Use of a RT-qPCR Method to Estimate Mycorrhization Intensity and Symbiosis Vitality in Grapevine Plants Inoculated with Rhizophagus irregularis.

Assessing the mycorrhization level in plant roots is essential to study the effect of arbuscular mycorrhizal fungi (AMF) on plant physiological responses. Common methods used to quantify the mycorrhization of roots are based on microscopic visualization of stained fungal structures within the cortical cells. While this method is readily accessible, it remains time-consuming and does not allow checking of the symbiosis vitality. The aim of this work is thus to develop an efficient method for assessing the intensity and vitality of mycorrhiza associated with grapevine through gene expression analyses by RT-qPCR. To this end, grapevine plants were inoculated with the AMF Rhizophagus irregularis (Ri). The relationship between mycorrhization level, assessed by microscopy, and expression of several fungus and grapevine genes involved in the symbiosis was investigated. In AMF-inoculated plants, transcript amounts of fungal constitutively-expressed genes Ri18S, RiTEF1α and RiαTub were significantly correlated to mycorrhization intensity, particularly Ri18S. Grapevine (VvPht1.1 and VvPht1.2) and AMF (GintPT, Ri14-3-3 and RiCRN1) genes, known to be specifically expressed during the mycorrhizal process, were significantly correlated to arbuscular level in the whole root system determined by microscopy. The best correlations were obtained with GintPT on the fungal side and VvPht1.2 on the plant side. Despite some minor discrepancies between microscopic and molecular techniques, the monitoring of Ri18S, GintPT and VvPht1.2 gene expression could be a rapid, robust and reliable method to evaluate the level of mycorrhization and to assess the vitality of AMF. It appears particularly useful to identify AMF-inoculated plants with very low colonization level, or with non-active fungal structures. Moreover, it can be implemented simultaneously with the expression analysis of other genes of interest, saving time compared to microscopic analyses.

Pathogenicity Factors of Botryosphaeriaceae Associated with Grapevine Trunk Diseases: New Developments on Their Action on Grapevine Defense Responses.

Botryosphaeriaceae are a family of fungi associated with the decay of a large number of woody plants with economic importance and causing particularly great losses in viticulture due to grapevine trunk diseases. In recent years, major advances in the knowledge of the pathogenicity factors of these pathogens have been made possible by the development of next-generation sequencing. This review highlights the knowledge gained on genes encoding small secreted proteins such as effectors, carbohydrate-associated enzymes, transporters and genes associated with secondary metabolism, their representativeness within the Botryosphaeriaceae family and their expression during grapevine infection. These pathogenicity factors are particularly expressed during host-pathogen interactions, facilitating fungal development and nutrition, wood colonization, as well as manipulating defense pathways and inducing impacts at the cellular level and phytotoxicity. This work highlights the need for further research to continue the effort to elucidate the pathogenicity mechanisms of this family of fungi infecting grapevine in order to improve the development of control methods and varietal resistance and to reduce the development and the effects of the disease on grapevine harvest quality and yield.

Arbuscular Mycorrhizal Symbiosis Triggers Major Changes in Primary Metabolism Together With Modification of Defense Responses and Signaling in Both Roots and Leaves of Vitis vinifera.

