induced by JA we named them JASMONATE-INDUCED OXYGENASEs ( JOXs ). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae . In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in over-accumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses. SIGNIFICANCE STATEMENT: In plants, the hormone jasmonic acid (JA) is synthesized in response to attack by pathogens and herbivores, leading to activation of defense responses. Rapidly following JA accumulation, the hormone is metabolized, presumably to prevent inhibitive effects of high JA levels on growth and development. The enzymes that directly inactivate JA were so far unknown. Here, we identify four jasmonate-induced oxygenases (JOXs) in Arabidopsis that hydroxylate jasmonic acid to form inactive 12-OH-JA. A mutant that no longer produces the four enzymes hyperaccumulates JA, exhibits reduced growth, and is highly resistant to attackers that are sensitive to JA-dependent defense. The JOX enzymes thus play an important role in determining the amplitude and duration of JA responses to balance the growth-defense tradeoff.

Several pathogens continuously threaten viticulture worldwide. Until now, the investigation on resistance loci has been the main trend to understand the interaction between grapevine and mildew causal agents. Dominantly inherited gene-based resistance has shown to be race-specific in some cases, to confer partial immunity and to be potentially overcome within a few years since its introgression. Recently, on the footprint of research conducted on Arabidopsis, the putative hortologues of genes associated with downy mildew susceptibility in this species, have been discovered also in the grapevine genome. In this work, we deep-resequenced four putative susceptibility genes in 190 highly genetically diverse grapevine genotypes to discover new sources of broad-spectrum recessively inherited resistance. The scouted genes areVvDMR6-1, VvDMR6-2, VvDLO1, VvDLO2and predicted to be involved in susceptibility to downy mildew. From all identified mutations, 56% were Single Nucleotide Polymorphisms (SNPs) in heterozygosity, while the remaining 44% were homozygous. Regarding the identified mutations with putative impact on gene function, we observed ~4% genotypes mutated inVvDMR6-1and ~8% mutated inVvDMR6-2, only a handful of genotypes that were mutated in both genes. ~2% and ~7% genotypes showed mutations inVvDLO1andVvDLO2respectively, and again a few genotypes resulted mutated in both genes. In particular, 80% of impacting mutations were heterozygous while 20% were homozygous. The current results will inform grapevine genetics and corroborate genomic-assisted breeding programs for resistance to biotic stresses.Significance statement: A survey on the genetic diversity of downy mildew susceptibility genes in grapevine varieties and wild species reveals a potential valuable for genomic-assisted breeding as well as tailored gene editing to induce disease resistance.