Identification, isolation and characterization of a CC-NBS-LRR candidate disease resistance gene family in grapevine

Abstract

Plasmopara viticola causes downy mildew of grapevine, one of the most important diseases in viticulture. Resistance to this oomycete is present in American and Asian Vitis species, while traditional European Vitis vinifera cvs. for wine and table grape production are susceptible. Breeding aims to achieve resistance through introgression, but the molecular mechanisms are still unknown. Therefore, the differential display approach was used to detect grapevine genes involved in defense. P. viticola sporangia were applied to the lower leaf surface of in vitro plants of the resistant Vitis riparia selection ‘Gloire de Montpellier’ and susceptible cv. ‘Riesling’. Controls were treated with sterile water. Messenger RNAs extracted 12 h post infection were subjected to differential display. Seven transcripts appeared specifically induced during the incompatible interaction. Sequencing showed that they build three classes. One of them, named VRP1, represented by three transcripts of almost identical sequence but differing lengths, showed clear homology to resistance genes of the NBS-LRR type from other plants. Northern hybridizations confirmed its elevated expression in the resistant ‘Gloire de Montpellier’. Redundancy of VRP1 PCR products from V. riparia prevented PCR-walking, so the VRP1 genes were isolated from a BAC-library of the resistant cv. ‘Regent’. Three genes matching the original VRP1 sequences were found within a BAC clone carrying a 134,392 bp insertion. These were referred to as VRP1-1, 1-2 and 1-3. They encode proteins of 798, 811 resp. 813 amino acids and exhibit the structure of CC-NBS-LRR resistance genes. They were genetically mapped to linkage group 10.