Showing papers on "Elicitor published in 1997"

Elicitor-stimulated ion fluxes and O2− from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley

Abstract: Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca2+ from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2− but not H2O2 stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2− rather than H2O2 appears to trigger the subsequent reactions.

Two Distinct Sources of Elicited Reactive Oxygen Species in Tobacco Epidermal Cells.

Abstract: Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured by dichlorofluorescein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death.

Elicitor-stimulated ion fluxes and O2 from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley (furanocoumarinsyion channelsypeptide elicitoryreactive oxygen speciesysignal transduction)

Abstract: Fungal elicitor stimulates am ulticomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the produc- tionofreactiveoxygenspecies(ROS),sequentiallyfollowedby defense gene activation and phytoalexin accumulation. Omis- sion of Ca 21 from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers pre- vented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes.Inhibitionofelicitor-stimulatedROSproductionusing diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2 but not H2O2stimulated phy- toalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activa- tion of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2 ratherthanH2O2appearstotriggerthesubsequentreactions.

Early events induced by the elicitor cryptogein in tobacco cells: involvement of a plasma membrane NADPH oxidase and activation of glycolysis and the pentose phosphate pathway

Abstract: Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.

A Gene Encoding a Protein Elicitor of Phytophthora infestans Is Down-Regulated During Infection of Potato

Abstract: Most species of the genus Phytophthora produce 10-kDa extracellular protein elicitors, collectively termed elicitins. Elicitins induce hypersensitive response in a restricted number of plants, part...

The structure and function of a soybean beta-glucan-elicitor-binding protein.

Abstract: β-Glucan elicitor (GE), released from the cell wall of the phytopathogenic fungus Phytophthora megasperma by soybean glucanases, causes defense reactions in soybean. A GE-binding protein (GEBP) was purified from the membrane fraction of soybean root cells, and its cDNA was isolated. Expression of the cDNA clone in tobacco suspension cultured cells and in Escherichia coli conferred GE-binding activity to both. An antibody against the recombinant protein was found to inhibit the GE binding with the soybean cotyledon membrane fraction as well as the resulting accumulation of phytoalexin. Immunolocalization assays indicated that the GEBPs are located in the plasma membrane of root cells. These results suggest that the cDNA encodes a GE receptor and may mediate the signaling of the elicitor.

Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense

Abstract: Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley.

Activation of plant plasma membrane Ca2+-permeable channels by race-specific fungal elicitors

Abstract: The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.

Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species

Abstract: A strategy for elucidating specific molecular targets of calcium and calmodulin in plant defense responses has been developed. We have used a dominant-acting calmodulin mutant (VU-3, Lys to Arg115) to investigate the oxidative burst and nicotinamide co-enzyme fluxes after various stimuli (cellulase, harpin, incompatible bacteria, osmotic and mechanical) that elicit plant defense responses in transgenic tobacco cell cultures. VU-3 calmodulin differs from endogenous plant calmodulin in that it cannot be methylated post-translationally, and as a result it hyperactivates calmodulin-dependent NAD kinase. Cells expressing VU-3 calmodulin exhibited a stronger active oxygen burst that occurred more rapidly than in normal control cells challenged with the same stimuli. Increases in NADPH level were also greater in VU-3 cells and coincided both in timing and magnitude with development of the active oxygen species (AOS) burst. These data show that calmodulin is a target of calcium fluxes in response to elicitor or environmental stress, and provide the first evidence that plant NAD kinase may be a downstream target which potentiates AOS production by altering NAD(H)/NADP(H) homeostasis.

The biotrophic fungus Cladosporium fulvum circumvents Cf-4-mediated resistance by producing unstable AVR4 elicitors.

