Dissection of the Rpv12 locus: from the genetic basis to the phenotype

Abstract

Modern viticulture relies predominantly on traditional European grapevine cultivars (Vitis vinifera) and is therefore heavily dependent on fungicide use to mitigate crop losses from downy (Plasmopara viticola) and powdery mildew (Erysiphe necator), the two most widespread grapevine diseases. The breeding of new fungus-resistant grapevine cultivars by crossing European elite cultivars with wild Vitis species, that possess genetic resistance loci, is one chance to reduce fungicide applications in viticulture.

To date, over thirty loci mediating resistance to downy mildew are known. However, only few of them are used in the breeding programs and little is known about their defense mechanisms and the genes responsible for them. Since the long-term goal of resistance breeding is to pyramid multiple resistance loci, these loci should rely on different resistance mechanisms to enhance durability in the vineyard. Therefore, detailed knowledge of the defense mechanisms and resistance genes associated with the respective Rpv loci is essential for sustainable resistance management and for achieving durable and stable resistance in breeding programs.

Rpv12 originating from Vitis amurensis on chromosome 14 is one of the strongest resistance loci against downy mildew. The defense mechanism mediated by this locus was compared to Rpv3 and Rpv10 carrying cultivars through assessment of the downy mildew sporulation, onset of programmed cell death (PCD) and production of defense metabolites like hydrogen peroxide and stilbenes. Additionally, resistance breaking isolates of P. viticola were used to prove that the resistance loci rely on different resistance mechanisms. The carried out experiments revealed a clear temporal difference between Rpv10 and Rpv12 cultivars, which exhibited an early and localized defense response in comparison to Rpv3 cultivars that displayed a delayed and diffuse response. These temporal differences were associated with increased trans-resveratrol accumulation and hydrogen peroxide formation shortly before the onset of PCD and may explain the observed cultivar specific variation in hyphal growth, sporulation and in the vineyard.

We were able to narrow down, the originally described Rpv12-locus (Venuti et al. 2013) through new resistance markers. Bioinformatic analysis of an Rpv12-carrier (Frommer et al. 2023) led to the identification of two putative diseases resistance R-genes. For functional characterization the two candidate genes will be cloned into expression vectors for stable and temporary expression. Additionally, we were able to design CRISPR-Cas9 knock-out constructs specifically for the two candidate genes. Infiltration experiments will show which of the genes is responsible for resistance.