A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl(3) treatment.     

beta-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling

beta-Aminobutyric acid (BABA) was used to induce resistance in grapevine (Vitis vinifera) against downy mildew (Plasmopara viticola). This led to a strong reduction of mycelial growth and sporulation in the susceptible cv. Chasselas. Comparing different inducers, the best protection was achieved with BABA followed by jasmonic acid (JA), whereas benzo (1,2,3)-thiadiazole-7-carbothionic acid-S-methyl ester (a salicylic acid [SA] analog) and abscisic acid (ABA) treatment did not increase the resistance significantly. Marker genes for the SA and JA pathways showed potentiated expression patterns in BABA-treated plants following infection. The callose synthesis inhibitor 2-deoxy-D-glucose partially suppressed BABA- and JA-induced resistance against P viticola in Chasselas. Application of the phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid and the lipoxygenase (LOX) inhibitor 5, 8, 11, 14-eicosatetraynoic acid (ETYA) also led to a reduction of BABA-induced resistance (BABA-IR), suggesting that callose deposition as well as defense mechanisms depending on phenylpropanoids and the JA pathways all contribute to BABA-IR. The similar phenotype of BABA- and JA-induced resistance, the potentiated expression pattern of JA-regulated genes (LOX-9 and PR-4) following BABA treatment, and the suppression of BABA-IR with ETYA suggest an involvement of the JA pathway in BABA-IR of grapevine leading to a primed deposition of callose and lignin around the infection sites.     

Development of a transient expression system in grapevine via agro-infiltration

Transient expression of genes using Agrobacterium is a powerful tool for the analysis of gene function in plants. We have developed this method for the analysis of genes involved in disease resistance in grapevine leaves. Our research showed that the quality of the plant material, the plant genotype used for agro-infiltration and the presence of additional virulence factors (carried on plasmid pCH32) in the Agrobacterium strain are all important factors for success of the procedure. After optimising these factors, we consistently achieve sufficient acceptable levels of expression of the markers beta-glucuronidase (GUS) and green fluorescent protein (GFP) using vacuum infiltration of grapevine leaves from plants grown in vitro. We used this procedure to investigate the proposed role of stilbenes in defense against grapevine downy mildew (Plasmopara viticola) by transiently overexpressing stilbene synthase in grapevine leaves, before infection with P. viticola. We found that agro-infiltration itself induces the synthesis of stilbenes in grapevine leaves, thus preventing us to test the effect of the overexpression of stilbene synthase in defense. However, our results revealed that agro-infiltration before P. viticola inoculation had an effect on the development of the infection. Further research is required to show whether stilbenes or some other factor are the causal agent restricting pathogen development. The method described here provides and excellent tool to exploit at the many grapevine genomic resources now available, and will contribute to a better understanding of many areas of grapevine biology.     

Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen.

The expression of PR protein encoding genes and genes involved in the phenylpropanoid metabolism was analysed on grapevine leaves of susceptible and resistant cvs. in response to inoculation with the host-pathogen Plasmopara viticola and the non-host pathogen Pseudoperonospora cubensis, the downy mildew pathogen of cucumber. These experiments were conducted to elucidate whether or not grapevine plants susceptible to downy mildew exhibit an identical defence response after inoculation with the non-host pathogen. Expression analysis of defence-related genes revealed marked differences between the susceptible cultivar "Riesling" (Vitis vinifera) and the resistant cultivar "Gloire de Montpellier" (Vitis riparia). Whereas some genes seem to be expressed constitutively in "Gloire" or induced after an inoculation with both pathogens, expression of defence-related genes in Riesling was influenced mainly after inoculation with the non-host pathogen: PR-2, PR-3, PR-4, a PGIP gene, and especially genes encoding enzymes involved in anthocyanin biosynthesis (DFR, F3H, LDOX) were affected. Therefore, the occurrence of the respective products (flavans and other phenolics) in inoculated leaves was investigated with appropriate histological staining techniques. These stainings revealed a production of catechins and related phenolic compounds within the first 48 hai (hours after inoculation) with Ps. cubensis but not with P. viticola in Riesling, whereas in Gloire no further production was seen, which may be due to the high content of polyphenolics as observed in control leaves. In addition to the staining procedures, sporulation intensity was monitored on leaf discs. Pretreatments of leaf discs with Ps. cubensis led to a reduced browning reaction (as a result of a hypersensitive reaction) in Gloire and significantly reduced the intensity of sporulation in Riesling after a subsequent inoculation with P. viticola.     