Abstract: The avirulence gene Avr4 conditions avirulence of the biotrophic fungus Cladosporium fulvum on tomato genotypes carrying resistance gene Cf-4 (MM-Cf4). Strains of the fungus that circumvent Cf-4-specific resistance show various single point mutations in the coding region of the Avr4 gene. Similar to expression of the Avr4 gene, expression of the various virulent avr4 alleles is specifically induced during pathogenesis. Polyclonal antibodies raised against the AVR4 elicitor, however, did not detect AVR4 isoforms in MM-Cf4 plants infected by the different virulent strains, indicating that these isoforms are unstable. To analyze whether the AVR4 isoforms still possess specific elicitor activity, the avr4 alleles were expressed in MM-Cf4 plants by using the potato virus X (PVX)-based expression system. Inoculation with PVX::Avr4 resulted in the development of spreading lesions, eventually leading to plant death, whereas the various PVX::avr4 derivatives induced symptoms ranging from severe necrosis to no lesions at all. We conclude that instability of the AVR4 isoforms that are produced by virulent strains is a crucial factor in circumvention of Cf-4-mediated resistance.

Differential Expression of Chitinases in Vitis vinifera L. Responding to Systemic Acquired Resistance Activators or Fungal Challenge

Abstract: The concept of systemic acquired resistance (SAR) enables a novel approach to crop protection, and particular pathogenesis-related proteins, i.e. an acidic chitinase, have been classified as markers of the SAR response. Basic class I (VCHIT1b) and a class III (VCH3) chitinase cDNAs were cloned from cultured Vitis vinifera L. cv Pinot Noir cells and used to probe the induction response of grapevine cells to salicylic acid or yeast elicitor. Furthermore, the cells were treated with the commercial SAR activators 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester. Elicitor or salicylic acid induced both VCHIT1b and VCH3 transcript abundances, whereas 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester enhanced exclusively the expression of VCH3. To assess the systemic sensation of chitinase expression, single leaves of Vitis vinifera L. cv Pinot Noir or Vitis rupestris plants were inoculated with Plasmopara viticola spore suspensions, and the VCH3 and VCHIT1b mRNA amounts in the infected versus the adjacent, healthy leaf were monitored. Two VCH3 mRNA maxima were observed 2 and 6 d postinoculation in the infected, susceptible V. vinifera tissue, whereas in the healthy leaf the transcript increased from low levels d 2 postinoculation to prominent levels d 6 to 8 postinoculation. The level of VCH3 mRNA increased also over 4 d in the inoculated, resistant V. rupestris tissue. However, necrotic spots rapidly limited the infection, and the VCH3 transcript was undetectable in the upper-stage, healthy leaf. The expression of VCHIT1b remained negligible under either experimental condition. Overall, the results suggest that the selective expression of VCH3 might be a reliable indicator of the SAR response in V. vinifera L.

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling.

Abstract: A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [125I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([125I]-GN8-AzPEA) and a [125I]-labeled 2-(4-aminophenyl)ethylamino conjugate ([125]-GN8-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligosaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)8 showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)8, cellopentaose nor alpha-1,4 linked N-acetylgalactosamine octamer (GalNAc)8 at concentrations as high as 25 microM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the 'high-affinity binding site' previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.

Fungal pathogenesis in plants and crops: molecular biology and host defense mechanisms.

