Breeding for inducible resistance against pests and diseases

27-29 April 2006, Heraklio, Crete at

Santa Marina Beach Hotel 106, A. Papandreou Str.

Heraklio, Ammoudara

Abstracts oral presentations

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Thurs 27 April 2006

Session 1

‘Mechanisms involved in inducible and constitutive resistance to pests and diseases’

Priming as a mechanism of induced resistance against pathogens, insects and abiotic stress. Jurriaan Ton1,3,4, Sjoerd van der Ent1, Marieke van Hulten1, Maria Pozo1,2, Vivian van Oosten1, L.C. van Loon1, Brigitte Mauch-Mani3, Ted C. J. Turlings4, and Corné M.J. Pieterse1. 1 Institute of Environemtal Biology, Section Phytopathology, Utrecht University, Faculty of Biology, PO Box 800.84, 3508 TB Utrecht, The Netherlands. 2 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, Spain. 3 Biochemistry and Molecular Biology, Institute of Botany, University of Neuchâtel, Neuchâtel, Switzerland. 4 Evolutionary Entomology, Institute of Zoology, University of Neuchâtel, Neuchâtel, Switzerland. Upon specific stimulation, plants acquire an enhanced defensive capacity that results in a faster and/or stronger defence reaction once the plant is exposed to biotic or abiotic stress. This phenomenon is commonly known as priming and has been associated with different forms of induced resistance. Priming accelerates and increases the plant’s ability to activate the defence that is best adapted to resist the stress situation encountered. Under conditions of disease pressure, primed plants exhibit a higher fitness than non-primed plants or defence-expressing plants (Van Hulten et al., 2006; PNAS). Hence, the benefits of priming outweigh its costs in environments where disease occurs. Although priming has been known to occur in plants for decades, most progress in the understanding of this phenomenon has been made over the past few years. Recent insights in the mechanisms behind systemic acquired resistance (SAR), β-amino-butyric acid-induced resistance (BABA-IR), rhizobacteria-mediated induced systemic resistance (ISR), and volatile-induced resistance against insects have revealed various priming mechanisms that protect against different stresses. Whereas SAR and BABA-IR are associated with priming for salicylate (SA)-dependent defence that acts against biotrophic pathogens, ISR and volatile-induced resistance function through priming for jasmonate (JA)-dependent defence against pathogens and insects. Expression of BABA-IR and ISR against pathogenic fungi and oomycetes is also associated with a potentiated formation of callose-rich papillae. This priming response depends on a yet unknown defence pathway, which involves abscisic acid (ABA) and phosphoinositide (PtdIns) signalling, and targets the cellular secretory pathway. Induction of the primed state may be mediated by an enhanced accumulation of signalling compounds, such as transcription factors (TF), that remain inactive until the plant is exposed to stress. A Q-PCR-based transcription profiling of ~2.200 TF genes in Arabidopsis has revealed consistent changes in the expression of certain TF genes directly upon activation of ISR and BABA-IR. We are

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currently investigating the contribution of these transcription factors to the various priming responses.

Priming: It’s all the world to induced disease resistance Beckers, G.1, Schmitz, G.1, Köhle, H.2, Reinhold, T.3, Neuhaus, E.3, and Conrath, U.1 1 Plant Biochemistry & Molecular Biology Unit, Department of Plant Physiology, RWTH Aachen University, 52056 Aachen, Germany 2 BASF Corporation, 26 Davis Drive, Research Triangle Park, NC 27709, USA 3Department of Plant Physiology, TU Kaiserslautern University, PO Box 3049, 67653 Kaiserslautern, Germany Plants can acquire enhanced resistance to pathogens after treatment with necrotizing attackers, various natural and synthetic compounds (which include some commercial fungicides) and upon inhibition of a plastid ATP/ADP transporter protein. The induced resistance is often associated with an enhanced capacity to mobilize infection-induced cellular defense responses - a process called ‘priming’ (‘sensitization’). Although the phenomenon has been known for years, most progress in the understanding of priming has been made over the past years. These studies show that priming often depends on the induced disease resistance key regulator protein NPR1, and that priming is likely to affect the regulation of cellular defense responses by enhancing the cellular level of mitogen-activated protein (MAP) kinases.

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An extract of Penicillium chrysogenum elicits early defense-related responses and induces resistance in Arabidopsis thaliana independently of known signalling pathways Thuerig B a, Felix G b, Binder A b, Boller T b and Tamm L a

a Research Institute of Organic Agriculture, Frick, Switzerland b University of Basel, Botanical Institute, Switzerland

An aqueous extract of the mycelium of Penicillium chrysogenum (further called ‘Pen’) protected A. thaliana from a broad range of pathogens, including an oomycete (Hyaloperonospora parasitica), two ascomycetes (Botrytis cinerea, Alternaria brassicicola) and a bacterium (Pseudomonas syringae pv. tomato DC3000) without having a direct antimicrobial effect. Various mutants of A. thaliana were used to test whether Pen induces resistance on one of the known signaling pathways: Pen was fully protective against B. cinerea in A. thaliana transgenes or mutants impaired in the salicylic acid (NahG, npr1), jasmonic acid (coi1-1), and ethylene (ein2-1) signalling pathway. Similarly, Pen-mediated resistance against H. parasitica was not affected in the mutants npr1, coi1-1 or ein2-1. However, its efficacy was reduced in the transgene NahG.

Pen not only induced resistance in plants but also early defense-related responses such as an extracellular alkalinisation in cell cultures and ethylene production in leaf slices of numerous mono- and dicotyledon plant species, including Arabidopsis thaliana, tomato, tobacco and rice. The Pen-elicitor was sensitive to protease digestion but insensitive to other enzymes, suggesting that the elicitor-active region is a protein or a peptide. Reversed phase, ion exchange and size exclusion chromatography revealed that the Pen-elicitor is heterogeneous, preventing further identification of the elicitor.

From these data on cell cultures and on A. thaliana we conclude that Pen contains at least one unidentified elicitor, most likely a protein or a glycoprotein, inducing resistance via signal transduction pathways different from classical SA/NPR1- or JA/ethylene-dependent pathways.

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The identification of PAD2 as g-glutamylcysteine synthetase highlights the importance of glutathione in plant disease resistance Vincent Parisy1, Benoit Poinssot1, Lucas Owsianowski1, Antony Buchala1, Jane Glazebrook2

and Felix Mauch1

1Department of Biology, University of Fribourg, Switzerland 2Department of Plant Biology, University of Minnesota, USA The pad2 mutant of Arabidopsis was originally identified in a biochemical screen for phytoalexin deficiency and showed enhanced susceptibility towards virulent isolates of Pseudomonas syringae. The pad2 mutant was later found to be hypersusceptible to the oomycete pathogen Phytophthora brassicae. The Arabidopsis/Phytophthora pathosystem was used to identify the pad2 mutation by positional cloning. PAD2 was mapped on chromosome 4 as a point mutation in the gene coding for g-GLUTAMYLCYSTEINE SYNTHETASE (GSH1), the enzyme catalysing the first step of glutathione (GSH) biosynthesis. The pad2 mutant contained only 20-25% of the foliar wild-type GSH thus suggesting that the various phenotypes of pad2 were caused by a deficiency in the antioxidant tripeptide GSH. Complementation of pad2 by expression of the wild-type GSH1 cDNA restored normal GSH levels, resistance towards P. brassicae, camalexin deficiency and all other pad2-related phenotypes. In addition, another GSH deficient mutant, cad2-1, showed phenotypes similar to pad2. The GSH deficiency of pad2 did not only lead to enhanced susceptibility towards P. brassicae and P. syringae but also caused enhanced susceptibility towards a number of necrotrophic pathogens including Botrytis cinerea, Alternaria brassicicola and Plectospaerella cucumerina. Thus, GSH is identified as an important component of general disease resistance in Arabidopsis.

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Extracellular factors of early basal resistance induced against bacteria Eszter Besenyei(1), Erika Szabó(1), Ágnes Szatmári(1), Zoltán Bozsó(1), Katalin F. Medzihradszky(2,3), Csorba Attila(2), Péter G. Ott(1) (1) Department of Pathophysiology, Plant Protection Institute, Hungarian Academy of Sciences, 1022 Budapest, Pf. 102, Hungary; (2) Mass Spectrometry Facility, Biological Research Center of the Hungarian Academy of Sciences, 6701 Szeged, Pf. 521, Hungary; (3) Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143-0446, USA Corresponding author: Peter G. Ott, e-mail address: pott@nki.hu Early basal resistance (EBR) is a response of plants to general bacterial constituents (elicitors), amounting in suppression of the hypersensitive and partial suppression of the susceptible reaction. The quickness of EBR raises the possibility that plants have a non-specific system for inhibition of bacterial activity, for example to prevent deployment of pathogenicity factors. As bacterial pathogens remain outside living plant cells, cell wall proteins and/or structural modifications are likely to be involved in the above EBR effects. Using genomic and proteomic approaches in tobacco, Medicago truncatula and pepper, several general elicitor-inducible plant extracellular factors were found to be associated with EBR, including chitinases, peroxidases, proteases as well as structural proteins. Their potential roles in generating a harmfulenvironment for bacteria shall be discussed.

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Cytological aspects of elicitor-induced resistance against Plasmopara viticola in grapevine Trouvelot S1. , Allègre M1., Joubert2, J.-M., Pugin A1, Daire X.1 1 UMR INRA-Université de Bourgogne - CNRS Plante Microbe Environnement. Dijon. France 2 Laboratoires Goëmar SA, St Malo, France

An oligosaccharide (OS) has been shown to induce resistance in grapevine (Vitis vinifera) against Plasmopara viticola (downy mildew) under greenhouse conditions. Spray application reduced the severity of the disease by 70-80 %. However, the efficacy of the treatment appeared to be influenced by the age of the leaf, old leaves being more resistant to the disease than the young ones.

