Project description:Plasmopara viticola (Berk. and Curt.) Berl. and de Toni is the agent of the destructive disease known as grapevine downy mildew, for the control of which intensive fungicide treatments are required. Natural sources of resistance are available in several wild Vitis species, which are being used in traditional breeding approaches. However, molecular switches, signals and effectors involved in resistance are poorly understood. In this paper we report a microarray analysis of early transcriptional changes associated to P. viticola infection in both susceptible Vitis vinifera and resistant Vitis riparia plants (12 and 24 h post inoculation). To provide a biological basis to the choice of time points for transcriptome analyses, we performed microscopic examinations of infected tissues at 12, 24, 48 and 96 hpi. Data suggest that resistance in V. riparia is mainly a post-infectional event and involves a large reprogramming of host metabolism. Transcripts of signal transduction-related genes are specifically and often strongly accumulated in response to infection. Well known defence genes also show marked transcript increases, especially pathogenesis-related proteins PR-10 and stylbene synthases, and genes related to an hypersensitive reaction. On the other hand, V. vinifera mounts a much weaker transcriptional response, involving mainly defence genes, not effective enough in preventing pathogen infection. Leaves from one resistant (V. riparia cv. Gloire de Montpellier) and one susceptible (V.vinifera cv. Pinot Noir) grapevine cultivars grown in vitro were infected with the oomycete Plasmopara viticola, and transcriptome changes were investigated at 12h and 24h after infection. Three biological replicates were considered and each hybridization was performed twice. One color labeling was performed
Project description:Cultivated grapevine (Vitis vinifera) is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in elicitation of plant immunity by bio-molecules such as Pathogen Associated Molecular Patterns (PAMPs). We have demonstrated that the beta-glucan laminarin (Lam) and its sulfated derivative (PS3) induce a PAMP-triggered immunity in grapevine against downy mildew (Plasmopara viticola). However, if Lam elicits classical grapevine defenses, PS3 triggered grapevine resistance via a poorly understood priming phenomenon. The aim of this study was to discover the mechanism of the PS3-induced resistance. On uninfected grapevine, we first investigated defense signaling and performed microarray experiments to identify early events and genes directly triggered by PS3. Our results showed that PS3 i) was unable to elicit ROS and NO production, cytosolic Ca2+ variations, MAPK activation but triggered a long lasting plasma membrane depolarization in grapevine cells ii) up-regulated a stress-responsive transcriptome close to the one induced by Lam but only partly overlapping the ones triggered by salicylate (SA) or jasmonate (JA). Finally, in response to P. viticola infection, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine triggered immunity against this biotroph. Keywords: cell death, induced resistance, oomycete, priming, reactive oxygen species, salicylate, sulfated laminarin, transcriptomics, Vitis vinifera.
Project description:Cultivated grapevine (Vitis vinifera) is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in elicitation of plant immunity by bio-molecules such as Pathogen Associated Molecular Patterns (PAMPs). We have demonstrated that the beta-glucan laminarin (Lam) and its sulfated derivative (PS3) induce a PAMP-triggered immunity in grapevine against downy mildew (Plasmopara viticola). However, if Lam elicits classical grapevine defenses, PS3 triggered grapevine resistance via a poorly understood priming phenomenon. The aim of this study was to discover the mechanism of the PS3-induced resistance. On uninfected grapevine, we first investigated defense signaling and performed microarray experiments to identify early events and genes directly triggered by PS3. Our results showed that PS3 i) was unable to elicit ROS and NO production, cytosolic Ca2+ variations, MAPK activation but triggered a long lasting plasma membrane depolarization in grapevine cells ii) up-regulated a stress-responsive transcriptome close to the one induced by Lam but only partly overlapping the ones triggered by salicylate (SA) or jasmonate (JA). Finally, in response to P. viticola infection, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine triggered immunity against this biotroph. Keywords: cell death, induced resistance, oomycete, priming, reactive oxygen species, salicylate, sulfated laminarin, transcriptomics, Vitis vinifera. 6 samples (Adj, PS3, Lam, ctrl, SA, JA) were analized with 3 biological replicates each, Adj and ctrl samples are reference samples
Project description:Plasmopara viticola (Berk. and Curt.) Berl. and de Toni is the agent of the destructive disease known as grapevine downy mildew, for the control of which intensive fungicide treatments are required. Natural sources of resistance are available in several wild Vitis species, which are being used in traditional breeding approaches. However, molecular switches, signals and effectors involved in resistance are poorly understood. In this paper we report a microarray analysis of early transcriptional changes associated to P. viticola infection in both susceptible Vitis vinifera and resistant Vitis riparia plants (12 and 24 h post inoculation). To provide a biological basis to the choice of time points for transcriptome analyses, we performed microscopic examinations of infected tissues at 12, 24, 48 and 96 hpi. Data suggest that resistance in V. riparia is mainly a post-infectional event and involves a large reprogramming of host metabolism. Transcripts of signal transduction-related genes are specifically and often strongly accumulated in response to infection. Well known defence genes also show marked transcript increases, especially pathogenesis-related proteins PR-10 and stylbene synthases, and genes related to an hypersensitive reaction. On the other hand, V. vinifera mounts a much weaker transcriptional response, involving mainly defence genes, not effective enough in preventing pathogen infection.