Abstract: PERCEPTION AND TRANSDUCTION OF PLANT SIGNALS IN PATHOGENS Introduction Signaling and Transduction Systems in "First Touch" and Adhesion of Fungal Spores Signaling in Fungal Spore Germination Signaling in Differentiation of Germ Tubes into Infection Structures Signal Transduction in Fungal Pathogenesis Genes Involved in Formation of Infection Structures Signals in Fungal Infection Process Conclusion References PERCEPTION AND TRANSDUCTION OF PATHOGEN SIGNALS IN PLANTS Introduction What Are Elicitors? Oligosaccharide Elicitors Protein Peptide Elicitors Glycoprotein Elicitors Lipid Elicitors Toxins as Elicitor Molecules Plant Cell Wall-Degrading Enzymes as Elicitors Race-Specific and Cultivar-Specific Elicitors Specificity of General Elicitors Endogenous Oligogalacturonide Elicitors Multiple Elicitors May Be Needed to Activate Defense Responses Availability of Fungal Elicitors at the Site of Fungal Invasion in Plants Receptors for Elicitor Signals in Plant Cell Membrane Calcium Ion May Act as Second Messenger Phosphorylation of Proteins as a Component in Signal Transduction System Mitogen-Activated Protein Kinase Cascades in Signal Transduction Phospholipid-Signaling System Anion Channels in Signal Transduction Extracellular Alkalinization and Cytoplasmic Acidification in Signaling System Reactive Oxygen Species in Signal Transduction Nitric Oxide in Signal Transduction Salicylic Acid-Signaling System Jasmonate-Signaling Pathway Role of Systemin in Signal Transduction System Ethylene-Dependent Signaling Pathway Abscisic Acid Signaling Fatty Acids as Systemic Signal Molecules Other Signaling Systems Network and Interplay of Signaling Pathways Induction of Defense Genes May Require Different Signal Transduction Systems Perception and Transduction of Pathogen Signals in Plants Leading to Susceptibility Signaling Systems in Susceptible Interactions Conclusion References DISEASE RESISTANCE AND SUSCEPTIBILITY GENES IN SIGNAL PERCEPTION AND EMISSION Introduction Molecular Structure of Resistance Genes Classification of Resistance Genes based on Molecular Structure of R Gene-Encoded Proteins Molecular Structure of Recessive Genes Perception of Pathogen Signals by Resistance Genes Activation of R Protein and Emission of Signals to Other Components in the Cell Downstream Components of R Gene Signaling Systems Downstream Signaling Events in R Gene-Mediated Resistance Susceptibility Genes in Signal Transduction Conclusion References CELL DEATH PROGRAMS DURING FUNGAL PATHOGENESIS Introduction Cell Death in Resistant Interactions Molecular Mechanism of Induction of Hypersensitive Cell Death Molecular Mechanism of Induction of Spontaneous Cell Death Molecular Mechanism of Induction of Runaway Cell Death Role of Cell Death in Induction of Systemic Acquired Resistance Susceptibility-Related Cell Death Molecular Mechanisms in Induction of Cell Death in Susceptible Interactions What Is the Function of Cell Death in Fungal Pathogenesis? Conclusion References CELL WALL DEGRADATION AND FORTIFICATION Introduction Structure of Cuticle Penetration of Epicuticular Waxy Layer by Pathogens Production of Cutinases to Breach Cuticle Barrier Genes Encoding Cutinases Plant Signals Triggering Fungal Cutinases Importance of Cutinases in Penetration of Cuticle Cutinases as Virulence Pathogenicity Factors Melanins in Fungal Penetration of Cuticle Barrier Degradation of Pectic Polysaccharides Pathogens Produce Cellulolytic Enzymes to Breach Cell Wall Barrier Fungal Hemicellulases in Plant Cell Wall Degradation Degradation of Cell Wall Structural Proteins Requirement of Several Cell Wall-Degrading Enzymes to Degrade the Complex-Natured Cell Wall Production of Suitable Enzymes in Appropriate Sequence by Fungal Pathogens Reinforcement of Host Cell Wall during Fungal Invasion Papillae Suppress Fungal Penetration Callose Deposition in Cell Wall How Do Pathogens Overcome the Papillae and Callose Barriers? Cell Wall-Bound Phenolics and Lignins Suberization during Fungal Pathogenesis Deposition of Mineral Elements in Host Cell Wall in Response to Fungal Invasion Conclusion References INDUCTION AND EVASION OF PATHOGENESIS-RELATED PROTEINS Introduction Multiplicity of PR Proteins Classification of PR Proteins Induction of PR Proteins during Fungal Pathogenesis Genes Encoding PR Proteins Transcription of PR Genes Signals Involved in Transcriptional Induction of PR Genes PR Proteins Are Synthesized as Larger Precursors Secretion of PR Proteins PR Proteins May Be Involved in Inhibition of Pathogen Development PR Proteins May Be Involved in Triggering Disease Resistance How Do Pathogens Overcome Fungitoxic PR Proteins of the Host? Conclusion References EVASION AND DETOXIFICATION OF SECONDARY METABOLITES Introduction Chemical Structural Classes of Phytoalexins Biosynthesis of Isoflavonoid Phytoalexins Biosynthesis of Flavanone Phytoalexins Biosynthesis of Coumarin Phytoalexins Biosynthesis of Stilbene Phytoalexins Biosynthesis of Terpenoid Phytoalexins Biosynthesis of Indole-Based Sulfur-Containing Phytoalexins Biosynthesis of Alkaloid Phytoalexins Site of Synthesis of Phytoalexins Phytoalexins Are Fungitoxic How Do Pathogens Overcome the Antifungal Phytoalexins? Chemical Structural Classes of Phytoanticipins Phenolics as Phytoanticipins Toxicity of Phenolics to Pathogens How Does Pathogen Overcome the Antifungal Phenolics? Saponins as Phytoanticipins Glucosinolates as Phytoanticipins Cyanogenic Glucosides Dienes Conclusion References TOXINS IN DISEASE SYMPTOM DEVELOPMENT Introduction Importance of Toxins in Disease Development Toxins Suppress Host-Defense Mechanisms Toxins Cause Cell Membrane Dysfunction How Do Pathogens Induce Membrane Dysfunction only in Susceptible Hosts? Conclusion References Index