Following OS application, foliar tissues underwent significant ultrastructural and biochemical modifications, detectable only after inoculation with the pathogen. These included H202 production at the infection site, the deposition of phenolic compounds-enriched material and the formation of structural barriers that appeared to prevent pathogen development and sporulation in foliar tissues.

Our observations suggest that in grapevine OS act rather like a priming agent than a true elicitor.

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Session 2

Evolutionary and ecological aspects of plant resistance / Deployment strategies for durable resistance within Integrated Crop

Management

Linking aboveground and belowground induced resistance Bezemer T.M.1,2,3 1 Laboratory of Nematology, Wageningen University and Research Centre, PO Box 8123, 6700 ES Wageningen, The Netherlands 2 Laboratory of Entomology, Wageningen University and Research Centre, PO Box 8031, 6700 EH Wageningen, The Netherlands 3 Netherlands Institute of Ecology (NIOO-KNAW), Centre for Terrestrial Ecology, PO Box 40, 6666 ZG Heteren, The Netherlands Induced resistance of plants against pests and diseases via plant defense responses is well documented and can occur aboveground, in the leaves, and belowground in the roots. A number of recent studies have shown that soil-borne pests can also induce plant resistance aboveground and vice versa. Since plants are frequently exposed to aboveground and belowground pests simultaneously, interactions between induced plant defense responses in the foliage and in the roots can have major implications for induced resistance. On the one hand, induction in one plant part may result in systemically induced resistance in the other part. On the other hand, simultaneously occurring aboveground and belowground induced plant defenses may interfere, for example, when the activities of root feeders alter the effectiveness of induced resistance against foliar herbivores. I will review the literature to show similarities and dissimilarities in aboveground and belowground resistance. I will show how soil dwelling pests such as nematodes and root feeding insects can induce defense responses in aboveground plant parts, and how aboveground pests can influence plant defense responses in the roots. I will discuss the mechanisms involved and provide examples of the consequences of interference between aboveground and belowground plant defense responses for plant damage and insect herbivore performance. Finally, I will show how such aboveground-belowground interactions can influence the reliability of indirect plant defense responses, the attraction by the plant of natural enemies of the attacker.

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Inducible defenses in food webs: models and experimental data

Van der Stap, I.1, Vos, M.2* and Mooij, W.M. 1 Department of Food Web Studies, Netherlands Institute of Ecology (NIOO-KNAW), Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands 2 Department of Ecosystem Studies, Netherlands Institute of Ecology (NIOO-KNAW), Korringaweg 7, 4401 NT Yerseke, The Netherlands. *Present address: Department of Biology, University of Victoria, Victoria British Columbia, Canada We incorporated inducible defenses in food chain models and showed that these resolve the paradox of enrichment for a wide range of ecologically relevant conditions. The predicted stability occurs in both bi- and tritrophic food chains. Inducible defenses also affect trophic structure by causing a gradual instead of step-wise increase in the biomass of all trophic levels in response to enrichment. Such all-level responses have been observed in both aquatic and terrestrial ecosystems. We performed laboratory experiments to test the hypotheses of the theoretical studies. First, we studied the occurrence of colony formation within different strains of green algae Scenedesmaceae in response to grazing-released infochemicals from the herbivores Brachionus and Daphnia. Then, we observed the effect of inducible defenses on the population dynamics of a freshwater planktonic system using algal strains with different defense strategies and rotifers. Simple food webs were composed of green algae (Scenedesmaceae), herbivorous rotifers (Brachionus calyciflorus and/or Brachionus rubens) and carnivorous rotifers (Asplanchna brightwelli). In this system not only the green algae but also B. calyciflorus exhibits an inducible defense against predation by developing long spines, while B. rubens does not exhibit a defense in presence of Asplanchna. Our experimental results showed that inducible defenses, as opposed to the absence of defenses, can prevent population fluctuations and decrease the strength of trophic cascades.

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Exploring the barley-Magnaporthe oryzae pathosystem Zellerhoff N. and Schaffrath U. Department of Plant Physiology (Bio III), RWTH Aachen University, D-52056 Aachen, Germany

The fungus Magnaporthe grisea, the causal agent of rice blast disease, is a major pathogen of rice and is capable of producing epidemics on other cultivated cereals, including barley (Hordeum vulgare).

We wanted to assess the capacity of barley to resist this uncommon pathogen, so we started a survey investigating this plant-fungus interaction at multiple levels.

Interestingly, we identified the MLO-gene as a player in basic pathogen resistance in this pathosystem and mutations at the MLO-locus, i.e. mlo, which confer a durable resistance against powdery mildew, condition a hypersusceptible disease phenotype against blast. We have started a mutational screen in the mlo-genetic background and identified a mutant that exhibits enhanced resistance to blast. Resistance of this mutant (emr1, enhanced Magnaporthe resistance) is only altered against blast but not against rust or netblotch. Scanning electron microscopy showed a reduced content of epicuticular waxes on the leaf surface as compared to mlo-plants and this phenotype co-segregated in F2 and F3 populations together with blast resistance. This, in turn, may hint to an important function for epicuticular waxes in disease resistance of barley against blast.

Moreover, we have shown that SAR-inducing treatments enhance resistance of barley against M. oryzae, even in the ostensibly hypersusceptible interactions with mlo-genotypes. A more recent study focussed on the question as to whether SAR functions in roots of barley and rice against M. oryzae with the same efficacy as known from leaves. So far, it seems that this is not the case, and neither race-specific nor the induction of acquired resistance was able to protect roots from colonisation by the blast fungus.

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Identification and expression of genes related to herbivory Broekgaarden C.1 2, Voorrips R.E.1, Dicke M.2 and Vosman B.1 1Plant Research International B.V., Wageningen 2Wageningen University and Research Center, Laboratory of Entomology, Wageningen In their natural surrounding, plants are constantly attacked by all kinds of enemies. Among these are herbivores, like insects and mammals, as well as viruses, fungi and bacteria. To be able to survive these treads, plants have developed a wide spectrum of defense strategies. Besides pre-existing barriers, plants can also make use of inducible responses. These are activated upon damage or attack and can result in either direct or indirect defense. Direct defense is based upon non-volatile secondary metabolites including toxins, such as nicotine and glucosinolates, and digestibility reducers like proteinase inhibitors. Direct defense compounds are produced in order to reduce the growth rate and/or increase mortality of the attacking herbivore. In contrast to direct defense, indirect defense is based upon the production of volatile secondary metabolites. In this case the plant starts to produce volatiles that attract the natural enemy of the herbivore. Both indirect and direct defense are based upon signal transduction. Cross-talk between different pathways makes it possible to induce many different kinds of responses. Differences among insects may cause differences in defense response depending on their feeding strategy. A lot of research on inducible defense has been done in the model plant Arabidopsis thaliana. However, almost no data is available on coordination of attacker-induced signals into specific defense responses in Brassica oleracea plants. Two Brassica oleracea genotypes, Christmas Drumhead and Rivera were selected for this study of which Rivera was found to display a higher level of direct resistance when compared to Christmas Drumhead. Using an Arabidopsis whole genome oligonucleotide microarray, we characterized gene expression in damaged and undamaged leaves of then two Brassica oleracea genotypes in response to Pieris rapae. After 72h of feeding, we identified more than 400 and more than 800 insect-responsive genes in Christmas Drumhead and Rivera respectively. About 50% of the genes identified as differentially expressed were regulated in a genotype specific way When coupling analysis with jasmonic acid treatment 30% in Rivera and 37% of Pieris-induced genes in Christmas Drumhead were shown to be part of the jasmonate pathway. Finally, we compared transcript patterns in Christmas Drumhead and Rivera in response to larvae of Pieris rapae and to Brevicoryne brassicae aphids. Transcriptional responses to these insects seem to be highly unrelated.

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Integrated Control of Cucurbit Powdery Mildew, Podosphaera xanthii, (syn. Sphaerotheca fuliginea) using Resistant Cultivars, Resistance Inducing Agents and Hyperparasites Fanourakis N., Tampakaki A., Fanouraki, M.N. and Malathrakis N.E. Technological Education Institute of Crete,71004 Heraklio, Crete, Greece Powdery mildew, caused by Podosphaera xanthii, is a worldwide disease of cucucrbits causing severe losses in production. In the present work, an attempt was made to control the disease by integrating: a) resistant cultivars, b) inducers of resistance and c) hyperparasites. Genes for resistance to powdery mildew from the line WI 2757 were transferred to slicer cucumber imbreds developed in the Plant Breeding Laboratory. The cvs obtained were evaluated in preliminary experiments. Two harpins produced from suspensions of the bacterium Echericia coli were tested in a small scale experiment on young cucumber plants. The first, the commercial product Messanger, was encoded by the harpin gene of Erwinia amylovora and the other, a produce of our Laboratory, was encoded by the harpin gene of Pseudomonas syringae pv. phaseolicola. The hyperparasites Acremonium alternatum and Fusarium sp. isolated from the thallus of P. xanthii were also tested in preliminary experiments. Based on the results of the above experiments, a trial was set in a plastic greenhouse to test the potential of integrating the above control means against cucumber powdery mildew (P. xanthii) as following: resistant cv 3059 and susceptible cv 3060, inducing agent harpin (commercial product Messanger) in two rates, hyperparasites Acremonium alternatum and Fusarium sp., and combination of hyperparasites with harpin at the low rate. Results obtained indicated that resistant cultivar, harpin and hyperparasites significantly reduced disease severity. There were no statistically significant differences between foliar sprays (hyperparasites/harpin/combinations). Disease reduction by the resistant cv, regardless of foliar spray, was 88%. Disease reduction by hyperparasites, harpin and combinations, regardless of variety, varied from 42 to 58%. The higher reduction was obtained by A. alternatum. These data indicated that cucumber powdery mildew can be effectively controlled by the integration of the above means.

