Integrated signaling network involving calcium, nitric oxide, and active oxygen species but not mitogen-activated protein kinases in BcPG1-elicited grapevine defenses

We have already reported the identification of the endopolygalacturonase 1 (BcPG1) from Botrytis cinerea as a potent elicitor of defense responses in grapevine, independently of its enzymatic activity. The aim of the present study is the analysis of the signaling pathways triggered by BcPG1 in grapevine cells. Our data indicate that BcPG1 induces a Ca2+ entry from the apoplasm, which triggers a phosphorylation-dependent nitric oxide (NO) production via an enzyme probably related to a NO synthase. Then NO is involved in (i) cytosolic calcium homeostasis, by activating Ca2+ release from internal stores and regulating Ca2+ fluxes across the plasma membrane, (ii) plasma membrane potential variation, (iii) the activation of active oxygen species (AOS) production, and (iv) defense gene expression, including phenylalanine ammonia lyase and stilbene synthase, which encode enzymes responsible for phytoalexin biosynthesis. Interestingly enough, mitogen-activated protein kinase (MAPK) activation is independent of this regulation pathway that closely connects Ca2+, NO, and AOS.     

Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris

Five Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the V(max,app) ranged from 10 to 900 U mg(-1), while the K(m,app) ranged from 0.16 to 0.6 mg ml(-1). Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP < 7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50-85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea.     

Elicitor and resistance-inducing activities of beta-1,4 cellodextrins in grapevine, comparison with beta-1,3 glucans and alpha-1,4 oligogalacturonides

Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response.     

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.     

A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.     

Evolutionary analysis of endopolygalacturonase-encoding genes of Botrytis cinerea

Sequence analysis of five of the six endopolygalacturonase-encoding genes ( Bcpg1 , Bcpg2 , Bcpg3 , Bcpg4 , Bcpg5 ) from 32 strains of Botrytis cinerea showed marked gene to gene differences in the amount of among-strains diversity. Bcpg4 was almost invariable in all strains; Bcpg3 and Bcpg5 showed a moderate variability, similar to that of non-pathogenicity-associated genes examined in other studies. Conversely, Bcpg1 and Bcpg2 were highly variable and were shown to be under positive selection based on the McDonald–Kreitman test and likelihood ratio test. The evolution of the five endopolygalacturonase genes is explained by their different ecophysiological role. Diversification and balancing selection, as detected in Bcpg1 and Bcpg2 , can be used by the pathogen to escape recognition by the host and delay plant reaction in the early phases of infection. The analysis of the polymorphisms and the location of the sites with high probability of being positively selected highlighted the relevance of variability of the BcPG1 and BcPG2 proteins at their C-terminal end. By contrast, the absence of variability in Bcpg4 suggests that the efficiency of the product of this gene is critical for B. cinerea growth in late phases of infection or during intraspecific competition, thus markedly affecting strain fitness.     

The polygalacturonase-inhibiting protein PGIP2 of Phaseolus vulgaris has evolved a mixed mode of inhibition of endopolygalacturonase PG1 of Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungus that causes gray mold in >1,000 plant species. During infection, it secretes several endopolygalacturonases (PGs) to degrade cell wall pectin, and among them, BcPG1 is constitutively expressed and is an important virulence factor. To counteract the action of PGs, plants express polygalacturonase-inhibiting proteins (PGIPs) that have been shown to inhibit a variety of PGs with different inhibition kinetics, both competitive and noncompetitive. The PG-PGIP interaction promotes the accumulation of oligogalacturonides, fragments of the plant cell wall that are general elicitors of plant defense responses. Here, we characterize the enzymatic activity of BcPG1 and investigate its interaction with PGIP isoform 2 from Phaseolus vulgaris (PvPGIP2) by means of inhibition assays, homology modeling, and molecular docking simulations. Our results indicate a mixed mode of inhibition. This is compatible with a model for the interaction where PvPGIP2 binds the N-terminal portion of BcPG1, partially covering its active site and decreasing the enzyme affinity for the substrate. The structural framework provided by the docking model is confirmed by site-directed mutagenesis of the residues that distinguish PvPGIP2 from the isoform PvPGIP1. The finding that PvPGIP2 inhibits BcPG1 with a mixed-type kinetics further indicates the versatility of PGIPs to evolve different recognition specificities.     