Spatial and Temporal Induction of Cell Death, Defense Genes, and Accumulation of Salicylic Acid in Tobacco Leaves Reacting Hypersensitively to a Fungal Glycoprotein Elicitor

Abstract: We have analyzed the spatial and temporal expression of defense responses induced in attached tobacco leaves treated with a Phytophthora megasperma glycoprotein that was previously shown to be an efficient elicitor of the hypersensitive reaction The infiltrated tissue (zone 1), the surrounding tissue (zone 2), which is 5 mm in width, and tissue at a distance >2 cm from zone 2 (zone 3) were analyzed separately Cell death occurred only in zone 1 and was completed by 14 h Defense gene expression was induced in zones 1 and 2 with striking differential patterns, but not in zone 3 There was a rapid (1 to 4 h) induction of genes of the phenylpropanoid, salicylic acid, and ses-quiterpenoid pathways in zones 1 and 2 However, it was strong and transient in the former and of lesser extent but sustained in the latter High amounts of scopoletin, a phenylpropanoid metabolite, were found synthesized in zone 2 Pathogenesis-related (PR) transcripts and the corresponding PR proteins accumulated in high amounts in zo

Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways

Abstract: Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia-derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis-related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia, and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia. This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild-type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA-mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia. The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA-mediated response.

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels.

Abstract: The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.

Cloning and Characterization of a cDNA Encoding an Elicitor of Phytophthora parasitica var. nicotianae That Shows Cellulose-Binding and Lectin-Like Activities

Abstract: Phytophthora parasitica var. nicotianae produces a 34-kDa glycoprotein elicitor (CBEL) that is localized in the cell wall. A cDNA encoding the protein moiety of this elicitor was cloned and characterized. The deduced amino acid sequence consisted of two direct repeats of a cysteine-rich domain, joined by a Thr/Pro-rich region. Although having no general homology with published sequences, the positions of the cysteine residues in the two repeats show a conserved pattern, similar to that of the cellulose-binding domain of fungal glycanases. CBEL did not possess hydrolytic activity on a variety of glycans, but bound to fibrous cellulose and plant cell walls. In addition, it exerted a lectin-like hemagglutinating activity. Infiltration of tobacco leaves (cultivar 46–8) with this molecule elicited necrosis and defense gene expression at 150 nM. Elicitor pre-treatment of this tobacco cultivar resulted in protection against subsequent inoculation with an otherwise virulent race of P. parasitica var. nicotianae. ...

Differential induction of seven 1-aminocyclopropane-1-carboxylate synthase genes by elicitor in suspension cultures of tomato (Lycopersicon esculentum).

