Secondary and primary metabolites reveal putative resistance-associated biomarkers against Erysiphe necator in resistant grapevine genotypes

Numerous fungicide applications are required to control Erysiphe necator, the causative agent of powdery mildew. This increased demand for cultivars with strong and long-lasting field resistance to diseases and pests. In comparison to the susceptible cultivar ‘Teroldego’, the current study provides information on some promising disease-resistant varieties (mono-locus) carrying one E. necator-resistant locus: BC4 and ‘Kishmish vatkana’, as well as resistant genotypes carrying several E. necator resistant loci (pyramided): ‘Bianca’, F26P92, F13P71, and NY42. A clear picture of the metabolites’ alterations in response to the pathogen is shown by profiling the main and secondary metabolism: primary compounds and lipids; volatile organic compounds and phenolic compounds at 0, 12, and 48 hours after pathogen inoculation. We identified several compounds whose metabolic modulation indicated that resistant plants initiate defense upon pathogen inoculation, which, while similar to the susceptible genotype in some cases, did not imply that the plants were not resistant, but rather that their resistance was modulated at different percentages of metabolite accumulation and with different effect sizes. As a result, we discovered ten up-accumulated metabolites that distinguished resistant from susceptible varieties in response to powdery mildew inoculation, three of which have already been proposed as resistance biomarkers due to their role in activating the plant defense response

Semi-Targeted Profiling of the Lipidome Changes Induced by Erysiphe Necator in Disease-Resistant and Vitis vinifera L. Varieties

The ascomycete Erysiphe necator is a serious pathogen in viticulture. Despite the fact that some grapevine genotypes exhibit mono-locus or pyramided resistance to this fungus, the lipidomics basis of these genotypes’ defense mechanisms remains unknown. Lipid molecules have critical functions in plant defenses, acting as structural barriers in the cell wall that limit pathogen access or as signaling molecules after stress responses that may regulate innate plant immunity. To unravel and better understand their involvement in plant defense, we used a novel approach of ultra-high performance liquid chromatography (UHPLC)-MS/MS to study how E. necator infection changes the lipid profile of genotypes with different sources of resistance, including BC4 (Run1), “Kishmish vatkhana” (Ren1), F26P92 (Ren3; Ren9), and “Teroldego” (a susceptible genotype), at 0, 24, and 48 hpi. The lipidome alterations were most visible at 24 hpi for BC4 and F26P92, and at 48 hpi for “Kishmish vatkhana”. Among the most abundant lipids in grapevine leaves were the extra-plastidial lipids: glycerophosphocholine (PCs), glycerophosphoethanolamine (PEs) and the signaling lipids: glycerophosphates (Pas) and glycerophosphoinositols (PIs), followed by the plastid lipids: glycerophosphoglycerols (PGs), monogalactosyldiacylglycerols (MGDGs), and digalactosyldiacylglycerols (DGDGs) and, in lower amounts lyso-glycerophosphocholines (LPCs), lyso-glycerophosphoglycerols (LPGs), lyso-glycerophosphoinositols (LPIs), and lyso-glycerophosphoethanolamine (LPEs). Furthermore, the three resistant genotypes had the most prevalent down-accumulated lipid classes, while the susceptible genotype had the most prevalent up-accumulated lipid classes

Two-omics data revealed commonalities and differences between Rpv12- and Rpv3-mediated resistance in grapevine

Plasmopara viticola is the causal agent of grapevine downy mildew (DM). DM resistant varieties deploy effector-triggered immunity (ETI) to inhibit pathogen growth, which is activated by major resistance loci, the most common of which are Rpv3 and Rpv12. We previously showed that a quick metabolome response lies behind the ETI conferred by Rpv3 TIR-NB-LRR genes. Here we used a grape variety operating Rpv12-mediated ETI, which is conferred by an independent locus containing CC-NB-LRR genes, to investigate the defence response using GC/MS, UPLC, UHPLC and RNA-Seq analyses. Eighty-eight metabolites showed significantly different concentration and 432 genes showed differential expression between inoculated resistant leaves and controls. Most metabolite changes in sugars, fatty acids and phenols were similar in timing and direction to those observed in Rpv3-mediated ETI but some of them were stronger or more persistent. Activators, elicitors and signal transducers for the formation of reactive oxygen species were early observed in samples undergoing Rpv12-mediated ETI and were paralleled and followed by the upregulation of genes belonging to ontology categories associated with salicylic acid signalling, signal transduction, WRKY transcription factors and synthesis of PR-1, PR-2, PR-5 pathogenesis-related proteins

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

HILIC MS metabolic fingerprint changes in Jasmine and Bianca vine leaves induced by downy mildew

Downy mildew is a significant limitation for grape production in the absence of chemical protection of vineyards. To reduce sprayings, the selection of resistant varieties can be an alternative Anyway the mechanisms underlying the resistant phenotypes are for now poorly understood. Naturally plants have to live with a multitude of stress conditions and the biosynthesis of protective chemicals is one of the major strategies. Vine resistance is a result of multiple mechanisms, such as the regulation of processes associated with primary metabolism [1]. The role of primary metabolism during plant-pathogen interactions is to support cellular energy requirements for plant defense responses [2]. The aim of this project was to develop a mass spectrometry based untargeted method able to analyze polar primary metabolite fingerprint but also some secondary metabolites by using hydrophilic interaction liquid chromatography (HILIC) coupled to a QTof MS instrument. This method was applied to study the metabolic changes in vine leaves of two resistant varieties Jasmine and Bianca induced by the infection with downy mildew in the first 96 hours after pathogen inoculation (with sampling at time 0, 12, 24, 48 and 96 hours). The method was able to measure the behavior of a big number of known polar primary and secondary metabolites (including sugars, amino acids, organic acids, phenolics, etc) and different unknowns. The results pointed out various tentative markers metabolites of the plant defense mechanism. Acknowledgements This work was made in collaboration with FIRST school of Fondazione Edmund Mach. We thank Silvano Clementi for plant preparation

