Metabolomics and Transcriptomics: novel approaches to understand resistance in grape against Plasmopara viticola

The grapevine is the most economically important fruit crop worldwide. Among the species of fungi considered to be the main grapevine pathogens, downy mildew is considered to be an extremely destructive disease of the grapevine, caused by the oomycete Plasmopara viticola (Berk. et Curt.) Berl. et de Toni. Grapevine research is directed towards better understanding of plant defence mechanisms and characterisation of the particular plant-pathogen interactions affecting the species. One of the most promising future strategies to ensure plant protection against disease is to stop the use of chemical compounds and focus on the selection of varieties showing durable specific resistance. Understanding plant-pathogen interaction is important for the future of the breeding; indeed grapevine species can be crosses, including resistant traits using conventional breeding techniques. In the last few years, comprehensive studies called omics have been applied to model plant study and these have contributed enormously to plant science. The project aims to decipher the mechanisms responsible for resistance in vine plants, since the molecular bases of the defence mechanism against P. viticola are still poorly understood. In particular, early responses to the pathogen, occurring within the initial 96 hours post inoculation, have been investigated in grape varieties using metabolomic and transcriptomic data. The use of leaf discs is widely adopted in experiments regarding the effect of different types of biotic stress on the biochemical response of the grapevine. Since there is little knowledge regarding mechanical wounding of grapevine leaves, we analyzed changes in phenolic, lipid and carotenoid content in Bianca grapevine leaves subjected to mechanical wounding (leaf discs), testing two different sizes of leaf discs (1.1 cm and 2.8 cm in diameter). One of the most well-known defence responses in vine plants is the production of defence compounds, mainly secondary metabolites also known as phytoalexins. Primary metabolism is also involved in plant defense with the participation of different molecules including carbohydrates, organic acids, amines, amino acids and lipids not only as a source of energy but also as a source of signaling molecules to directly or indirectly trigger defense response. We developed a rapid and versatile method for the extraction, identification and quantification of different classes of grape lipids using liquid chromatographic tandem mass spectrometry (LC-MS/MS). We also validated a method for the identification and quantification of primary compounds belonging to different chemical classes: acids, amminoacids, amines/others and sugars using a GC-MS method of separation and identification, interesting in terms of elucidating the role of primary compounds in plant-microbe interaction in future work. In this project the primary and secondary metabolism were investigated after P. viticola infection, in Bianca grapevine leaves with the aim of covering all the most important classes of plant metabolites. Our results gave a picture of plant metabolome perturbation. Several molecules were altered in Bianca leaf discs compared to the control after P. viticola infection, and they could act as potential biomarkers in Bianca variety after infection with P. viticola. Since plant resistance and plant-pathogen interaction are complex biological processes involving many signalling pathways, the multi omic approach is most suitable for examining these traits. An integrated metabolomic and transcriptomic approach was also applied to correlate variation in gene expression and metabolic perturbation in resistant Jasmine grapevine leaves, with the aim of discovering a specific and early stage biomarkers related to Downy mildew resistance.