Grapevine (Vitis vinifera L.) is one of the most important crops worldwide but is subjected to multiple biotic and abiotic stresses, especially related to climate change. In this context, the grapevine culture could take advantage of symbiosis through association with arbuscular mycorrhizal fungi (AMF), which are able to establish symbiosis with most terrestrial plants. Indeed, it is well established that mycorrhization improves grapevine nutrition and resistance to stresses, especially water stress and resistance to root pathogens. Thus, it appears essential to understand the effect of mycorrhization on grapevine metabolism and defense responses. In this study, we combined a non-targeted metabolomic approach and a targeted transcriptomic study to analyze changes induced in both the roots and leaves of V. vinifera cv. Gewurztraminer by colonization with Rhizophagus irregularis (Ri). We showed that colonization of grapevine with AMF triggers major reprogramming of primary metabolism in the roots, especially sugar and fatty acid metabolism. On the other hand, mycorrhizal roots had decreased contents of most sugars and sugar acids. A significant increase in several fatty acids (C16:1, linoleic and linolenic acids and the C20 arachidonic and eicosapentaenoic acids) was also detected. However, a downregulation of the JA biosynthesis pathway was evidenced. We also found strong induction of the expression of PR proteins from the proteinase inhibitor (PR6) and subtilase (PR7) families in roots, suggesting that these proteins are involved in the mycorrhiza development but could also confer higher resistance to root pathogens. Metabolic changes induced by mycorrhization were less marked in leaves but involved higher levels of linoleic and linolenic acids and decreased sucrose, quinic, and shikimic acid contents. In addition, Ri colonization resulted in enhanced JA and SA levels in leaves. Overall, this study provides a detailed picture of metabolic changes induced by AMF colonization in a woody, economically important species. Moreover, stimulation of fatty acid biosynthesis and PR protein expression in roots and enhanced defense hormone contents in leaves establish first insight in favor of better resistance of grapevine to various pathogens provided by AMF colonization.

Comparative Study of Secreted Proteins, Enzymatic Activities of Wood Degradation and Stilbene Metabolization in Grapevine Botryosphaeria Dieback Fungi.

Botryosphaeriaceae fungi are plant pathogens associated with Botryosphaeria dieback. To better understand the virulence factors of these fungi, we investigated the diversity of secreted proteins and extracellular enzyme activities involved in wood degradation and stilbene metabolization in Neofusicoccumparvum and Diplodiaseriata, which are two major fungi associated with grapevine B. dieback. Regarding the analysis of proteins secreted by the two fungi, our study revealed that N. parvum, known to be more aggressive than D. seriata, was characterized by a higher quantity and diversity of secreted proteins, especially hydrolases and oxidoreductases that are likely involved in cell wall and lignin degradation. In addition, when fungi were grown with wood powder, the extracellular laccase and Mn peroxidase enzyme activities were significantly higher in D. seriata compared to N.parvum. Importantly, our work also showed that secreted Botryosphaeriaceae proteins produced after grapevine wood addition are able to rapidly metabolize the grapevine stilbenes. Overall, a higher diversity of resveratrol and piceatannol metabolization products was found with enzymes of N. parvum compared to D. seriata. This study emphasizes the diversity of secreted virulence factors found in B. dieback fungi and suggests that some resveratrol oligomers produced in grapevine wood after pathogen attack could be formed via pathogenic fungal oxidases.

Wood Metabolomic Responses of Wild and Cultivated Grapevine to Infection with Neofusicoccum parvum, a Trunk Disease Pathogen.

Grapevine trunk diseases (GTDs), which are associated with complex of xylem-inhabiting fungi, represent one of the major threats to vineyard sustainability currently. Botryosphaeria dieback, one of the major GTDs, is associated with wood colonization by Botryosphaeriaceae fungi, especially Neofusicoccum parvum. We used GC-MS and HPLC-MS to compare the wood metabolomic responses of the susceptible Vitis vinifera subsp. vinifera (V.v. subsp. vinifera) and the tolerant Vitis vinifera subsp. sylvestris (V.v. subsp. sylvestris) after artificial inoculation with Neofusicoccum parvum (N. parvum). N. parvum inoculation triggered major changes in both primary and specialized metabolites in the wood. In both subspecies, infection resulted in a strong decrease in sugars (fructose, glucose, sucrose), whereas sugar alcohol content (mannitol and arabitol) was enhanced. Concerning amino acids, N. parvum early infection triggered a decrease in aspartic acid, serine, and asparagine, and a strong increase in alanine and -alanine. A trend for more intense primary metabolism alteration was observed in V.v. subsp. sylvestris compared to V. v. subsp. vinifera. N. parvum infection also triggered major changes in stilbene and flavonoid compounds. The content in resveratrol and several resveratrol oligomers increased in the wood of both subspecies after infection. Interestingly, we found a higher induction of resveratrol oligomer (putative E-miyabenol C, vitisin C, hopeaphenol, ampelopsin C) contents after wood inoculation in V.v. subsp. sylvestris.

Overexpression of the VvSWEET4 Transporter in Grapevine Hairy Roots Increases Sugar Transport and Contents and Enhances Resistance to Pythium irregulare, a Soilborne Pathogen.