Elevated genetic variation within virulence-associated Botrytis cinerea polygalacturonase loci

Botrytis cinerea, or gray mold, is a necrotrophic fungal pathogen of hundreds of plant species. The genetic diversity of B. cinerea may contribute to its broad host range; however, the level and structure of genetic variation at pathogenesis-associated loci has not been described. B. cinerea possesses six distinct cell-wall-degrading polygalacturonases (PGs), enzymes of demonstrated importance to pathogenesis and interaction with host plant defenses. Sequencing a collection of 34 B. cinerea isolates at three PG-encoding loci, BcPG1, BcPG2, and BcPG3, revealed limited evidence of host-mediated genetic subdivision within loci, yet suggested differences in the action of evolutionary forces among loci. BcPG1 and BcPG2 are highly polymorphic, particularly when compared with previously published data from nonpathogenicity loci, whereas BcPG3 is relatively conserved. Sequence variation at BcPG1 and BcPG2 did not appear to be associated with virulence on Arabidopsis leaves; however, BcPG2 variation showed a statistically significant association with growth rate on pectin. Rather than providing evidence for host-mediated genetic subdivision at individual PG loci, our data support specialization among PGs and the potential diversification of PGs interacting directly with host defenses.     

Purification and characterization of two isozymes of polygalacturonase from Botrytis cinerea. Effect of calcium ions on polygalacturonase activity

The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl2, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.     

Bacterial rhamnolipids are novel MAMPs conferring resistance to Botrytis cinerea in grapevine

Rhamnolipids produced by the bacteria Pseudomonas aeruginosa are known as very efficient biosurfactant molecules. They are used for a wide range of industrial applications, especially in food, cosmetics and pharmaceutical formulations as well as in bioremediation of pollutants. In this paper, the role of rhamnolipids as novel molecules triggering defence responses and protection against the fungus Botrytis cinerea in grapevine is presented. The effect of rhamnolipids was assessed in grapevine using cell suspension cultures and vitro-plantlets. Ca²⁺ influx, mitogen-activated protein kinase activation and reactive oxygen species production form part of early signalling events leading from perception of rhamnolipids to the induction of plant defences that include expression of a wide range of defence genes and a hypersensitive response (HR)-like response. In addition, rhamnolipids potentiated defence responses induced by the chitosan elicitor and by the culture filtrate of B. cinerea . We also demonstrated that rhamnolipids have direct antifungal properties by inhibiting spore germination and mycelium growth of B. cinerea . Ultimately, rhamnolipids efficiently protected grapevine against the fungus. We propose that rhamnolipids are acting as microbe-associated molecular patterns (MAMPs) in grapevine and that the combination of rhamnolipid effects could participate in grapevine protection against grey mould disease.     

Exogenous application of a lipid transfer protein-jasmonic acid complex induces protection of grapevine towards infection by Botrytis cinerea.

Type I plant lipid transfer proteins (LTPs) are small, basic, cystein-rich proteins involved in plant defense mechanisms. Five type I LTPs isoforms, named VvLTP1, 2, 3, 4 and 5 (Vitis vinifera lipid transfer proteins 1-5) were purified to homogeneity from the culture media of 41B grapevine cell suspension. The full sequence of isoforms 1, 3, 4 and 5 could be determined from mass spectrometry measurements of the enzymatically hydrolyzed proteins and from available VvLTP sequences. Phylogenetic analysis revealed that these proteins form two subgroups, one with isoforms 1 and 4, and the second one with isoforms 3 and 5. The ability of the three most abundant ones (VvLTP1, 4 and 3) to interact with jasmonic acid (JA) was tested by fluorometric studies, showing that VvLTP4 was the most efficient to interact with this oxylipin. Exogenous application of the VvLTP4-JA complex on grapevine plantlets induced a high level (80.3+/-10.05%) of tolerance towards Botrytis cinerea, as compared with control plants (18.65+/-12.13%); whereas plants treated with JA or VvLTP4 alone exhibited a lower protection level (31.04+/-9.72% and 45.52+/-7.51% of protection, respectively). The results are discussed in the context of grapevine defense mechanisms.     

Characterization of a new, nonpathogenic mutant of Botrytis cinerea with impaired plant colonization capacity

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.     