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Session 3

‘Chemical ecology / Trophic interactions; associations of phenotypes and genotypes’

Communicating Plants: Ecological Consequences of Induced Responses to Herbivory Andre Kessler Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14450, USA The past decades have seen an intense development of organismic biology and genomics of individual species on one hand and population biology and evolutionary ecology on the other hand. While the great discoveries fueled by these systems will continue over the next decades, more and more discoveries will occur through comparative biology/genomics and in the interface between different biological disciplines. It is through such integrative approaches that mechanisms of evolution and adaptation will be revealed. The study of plant-insect interactions is exemplary among the integrative research fields and succeeds by unifying the research efforts on the cellular and organismal level with those on the whole plant and community level. The current focus on molecular and chemical ecology research when studying plant-insect interactions reflects this modern consolidation. Here, I highlight studies of herbivore-induced responses of the wild tobacco plant Nicotiana attenuata and emphasize both, the role of using genetic and molecular tools in ecological research and the importance of a profound knowledge of the natural history of species when studying plant-insect interactions. In particular I will focus on recent results on the role of herbivore-induced volatile organic compound emission in mediating organism interactions in nature.

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Plant Responses to Caterpillar Footsteps, Chewing and Secretions

Gary W. Felton1 and Michelle Peiffer1

1 Department of Entomology, Penn State University, University Park, PA, USA The induced response of the tomato plant to insect herbivory is one of the best studied systems in terms of understanding the signalling pathways that lead to the increased expression of scores of defensive proteins and physical defences such as glandular trichomes. In most cases, the role of the herbivore has focused on the mechanical damage inflicted by chewing and the release of regurgitant signals that may elicit the production of volatiles that act as indirect defenses. In this presentation we will present evidence that the tomato plant up-regulates the expression of defensive genes in response to caterpillar movement across the leaf. This induced response does not require the occurrence of chewing or the release of oral secretions. We will also show that caterpillar secretions from the foregut, salivary glands and other exocrine glands may partially suppress jasmonate-regulated defences. The characterization of the possible component(s) in caterpillar saliva that are responsible for suppressing these defensive genes will be discussed.

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Induction of systemic acquired resistance in Zea mays is compatible with plant-parasitoid mutualism

Rostás M.1,2 and Turlings, TCJ1

1 Université de Neuchâtel, Institut de Zoologie, Neuchâtel, Switzerland 2 Universität Würzburg, Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl für Botanik II, Würzburg, Germany

Corn (Zea mays) plants under attack by caterpillars emit a blend of volatile compounds that attracts the herbivore’s natural enemies. This plant response is considered to be an indirect induced defense. In corn, the production of volatiles is mediated by the phytohormon jasmonic acid (JA). In contrast, pathogen attack can up-regulate the salicylic acid (SA)-pathway and the expression of systemic acquired resistance (SAR) against plant diseases. Activation of the SA-pathway has often been found to repress JA-dependent direct defenses. But, little is known about the effects of SA on indirect defenses such as volatile emission and parasitoid attraction. We examined if induction of SAR, by chemical elicitation with the SA-mimic BTH (5 mM soil drench), attenuates the emission of plant volatiles induced by caterpillars of Spodoptera littoralis and exogenous JA, respectively. In addition, we observed the effects of the released volatiles on the host location behavior of the parasitoid Microplitis rufiventris in a six-arm-olfactometer. BTH treatment of corn seedlings resulted in significant systemic resistance against the necrotrophic leaf pathogen Setosphaeria turcica but had a negligible effect on volatile emission. Induction of SAR significantly reduced the emission rates of two compounds (indole and trans-β-caryophyllene) in JA-treated plants while no negative cross-talk was found in herbivore-induced corn seedlings. As expected, induction of SAR with BTH did not reduce the attractiveness of herbivore-damaged plants to host-searching parasitoids. We conclude that in the studied system, plant protection by SAR activation is compatible with indirect defense against herbivores

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Genotypic manipulation of infochemicals to study chemical ecology and community ecology of herbivore-induced plant volatiles

Snoeren T.A.L, Jong P. de and Dicke M.

Laboratory of Entomology, Wageningen University, The Netherlands

Insect herbivory induces the emission of plant volatiles and thereby alters a plant’s phenotype. This may have consequences for various organisms in the environment and for community ecology. Organisms that may be affected comprise e.g. herbivores and their carnivorous enemies. We investigate the effects of herbivore-induced plant volatiles on interactions with insects and community composition in a system based on crucifer plants using the model plant Arabidopsis. We use molecular, chemical, behavioral and community ecology approaches. We will gather molecular genetic knowledge about signal transduction involved in the induction of plant volatiles using well-characterized genotypes that are altered in a single trait (mutants and transgenics). The signal transduction route of Jasmonic Acid (JA) is currently our main interest. This Jasmonic Acid cascade is believed to have a major impact on the development of herbivore-induced plant volatiles resulting from herbivory by caterpillars. Within the signal transduction route, we are focusing on the production of the oxylipins oxo-Phytodienoic Acid (OPDA), dinor-oxo-Phytodienoic Acid (dnOPDA) and Jasmonic Acid. We expect that these oxylipins have a relatively broad effect in plants’ indirect defense system. At present the mutants opr3, dde2-2 and fad5, and their corresponding ecotypes, are analyzed for oxylipin expression with GC-MS. This has already demonstrated variation in the oxylipin profiles. In addition, behavioral studies with parasitoids are conducted to check for discrimination between these Arabidopsis lines after herbivory. Parasitoid behavior demonstrated variation in herbivore-induced plant volatile production. The combination of these two approaches provides us insight in the role of the Jasmonic Acid cascade in infochemical production and the effect of specific volatiles on the behavior of community members.

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Endophytic bacteria of maize plants induce the emission of 2,3-butanediol, which modifies the resistance of the plants against herbivores and pathogens Marco D’Alessandro1, Ton Jurriaan2, Zopfi Jakob3 and Ted C. J. Turlings1

1 University of Neuchâtel, Institute of Zoology, Laboratory of Evolutionary Entomology, Case Postale 2, CH-2007 Neuchâtel, Switzerland. 2 Utrecht University, Phytopathology, P.O.Box 800.84, 3508 TB, Utrecht, The Netherlands 3 University of Neuchâtel, Institute of Botany, Laboratory of Microbiology, Case Postale 2, CH-2007 Neuchâtel, Switzerland. Plants interact with a multitude of other organisms, ranging from neighbouring plants to herbivorous insects and from beneficial to pathogenic microorganisms. These multitrophic interactions are also reflected in a number of complex defence strategies. Depending on previous interactions with some organisms, a specific defence mechanism might be activated or primed, which enables the plants to respond more efficiently to following exposure to harmful organisms. Here we show that the maize endophytic bacterium, Enterobacter aerogenes, which we isolated from germinated maize seeds (Zea mays var. Delprim), induces the release of substantial amounts of 2,3-butanediol in maize seedlings. In addition, E. aerogenes enhances the resistance of maize seedlings to the fungal pathogen Setosphaeria turcica and has a slight positive effect on the performance of the larvae of the noctuid moth Spodoptera littoralis. By exposing maize seedlings to a synthetic isomeric mixture 2,3-butanediol or its precursor acetoin, we also show that these bacterial derived VOCs are at least partially responsible for the observed differences in the defence against pathogens and herbivorous insects. Gene expression studies of pathogen related defence genes and genes induced after herbivore attack suggest that the bacteria and the bacterial derived VOCs prime maize seedlings to respond faster to pathogen attack. We argue that bacterial VOCs, such as 2,3-butanediol and acetoin might be key compounds that help to regulate the resistance of plants against pathogens and this might have important consequences for multitrophic interactions.

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Factors influencing root colonization by the beneficial strain Pseudomonas chlororaphis O6. Anne Anderson1 and Y-C Kim2

1 Biology Department Utah State University. Logan Utah. USA 84322-5305 2 Agricultural Plant Stress Research Center and Environmental-Friendly Agricultural Research Center, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea Pseudomonas chlororaphis O6 (PcO6) is an aggressive colonizer of plant roots under laboratory and field conditions. Root colonization is beneficial to the plant, inducing systemic protection against bacterial, fungal and viral pathogens. Antimicrobial phenazines and hydrogen cyanide are produced by PcO6 under GacS-dependent acyl homoserine lactone regulation. Phenazines are produced predominantly in the seed zone of a colonized barley seedling. Mutations in dctA, encoding a transporter for carboxylic acids and in edd, eliminating catabolism of sugars through the Entner-Douderoff pathway and reduces colonization early and later in colonization. These findings suggest to us that both organic acids and sugars are used during colonization. Reduced colonization in both dctA and edd mutants correlated with lessened ability to induce systemic resistance to the soft rot pathogen. Systemic protection against soft rot also is correlated with production of butanediol, a presumed product of fermentation. Butanediol production correlated with increased leaf surface production and induced resistance and is dependent on gacS activity. GacS regulates negatively the production of IAA from tryptophan and the shift in phenotype to a highly hydrophobic cell variant that is effective in generating biofilms in minimal medium conditions. These observations illustrate several ways in which the Gac system in P. chlororaphis O6 regulates important ecological features in this bacterium.

