Cloning of soybean genes induced during hypersensitive cell death caused by syringolide elicitor

Syringolide elicitors produced by bacteria expressing Pseudomonas syringae pv. glycinea avirulence gene D (avrD) induce hypersensitive cell death (HCD) only in soybean (Glycine max [L.] Merr.) plants carrying the Rpg4 disease resistance gene. Employing a differential display method, we isolated 13 gene fragments induced in cultured cells of a soybean cultivar Harosoy (Rpg4) treated with syringolides. Several genes for isolated fragments were induced by syringolides in an rpg4 cultivar Acme as well as in Harosoy; however, the genes for seven fragments designated as SIH (for syringolide-induced/HCD associated) were induced exclusively or strongly in Harosoy. cDNA clones for SIH genes were obtained from a cDNA library of Harosoy treated with syringolide. Several sequences are homologous to proteins associated with plant defense responses. The SIH genes did not respond to a non-specific -glucan elicitor, which induces phytoalexin accumulation but not HCD, suggesting that the induction of the SIH genes is specific for the syringolide–Harosoy interaction. HCD and the induction of SIH genes by syringolides were independent of H₂O₂. On the other hand, Ca²⁺ was required for HCD and the induction of some SIH genes. These results suggest that the induction of SIH genes by syringolides could be activated through the syringolide-specific signaling pathway and the SIH gene products may play an important role(s) in the processes of HCD induced by syringolides.Abbreviations AOS active oxygen species - CHS chalcone synthase - DPI diphenylene iodonium - HCD hypersensitive cell death - HR hypersensitive response - PAL phenylalanine ammonia lyase - SID syringolide-induced/defense associated - SIG syringolide-induced/general - SIH for syringolide-induced/HCD associated - XET xyloglucan endotransglycosylase     

Implication of signaling pathways involving calcium, phosphorylation and active oxygen species in methyl jasmonate-induced defense responses in grapevine cell cultures

Perception of elicitors triggers plant defense responses via various early signal transduction pathways. Methyl jasmonate (MeJA) stimulates defense responses in grapevine (Vitis vinifera). We investigated the involvement of various partners (calcium, ROS, reversible phosphorylation) in MeJA-induced responses by using a pharmacological approach. We used specific calcium channel effectors and inhibitors of serine/threonine phosphatases, superoxide dismutase and NAD(P)H oxidase and investigated production of stilbenes (resveratrol and its glucoside, piceid, the major form), which are the grapevine phytoalexins. RNA accumulation of two genes encoding enzymes involved in stilbene synthesis (PAL and STS), three genes encoding pathogenesis-related proteins (CHIT4C, PIN and GLU) and one gene encoding an enzyme producing jasmonates (LOX) were also assessed. Calcium and its origin seemed to play a major role in MeJA-induced grapevine defense responses. Phytoalexin production was strongly affected if calcium from the influx plasma membrane was inhibited, whereas calcium from the intracellular compartments did not seem to be involved. ROS production seemed to interfere with MeJA-stimulated defense responses, and protein phosphorylation/dephosphorylation events also played a direct role.     

Rice gene expression in response to N-acetylchitooligosaccharide elicitor: comprehensive analysis by DNA microarray with randomly selected ESTs

N-acetylchitooligosaccharides are potent elicitors to suspension-cultured rice cells, inducing a set of defense reactions. Expression of defense-related genes is considered to play an important role in defense reactions, and we employed microarray analysis of 8987 randomly selected expressed sequence tags to analyze the changes in gene expression caused by N-acetylchitooctaose. In this experiment, 166 genes were significantly induced and 93 genes were repressed. RNA gel blot analysis of 16 of these genes confirmed the microarray results. Of the 259 ESTs identified as responsive to N-acetylchytooctaose, 18 genes are related to signal transduction, including five calcium-dependent protein kinases (CDPKs). Among these, three novel CDPKs responsive to N-acetylchitooctaose were isolated.     

