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: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: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: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:WRKY genes are transcription factors involved in plant response to pathogen attacks in many plant species. These proteins have been shown to activate expression of defence genes in a salicylic acid- and/or jasmonic acid-dependent signalling pathway. To understand the molecular mechanisms involved in grapevine defence, we previously identified a WRKY gene, VvWRKY1, which was able to enhance tolerance to fungal pathogens when overexpressed in tobacco. To elucidate its role in grapevine, we generated transgenic grapevines that overexpress VvWRKY1. Microarray analyses were performed to compare global gene expression in leaves of the transgenic and wild-type lines. Results showed that expression of genes encoding defence-related proteins was enhanced in the transgenic 35S::VvWRKY1 line. Quantitative RT-PCR analysis confirmed that three genes putatively involved in jasmonic acid signalling pathway, two genes encoding JASMONATE ZIM-domain (JAZ) proteins and one lipoxygenase, are over-expressed. The ability of VvWRKY1 to trans-activate their corresponding promoters was confirmed by transient expression assay in grape protoplasts. After challenging with the downy mildew pathogen Plasmopara viticola, resistance was enhanced in the transgenic line compared to the wild-type line. These results suggest that VvWRKY1 transcription factor is able to control plant disease resistance to one of the main grapevine pathogen by activating jasmonic acid signalling pathway in grapevine.
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.