Elicitation of Casbene Synthetase Activity in Castor Bean : THE ROLE OF PECTIC FRAGMENTS OF THE PLANT CELL WALL IN ELICITATION BY A FUNGAL ENDOPOLYGALACTURONASE

Endopolygalacturonase isolated from culture filtrates of the fungus Rhizopus stolonifer was shown previously to act as an elicitor of biosynthetic capacity for the antifungal agent, casbene, in castor bean (Ricinus communis L.) seedlings (S.-C. Lee, C.A. West 1981 Plant Physiology 67:633-639). Selective amidation of exposed carboxyl groups of the pure fungal endopolygalacturonase using intermediate activation with a water-soluble carbodiimide under mild conditions leads to inactivation of its enzymic activity. Tests of active and partially inactivated preparations of the enzyme reveal a close correlation between the levels of catalytic and elicitor activities. This suggests that the catalytic activity of the enzyme is necessary for its function as an elicitor. Treatment of the cell-free particulate fraction of homogenates of castor bean seedlings with the active fungal endopolygalacturonase results in the production of a heat-stable, water-soluble component which is highly active as an elicitor of casbene synthetase activity. Several additional lines of evidence, including the susceptibility of the heat-stable elicitor fraction to partial inactivation following prolonged treatment with endopolygalacturonase, indicate that the heat-stable elicitor is most likely a pectic fragment of the plant cell wall and that it is a required intermediate in the process of elicitation of casbene synthetase activity by the fungal endopolygalacturonase.     

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.     

Induction of Volatile Organic Compounds of Lycopersicon esculentum Mill. and Its Resistance to Botrytis cinerea Pers. by Burdock Oligosaccharide

In the present study, we investigated the induction of volatile organic compounds （VOCs） of Lycoperslcon esculentum Mill. and its resistance to Botrytis cinerea Pers. by burdock oligosaccharlde. The disease severity of L. esculentum was evaluated 48 h after treatment with 0.6% burdock oligosaccharlde, followed by inoculation with a spore suspension of B. cinerea. The formation of O2＇, the activity of lipoxygenases （LOX）, peroxidase （POD）, cataiase （CAT）, and superoxide dismutase （SOD）, and the quantity and quallty of changes In VOCs were determined a period of time after treatment with 0.6% burdock ollgosaccharide. The results demonstrated that the disease index in treated plants was decreased by 42.5% compared with control 96 h after Inoculation. The production of O2＇ reached a maximum 6 h after treatment （1.36-fold compared with control）. There was an increase in LOX, POD, CAT and SOD activity in response to burdock oligosaccharide treatment and the enzymes showed different trends in the time-course of induction. At 120 h after treatment, （E）-2-hexenal was increased by 92% compared with control, whereas methyl salicylate showed a gradual Increase with induction period. Previous results had demonstrated that chitosan elicitor enhanced the production VOCs of L. esculentum and decreased plant susceptibility towards B. clnerea. Together, these findings suggest that increasing the production of VOCs in response to burrdock oligosaccharide may be an important mechanism for L. esculentumin its defense against pathogens, in addition, burrdock oligosaccharlde may act as a potent elicitor of resistance to disease in L. esculentum.     

Botrytis cinerea manipulates the antagonistic effects between immune pathways to promote disease development in tomato

Plants have evolved sophisticated mechanisms to sense and respond to pathogen attacks. Resistance against necrotrophic pathogens generally requires the activation of the jasmonic acid (JA) signaling pathway, whereas the salicylic acid (SA) signaling pathway is mainly activated against biotrophic pathogens. SA can antagonize JA signaling and vice versa. Here, we report that the necrotrophic pathogen Botrytis cinerea exploits this antagonism as a strategy to cause disease development. We show that B. cinerea produces an exopolysaccharide, which acts as an elicitor of the SA pathway. In turn, the SA pathway antagonizes the JA signaling pathway, thereby allowing the fungus to develop its disease in tomato (Solanum lycopersicum). SA-promoted disease development occurs through Nonexpressed Pathogen Related1. We also show that the JA signaling pathway required for tomato resistance against B. cinerea is mediated by the systemin elicitor. These data highlight a new strategy used by B. cinerea to overcome the plant's defense system and to spread within the host.     

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.     

Antisense expression of the Arabidopsis thaliana AtPGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea

Polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall pectin and are important virulence factors of several phytopathogenic fungi. In response to abiotic and biotic stress, plants accumulate PG-inhibiting proteins (PGIPs) that reduce the activity of fungal PGs. In Arabidopsis thaliana, PGIPs with comparable activity against BcPG1, an important pathogenicity factor of the necrotrophic fungus Botrytis cinerea, are encoded by two genes, AtPGIP1 and AtPGIP2. Both genes are induced by fungal infection through different signaling pathways. We show here that transgenic Arabidopsis plants expressing an antisense AtPGIP1 gene have reduced AtPGIP1 inhibitory activity and are more susceptible to B. cinerea infection. These results indicate that PGIP contributes to basal resistance to this pathogen and strongly support the vision that this protein plays a role in Arabidopsis innate immunity.