Characterization of Plasmopara-resistance in grapevine using in vitro plants

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in vitro-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order. An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection. The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in vitro-plants and the resistance of grapevine plants to P. viticola in the field.     

Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola

Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P. viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and P. viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.     

An ethoxylated surfactant enhances the penetration of the sulfated laminarin through leaf cuticle and stomata,leading to increased induced resistance against grapevine downy mildew

Some β‐1,3‐glucans and particularly sulfated laminarin (PS3) are known as resistance inducers (RIs) in grapevine against the downy mildew. However, their efficacy in vineyard is still often too low, which might be caused by a limited penetration through the leaf cuticle following spray application. We used ¹⁴C‐sucrose uptake experiments with grapevine leaves in order to select a surfactant as saccharide penetration enhancer. Our results showed that although sucrose foliar uptake was low, it was strongly enhanced by Dehscofix CO125 (DE), a highly ethoxylated surfactant. Fluorescent saccharides were then produced and laser scanning microscopy was used to analyze their foliar diffusion pattern in Arabidopsis thaliana and grapevine. Interestingly, sucrose and PS3 were seemingly able to penetrate the leaf cuticle only when formulated with DE. Diffusion could preferentially occur via stomata, anticlinal cell walls and trichomes. In grapevine, PS3 penetration rate was much higher on the stomateous abaxial surface of the leaf than on the adaxial surface. Finally, using DE allowed a higher level of downy mildew control by PS3, which corroborated diffusion observations. Our results have practical consequences for the improvement of treatments with saccharidic inducers on grape. That is, formulation of such RIs plays a critical role for their cuticular diffusion and consequently their efficacy. Also, spray application should preferentially target the abaxial surface of the leaves in order to maximize their penetration.     

Association of RGA-SSCP markers with resistance to downy mildew and anthracnose in grapevines

Downy mildew (Plasmopara viticola) and anthracnose (Sphaceloma ampelinum) are two major diseases that severely affect most grapevine (Vitis vinifera) cultivars grown commercially in Thailand. Progress of conventional breeding programs of grapevine for improved resistance to these diseases can be speeded up by selection of molecular markers associated with resistance traits. We evaluated the association between 13 resistance gene analog (RGA)-single-strand conformation polymorphism (SSCP) markers with resistance to downy mildew and anthracnose in 71 segregating progenies of seven cross combinations between susceptible cultivars and resistant lines. F(1) hybrids from each cross were assessed for resistance to downy mildew and anthracnose (isolates Nk4-1 and Rc2-1) under laboratory conditions. Association of resistance traits with RGA-SSCP markers was evaluated using simple linear regression analysis. Three RGA-SSCP markers were found to be significantly correlated with anthracnose resistance, whereas significant correlation with downy mildew resistance was observed for only one RGA-SSCP marker. These results demonstrate the usefulness of RGA-SSCP markers. Four candidate markers with significant associations to resistance to these two major diseases of grapevine were identified. However, these putative associations between markers and resistance need to be verified with larger segregating populations before they can be used for marker-assisted selection.     

Identification of effector genes from the phytopathogenic Oomycete Plasmopara viticola through the analysis of gene expression in germinated zoospores

Grapevine downy mildew caused by the Oomycete Plasmopara viticola is one of the most important diseases affecting Vitis spp. The current strategy of control relies on chemical fungicides. An alternative to the use of fungicides is using downy mildew resistant varieties, which is cost-effective and environmentally friendly. Knowledge about the genetic basis of the resistance to P. viticola has progressed in the recent years, but little data are available about P. viticola genetics, in particular concerning the nature of its avirulence genes. Identifying pathogen effectors as putative avirulence genes is a necessary step in order to understand the biology of the interaction. It is also important in order to select the most efficient combination of resistance genes in a strategy of pyramiding. On the basis of knowledge from other Oomycetes, P. viticola effectors can be identified by using a candidate gene strategy based on data mining of genomic resources. In this paper we describe the development of Expressed Sequence Tags (ESTs) from P. viticola by creating a cDNA library from in vitro germinated zoospores and the sequencing of 1543 clones. We present 563 putative nuclear P. viticola unigenes. Sequence analysis reveals 54 ESTs from putative secreted hydrolytic enzymes and effectors, showing the suitability of this material for the analysis of the P. viticola secretome and identification of effector genes. Next generation sequencing of cDNA from in vitro germinated zoospores should result in the identification of numerous candidate avirulence genes in the grapevine/downy mildew interaction.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5-6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.     