Abstract: The key enzyme of ethylene biosynthesis, ACC synthase, is encoded by a multigene family. We describe three new DNA sequences encoding members of the ACC synthase family of the tomato. One of these sequences encodes a novel ACC synthase, LE-ACS6, which is phylogenetically related to the ACC synthases LE-ACS1A and LE-ACS1B. Gene-specific probes for seven tomato ACC synthase genes were prepared. They were used for RNase protection assays to study the accumulation of ACC synthase transcripts in suspension-cultured tomato cells after the addition of an elicitor. The ACC synthase genes LE-ACS2, LE-ACS5 and LE-ACS6 were strongly induced by the elicitor. In contrast, the genes LE-ACS1B, LE-ACS3 and LE-ACS4 were constitutively expressed and LE-ACS1B was present at all times at a particularly high level. Thus, there are two groups of ACC synthase transcripts expressed in these cells, either elicitor-induced or constitutive. A transcript of LE-ACS1A was not detected. Despite the presence of LE-ACS1B, LE-ACS2, LE-ACS3, LE-ACS4 and LE-ACS5, there was only little ethylene produced in the absence of the elicitor. Increased ethylene production is usually correlated with the accumulation of ACC synthase transcripts, indicating that ethylene production is controlled via the transcriptional activation of ACC synthase genes. However, the abundance of several ACC synthase mRNAs studied was not strictly correlated with the rate of elicitor-induced ethylene production. Our data provide evidence that the activity of these ACC synthases may not solely be controlled by the transcriptional activation of ACC synthase genes.

Elicitor Actions of N-Acetylchitooligosaccharides and Laminarioligosaccharides for Chitinase and l-Phenylalanine Ammonia-lyase Induction in Rice Suspension Culture

Abstract: When a series of chitin oligosaccharides was added into a rice suspension culture, N-acetylchitohexaose, N-acetylchitopentaose, and N-acetylchitotetraose caused an increase in extracellular chitinase activity, mainly due to induction of a class III chitinase. In the case of N-acetylchitohexaose, a substantial increase in the chitinase activity was observed at a concentration higher than 0.01 micrograms/ml, and a maximum effect was reached at 1 microgram/ml. In contrast, N-acetylchitotriose, N-acetylchitobiose, N-acetyl-D-glucosamine, and chitohexaose (a chitosan oligosaccharide) were not very effective. Chitinase induction was also observed with laminarihexaose (a beta-1,3-glucan oligosaccharide), but about a 10-fold higher concentration, compared with N-acetylchitohexaose, was needed to get the maximum effect. beta-1,3-Glucanase activity was found in cells (but not in medium), and the activity was increased by neither N-acetylchitohexaose nor laminarihexaose. When cells were incubated with N-acetylchitohexaose, L-phenylalanine ammonia-lyase (PAL) activity increased promptly. A biphasic profile was obtained when a dose-dependent effect of the elicitor on the PAL induction was examined; the first phase was observed in a range from 0.01 to 1 microgram/ml and the second phase from 3 to 300 micrograms/ml. Laminarihexaose also acted as an elicitor for PAL induction.

Differential Induction of Lipoxygenase Isoforms in Wheat upon Treatment with Rust Fungus Elicitor, Chitin Oligosaccharides, Chitosan, and Methyl Jasmonate

Abstract: A glycopeptide elicitor prepared from germ tubes of the rust fungus Puccinia graminis Pers f sp tritici Erikss & Henn (Pgt), as well as chitin oligosaccharides, chitosan, and methyl jasmonate (MJ) stimulated lipoxygenase (LOX) activity (EC 1131112) in wheat (Triticum aestivum) leaves Immunoblot analysis using anti-LOX antibodies revealed the induction of 92- and 103-kD LOX species after Pgt elicitor treatment In contrast, MJ treatment led to a significant increase of a 100-kD LOX species, which was also detected at lower levels in control plants The effects of chitin oligomers and chitosan resembled those caused by MJ In conjunction with other observations the results suggest that separate reaction cascades exist, and that jasmonates may not be involved in Pgt elicitor action LOX-92 appears to be mainly responsible for the increase in LOX activity after Pgt elicitor treatment because its appearance on western blots coincided with high LOX activity in distinct anion-exchange chromatography fractions It is most active at pH 55 to 60, and product formation from linoleic and [alpha]-linolenic acid is clearly in favor of the 9-LOOHs It is interesting that a 92-kD LOX species, which seems to correspond to the Pgt elicitor-induced LOX species, was also detected in rust-inoculated leaves

Characterization and Expression of Caffeoyl-Coenzyme A 3-O-Methyltransferase Proposed for the Induced Resistance Response of Vitis vinifera L