Sugar transport and partitioning play key roles in the regulation of plant development and responses to biotic and abiotic factors. During plant/pathogen interactions, there is a competition for sugar that is controlled by membrane transporters and their regulation is decisive for the outcome of the interaction. SWEET sugar transporters are the targets of extracellular pathogens, which modify their expression to acquire the sugars necessary to their growth (Chen et al., 2010). The regulation of carbon allocation and sugar partitioning in the interaction between grapevine (Vitis vinifera) and its pathogens is poorly understood. We previously characterized the SWEET family in V. vinifera and showed that SWEET4 could be involved in resistance to the necrotrophic fungus Botrytis cinerea in Arabidopsis (Chong et al., 2014). To study the role of VvSWEET4 in grapevine, we produced V. vinifera cv. Syrah hairy roots overexpressing VvSWEET4 under the control of the CaMV 35S promoter (VvSWEET4 OX). High levels of VvSWEET4 expression in hairy roots resulted in enhanced growth on media containing glucose or sucrose and increased contents in glucose and fructose. Sugar uptake assays further showed an improved glucose absorption in VvSWEET4 overexpressors. In parallel, we observed that VvSWEET4 expression was significantly induced after infection of wild type grapevine hairy roots with Pythium irregulare, a soilborne necrotrophic pathogen. Importantly, grapevine hairy roots overexpressing VvSWEET4 exhibited an improved resistance level to P. irregulare infection. This resistance phenotype was associated with higher glucose pools in roots after infection, higher constitutive expression of several genes involved in flavonoid biosynthesis, and higher flavanol contents. We propose that high sugar levels in VvSWEET4 OX hairy roots provides a better support to the increased energy demand during pathogen infection. In addition, high sugar levels promote biosynthesis of flavonoids with antifungal properties. Overall, this work highlights the key role of sugar transport mediated by SWEET transporters for secondary metabolism regulation and pathogen resistance in grapevine.

Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization.

Grapevine trunk diseases: Eutypa dieback, esca and Botryosphaeria dieback, which incidence has increased recently, are associated with several symptoms finally leading to the plant death. In the absence of efficient treatments, these diseases are a major problem for the viticulture; however, the factors involved in disease progression are not still fully identified. In order to get a better understanding of Botryosphaeria dieback development in grapevine, we have investigated different factors involved in Botryosphaeriaceae fungi aggressiveness. We first evaluated the activity of the wood-degrading enzymes of different isolates of Neofusicoccum parvum and Diplodia seriata, two major fungi associated with Botryosphaeria dieback. We further examinated the ability of these fungi to metabolize major grapevine phytoalexins: resveratrol and δ-viniferin. Our results demonstrate that Botryosphaeriaceae were characterized by differential wood decay enzymatic activities and have the capacity to rapidly degrade stilbenes. N. parvum is able to degrade parietal polysaccharides, whereas D. seriata has a better capacity to degrade lignin. Growth of both fungi exhibited a low sensitivity to resveratrol, whereas δ-viniferin has a fungistatic effect, especially on N. parvum Bourgogne S-116. We further show that Botryosphaeriaceae are able to metabolize rapidly resveratrol and δ-viniferin. The best stilbene metabolizing activity was measured for D. seriata. In conclusion, the different Botryosphaeriaceae isolates are characterized by a specific aggressiveness repertory. Wood and phenolic compound decay enzymatic activities could enable Botryosphaeriaceae to bypass chemical and physical barriers of the grapevine plant. The specific signature of Botryosphaeriaceae aggressiveness factors could explain the importance of fungi complexes in synergistic activity in order to fully colonize the host.

Vitamins for enhancing plant resistance.