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Fri 28 April 2006

Session 4

‘Types of resistance important for plant breeders and possible

contribution of inducible resistance’

Breeding for inducible resistance against insects – applied plant breeding aspects Åhman, Inger M. Svalöf Weibull AB, SE-268 81 Svalöv, Sweden Many of the pre-requisites necessary for breeding plants with inducible resistance to pests are no different from breeding for constitutive resistance. It is necessary to have resistance genes giving high enough pest protection and yield gains, efficient selection methods and means of introducing resistance genes into agronomically acceptable plant material. In both constitutive and induced resistance, resistance traits need to reduce negative effects of target pests and be neutral or positive to non-target organisms. In inducible resistance there is also the need for proper timing and specificity of induction. Tentatively the ideal inducible resistance requires specific cues for induction which rapidly induce resistance traits with long duration relative to the sensitive period of the crop and which give a systemic plant response. It takes knowledge to develop appropriate selection methods for resistance and in the case of inducible resistance we also need knowledge about how the inducing factors are operating. Inducing cues may come from insect activities on the plant (e.g. feeding, oviposition), from neighbouring plants or from manmade chemical formulations of elicitors. Plant selections can be based on plant damage levels, insect numbers, insect responses, plant resistance traits and/or molecular characteristics of the plant genome.

Breeding for insect-inducible resistance to insects has been applied in the form of rapidly induced highly specific resistance in insect - crop combinations where there are gene-for-gene relationships, such as with the Hessian fly and the Russian wheat aphid in wheat. To my knowledge there are no examples of traditional breeding where less specific types of resistance; induced by insects, by plant neighbours or by chemical formulations; have been deliberately bred into commercial cultivars. It is likely that the accumulating knowledge about the mechanisms of induced resistance will find applications in cultivars produced by genetic engineering.

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Deployment strategies for crops with inducible resistance

Newton A.C.,1 Lyon G.D.,1 Begg G.,1 Zhan J.,1 Guy D.C.1 and Walters D.R.2 1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK 2 Scottish Agricultural College, West Mains Road, Edinburgh EH9 3JG, Scotland, UK In the field induced resistance in current varieties of crop plants is a normal defence component but it can be further exploited either by a) treating with a resistance elicitor, b) using genotypes which possess a stronger Inducible response, or c) selectively encouraging pathotypes that induce it.

Characteristically, resistance elicitors are unreliable as unknown environmental factors sometimes cause substantial loss of efficacy. Furthermore, different crop genotypes, such as cereal varieties, express differential degrees of inducible resistance with given elicitors. As elicitors may act via different receptors it seems advisable to use a combination of elicitors to combine different modes of action to improve efficacy, although trial data to validate this is lacking. However, gene expression data from field-grown plants indicates considerable activation of defence-related pathways so opportunities for further specific pathway induction may be limited.

Growing mixed genotypes of plants would be the norm in many natural ecosystems, but is uncommon in most crops grown as high input monocultures. Nevertheless, there is considerable evidence that cultivar mixtures reduce disease, increase yield, and deliver stability in yield and quality. This is through the spatial effects of dilution of susceptible plants and barriers of resistance plants, (disruptive selection?), and through induced resistance. The latter is achieved through cross-over of spores virulent on one component but avirulent on another, and is thus most effective for pathogens with a 'simple' and specific race structure such as some obligate biotrophs on cereals where its contribution has been estimated. However, mixtures are effective against hemi-biotrophic pathogens with little race-specificity too, and critical to all mixture efficacy is component number, proportions and spatial deployment strategy. For example, a multi-component 'patchy' mixture is likely to be more efficacious than just two or three components in a homogeneous mixture. The scale and degree of patchiness must be a compromise between the parameters required for individual pathogen species control and resource exploitation, the former being determined by pathogen dispersal gradient.

The stability of disease control through exploitation of induced resistance is dependent on its costs, both to the pathogen and to the plant. Modelling demonstrated how the balance can be tipped towards disease control through spatial resistance deployment strategies in mixtures. It can be argued that resistance elicitors will not select for pathotypes that will erode their efficacy as they do not act directly against the pathogen, although experimental evidence indicates this may not always be the case.

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Response of Solanum stoloniferum to infestation by the green peach aphid: Induced resistance and gene expression Alvarez, A.E. 1,2,3, Broglia V. 2, Alberti D’Amato, A.M. 2, Tjallingii, W. F1., Dicke, M. 1 and Vosman, B. 3 1Wageningen University, Laboratory of Entomology, P.O. Box 8031, 6700 EH Wageningen, NL. 2Universidad Nacional de Salta, Facultad de Ciencias Naturales, Buenos Aires 177, 4400 Salta, Argentina. 3Plant Research International, Department of Biodiversity and Breeding P.O. Box 16, 6700 AA, Wageningen, NL.

Wild tuber-bearing potato Solanum stoloniferum (Schlechtd) possesses high constitutive resistance to the green-peach aphid, Myzus persicae (Sulzer). This was evident from our previous aphid probing behavior studies. We could localize a resistance factor at the epidermal/mesophyll level, disturbing the mechanical activities of aphid stylets. We extended this study by testing S. stoloniferum for induced responses and by analyzing gene expression at the local and systemic level. The plants were pre-infested for 96 h with 40 young adult aphids at fully expanded leaves (leaf numbers 5 and 6 from the apex). An aphid settling behavior test was performed to study induced resistance on the previously infested leaf (local responses), and on a higher non-infested leaf (systemic response). To study the differential gene expression, we used a dedicated cDNA microarray containing 3888 defense-related potato genes. The following binary comparisons were made: (1) pre-infested local leaf vs. non-infested systemic leaf of the same plant individual, and (2) pre-infested leaf vs. non-infested leaf (control plant) of different plant individuals, and using leaves at similar position as the systemic and local leaves in the previous test. The results will be discussed during the meeting.

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Increased fungal resistance of crops by pathogen-induced overexpression of plant resistance genes Schmidt, K.1, Pflugmacher, M.1, Brieß, W.1, Klages, S.1, Rohlf, C.1, Mäser, A.1, Kurrasch, J.1, Holtschulte, B.2, Truberg, B.2, Nehls., R.1, and Stahl, D. J.1

1Planta GmbH, Grimsehlstraße 31, D-37555 Einbeck, Germany, 2KWS SAAT AG, Grimsehlstraße 31, D-37555 Einbeck, Germany Plant diseases caused by fungal infection result in serious yield and quality losses of crops. Here we present a new strategy to improve the general fungal resistance of crops by genetic engineering. The activation of resistance gene dependent defence reactions is one of the most effective protection mechanism of a plant against invading pathogens. These defence reactions are often associated with the hyper-sensitive reaction (HR). The regulated over expression of resistance genes will be used to trigger the induced defence responses of crops. cDNA clones of resistance gene analogous (RGA) of the CC-NBS-LRR type have been isolated from potato, sugar beet and wheat. Transient over expression of some RGA triggers a HR in the transformed leaf tissue which indicates that the RGAs are functional elements of a gene-for-gene interaction. In order to circumvent the detrimental effect of a constitutive expression of R genes in transgenic plants the R genes were combined with pathogen inducible synthetic promoters. Synthetic pathogen-inducible promoters exclusively containing well defined regulatory elements of PR genes have been described for A. thaliana, parsley and sugar beet (Rushton et al., 20023, Klages et al., 2004). Depending on the combination and number of cis-elements the promoters differed in mediating the strength of gene expression, pathogen inducibility and basal activity. Selected combinations of synthetic promoters and R genes were transformed into sugar beet and transgenic plants were generated. Resistance assays which have been performed under greenhouse conditions revealed an increased resistance of the plants against Cercospora beticola, the major fungal pathogen of sugar beet. 3Rhuston et al., 2002. Plant Cell 14, 749-762.

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Session 5

‘Biotechnology approaches to breeding for (inducible) resistance /

Tools for biotechnology’

Critical analysis of Bt-crops as essential element of sustainable insect control

Ponti, O.M.B. de Nunhems B.V., Haelen, the Netherlands Today millions of hectares are planted to varieties of so-called Bt-crops, both in developed and developing countries. These crops are cotton, corn and rice. Others will follow in the near future. Bt-varieties can contribute to the sustainability of insect control, provided that durability strategies are seriously taken into account, such as the incorporation of multiple Bt-genes and the release of these varieties in IPM/ICM systems. The development and commercialization of Bt-varieties, or rather transgenic varieties in general, require substantial efforts in time and budget. Therefore, the very need of a transgenic solution should be carefully analyzed, both in agronomic and socio-economic terms. The discovery process is far more complex and expensive than generally assumed, with a rather low success rate. After the effect of a transgene had been confirmed (proof of concept, proof of product), the development of a transgenic variety is rather straightforward. However, time and budget required for the development of the regulatory dossier, to acquire the required permits for commercial release, can surpass the variety development costs with a factor of ten or more. This is the major hurdle for the advent of transgenic crops, in particular for the so-called orphan crops, which are not restricted to the developing countries only.

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Using Pathogen Genomics to Investigate the Induction and Manipulation of Inducible Plant Disease Resistance Paul R J Birch Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. (pbirch@scri.ac.uk) Genomics has made a hugely beneficial impact on our understanding of many aspects of biology, and plant pathology is no exception. To be successfully infect, invading pathogens need to manipulate, suppress or evade plant host defences, requiring the delivery of proteins that are targeted to interact with host proteins. The complete genome sequences of plant pathogens are providing blueprints of the entire repertoire of genes required for successful infection. Moreover, they are revealing insights into the lives of pathogens when they are not infecting their host plants. Similarly, genomic approaches are informing us about how plants defend themselves from pathogens. Armed with this knowledge, we can better direct the efforts of conventional disease resistance breeding programmes to utilise the natural defence strategies of plants. Moreover, as we discover more about the natural defences of plants we can find ways to manipulate or induce those defences to the detriment of invading pathogens. I will illustrate the potential of genomics in developing disease control strategies for two pathogens of potato: the bacterium Erwinia carotovora subsp. atrosepticum, which causes soft rot and blackleg and the oomycete Phytophthora infestans, which causes late blight.