Lipid, phenol and carotenoid changes in 'Bianca' grapevine leaves after mechanical wounding: a case study

Metabolic changes can occur in plants in response to abiotic stress. Extensive use of leaf discs (mechanical wounding) in studies regarding the effect on the biochemical response of the grapevine to different types of biotic stress makes it necessary to understand metabolic perturbation after injury. In this study, we investigate how mechanical wounding can affect the metabolism of grapevine leaf tissue using Bianca variety as case study. Two sizes of leaf discs (1.1 and 2.8 cm in diameter) were excised from leaves, and phenol, lipid and carotenoid perturbation were investigated 0, 6, 12, 24, 48, 96 and 120 h post cutting. In our study, we found an accumulation of molecules belonging to stilbenoid and stilbene classes such as trans-resveratrol, trans-piceide, Z-miyabenol C, E-cis-miyabenol C and ampelopsin D + quadrangularin A after abiotic stress. The increase in fatty acids such as linoleic acid, linolenic acid and oleic + cis-vaccenic acid during the first 12 h after injury, followed by a return to basal level, allowed us to surmise their role in response to abiotic stress, in particular to mechanical wounding in grapevine leaves. Different-sized discs caused a different response to the tissue, with a higher accumulation in 1.1-cm-diameter discs, especially of phenol compounds. The results of this work can be used to better understand metabolic changes due to biotic stress, having previous knowledge about the perturbation caused by abiotic stress

Grape lipidomics: An extensive profiling thorough uhplc–ms/ms method

Lipids play many essential roles in living organisms, which accounts for the great diversity of these amphiphilic molecules within the individual lipid classes, while their composition depends on intrinsic and extrinsic factors. Recent developments in mass spectrometric methods have significantly contributed to the widespread application of the liquid chromatography‐mass spectrometry (LC–MS) approach to the analysis of plant lipids. However, only a few investigators have studied the extensive composition of grape lipids. The present work describes the development of an ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method that includes 8098 MRM; the method has been validated using a reference sample of grapes at maturity with a successful analysis and semi‐quantification of 412 compounds. The aforementioned method was subsequently applied also to the analysis of the lipid profile variation during the Ribolla Gialla cv. grape maturation process. The partial least squares (PLS) regression model fitted to our experimental data showed that a higher proportion of certain glycerophospholipids (i.e., glycerophosphoethanolamines, PE and glycerophosphoglycerols, PG) and of some hydrolysates from those groups (i.e., lyso‐glycerophosphocholines, LPC and lysoglycerophosphoethanolamines, LPE) can be positively associated with the increasing °Brix rate, while a negative association was found for ceramides (CER) and galactolipids digalactosyldiacylglycerols (DGDG). The validated method has proven to be robust and informative for profiling grape lipids, with the possibility of application to other studies and matrices

Metabolomic Approach for understanding Biochemical Mechanism of Grapevine Resistance to Plasmopara viticola

Downy mildew is one of the most important diseases of grapevines (Vitis vinifera L.), especially for the European varieties caused by the oomycete pathogen Plasmopara viticola (Berk. et Curt.) Berl. et de Toni. This pathogen was reported for the first time in Europe in 1878, where it was probably imported from North America. To reduce sprayings, V. vinifera cultivars were crossed in the past with resistant Vitis spp, to select resistant hybrids, but the biochemical mechanisms underlying the resistant phenotypes are poorly understood. Plants respond naturally to a multitude of stress conditions and the biosynthesis of protective chemicals is one of their major strategies (1). The metabolome, typically defined as the collection of small molecules produced by cells, offers a window for interrogating how mechanistic biochemistry relates to cellular phenotype (2). A metabolomic approach was applied, enabling the analysis of hundreds of biomarker compounds of different chemical classes allowing a better understanding of defense response. Particularly primary and secondary metabolites and lipids were analyzed. The aim was to cover all important classes of plant metabolites and to aim at identifying early stage biomarkers within the first 96 hours after pathogen inoculation

Untargeted lipidomic profiling of grapes highlights the importance of modified lipid species beyond the traditional compound classes

The aim of this paper is to provide a detailed characterisation of grape lipidome. To achieve this objective, it starts by describing a pipeline implemented in R software to allow the semi-automatic annotation of the detected lipid species. It also provides an extensive description of the different properties of each molecule (such as retention time dependencies, mass accuracy, adduct formation and fragmentation patterns), which allowed the annotations to be made more accurately. Most annotated lipids in the grape samples were (lyso)glycerophospholipids and glycerolipids, although a few free fatty acids, hydroxyceramides and sitosterol esters were also observed. The proposed pipeline also allowed the identification of a series of methylated glycerophosphates never previously observed in grapes. The current results highlight the importance of expanding chemical analyses beyond the classical lipid categories