MAIN CONCLUSION: This paper provides an overview on vitamins with inducing activities in plants, the molecular and cellular mechanisms implicated, and the hormonal signalling-network regulating this process. Moreover, it reports how vitamins might be part of the molecular events linked to induced resistance by the conventional elicitors. Induced resistance (IR), exploiting the plant innate-defense system is a sustainable strategy for plant disease control. In the last decade, vitamins have been proven to act as inducers of disease resistance, and these findings have received an important attention owing to their safety and cost effectiveness. Vitamins, including thiamine (TH, vitamin B1), riboflavin (RF, vitamin B2), menadione sodium bisulfite (MSB, vitamin K3), Para-aminobenzoic acid (PABA, vitamin Bx), and folic acid (FA, vitamin B9) provided an efficient protection against a wide range of pathogens through the modulation of specific host-defense facets. However, other vitamins, such as ascorbic acid (AA, vitamin C) and tocopherols (vitamin E), have been shown to be a part of the molecular mechanisms associated to IR. The present review is the first to summarize what vitamins are acting as inducers of disease resistance in plants and how could they be modulated by the conventional elicitors. Thus, this report provides an overview on the protective abilities of vitamins and the molecular and cellular mechanisms underlying their activities. Moreover, it describes the hormonal-signalling network regulating vitamin-signal transduction during IR. Finally, a biochemical model describing how vitamins are involved in the establishment of IR process is discussed.

Characterization of triterpenoid profiles and triterpene synthase expression in the leaves of eight Vitis vinifera cultivars grown in the Upper Rhine Valley.

Plant triterpenoids are a diverse group of secondary metabolites with wide distribution, high chemical diversity and interesting pharmacological and antimicrobial properties. The first step in the biosynthesis of all triterpenoids is the cyclization of the 2,3-oxidosqualene precursor, catalyzed by oxidosqualene cyclases (OSCs), which have characteristic product specificities. Biosynthesis and functions of pentacyclic triterpenes have been poorly studied in grapevine. In this study, we first investigated the profile of triterpenoids present in leaf cuticular waxes from eight Vitis vinifera cultivars cultivated in the Upper Rhine Valley. Further quantification of triterpenoids showed that these cultivars can be divided into two groups, characterized by high levels of lupeol (e.g., Pinot noir) or taraxerol (e.g., Gewurztraminer) respectively. We further analyzed the OSC family involved in the synthesis of pentacyclic triterpenes (called VvTTPSs) in the sequenced V. vinifera 40024 genome and found nine genes with similarity to previously characterized triterpene synthases. Phylogenetic analysis further showed that VvTTPS1-VvTTPS3 and VvTTPS5-VvTTPS9 belong to the ß-amyrin synthase and multifunctional triterpene synthase clade, whereas VvTTPS10 belongs to the lupeol synthase clade. We studied the expression of several members of the VvTTPS family following biotic and abiotic stresses in V. vinifera 40024 as well as in the eight healthy cultivars. This study further revealed that one candidate gene, VvTTPS5, which does not belong to the lupeol synthase clade, is highly expressed in lupeol-rich cultivars. VvTTPS3, VvTTPS5, VvTTPS6, VvTTPS7 and VvTTPS10 were highly upregulated by UV stress, but only VvTTPS3, VvTTPS5, VvTTPS6 and VvTTPS10 were upregulated following downy mildew and gray mold infections respectively. These results suggest differential roles of VvTTPS against environmental stresses in grape leaves.

Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine.

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, (-)-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) (-)-4-chloro-terremutin hydrate (4) and(+)-4-hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-(-)-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)(-)-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with (-)-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxins.

The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea.

During plant development, sugar export is determinant in multiple processes such as nectar production, pollen development and long-distance sucrose transport. The plant SWEET family of sugar transporters is a recently identified protein family of sugar uniporters. In rice, SWEET transporters are the target of extracellular bacteria, which have evolved sophisticated mechanisms to modify their expression and acquire sugars to sustain their growth. Here we report the characterization of the SWEET family of sugar transporters in Vitis vinifera. We identified 17 SWEET genes in the V. vinifera 40024 genome and show that they are differentially expressed in vegetative and reproductive organs. Inoculation with the biotrophic pathogens Erysiphe necator and Plasmopara viticola did not result in significant induction of VvSWEET gene expression. However, infection with the necrotroph Botrytis cinerea triggered a strong up-regulation of VvSWEET4 expression. Further characterization of VvSWEET4 revealed that it is a glucose transporter localized in the plasma membrane that is up-regulated by inducers of reactive oxygen species and virulence factors from necrotizing pathogens. Finally, Arabidopsis knockout mutants in the orthologous AtSWEET4 were found to be less susceptible to B. cinerea. We propose that stimulation of expression of a developmentally regulated glucose uniporter by reactive oxygen species production and extensive cell death after necrotrophic fungal infection could facilitate sugar acquisition from plant cells by the pathogen.