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Activation of defense responses in Arabidopsis thaliana by oxylipins and yeast elicitors

Raacke I.C., Müller M.J. and Berger S. Lehrstuhl für Pharmazeutische Biologie, Julius-von-Sachs-Institut für Biowissenschaften, Würzburg

Defense mechanism in plants can be activated by microorganism and endogenous or exogenous elicitors. Cell wall and glucopeptide components of yeast have been reported to exhibit elicitor activity. The mode of action of defense activation by yeast is not known so far. We used the model plant Arabidopsis thaliana to investigate the activation of defense responses by yeast, the effect on resistance against different pathogens and the mode of action. Treatment of Arabidopsis plants with an autoclaved yeast suspension induced the expression of SAR-related genes and accumulation of the phytoalexin camalexin. Symptom development and bacterial growth after infection with a virulent strain of the pathogen Pseudomonas syringae was reduced in yeast pre-treated plants. No protection was detectable in mutants affected in the salicylate pathway while mutants in the jasmonate or camalexin pathway were protected by yeast indicating that the salicylate pathway is necessary for the yeast induced resistance against P. syringae. Pretreatment with yeast also reduced symptom development after challenge with the fungal pathogen Botrytis cinerea. This protection was detectable in mutants of the salicylate, jasmonate and camalexin pathway indicating that it is independent of these defense mechanisms.

Enzymatically formed oxylipins like jasmonic acid and OPDA activate defense responses in plants. We investigated the biological activity of phytoprostanes, non-enzymatically formed oxylipins. Phytoprostanes are structurally similar to OPDA and accumulate upon pathogen infection. Phytoprostanes inhibited root growth and induced camalexin accumulation and expression of genes involved in detoxification.

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Inducible Disease Resistance Involves an Apoplastic Cysteine Protease Cathepsin B Gilroy, E.M 1,2, Venter, E1, McLellan, H1,2, Hein, I1, Hrubikova, K1, Holeva, M.C1, Boevink, P.C1, Loake, G.J2, Lacomme, C.L1 and Birch, PRJ 1. eleanor.gilroy@scri.ac.uk 1 Scottish Crop Research Institute, Invergowrie, Dundee, DD2-5DA 2Institute of Molecular Plant Sciences, Edinburgh University, EH9 3JR

The hypersensitive response (HR) is an important inducible defence response effective against biotrophic pathogens in the early stages of an incompatible interaction. The HR is a rapid form of genetically programmed cell death (PCD) triggered in host cells through R protein signalling and activation of countless proteases. Suppression subtractive hybridisation (SSH) revealed the up-regulation of a plant cysteine protease, cathepsin B (CathB) from Phytophthora infestans challenged potato undergoing R gene-mediated HR. A role for cathepsin B during the HR was supported by previous reports of its requirement in some animal PCD processes. Nicotiana benthamiana, a close relative of potato, represents a suitable model for investigating many potato traits and is amenable for rapid functional investigation of candidate genes using virus-induced gene silencing (VIGS). Therefore, the VIGS vector, tobacco rattle virus (TRV), was utilised for investigating the role of cathepsin B during the hypersensitive response (HR). We demonstrate that the induction of cathepsin B expression and activity precedes cell death in response to a non-host bacterium, Erwinina amylovora (Eam) and that this cell death can be suppressed at the cellular level in TRV::CathBas N. benthamiana shown by trypan blue staining. HR induced with non-host bacteria, agro-mediated expression of cytoplasmic R3a and Avr3a and the HR signalling molecule hydrogen peroxide was perturbed in CathB-silenced N. benthamiana. In addition, silencing of CathB and co-inoculation of Eam with commercial CathB inhibitors suppressed CathB activity and caused an increase in bacterium growth. Fluorescent tagging with mRFP indicates that CathB has an apoplastic cellular location. Interestingly, the HR was not suppressed in response to agro-mediated expression of apoplastic Cf-4 and Avr4, suggesting that CathB is not required for HR induction in all R gene-mediated HRs. We therefore provide evidence that cathepsin B has both a conserved role during a PCD mechanism shared by plants and animals and that it is specific to a subset of triggered HRs in N. benthamiana.

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Proteomic Analyses To Understand Pathways Of Resistance To Aflatoxin Accumulation In Developing Maize Ears Luthe, D.S.1, Pechanova, O.2, Bridges, S.M.3, Pechan, T.4, and Williams, W. P.5 1Department of Crop and Soil Sciences, Pennsylvania State University 2Deparment of Biochemistry and Molecular Biology, Mississippi State University 3Department of Computer Science and Engineering, Mississippi State University 4Life Sciences and Biology Institute, Mississippi State University 5Corn Host Plant Resistance Laboratory, Mississippi State University In maize, the first line of defense against kernel infection by the fungus Aspergillus flavus and subsequent aflatoxin accumulation are tissues of the developing ear. We are examining the proteomes of three maternally-derived ear tissues, the rachis (cob), silk and pericarp. The rachis, the structure that transports nutrients from the leaves to the kernels, is particularly important in resistance. The proteome of the developing maize cob was mapped and approximately 1400 unique cob proteins were identified using AgBase tools. The proteomes of several maize genotypes that are resistant (R) or susceptible (S) to A. flavus infection were compared. Changes in proteomes following inoculation with the fungus also were examined. These results showed that both R and S cobs have a group of defensive proteins called pathogenesis-related (PR) proteins that are expressed in response fungal infection. However, R cobs appear to preferentially express some enzymes in the phenolic pathway that lead to lignin accumulation, which can slow fungal growth. In addition, R cobs express a suite of heat shock proteins (HSPs) and antioxidant enzymes that are less prevalent in the S cobs. These proteins are likely to provide greater resistance to the abiotic stresses of heat and drought that exacerbate A. flavus infection and aflatoxin production in the ear. This systems biology approach has allowed us to formulate models for resistance that can be experimentally tested in the future.

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POSTER ABSTRACTS

Activation of defense responses in Arabidopsis thaliana by oxylipins and yeast elicitors

Raacke I.C., Müller M.J. and Berger S. Lehrstuhl für Pharmazeutische Biologie, Julius-von-Sachs-Institut für Biowissenschaften, Würzburg

Defense mechanism in plants can be activated by microorganism and endogenous or exogenous elicitors. Cell wall and glucopeptide components of yeast have been reported to exhibit elicitor activity. The mode of action of defense activation by yeast is not known so far. We used the model plant Arabidopsis thaliana to investigate the activation of defense responses by yeast, the effect on resistance against different pathogens and the mode of action. Treatment of Arabidopsis plants with an autoclaved yeast suspension induced the expression of SAR-related genes and accumulation of the phytoalexin camalexin. Symptom development and bacterial growth after infection with a virulent strain of the pathogen Pseudomonas syringae was reduced in yeast pre-treated plants. No protection was detectable in mutants affected in the salicylate pathway while mutants in the jasmonate or camalexin pathway were protected by yeast indicating that the salicylate pathway is necessary for the yeast induced resistance against P. syringae. Pretreatment with yeast also reduced symptom development after challenge with the fungal pathogen Botrytis cinerea. This protection was detectable in mutants of the salicylate, jasmonate and camalexin pathway indicating that it is independent of these defense mechanisms.

Enzymatically formed oxylipins like jasmonic acid and OPDA activate defense responses in plants. We investigated the biological activity of phytoprostanes, non-enzymatically formed oxylipins. Phytoprostanes are structurally similar to OPDA and accumulate upon pathogen infection. Phytoprostanes inhibited root growth and induced camalexin accumulation and expression of genes involved in detoxification.

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Quantifying cost and benefits of Induced Systemic Resistance in a clonal plant network Gómez S. and Stuefer J.F. Institute for Water and Wetland Research. Radboud University Nijmegen. The Netherlands.

Plants lack the advantage of mobility to escape from herbivores. Nevertheless, they are not helpless as they have developed a wide array of defense mechanisms to cope with their attackers. Defense induction and defense expression are assumed to be costly processes. Plant defense theory suggests that inducible defensive phenotypes have evolved to save costs allowing allocation of finite resources to other important functions when defense is not needed (Herms and Mattson 1992, Zangerl and Bazzaz 1992, Agrawal 1999).

Clonal plant networks consist of interconnected individuals (ramets) that share not only resources but also information, such as defense induction agents (Gómez and Stuefer 2006). This allows clonal plants to activate an early-warning system alerting network members about the presence of enemies, after herbivores have attacked one or a few of the ramets. This can translate into obvious benefits. However, due to the large spatial extent of many clonal plants, upregulating defense traits in the whole network could result in large costs if defense induction does not closely match herbivore feeding dynamics in time and in space (Stuefer et al. 2004).

In this study we used natural genotypes of the stoloniferous herb Trifolium repens to quantify costs and benefits of induced systemic resistance. To do this we grew induced and uninduced clonal fragments of the same genotype in competition with each other. Benefits and costs of induced resistance were assessed in the presence and absence of herbivores, respectively.

Induced plants produced significantly fewer ramets on the main stolon and developed shorter petioles than control plants. Induction led to a shift of biomass allocation, especially to a reduced biomass investment into roots. These changes can be interpreted as costs of defense induction because they are likely to reduce the vegetative spread and impair the competitive ability of plants. When herbivores were present, induced plants suffered 50% less damage than uninduced plants. These benefits of defense induction were particularly pronounced for vulnerable and yet very valuable young ramets, which were strongly preferred by the insects over the rest of the ramets.