Abstract: Cell-suspension cultures of Vitis vinifera L cv Pinot Noir accumulated resveratrol upon fungal elicitation, and the activity of S-adenosyI-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyl-transferase (CCoAOMT), yielding feruloyl-CoA, increased to a transient maximum at 12 to 15 h CCoAOMT cDNA was cloned from the elicited cells and was shown to encode a polypeptide highly homologous to CCoAOMTs from cells of Petroselinum species or Zinnia species The expression of the cDNA in Escherichia coli revealed that grapevine CCoAOMT methylates both caffeoyl-and 5-hydroxyferuloyl-coenzyme A and is probably involved in phenolic esterification and lignification Commercial plant activators induce the disease-resistance response of test plants and are considered to mimic the action of salicylic acid Among these chemicals, 2,6-dichloroisonicotinic acid and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester provoke systemic acquired resistance (SAR) and were also shown to induce the expression of class III chitinase in grapevine The SAR response is classified by an unchanged phenotype of tissues, but the mechanistic basis is unknown Treatment of the cultured V vinifera cells with either fungal elicitor or low concentrations of salicylic acid and 2,6-dichloroisonicotinic acid, respectively, raised the CCoAOMT or stilbene synthase transcript abundance, suggesting that grapevine is capable of the SAR response, whereas benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester was ineffective The data imply for the first time (to our knowledge) that the expression of phenylpropanoid genes in grapevine is induced by SAR activators without phenotypic consequences and suggest a role for CCoAOMT and stilbene synthase in the disease-resistance response leading beyond the level of pathogenesis-related proteins as markers of the SAR

Formation of Di-Isodityrosine and Loss of Isodityrosine in the Cell Walls of Tomato Cell-Suspension Cultures Treated with Fungal Elicitors or H2O2.

Abstract: About 84% of the hydroxyproline residues in a cell culture of tomato (Lycopersicon esculentum x Lycopersicon peruvianum) were present in phenol-inextractable (i.e. covalently wall-bound) material. Treatment of the cells with any of three fungal elicitors (wall fragments from Phytophthora megasperma and Pythium aphanidermatum and xylanase from Aureobasidium pullulans) or with 1 mM H2O2 had little effect on the quantity of phenolinextractable hydroxyproline per milligram of freeze-dried cells. However, each treatment induced a decrease in the content of phenol-inextractable isodityrosine (Idt) residues. Each treatment, except with the P. megasperma fragments, also induced an increase in phenol-inextractable di- (Di-Idt). The increase in Di-Idt partly accounted for the loss of Idt. We conclude that the elicitors and H2O2 acted to reinforce the existing cross-linking of cell wall (glyco)proteins by evoking oxidative coupling reactions to convert Idt to Di-Idt plus unidentified products. The promotion of cross-linking by elicitor treatment is proposed to be a defensive response that restricts the penetration of pathogens.

Molecular characterization and expression of the Erwinia carotovora hrpNEcc gene, which encodes an elicitor of the hypersensitive reaction

Abstract: The nucleotide sequence of hrpNEcc DNA, cloned from Erwinia carotovora subsp. carotovora strain Ecc71, reveals a coding region of 1,068 bp which matches the size of hrpNEcc transcripts. hrpNEcc is predicted to encode a glycine-rich protein of approximately 36 kDa. Like the elicitors of the hypersensitive reaction (HR) produced by E. chrysanthemi (HarpinEch) and E. amylovora (HarpinEa), the deduced 36-kDa protein does not possess a typical signal sequence, but it contains a putative membrane-spanning domain. In Escherichia coli strains overexpressing hrpNEcc , the 36-kDa protein has been identified as the hrpNEcc product by Western blot analysis using anti-HarpinEch antibodies. The 36-kDa protein fractionated from E. coli elicits the HR in tobacco leaves. Moreover, a HrpN- and RsmA- double mutant (RsmA = regulator of secondary metabolites) does not produce this 36-kDa protein or elicit the HR, although this strain, like the RsmA- and HrpN+ bacteria, overproduces extracellular enzymes and macerates celery p...