Changes in the triterpenoid content of cuticular waxes during fruit ripening of eight grape (Vitis vinifera) cultivars grown in the Upper Rhine Valley.

Triterpenoids present in grape cuticular waxes are of interest due to their potential role in protection against biotic stresses, their impact on the mechanical toughness of the fruit surface, and the potential industrial application of these biologically active compounds from grape pomace. The determination of the triterpenoid profile of cuticular waxes reported here supplements existing knowledge of the chemical diversity of grape, with some compounds reported in this species for the first time. Common compounds identified in eight examined cultivars grown in the Upper Rhine Valley include oleanolic acid, oleanolic and ursolic acid methyl esters, oleanolic aldehyde, α-amyrin, α-amyrenone, ß-amyrin, cycloartanol, 24-methylenecycloartanol, erythrodiol, germanicol, lupeol accompanied by lupeol acetate, campesterol, cholesterol, sitosterol, stigmasterol, and stigmasta-3,5-dien-7-one, whereas 3,12-oleandione was specific for the Muscat d'Alsace cultivar. Changes in the triterpenoid content of cuticular waxes were determined at three different phenological stages: young grapes, grapes at véraison (the onset of ripening), and mature grapes. The results reveal a characteristic evolution of triterpenoid content during fruit development, with a high level of total triterpenoids in young grapes that gradually decreases with a slight increase in the level of neutral triterpenoids. This phenomenon may partially explain changes in the mechanical properties of the cuticle and possible modulations in the susceptibility to pathogens of mature grapes.

Differential responses of three grapevine cultivars to Botryosphaeria dieback.

Botryosphaeria dieback is a fungal grapevine trunk disease that represents a threat for viticulture worldwide due to the decreased production of affected plants and their premature death. This dieback is characterized by a typical wood discoloration called brown stripe. Herein, a proteome comparison of the brown striped wood from Botryosphaeria dieback-affected standing vines cultivars Chardonnay, Gewurztraminer, and Mourvèdre was performed. The transcript analysis for 15 targeted genes and the quantification of both total phenolics and specific stilbenes were also performed. Several pathogenesis-related proteins and members of the antioxidant system were more abundant in the brown striped wood of the three cultivars, whereas other defense-related proteins were less abundant. Additionally, total phenolics and some specific stilbenes were more accumulated in the brown striped wood. Strongest differences among the cultivars concerned proteins of the primary metabolism, which looked to be particularly impaired in the brown striped wood of 'Chardonnay'. Low abundance of some proteins involved in defense response probably contributes to make global response insufficient to avoid the symptom development. The differential susceptibility of the three grapevine cultivars could be linked to the diverse expression of various proteins involved in defense response, stress tolerance, and metabolism.

Methionine elicits H2O2 generation and defense gene expression in grapevine and reduces Plasmopara viticola infection.

Methionine (Met) is a nutritionally essential sulfur-containing amino acid (SAA) known for its preponderant role as initiator in protein synthesis. However, other functions for Met in plants are not well described. The implication of this SAA in oxidative stress tolerance has been recently reported, however the mode of action of Met is still poorly understood. Here, we analyzed the elicitor activity of Met in grapevine as well as its effect on Plasmopara viticola resistance. The results show that Met induces hydrogen peroxide (H2O2) generation, a key element in plant defense signaling, and upregulates the expression of a battery of defense-related genes. Transcript levels of these genes were not further modulated by P. viticola inoculation of Met-pretreated plants, suggesting an elicitor role rather than a priming role for Met in grapevine. Met treatment also reduces P. viticola development in grapevine plants grown under glasshouse controlled-conditions. Fungitoxicity assays revealed that Met possesses a moderate antifungal activity compared with cysteine (Cys), another SAA known for its toxic effect to a large spectrum of fungi.

Thiamine induced resistance to Plasmopara viticola in grapevine and elicited host-defense responses, including HR like-cell death.