Oligogalacturonide signal transduction,induction of defense-related responses and protection of grapevine against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H₂O₂, followed by differential expression of nine defense-related genes and stimulation of chitinase and -1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and -1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55–65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.Abbreviations AOS Active oxygen species - Chit Chitinase - DPI Diphenylene iodonium - -Glu -1,3-Glucanase - GST Glutathione-S-transferase - MAP Mitogen-activated protein - OGA Oligogalacturonides - PAL Phenylalanine ammonia-lyase - PR Pathogenesis-related - PGIP Polygalacturonase inhibiting protein - PIN Serine-proteinase inhibitor - STS Stilbene synthase     

Activity of defense related enzymes and gene expression in pigeon pea (Cajanus cajan) due to feeding of Helicoverpa armigera larvae

The biochemical and molecular basis of the defense in a mild tolerant (ICPL-332) and susceptible (ICPL-87) cultivars of pigeon pea (Cajanus cajan) due to Helicoverpa. armigera infestation was studied. We found that feeding by the larvae generated H₂O₂ in a localized manner and activity was observed upto 12?h (hrs) of with a sharp decline within 24?h. Similarly, PPO activity was also detected till 12?h of treatment, which decreased after 24?h of feeding by larvae. The activity of trypsin inhibitor was detected in all the treatments when assayed at 12 and 24?h after larval feeding. The expression of defense genes like the Pre-hevein-like protein PR-4 precursor (PR-4), protease inhibitor/seed storage/LTP family protein (Ltp) were significantly up-regulated in ICPL-332 upon infestation after 12?h as compared to ICPL-87, whereas the endo 1, 4 -β-glucanase (Kor-1) gene was expressed in both the cultivars after 24?h of infestation. Both the cultivars varied with respect to the induction of defense-related genes during larval feeding, both the PR-4 and Ltp genes appeared to be important for defense against H. armigera in pigeon pea. Thus, the present study revealed an insight of herbivore-induced biochemical and molecular changes in pigeon pea.     

Effect of auxins and ectomycorrhizal elicitors on wall-bound proteins and enzymes of spruce [Picea abies (L.) Karst.] cells

Summary Elicitors of the ectomycorrhizal fungus Hebeloma crustuliniforme and auxins (IAA, NAA and 2,4-D) were tested for their effects on apoplastic proteins and enzymes of suspension cultured cells of Picea abies (L.) Karst. The ectomycorrhizal elicitor increased the amount of some ionically wall-bound proteins (36, 28, 24, 21 kDa) and decreased the amount of others (61, 22 kDa). The elicitor triggered an H₂O₂ burst and enhanced the peroxidase (EC 1.11.1.7) activity of the Picea cells by increasing one of the two wall-bound peroxidase isoforms. Auxins significantly suppressed the elicitor induction of peroxidase but did not influence the elicitor-triggered H₂O₂ burst. The elicitors and auxin did not change the amount and the pattern of wall-bound invertase isoforms (EC 3.2.1.26) of spruce cells. However, auxin reduced the uptake of glucose by spruce cells and increased the acidification of the cell culture medium. Since Hebeloma lacks apoplastic invertase as well as a sucrose uptake system, utilization of plant-derived sucrose depends on the apoplastic plant invertase activity. Although the host invertase is constitutive, the fungus might be able to increase this invertase activity within a mycorrhiza by lowering the pH of the interface towards the pH optimum of the enzyme via the action of auxin. This fungus-released hormone could increase the H⁺ extrusion of plant cells by activation of the plant membrane H⁺-ATPases. Additionally, an auxin-dependent suppression of glucose uptake by cortical root cells could improve the glucose supply for the fungus. Furthermore, the fungal auxin might suppress the elicitor induced formation of defense enzymes, such as peroxidase.     