Trifolium repens is a valuable species from an economical point of view due to its common use as a forage legume in pastures. In our short-term study induction did not affect total biomass production but it did confer large benefits. This suggests that the use of highly inducible cultivars in pastures could be a good strategy to confer protection against insect pests without compromising the yield.

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Using Q-RT-PCR to determine a defence-related signal signature of Maize Danielle Karlen1,2, Marco D’Alessandro1, Brigitte Mauch-Mani2, Ted Turlings1 and Jurriaan Ton3 1 Institute of Zoology, Laboratory of evolutionary Entomology, University of Neuchâtel, Switzerland

2 Institute of Botany, Laboratory of Cellular and Molecular Biology, University of Neuchâtel, Switzerland. 3 Institute of Environmental Biology, Section Phytopathology, Utrecht University, The Netherlands. Plants protect themselves against biotic and abiotic stress by inducible defence responses, which involve the activation of distinct signalling pathways. The plant hormones jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA) often play crucial roles in these defence pathways. Marker genes, whose expression is specifically induced by these hormones, are convenient tools to quantify the activity of these pathways. Here, we describe a relatively fast method to simultaneously determine the activity the JA-, SA-, ET-, and ABA-inducible defence responses, using a dedicated set of marker genes. To obtain transcription profiles that specifically mark activity of the four different defense pathways, 2-week-old maize plants were treated with JA, BTH, ACC, or ABA, and analyzed for marker gene expression by Q-RT-PCR. Currently, we are comparing these hormone-specific transcription profiles with the transcriptional responses to infection by the fungal pathogen Setosphaeria turcica, infestation by the generalist herbivore Spodoptera littoraris, and exposure to salt stress. Using hierarchical cluster analysis, we will investigate whether these transcription profiles provide a reliable signature of the signaling state in the plant.

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Changes in free amino acids induced in cucumber plants by spider mites and plant growth promoting rhizobacteria (PGPR) Tomczyk A. Department of Applied Entomology, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Tomczyk@alpha.sggw.waw.pl The experiment was conducted on glasshouse cucumber cv. Corona. Plants were cultivated either in the presence of Pseudomonas fluorescens (PGPR) or without these bacteria. Half of the bacterized, as well as non bacterized plants were infested with Tetranychus cinnabarinus Boisd. In all groups of plants, free amino acids were identified and their content was estimated. The total content of free amino acids strongly increased in both spider mite damaged and in PGPR treated plants. Spider mite feeding on bacteria treated plants caused a lower increase in free amino acids as compared to untreated plants, however the density of the spider mite population was also lower on bacterized plants as compared to non bacterized. Amounts of all free protein amino acids increased, as a result of spider mite feeding on the leaves of not bacterized plants as compared to bacterized, in contrast to non-protein amino acids. The highest increase was observed for aromatic amino acids. In contrast, on the leaves of plants treated with bacteria, the ratio between aromatic and other free amino acids was the lowest. The difference in the concentration of proline between spider mite-damaged and not- infested bacteria treated plants was evidently the main reason for this phenomenon.

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Impact of transgenic cucumbers expressing the thaumatin II gene on the occurrence of arthropod fauna Kielkiewicz M.1, Gajc-Wolska J.2, Szwacka M.3, Malepszy S.3 1Department of Applied Entomology, Warsaw Agricultural University 2Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural University 3Department of Vegetable and Medicinal Plants, Warsaw Agricultural University, Warsaw, Poland Incorporation of thaumatin II gene into genome of strawberry, cucumber, tomato, carrot or pear improves the taste property of the fruits. Sweet-testing protein thaumatin II also serves as an effective defence factor against some abiotic and biotic stresses. The objective of the present study was to determine the impact of genetically modified cucumbers expressing the thaumatin II gene on the occurrence of arthropod fauna. Four lines of transgenic cucumbers (T224, T225, T212, T210) derived from the inbred line I18 of Cucumis sativus L. cv. Borszczagowski (line B) and exhibited high, moderate or low level of thaumatin II protein in the leaf were evaluated under field conditions. Insect and mite pests, as well as their natural enemies were monitored weekly. We found that fewer onion thrips (Thrips tabaci), cotton aphids (Aphis gossypii) and two-spotted spider mites (Tetranychus urticae) settled and developed on transgenic lines than on line B. More leaves damaged by tarnished plant bug (Lygus rugulipennis) were recorded on non-transgenic plants in comparison with transgenic ones. However, only density of aphids showed moderately strong correlation with the accumulation of thaumatin II in the leaf. The coexistence of herbivores and their natural enemies as Coccinella septempunctata, Aphidoletes sp., Chrysoperla sp., Aeolothrips intermedius and Orius minutus on all cucumber lines was observed. Significant association between the number of eggs of Chrysoperla sp. and the number of aphids was found. The results of these studies showed that transgenic cucumber plants expressing the thaumatin II gene affect the abundance of some sucking insects and mites. This result is likely more of the consequence of individual and specific chemical properties of transgenic cucumber lines than the presence of thaumatin II protein itself. We conclude that variation in nutritional quality and possibly susceptibility among tested cucumber lines are potentially important factors worthy of further studies. This work was partially supported by a grant No. P06R 017 29 from the Ministry of Education and Science, Poland.

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BABA induced resistance in grapevine

Slaughter A.R.1 , Hamiduzzaman M.M.1, Jakab G.1, Mauch-Mani B.1 and Neuhaus J-M.1 1 University of Neuchâtel, Institute of Botany, Department of Molecular and Cell Biology, Rue Emile-Argand 11, Case Postale 158, CH-2009 Neuchâtel, Switzerland

Grapevine (Vitis vinifera) is a major fruit crop worldwide and is affected by many diseases. Downy mildew, caused by the oomycete Plasmopara viticola leads to great damage and yield losses in grapevine if no protective measures are taken. The majority of the traditional cultivars that are planted are susceptible to this disease, necessitating the intensive use of chemicals to limit the damage in vineyards. One possible solution would be the activation of a plants own defense system, known as induced resistance. b-Aminobutyric acid (BABA) has previously been shown to induce resistance against many oomycetes. It was observed that the protective effect of BABA in Arabidopsis was due to the potentiation of natural defense mechanisms against biotic stresses, a phenomenon refered to as priming. Priming is the capacity of a plant to express a faster and stronger basel defense response upon pathogen infection. BABA was used to induce resistance in grapevine. The resistance depended to a large extent on the deposition of callose. A strong reduction of mycelial growth and sporulation in the susceptible cultivar Chasselas was observed. Initial expression profile by microarray of BABA-treated Chasselas will be discussed.

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Quantification of oomycete biomass in Grapevine Dubresson R., Slaughter A., Neuhaus J.M. and Mauch-Mani B. University of Neuchâtel, Institute of Botany, Laboratory of Cellular and Molecular Biology, Emile-Argand 11, Case Postal 158, CH-2007 Neuchâtel, Switzerland. Examination of disease progression by quantifying the amount of pathogen biomass in infected host tissues is important in the study of fungal/oomycete-plant interactions and in the quantification of induced resistance. Several methods of quantification will be investigated. The first method uses the green fluorescent protein to quantify the growth of Plasmopara viticola during infection of grapevine. To develop a quantitative assay of pathogen growth in plant tissues, the NCCR1 strain of Plasmopara viticola, the causal agent of downy mildew in grapevine, was transformed with a modified green fluorescent protein (GFP) gene fused with a ham34 promoter from Bremia lactucae (Si-Ammour et al., 2003).The transformation protocol will be discussed. GFP accumulation in inoculated leaves of grapevine will be quantified in leaf discs using a fluorescence microplate reader.

The second method is based on the in vivo monitoring of obligate biotrophic pathogen growth by Real Time PCR. A rapid high-throughput method was previously developed for the relative quantification of P.viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative PCR with TaqMan chemistry (Valsesia et al., 2005). This method allows for the simultaneous amplification and for the independent detection of pathogen and host DNA by using species-specific primers and TaqMan probes that are labelled with different fluorescent dyes. This method is highly sensitive and specific for the detection of P. viticola DNA in plant tissue.

Results obtain from these two methods will be discussed and compared to microscopic analysis of oomycete growth in grapevine.

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Inducible defence of Bt oilseed rape and global climate change

Himanen S.J.1, Nissinen A.2, Nerg A.-M.1, Poppy G.M.3, Stewart C.N. Jr4 and Holopainen J.K.1 1 Department of Ecology and Environmental Science, University of Kuopio, Finland 2 Agrifood Finland, Plant Protection, Jokioinen, Finland 3 School of Biological Sciences, University of Southampton, U.K. 4 Department of Plant Sciences, University of Tennessee, USA Cruciferous plants possess a distinctive secondary defence against biotic stresses in the form of glucosinolates, i.e. sulphur and nitrogen containing β-thioglycoside-hydroxysulfates. They are hydrolyzed upon herbivore or mechanical damage into various degradation products such as isothiocyanates and nitriles, of which some are also volatile. Terpenoids are another group of secondary compounds involved in inducible defence, emitted also from Brassica plants. Genetically modified crop varieties, which are resistant against pests or pathogens, are actively under research. Bt varieties of maize and cotton are common, and the application of Bt toxin production is underway for many new crops also. At the same time, the risk analysis of GM crops is emphasized, and possible unintended changes in plant secondary defence need to be examined thoroughly. The global climate change is inevitably changing the atmospheric CO2 level and ozone episodes are a major problem especially in urban areas. Therefore also effects of these abiotic factors for plant secondary defence and pest control are of growing concern. We studied the inducible defence, in the form of glucosinolates and volatile organic compounds (VOCs) emitted from Bt oilseed rape (Brassica napus, OSR) leaves in elevated CO2 and ozone treatments, and the induction of these defences after Bt target herbivore diamond back moth (DBM, Plutella xylostella) damage.