Defense and resistance-inducing activities in tobacco of the sulfated beta-1,3 glucan PS3 and its synergistic activities with the unsulfated molecule

Laminarin, a beta-1,3 glucan with single beta-glucose branches at position 6, was chemically sulfated to produce PS3 with a degree of sulfation of 2.4. PS3 has previously been shown to activate the salicylic acid (SA) signaling pathway in infiltrated tobacco and Arabidopsis thaliana leaf tissues. Here, we investigated whether PS3 induces systemic defense and resistance responses in tobacco. Using a radiolabeled compound, it was first demonstrated that PS3 remains strictly localized to the infiltrated tissues. PS3 is also resistant to beta-glucanase degradation. In transgenic PR1-beta-glucuronidase (GUS) tobacco plants, PS3 causes a strong increase in GUS activity in treated tissues but none in untreated leaves. PS3-infiltrated tissues challenged with tobacco mosaic virus (TMV) 8 d after elicitor application show a decrease in both the lesion number and the lesion size, whereas treatment with laminarin, the unsulfated native glucan, affected only the lesion number. PS3 does not induce systemic acquired resistance to TMV. PS3 and laminarin show synergistic effects in promoting the oxidative burst in tobacco cell suspensions and in increasing the expression of genes encoding O-methyltransferases of the phenylpropanoid pathway in tobacco plants. No synergistic effect was observed on the expression of either the SA-dependent acidic PR1 gene or the ethylene-dependent basic PR5 gene in tobacco plants.     

The host guides morphogenesis and stomatal targeting in the grapevine pathogen <Emphasis Type="Italic">Plasmopara viticola</Emphasis>

The oomycete grape downy mildew (Plasmopara viticola Berk. & Curt. Ex de Bary) is a serious pathogen of grapevine and spreads by extremely efficient cycles of asexual propagation. The high efficiency must involve efficient sensing of the host. We therefore analyzed the time course and morphology of the early development of this pathogen in a host system, by infection of leaf discs of grapevine (Vitis vinifera L. cv. Müller-Thurgau), and in a host-free system. Host factors were demonstrated to influence pathogen development in the following ways: (i) the release of zoospores from mature sporangia was accelerated, (ii) the morphogenesis of the germ tube was coordinated, and (iii) the zoospores were targeted to the stomata by factors that depended on stomata closure. The findings show that the early development of P. viticola is regulated, specifically and coordinately, by factors originating from the host plant.     

More years of field trials against Plasmopara viticola in organic viticolture

The organic viticulture assumes a decisive role in the national agricultural sector. More impelling problems in the management of organic vineyards are represented from the plants pathology defence and particularly of Plasmopara viticola containment. Copper represents one of the few usable fungicides in the organic farming and the only effective against downy mildew. With Regulation EC n. 473/2002, fixed maximum quantity usable of copper compounds, owing to the environment problems due to the copper accumulation in the soil. To reduce quantity of metal copper or replace it with natural products, are conducted field trials with copper compounds at a low rate or alternative to copper products. Besides, we are estimating possibility to reduce the operations against P. viticola optimizing fungicidal treatments. Field trials in the organic farms located near Rome, have been carried out. Guidelines EPPO/OEPP PP 1/31 (3) have been carried out. The results of the trials have showed that, using cupric products with low metallic content, to reduce copper quantities used, always allowed to respect the limits established by Regulation EC. The alternative products that were investigated have not guaranteed, instead, an adequate protection in high pressure of grapevine downy mildew. It was possible to reduce treatments against P. viticola through control of different environmental parameters. The trials confirm that the copper is indispensable for plant protection in organic farming as it is not possible to replace it with natural extracts substances. We can reduce, instead, the copper quantities used trough the use of new products with low quantity metal copper or through the evaluation of climatic and pedologic data that allow to rationalize the fungicidal treatments.