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Crude extracts of Vitis vinifera canes represent a natural source of stilbene compounds with well characterized antifungals properties. In our trials, exogenous application of a stilbene extract (SE) obtained from grape canes on grapevine leaves reduces the necrotic lesions caused by Botrytis cinerea. The SE showed to possess a direct antifungal activity by inhibiting the mycelium growth. The activation of some grapevine defense mechanism was also investigated. H 2 O 2 production and activation of mitogen-activated protein kinase (MAPK) phosphorylation cascades as well as accumulation of stilbenoid phytoalexins were explored on grapevine cell suspension. Moreover, the transcription of genes encoding for proteins affecting defense responses was analyzed on grapevine plants. The SE induced some grapevine defense mechanisms including MAPK activation, and the expression of pathogenesis-related (PR) genes and of a gene encoding the glutathione-S-transferase 1 ( GST1) . By contrast, treatment of grapevine leaves with SE negatively regulates de novo stilbene production.

Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea, while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus spreading. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes upregulated during infection of MB are enriched in functional categories related to necrotrophy, such as degradation of the plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation, and detoxification. Quantitative-polymerase chain reaction on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but is stopped at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathway switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin, and cell-wall strengthening. On the contrary, in infected MB, the jasmonate-dependent pathway was activated, which did not stop the fungal necrotrophic process.

Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. In this study, we investigated the production and/or function of NO in Arabidopsis thaliana leaf discs and plants elicited by oligogalacturonides (OGs) and challenged with Botrytis cinerea. We provided evidence that OGs triggered a fast and long lasting NO production which was Ca(2+) dependent and involved nitrate reductase (NR). Accordingly, OGs triggered an increase of both NR activity and transcript accumulation. NO production was also sensitive to the mammalian NO synthase inhibitor L-NAME. Intriguingly, we showed that L-NAME affected NO production by interfering with NR activity, thus questioning the mechanisms of how this compound impairs NO synthesis in plants. We further demonstrated that NO modulates RBOHD-mediated reactive oxygen species (ROS) production and participates in the regulation of OG-responsive genes such as anionic peroxidase (PER4) and a β-1,3-glucanase. Mutant plants impaired in PER4 and β-1,3-glucanase, as well as Col-0 plants treated with the NO scavenger cPTIO, were more susceptible to B. cinerea. Taken together, our investigation deciphers part of the mechanisms linking NO production, NO-induced effects and basal resistance to B. cinerea., (© 2012 Blackwell Publishing Ltd.)

Botrytis cinerea is a necrotrophic pathogen that attacks more than 200 plant species. Here, the nonpathogenic mutant A336, obtained via insertional mutagenesis, was characterized. Mutant A336 was nonpathogenic on leaves and fruits, on intact and wounded tissue, while still able to penetrate the host plant. It grew normally in vitro on rich media but its conidiation pattern was altered. The mutant did not produce oxalic acid and exhibited a modified regulation of the production of some secreted proteins (acid protease 1 and endopolygalacturonase 1). Culture filtrates of the mutant triggered an important oxidative burst in grapevine (Vitis vinifera) suspension cells, and the mutant-plant interaction resulted in the formation of hypersensitive response-like necrosis. Genetic segregation analyses revealed that the pathogenicity phenotype was linked to a single locus, but showed that the mutated gene was not tagged by the plasmid pAN7-1. Mutant A336 is the first oxalate-deficient mutant to be described in B. cinerea and it differs from all the nonpathogenic B. cinerea mutants described to date.

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.

A purified glycoprotein from Botrytis cinerea (strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

Nonspecific lipid transfer proteins (nsLTPs) are small, basic cystein-rich proteins believed to be involved in plant defense mechanisms. Three cDNAs coding nsLTPs from grape (Vitis vinifera sp.) were cloned by reverse-transcriptase-polymerase chain reaction (RT-PCR) and PCR. The expression of nsLTP genes was investigated in 41B-rootstock grape cell suspension, in response to various defense-related signal molecules. Ergosterol (a fungi-specific sterol) and a proteinaceous elicitor purified from Botrytis cinerea strongly and rapidly induced the accumulation of nsLTP mRNAs. Jasmonic acid, cholesterol, and sitosterol also promoted nsLTPs mRNA accumulation, although to a lesser extent, whereas salicylic acid had no effect. High performance liquid chromatography analysis indicated that the amounts of three LTP isoforms (previously named P1, P2, and P4) were increased by ergosterol. None of the four isoforms displayed any significant antifungal properties, with the exception of the P4 isoform, which reduced Botrytis mycelium growth in vitro, but only in calcium-free medium. The results are discussed in the context of plant-pathogen interactions.

Resveratrol is a major phytoalexin in grapevine but its synthesis in response to phytopathogen attack decreases with grape berry ripening. A chimeric gene combining an alfalfa PR 10 promoter and Vst1 (Vitis stilbene synthase 1) gene was introduced into the genome of 41B rootstock. Transgenic plants were analysed for resveratrol production in leaves infected with Botrytis using an in vitro test. Among the 50 transgenic lines analysed, some exhibited a production lower than the non-transgenic control, but others accumulated resveratrol from 5-100-fold. Moreover, in the latter clones, symptoms were highly reduced in response to infection. These results were a good indication that the combination of a pathogen-inducible promoter and a defence gene may increase tolerance against fungi in grapevine. The efficacy of this approach should be further tested by experiments conducted in the vineyard.