Constitutive glucosinolate (GS) defence, measured as total GS concentration per foliage dry weight, was lower in OSR plants grown in elevated CO2 or elevated ozone than in ambient air. Individual GSs varied in their response to both CO2 and ozone enrichment. Typically, concentrations of indolyl GSs were lower and some aliphatic GSs were higher in elevated CO2 and ozone treatments compared to ambient air. The induction of GS response (indolyl GSs) after DBM herbivory was higher in elevated than in ambient CO2. GS concentration of Bt and non-Bt plants responded similarly to CO2, ozone and herbivory.

Elevated CO2 did not change the profile of VOCs emitted constitutively by OSR. DBM damage induced the emission of several terpenes. Some terpenes were emitted in lower amounts from Bt transformant but this was largely attributed to lowered feeding damage by DBM larvae. Ozone treatment inhibited the induction of terpene emission.

The production of Bt toxin by Bt-OSR remained stable also during the elevated CO2 and medium ozone treatments, which indicated efficient pest control. High ozone resulted in lower Bt concentration in plant leaves at a stage where the growth and primary metabolism of the plants were also affected.

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Derailed stylet mechanics during plant penetration by aphids and its potential as a mechanism of host plant resistance Tjallingii, W.F.1, Alvarez, A.E.1,2,3, Vosman, B. 2, and Hogen Esch, Th. 1

1Wageningen University, Laboratory of Entomology, P.O. Box 8031, 6700 EH Wageningen, NL. 2Plant Research International, Department of Biodiversity and Breeding P.O. Box 16, 6700 AA, Wageningen, NL. 3Universidad Nacional de Salta, Facultad de Ciencias Naturales, Buenos Aires 177, 4400 Salta, Argentina.

A Myzus persicae (Sulzer) resistant wild ‘potato’ Solanum stoloniferum (Schlechtd) showed strongly increased occurrence of the electrical penetration graph (EPG) waveform F activity, which contributes substantially to the plant’s resistance we think. The underlying mechanism is derailed stylet mechanics presumably due to an anatomical or chemical tissue property enhancing this phenomenon substantially more than on Myzus susceptible cultivated potatoes.

Aphid stylets include mandibular (outer) and 2 maxillary (inner) stylets together forming the functional stylet bundle. When the stylets penetrate the left and right mandibular stylets make alternating protraction and retraction movements between the cellulose fibers of secondary cell walls thus following an intercellular track towards the vascular target tissues, phloem and xylem. Periods of stylet penetration and intermittent salivary sheath secretion are recorded as alternating EPG waveforms C and B, respectively. Rather often aphids show a waveform labeled F, a high frequency waveform which also occurs in whiteflies and mealy bugs. Stylectomy during waveform F and subsequent ultra thin sectioning of plant tissue and embedded stylets showed that one of the maxillary stylets appeared out of its normal position in relation to the other 3 stylets. Its tip being protruded much further it was no longer supported by the other 3 stylets and therefore, the aphid lost control of its normal stylet penetration. We have called waveform F ‘penetration difficulties’, recently changed to ‘derailed stylet mechanics’ which describes it better. Electron micrographs of the stylet position during waveform F are shown and the impact is discussed.

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Transgenic Plantlets of Grapevine (Vitis spec.) provide a look insight of host-pathogen interaction Seibicke T. and Kassemeyer H.-H. Staatliches Weinbauinstitut Freiburg, Merzhauser-Str. 119 D-79100 Freiburg (Germany) Plants defend against pathogen infection through a wide variety of mechanisms that can be either local or systemic, constitutive or inducible. Similar plant responses can also be triggered by certain natural and synthetic chemical compounds designated as elicitors. The induction of pathogenesis related (PR) proteins is a well known response found in a number of plant species after elicitor treatment or pathogen attack. Transcription of a b-1,3 glucanase gene which product belongs to the PR-protein family was found to be induced in grape leaves upon infection with Plasmopara viticola. A chimeric gene composed of the glucanase-promoter fused to the firefly luciferase-coding region was found to be induced by addition of salicylic acid (SA) in transiently transformed protoplasts. The same construct including a neomycin phosphotransferase II (NPTII) was used to create a stably transformed Vitis suspension cell culture for screening and detecting compounds acting as elicitors effectively inducing this PR-gene promoter. Furthermore, embryogenic cell suspensions were bombarded via particle gun and regeneration of transgenic Vitis plantlets could be observed by transfer on kanamycin-containing media. Promoter efficiency dependent luciferase activity was recorded in a luminometer. SA was found to induce luciferase activity more than tenfold compared to the buffer treated control in stable transformed cell suspension cultures, which confirms results that the compound is involved in PR-gene induction. In order to test compounds on their ability to induce PR-gene expression and perhaps the plant’s pathogen defense, this assay might represent a powerful screening system. Besides screening for PR-gene expression compounds using the stable transformed cell suspensions culture, the transgenic plantlets allow to study PR-gene expression in planta after elicitation or pathogen attack. The tissue-specific distribution of luciferase activity was visualized by a photon-counting camera (PCC) and the in vivo pattern of light was examined after local infection with Plasmopara viticola. However, plants showed only a local induction of luciferase expression at the infection site and no systemic expression of PR-genes. Consequently, the intriguing SAR (systemic acquired resistance) concept plays not a general role in Vitis spec. All data was also proven by northern blot analysis with same results. The observed differences in luciferase activity within a plant thus truly reflect differences in luciferase gene expression. This simple system might be useful in attempts to elucidate the temporal an spatial pattern of PR-gene expression in grapevine after pathogen attack.

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Activation of plant defence response contributes to the antiviral activity of Diocin2 from Phytolacca dioica Iriti M.1,2, Conforto B.3, Parente A.3 and Faoro F 1,2

1CNR, Istituto di Virologia Vegetale, Sezione di Milano and 2Istituto di Patologia Vegetale, Università di Milano, 3Dipartimento di Scienze della Vita, II^ Università di Napoli.

Dioicin 2 (D2) is a single-chain type-1 ribosome-inactivating protein (RIP), constitutively expressed in Phytolacca dioica leaves, with no seasonal or ontogenetic constraint. Besides its activity in deadenylating rRNAs, D2 can depurinate DNA as well and possesses a high homology with pokeweed antiviral protein (PAP II) from P. americana. Though antiviral effect of RIPs has been known for almost a century, the mechanisms underlying this activity are still matter of debate.

Using the patosystems Nicotiana tabacum, cv. Samsun NN - tobacco mosaic virus (TMV) and Phaseolus vulgaris - tobacco necrosis virus (TNV), we demonstrated that D2 possesses a strong antiviral activity, that is expressed only in vivo, when both RIP and virus are contemporaneously inoculated in the same leaf (either in the same surface, or, separately, in the adaxial and abaxial surfaces) but not in the upper leaves. This suggests that D2 cannot move into the phloem, nor induce SAR signals. Similar results have been obtained with both pathosystems.

Cytochemical and ultrastructural investigations showed that D2 alone induce localized H2O2 accumulation in treated tissues but not cell death, which is instead elicited by the concomitant presence of the virus. In this case, some ultrastructural features, such as mitochondrial swelling and nuclear disorganization seem to indicate that programmed cell death events are undergoing. Furthermore, in presence of the RIP, virus replication still occurs, but is restricted to very few cells and virus small necrotic lesions remain invisible. Thus it is likely that D2 antiviral activity is the result of the combined effect of its deadenylation properties on cell nucleic acid and the activation of plant defense response, rather than a direct effect on viral RNA. This conclusion is also supported by the fact that antiviral activity is exerted both on plant infected with TMV, which has a 5’ terminal m7GpppG cap, a target for depurination, and the uncapped TNV RNA.

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Different Chitinase Expression In Sugar Beet Plants After Acibenzolar-S-Methyl Applications

Marinello S., Burzi P.L., Sala E., Galletti S., Cerato C. C.R.A. - Istituto Sperimentale per le Colture Industriali Bologna (Italy) Chitinases are hydrolytic enzymes (EC 3.2.1.14) that catalyse the hydrolysis of chitin, a polymer of N-acetyl-D-glucosamine. Plant chitinases belong to relatively large gene families subdivided in classes that suggest class-specific functions. They are commonly induced upon the attack of pathogens and by various sources of stress. Chitinases are therefore also classified pathogenesis-related (PR) protein, which increase when systemic acquired resistance (SAR) is challenged. The plant defence activator, acybenzolar-S-methyl (ASM), a functional analogue of salicylic acid and SAR inducer, was shown to be able to protect sugar beet plant against Cercospora leaf spot. The aim of this study was to analyse the expression of different chitinases in sugar beet, induced by ASM application, by RT-PCR. Plants of sugar beet cv Aaron were grown until sixth leaf stage under greenhouse conditions. A suspension of ASM at the concentration of 60 mg L-1 in distilled water was applied on one leaf per plant until run-off and was compared to a water treated control. One, 2, 3 days after treatment, untreated leaves were excised and plant RNA was extracted for RT-PCR study. An alignment of amino acid sequence of several chitinases proteins was used to design two set of degenerate PCR primers in highly conserved regions. PCR on cDNA oligo dt in all samples produced the amplification of 621 bp for Chitinase IV and 550 bp for Chitinase. The analyse of the number of copies at different cycles of PCR revealed differences in the level of the transcripts. The quantification of the transcripts at different number of cycle of PCR, showed different levels of these chitinases in the samples. In the plant treated with ASM the activation of transcriptions of chitinases already started after 24h, and maintained at higher level than in plant untreated until 96h after treatment. Systemic nature of ASM action in sugar beet was already demonstrated. In the present study it has been observed that at least two of the five known chitinases classes accumulated in the ASM treated sugar beet leaves more quickly than in untreated plants.