Recently, thiamine (VitaminB1) has been shown to induce resistance against Pseudomonas syringae in Arabidopsis plants through priming of defense responses. In this paper, we have demonstrated the efficiency of thiamine to induce resistance against downy mildew caused by the oomycete Plasmopara viticola in a susceptible Vitis vinifera cultivar "Chardonnay" under glasshouse controlled conditions by providing a dual mode of action involving direct antifungal activity and elicitation of host-defense responses. Thiamine-induced defense responses included the generation of hydrogen peroxide (H(2)O(2)) in both grapevine suspension cultured cells (SCC) and plant leaves, upregulation of an array of defense-related genes and the induction of other defense responses at subcellular level such as callose deposition in stomata cells, phenolic compounds accumulation and hypersensitive response (HR) like-cell death. Epifluorescence microscopy studies revealed dramatic changes in P. viticola individual developmental stages during its colonization of the intercellular space of the leaf mesophyll in thiamine-treated plants. Collectively, our report evidenced the efficiency of thiamine in the control of downy mildew in grapevine by direct and indirect effects, suggesting that thiamine could be an attractive alternative to chemical fungicides in disease management in vineyards.

FGF-2- and TGF-ß1-induced downregulation of lumican and keratocan in activated corneal keratocytes by JNK signaling pathway.

PURPOSE: Downregulation of lumican and keratocan expression is an undesirable phenotypic change that occurs during corneal wound healing. The present study was intended to determine whether the activation of Jun N-terminal kinase (JNK)-signaling pathway is involved in their downregulation in TGF-ß1- and FGF-2-activated keratocytes. METHODS: Keratocytes, isolated from rabbit corneal stroma, and cultured in a serum-free medium, pretreated or not treated with JNK inhibitor (SP600125), were activated with FGF-2/heparin sulfate (HS) or TGF-ß1 in the presence or absence of SP600125. In another set of experiments, keratocytes were transfected with JNK1/2 Dicer-substrate RNA (DsiRNA) and then activated with TGF-ß1 or FGF-2/HS. Specific phenotypic changes were analyzed immunocytochemically and correlated with Western blot analyses. The relative levels of specific mRNAs were estimated by quantitative RT-PCR using specific reagents. RESULTS: The FGF-2/HS- or TGF-ß-induced activation of corneal stromal keratocytes to fibroblast- or myofibroblast-phenotype, respectively, resulted in marked decreases in cell surface-associated and secreted keratan sulfate proteoglycans (KSPGs). Both keratocan and lumican proteins and their mRNAs were downregulated in the activated keratocytes. However, JNK inhibition during the activation of keratocytes, pretreated with the JNK inhibitor, suppressed the reduction in the cell-surface associated and secreted KSPGs (lumican and keratocan), and their mRNA transcripts. Downregulation of total KSPGs and their mRNAs was also inhibited by decreasing JNK1 and JNK2 levels via JNK1/2 DsiRNA transfection of keratocytes before their activation. CONCLUSIONS: Extrapolating from the present study, FGF-2- and TGF-ß1-activation of JNK signaling pathway may be partly responsible for the downregulation of keratocan and lumican expression in activated corneal keratocytes observed during corneal stromal wound healing.

Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PR genes and enhanced resistance to powdery mildew.

Studying grapevine (Vitis vinifera) innate defense mechanisms is a prerequisite to the development of new protection strategies, based on the stimulation of plant signaling pathways to trigger pathogen resistance. Two transcriptional coactivators (VvNPR1.1 and VvNPR1.2) with similarity to Arabidopsis thaliana NPR1 (Non-Expressor of PR genes 1), a well-characterized and key signaling element of the salicylic acid (SA) pathway, were recently isolated in Vitis vinifera. In this study, functional characterization of VvNPR1.1 and VvNPR1.2, including complementation of the Arabidopsis npr1 mutant, revealed that VvNPR1.1 is a functional ortholog of AtNPR1, whereas VvNPR1.2 likely has a different function. Ectopic overexpression of VvNPR1.1 in the Arabidopsis npr1-2 mutant restored plant growth at a high SA concentration, Pathogenesis Related 1 (PR1) gene expression after treatment with SA or bacterial inoculation, and resistance to virulent Pseudomonas syringae pv. maculicola bacteria. Moreover, stable overexpression of VvNPR1.1-GFP in V. vinifera resulted in constitutive nuclear localization of the fusion protein and enhanced PR gene expression in uninfected plants. Furthermore, grapevine plants overexpressing VvNPR1.1-GFP exhibited an enhanced resistance to powdery mildew infection. This work highlights the importance of the conserved SA/NPR1 signaling pathway for resistance to biotrophic pathogens in V. vinifera.