BTH-Induced Accumulation of Extracellular Proteins and Blackspot Disease in Rose

Treatment of rose shoots with 50 µM acibenzolar-S-methyl (BTH) resulted in increased protection against Diplocarpon rosae. This was accompanied by the induction and accumulation of a set of extracellular proteins as shown by SDS-PAGE and 2D-PAGE. Some of these proteins have been identified as PR-1, PR-2, PR-3 and PR-5 proteins by immunoblot analysis probed with tobacco antisera against PR-1c, PR-N, PR-Q and PR-S protein. Most of the extracellular proteins activated by BTH were also induced and found to accumulate in leaves upon infection with Diplocarpon rosae. However, their accumulation was much more pronounced in BTH-pretreated leaves than in water-pretreated leaves upon a challenge inoculation with D. rosae, particularly, the 15 kD PR-1, 36 and 37 kD PR-2 proteins. They may be more important in the expression of disease resistance.     

Preparation of mixed alginate elicitors with high activity for the efficient production of 5′-phosphodiesterase by Catharanthus roseus cells

When various autoclaved microbial cells suspensions (exogenous elicitors) were added to Catharanthus roseus cell cultures, its growth was inhibited but 5′-phosphodiesterase (PDase) production was stimulated. The greatest effect was with autoclaved Alteromonas macleodii: the dry cell concentration decreased from 13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen species) minimized the cell growth inhibition but enhanced PDase production (0.474 U/ml) about 20 times higher than the control (no addition). The method for the preparation of mixed alginate elicitors with high activities containing exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and trans-4,5-dihydroxy-2-cyclopenten-1-one was developed. The mixed alginate elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and the productivity was increased 120-fold compared to the control without cell growth inhibition.     

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.     

Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells

The interaction between Botrytis cinerea Pers. and grapevine (Vitis vinifera L.) was studied in a model system of reduced complexity. Cultured plant cells and fragments of fungal cell wall were used to simulate some of the processes taking place upon infection of grapevine with B. cinerea. A soluble glucan elicitor was prepared from the fungal cell wall by acid hydrolysis. Like the insoluble wall preparation, the soluble fragment derived from the cell wall acted upon plant cells in eliciting stilbene formation. In grapevine cells, the interaction with the fungus led to a dramatic shut-off general protein synthesis and to the selective formation of a small set of proteins involved in induced resistance. The proteins synthesized de novo with highest rates were stilbene synthase (StiSy) and l-phenylalanine ammonia-lyase (PAL). Stilbene synthase was purified to apparent homogeneity and its molecular properties were characterized. The enzyme is a homodimer with subunit M_r 43 000 and pl = 5.4. Although there were indications of the presence of isoenzymes, these were not distinguished by charge differences. In size, the grapevine StiSy shows microheterogeneity and differs from the appreciably larger enzyme prepared from peanut. Prior to induction by fungal attack, virtually no stilbenes are formed in the plant cell. Upon induction of the pathway leading to the stilbene resveratrol, StiSy activity determines the ratelimiting step in the metabolic sequence. The highly induced grapevine cells produce and secrete resveratrol and derivatives which are known to be fungistatic.Abbreviations PAL l-phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamine gel electrophoresis - StiSy stilbene synthase (resveratrol forming) The authors thank Dr. Blaich, Bundesforschungsanstalt Geilweilerhof, Siebeldingen, F.R.G., for provision of callus culture. This paper is based on research supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Cerebroside mediated elicitation of defense response in chilli (Capsicum annuum L.) against Colletotrichum capsici infection

Abstract

Enhancing the host resistance by using naturally occurring elicitors derived from pathogenic organisms is emerging as an ecofriendly approach in plant disease management. Cerebrosides, categorized as glycosphingolipids, were isolated and partially purified from the wilt causing fungus (Fusarium oxysporum f. sp. lycopersici). Cerebroside treatment significantly reduced the anthracnose disease incidence under greenhouse conditions. Cerebroside elicitors were found to stimulate the early H₂O₂ accumulation followed by the production of plant defense-related enzymes such as Phenylalanine ammonia lyase (PAL), Peroxidase (POX), Polyphenol oxidase (PPO), and Lipoxygenase (LOX) when applied to chilli (Capsicum annuum L.) plants by spray treatment and also induced the accumulation of capsidiol. Defense-related enzyme activities were increased by the elicitor treatment and an high level in activity was maintained during the experimental period. Under greenhouse conditions, the cerebroside elicitors effectively protected chilli plants against infection by anthracnose causing organism, Colletotrichum capsici.     