The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Abstract: Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al, 1992, Plant Physiol Biochem 30: 579–584) The role of this compound in the induction of this and of other defence reactions is further studied in this paper Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions

Superoxide generation in extracts from isolated plant cell walls is regulated by fungal signal molecules.

Abstract: Fractions solubilized with NaCl from cell walls of pea and cowpea plants catalyzed the formation of blue formazan from nitroblue tetrazolium. Because superoxide dismutase decreased formazan production by over 90%, superoxide anion (O2¯) may participate in the formation of formazan in the solubilized cell wall fractions. The formazan formation in the fractions solubilized from pea and cowpea cell walls was markedly reduced by exclusion of NAD(P)H, manganese ion, or p-coumaric acid from the reaction mixture. The formazan formation was severely inhibited by salicylhydroxamic acid and catalase, but not by imidazole, pyridine, quinacrine, and diphenyleneiodonium. An elicitor preparation from the pea pathogen Mycosphaerella pinodes enhanced the activities of formazan formation nonspecifically in both pea and cowpea fractions. The suppressor preparation from M. pinodes inhibited the activity in the pea fraction in the presence or absence of the elicitor. In the cowpea fraction, however, the suppressor d...

High-affinity binding site for ethylene-inducing xylanase elicitor on Nicotiana tabacum membranes

Abstract: Summary Challenge of Nicotiana tabacum cv Xanthi with the ethylene-inducing xylanase (EIX) from Trichoderma viride causes rapid induction of plant defense responses leading to hypersensitive necrosis. This phenomenon is cultivar-specific; no response is detected when N. tabacum cv Hicks is similarly treated. The responsiveness is determined in tobacco and tomato by a single dominant gene. EIX was labeled with fluorescein-isothiocyanate and incubated with cell suspension cultures, protoplasts or microsomal membranes. Binding of EIX to the microsomal membranes was found to be specific and saturable, with a dissociation constant of 6.2 nM. Using confocal laser microscopy, the EIX binding site was localized to the plasma membrane. Binding of EIX to its high-affinity site occurred in responsive species. These results demonstrate the existence of a high-affinity binding site for EIX on the plasma membrane of responsive cultivars. Chemical cross-linking of EIX to microsomal membranes from responding plants revealed a 66 kDa protein complex. This protein may function as the receptor that mediates the hypersensitive response induced by EIX binding.

Molecular evolution and functional relevance of the chalcone synthase genes of pea.

Abstract: We have isolated seven genomic chalcone synthase (CHS) genes and six classes of CHS cDNA from elicitor-treated pea tissues. Comparison of the nucleotide sequences of the coding regions revealed the existence of eight members of the CHS gene family in pea. These can essentially be divided into three groups (PSCHS1, 2 and 8; PSCHS3, 4 and 5; and PSCHS6 and 7) on the basis of nucleotide and/or amino acid sequence comparisons of the coding regions, introns and promoter regions. We previously reported that the accumulation of CHS mRNAs is induced by elicitor treatment. Accumulation of CHS mRNA was observed mainly in roots and very little was found in floral organs. To specifically detect expression of each CHS gene in various types of pea cells, S1 nuclease protection assays were performed. Interestingly, the classification of the eight members of the CHS gene family based on the sequence identity was found to reflect their expression patterns as determined by the S1 nuclease protection assay. The first group of CHS genes, PSCHS1, 2 and 8, was strongly induced not only by elicitor treatment and UV irradiation but is also constitutively expressed in root and flower tissues. The second group, PSCHS3, 4 and 5, was also strongly induced by elicitor treatment and UV irradiation but is constitutively expressed only in root. Expression of the third group, PSCHS6 and 7 was barely detectable in any of the organs tested and was not influenced by environmental stimuli such as elicitor or UV. Furthermore, sequence analysis of the promoter region of each member of the CHS gene family revealed that putative cis-regulatory elements, such as Box-I, Box-II and G-Box, were conserved only in PSCHS1, 2, 3, 4 and 5. From these results we propose that an ancestral CHS gene might have given rise to defense response-related (UV irradiation- and elicitor-responsive) and -unrelated (unresponsive) genes at an early stage of evolution, followed by divergence within these subclasses based upon the developmental program in pea.