Impact of Quillaja saponaria saponins on grapevine ecosystem organisms.

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.

Characterization of Vitis vinifera NPR1 homologs involved in the regulation of pathogenesis-related gene expression.

BACKGROUND: Grapevine protection against diseases needs alternative strategies to the use of phytochemicals, implying a thorough knowledge of innate defense mechanisms. However, signalling pathways and regulatory elements leading to induction of defense responses have yet to be characterized in this species. In order to study defense response signalling to pathogens in Vitis vinifera, we took advantage of its recently completed genome sequence to characterize two putative orthologs of NPR1, a key player in salicylic acid (SA)-mediated resistance to biotrophic pathogens in Arabidopsis thaliana. RESULTS: Two cDNAs named VvNPR1.1 and VvNPR1.2 were isolated from Vitis vinifera cv chardonnay, encoding proteins showing 55% and 40% identity to Arabidopsis NPR1 respectively. Constitutive expression of VvNPR1.1 and VvNPR1.2 monitored in leaves of V. vinifera cv chardonnay was found to be enhanced by treatment with benzothiadiazole, a SA analog. In contrast, VvNPR1.1 and VvNPR1.2 transcript levels were not affected during infection of resistant Vitis riparia or susceptible V. vinifera with Plasmopara viticola, the causal agent of downy mildew, suggesting regulation of VvNPR1 activity at the protein level. VvNPR1.1-GFP and VvNPR1.2-GFP fusion proteins were transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, where they localized predominantly to the nucleus. In this system, VvNPR1.1 and VvNPR1.2 expression was sufficient to trigger the accumulation of acidic SA-dependent pathogenesis-related proteins PR1 and PR2, but not of basic chitinases (PR3) in the absence of pathogen infection. Interestingly, when VvNPR1.1 or AtNPR1 were transiently overexpressed in Vitis vinifera leaves, the induction of grapevine PR1 was significantly enhanced in response to P. viticola. CONCLUSION: In conclusion, our data identified grapevine homologs of NPR1, and their functional analysis showed that VvNPR1.1 and VvNPR1.2 likely control the expression of SA-dependent defense genes. Overexpression of VvNPR1 has thus the potential to enhance grapevine defensive capabilities upon fungal infection. As a consequence, manipulating VvNPR1 and other signalling elements could open ways to strengthen disease resistance mechanisms in this crop species.

Identification, expression analysis and characterization of defense and signaling genes in Vitis vinifera.

The reduction of phytochemicals applied to grapevine relies on the development of alternative strategies involving activation of the plant's own defense system. The aim of this work was to study the signaling of defense responses to pathogens in Vitis vinifera. We identified in V. vinifera cv. Chardonnay two putative regulatory elements, VvNHL1 and VvEDS1, with similarity to Arabidopsis defense regulators NDR1 and EDS1. Expression studies of these putative signaling genes together with other known grape defense genes show that they are differentially regulated by salicylic acid and jasmonate-ethylene treatments, as well as by inoculation with different types of pathogens. The expression of VvEDS1 was stimulated by salicylic acid treatment, Botrytis cinerea and Plasmopara viticola inoculation, whereas VvNHL1 was repressed by B. cinerea. VvNHL1 overexpression introduced in Arabidopsis ndr1 mutant did not complement the mutation in terms of sensitivity to avirulent Pseudomonas syringae pv. tomato. Moreover, we observed a weakened resistance to B. cinerea of ndr1 mutants overexpressing VvNHL1, which may be related to cell death enhancement. Together, our results identify two new pathogen-responsive regulatory elements in Vitis vinifera, with potential roles in pathogen defense.