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.     

PR-1 gene family of grapevine: a uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco

A functional contribution of pathogenesis-related 1 (PR-1) proteins to host defense has been established. However, systematic investigation of the PR-1 gene family in grapevine (Vitis spp.) has not been conducted previously. Through mining genomic databases, we identified 21 PR-1 genes from the Vitis vinifera genome. Polypeptides encoded by putative PR-1 genes had a signal sequence of about 25 residues and a mature protein of 10.9–29 kDa in size. PR-1 mature proteins contained a highly conserved six-cysteine motif and pI values ranging from 4.6 to 9. A major cluster with 14 PR-1 genes was mapped to a 280-kb region on chromosome 3. One particular PR-1 gene within the cluster encoding a basic-type isoform (pI 7.77), herein named VvPR1b1, was isolated from various genotypes of grapevine (Vitis spp.) for functional studies. Sequence analysis of PCR-amplified DNA revealed that all genotypes contained a single VvPR1b1 gene except for a broad-spectrum bacterial and fungal disease resistant Florida bunch grape hybrid, ‘BN5-4’, from which seven different homologues were identified. Duplication of VvPR1b1-related genes encoding acidic-type PR-1 isoforms was also observed among several genotypes. However, transgenic expression analysis of grapevine PR-1 genes under strong constitutive promoters in transgenic tobacco revealed that only the basic-type VvPR1b1 gene duplicated in ‘BN5-4’ was capable of conferring high level resistance to bacterial disease caused by Pseudomonas syringae pv. tabaci.     

Isolation of putative defense-related genes from Arabidopsis thaliana and expression in fungal elicitor-treated cells

Numerous Arabidopsis genes have been cloned that correspond to putative pathogen defense-related genes identified in parsley (Petroselinum crispum). Treatment of Arabidopsis cells with fungal elicitor leads to rapid accumulation of the respective mRNAs with time courses comparable to those observed for their counterparts in parsley. Evolutionary sequence conservation of many of these genes in several plant species suggests they code for important plant functions.     

Induction of some defense-related genes and oxidative burst is required for the establishment of systemic acquired resistance in <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The inoculation of primary pepper leaves with an avirulent strain of Xanthomonas campestris pv. vesicatoria induced systemic acquired resistance (SAR) in the non-inoculated, secondary leaves. This SAR response was accompanied by the systemic expression of the defense-related genes, a systemic microoxidative burst generating H₂O₂, and the systemic induction of both ion-leakage and callose deposition in the non-inoculated, secondary leaves. Some defense-related genes including those encoding PR-1, chitinase, osmotin, peroxidase, PR10, thionin, and SAR8.2 were markedly induced in the systemic leaves. The conspicuous systemic accumulation of H₂O₂ and the strong increase in peroxidase activity in the pepper leaves was suggested to play a role in the cell death process in the systemic micro-hypersensitive responses (HR), leading to the induction of the SAR. Treatment of the primary leaves with diphenylene iodinium (DPI), an inhibitor of oxidative burst, substantially reduced the induction of some of the defense-related genes, and lowered the activation of the oxidative bursts in the systemic leaves distant from the site of the avirulent pathogen inoculation and subsequently SAR. Overall, these results suggest that the induction of some defense-related genes as well as a rapid increase in oxidative burst is essential for establishing SAR in pepper plants.