Project description:Study of gene expression during Plasmopara viticola infection in the resistant Vitis vinifera cultivar 'Regent'. The oomycete fungus Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is responsible for grapevine downy mildew disease. Most of the cultivated grapevines are sensitive to this pathogen, thus requiring intensive fungicide treatments. The molecular basis of resistance to this pathogen is poorly understood. We have carried out a cDNA microarray transcriptome analysis to identify grapevine genes associated with resistance traits. Early transcriptional changes associated with downy mildew infection in the resistant Vitis vinifera cultivar ‘Regent’, when compared to the susceptible cultivar ‘Trincadeira’, were analyzed. Transcript levels were measured at three time-points: 0, 6 and 12 hours post inoculation (hpi). Our data indicate that resistance in V. vinifera ‘Regent’ is induced after infection. This study provides the identification of several candidate genes that may be related to ‘Regent’ defense mechanisms, allowing a better understanding of this cultivar's resistance traits.
Project description:Downy mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead shown resistance to this pathogen and they have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity has been strictly related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling x Teroldego cross by profiling the stilbenoid content of the leaves of the entire population and the transcriptome of resistant and susceptible individuals following infection. A three-year analysis of the population?s response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine. Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response. A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis. A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport.This set of genes should be taken into account in future breeding programs. Leaves from one resistant (F1 21/66) and two susceptible (F1 22/73 and Teroldego) genotypes were infected with Plasmopara viticola, and transcriptome changes were investigated at 12h and 96h after infection (treated samples) and at 0h after spraying with water (control samples) Two biological replicates were considered and each hybridization was performed three times. One color labeling was performed.
Project description:Study of gene expression during Plasmopara viticola infection in the resistant Vitis vinifera cultivar 'Regent'. The oomycete fungus Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is responsible for grapevine downy mildew disease. Most of the cultivated grapevines are sensitive to this pathogen, thus requiring intensive fungicide treatments. The molecular basis of resistance to this pathogen is poorly understood. We have carried out a cDNA microarray transcriptome analysis to identify grapevine genes associated with resistance traits. Early transcriptional changes associated with downy mildew infection in the resistant Vitis vinifera cultivar M-bM-^@M-^XRegentM-bM-^@M-^Y, when compared to the susceptible cultivar M-bM-^@M-^XTrincadeiraM-bM-^@M-^Y, were analyzed. Transcript levels were measured at three time-points: 0, 6 and 12 hours post inoculation (hpi). Our data indicate that resistance in V. vinifera M-bM-^@M-^XRegentM-bM-^@M-^Y is induced after infection. This study provides the identification of several candidate genes that may be related to M-bM-^@M-^XRegentM-bM-^@M-^Y defense mechanisms, allowing a better understanding of this cultivar's resistance traits. 3 time points: 0, 6 and 12 hours post inoculation by P. viticola. Two cultivars: control (Trinacedira) and test (Regent). Two biological replicates were performed at 0 hpi, and 3 biological replicates at 6 and 12hpi. At 12hpi, three technical replicates also were performed.
Project description:Grapevine downy mildew is an important disease affecting crop production and causing severe losses. To identify genotype-dependent responses towards this pathogen and to explore the molecular mechanisms involved in grapevine-P. viticola resistance, we have conducted a proteomic analysis of leaf samples from resistant and susceptible grapevine genotypes prior and post-inoculation with the pathogen. Proteins were analyzed by quantitative two-dimensional differential gel electrophoresis (2D-DIGE). The analysis able to identified 50 unique proteins. Functional analysis showed that photosynthesis and metabolism were the main categories differentiating genotypes at 0h and that P. viticola-responsive proteins were mainly involved in photosynthesis, carbohydrate metabolism, stress and defense responses and redox homeostasis. ROS production, total antioxidant capacity and lipid peroxidation on both genotypes were determined and together with the proteome data suggest that Regent presents a strict balance between ROS control and signaling leading to plant cell death activation. Our data reveals the genotype-dependent modulation of plant metabolism and defense responses providing new insights into underlying molecular processes of grapevine resistance against the downy mildew fungus.
Project description:European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance to P. viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance. In this study, Rpv3-mediated defense responses were studied in Rpv3+ and Rpv3ˉ grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3+ and avrRpv3ˉ, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses.
Project description:Downy mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead shown resistance to this pathogen and they have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity has been strictly related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling x Teroldego cross by profiling the stilbenoid content of the leaves of the entire population and the transcriptome of resistant and susceptible individuals following infection. A three-year analysis of the population?s response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine. Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response. A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis. A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport.This set of genes should be taken into account in future breeding programs.