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Possible Induction Of Systemic Acquired Resistance Against Cercospora Leaf Spot In Sugar Beet By Trichoderma Foliar Applications

Burzi P.L., Marinello S., Sala E., Galletti S., Cerato C. C.R.A. - Istituto Sperimentale per le Colture Industriali Bologna (Italy) Cercospora leaf spot, caused by Cercospora beticola Sacc. is considered one of the main sugar beet disease in southern Europe, negatively affecting root yield and sucrose content. Since resistance is only partial and the control of the disease is mainly chemical, today there is a growing interest for alternative and integrated ways of control. Systemic acquired resistance (SAR) is a mechanism of induced defence that confers long-lasting protection against a broad spectrum of microorganisms. Molecularly, SAR is characterized by the increased expression of a large number of pathogenesis-related genes (PR genes). This study reports possible relations between Cercospora leaf spot incidence and PR-protein accumulation after Trichoderma treatment. Sugar beet susceptible to C. beticola (cv Aaron) and partially resistant (cv Monodoro), were treated twice with Trichoderma koningii (isolate Bf2) on one leaf and inoculated with the pathogen on the other leaves, 2 days after the second treatment (T+I). Controls were represented by: i) untreated and uninoculated plants (NT); ii) untreated and inoculated plants (I); iii) treated and uninoculated plants (T). Inoculated leaves were harvested at different times after inoculation for protein extraction. Peroxidase and chitinase activities were determined after isoelectrofocusing (IEF). Cercospora leaf spot symptoms were evaluated 20 days after inoculation by a disease index (0-9 KWS scale). Plants of the susceptible cv (Aaron) treated with Trichoderma showed significantly reduced symptoms if compared with the untreated controls. In this cv a peroxidase isoform (pI 4.0) was induced twelve days after the inoculation in plants treated with Trichoderma (T+I). This isoform was already present in the partially resistant cv (Monodoro) and any increasing was detected after the treatment or inoculation. Another isoform (pI 3.6) accumulated in T and T+I plants both in susceptible and partially resistant cvs. Moreover three chitinase isoforms (pI 3.3, pI 3.4, pI 3.6) increased in T and T+I plants in comparison to inoculated and untreated plants. This results suggest a possible role of a SAR mechanism in controlling Cercospora leaf spot damage in sugar beet.

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Involvement of phospholipid signalling system in early stages of SA perception

Krinke O.1,2,3, Burketová L.2, Ruelland E.3, Collin S. 3, Valentová O.1 and Zachowski A.3 1 Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Czech Republic 2 Institute of experimental botany ASCS, Prague, Czech Republic 3 FRE 2846, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Université Pierre et Marie Curie, Paris, France

The involvement of phospholipid signalling system in stress cues is much less elucidated in plants than in animals, nevertheless recent results indicate its role in early events in plant defence response to pathogens and processes leading to systemic acquired resistance (SAR). The role of salicylic acid (SA) in the establishment of SAR is undisputed and a lot of work has been done on PR gene expression, however the data dealing with SA perception are rather scarce.

We investigated the role of phospholipid signalling in early stages of defence response triggered by exogenous application of SA in Arabidopsis cell suspension culture. Expression profiles of genes engaged in the phosphoinositide metabolism (PIS1, PIS2, PI4Kα1, PI4Kα2, PI4Kβ1, PI4Kβ2) and SAR (GST6, NPR1, PR1) were analyzed. Both PIS isoforms and PI4Kβ genes were induced upon SA treatment. All three marker genes of SAR followed the expected trends and showed three phases of expression according to the well known scheme. SA-treated cells were metabolically labelled by 33Pi and radioactive phospholipids were analyzed by TLC. Phospholipid analysis revealed a rapid and sustained decrease of phosphatidylinositol (PI) pool and a concomitant increase of phosphatidylinositolmonophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) after SA application. This phenomenon was dose dependent and points to the involvement of phosphoinositide metabolism in the early signal transduction triggering the SAR.

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Breeding for inducible resistance against Potato virus disease Masoomeh Peiman Islamic Azad University Takestan Branch, Iran Research and Science Campus, I.A University, Tehran, Young Researcher Club Millions in developing countries depend on potatoes as a primary dietary component. Potato production is limited by many factors, but diseases, and viruses in particular, are especially important. Because virus-infected crops cannot be cured, viruses must be controlled through prevention and sanitation, i.e. the use of resistant varieties and healthy seed production. In other words, one of the challenging problems faced by plant breeders is finding the virus resistance sources. For this reason potato pathologists and breeders have paid important attention to the introduction, evaluation, identification, innovation and utilization of resistance potatoes. In brief, potato viruses are challenging for researchers as they are for farmers and using the chemical methods for controling them is a serious danger for environmental. In this paper will be focused on potato viruses available in Iran, their dammage and the resistance material have been found to Potato virus X (PVX) and Potato virus Y (PVY), Potato leaf roll virus(PLRV). Key words: Potato- virus-yield-resistance-detection

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Effect of resistance inducing agents on seed borne Didymella lycopersici in tomato Fanourakis N., Tampakaki A., Fanouraki, M.N. and Malathrakis N.E. Technological Education Institute of Crete,71004 Heraklio, Crete, Greece

Didymella lycopersici is a seed borne pathogen of tomato. To eliminate seed inoculum, different concentrations of the resistance inducing agents: a)β-aminobutyric acid, min 95% (BABA), b) chitosan (85% deacetylated) and c) Tillecur were applied to the seeds by dipping, before sowing. Treated seeds were sown in pots and placed in the growth chamber (12hL/12hD, 20oC). Percentage of infected seedlings (damping-off) was assessed 30 days later. Chitosan and Tillecur were very effective, while BABA was less effective in all 3 concentrations tested. Chitosan at 0.005g/ml was 94% effective at controlling the disease, while Tillecur at 0.05g/ml completely inhibited post germination infection (post emergence damping off) but also controlled significantly pre-germination death. Results indicate that both tillecur and chitosan could be used as alternatives to thiram, currently applied against seed borne Didymella lycopersici.

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Early events of the signalling process leading to defence responses in grapevine Vandelle E., Poinssot B., Wendehenne D. , Pugin A. UMR INRA-Université de Bourgogne - CNRS Plante Microbe Environnement. Dijon. France

It is now established that plants possess pathogen-activated defense components comparable to those of the animal immune system. In this context, the endopolygalacturonase I of Botrytis cinerea (BcPG1) has emerged as an elicitor of grapevine defense reactions (Poinssot et al., 2003). We have demonstrated that the elicitor activity of BcPG1 is not due to released oligogalacturonides from its enzyme activity but to specific motifs of the protein recognized by grapevine. Hence, BcPG1, a virulence factor participating in B. cinerea pathogenicity, possesses an avirulence function responsible for grapevine defense activation. The events induced by BcPG1 in grapevine cell suspensions and the links in-between these events are reported here (Vandelle et al., 2006). BcPG1 induces a calcium influx from the extracellular medium which, together with a calcium release from internal stores, leads to a large and transient rise of the cytosolic free calcium concentration ([Ca2+]cyt) monitored using cells expressing the jellifish luminescent protein aequorin. Calcium activates an NADPH-oxidase and a NOS-like enzyme leading to the production of reactive oxygen species (ROS) and NO respectively. The activation of calcium influx by protein kinase inhibitors suggests a regulation of calcium channels by protein kinases/phosphatases. ROS and NO production also depends on the activation of protein kinase(s). Moreover, NO has been shown to be involved in [Ca2+]cyt variations i) by promoting calcium release from internal stores through the activation of cADPR-dependent calcium channels and, 2) by inhibiting the calcium influx. NO is also responsible for NADPH-oxidase activation. Two Mitogen Activated Protein Kinases (MAPK) are rapidly and transiently activated in response to BcPG1 independently of calcium, AOS and NO. Taken together, these data indicate that grapevine exhibits elicitor-activated defense mechanisms close to those described in other plant species (Garcia-Brugger et al., 2006) but differently connected. References

• Poinssot B., Vandelle E., Bentéjac M., Adrian M., Levis C., Brygoo Y., Garin J., Sicilia F., Coutos-Thévenot P., and A. Pugin (2003). The endopolygalacturonase I from Botrytis cinerea activates grapevine defence reactions unrelated to its enzymatic activity. Mol. Plant Microbe Interact., 6: 553-564.

• Vandelle E., Poinssot B., Wendehenne D., Bentéjac M. and Pugin A. (2006). Integrated signaling network involving calcium, nitric oxide, active oxygen species but not mitogen-activated protein kinases in BcPG1-elicited grapevine defenses. Mol. PlantMicrobe Interact. 19: 429-440.

• Garcia-Brugger A., Lamotte O., Vandelle E., Bourque S., Lecourieux D., Poinssot B., Wendehenne D. and A. Pugin (2006). Signaling pathways activated by elicitors of plant defenses. Mol. Plant Microbe Interact., in press

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Activation of defense responses in Arabidopsis by non enzymatically formed oxylipins

Grun C., Müller M.J. and Berger S. Lehrstuhl für Pharmazeutische Biologie, Julius-von-Sachs-Institut für Biowissenschaften, Würzburg

Plant oxylipins are derived from linolenic acid by enzymatic or non-enzymatic pathways.

The best characterised enzymatically formed oxylipins are 12-oxophytodienoic acid (OPDA) and jasmonic acid. Both are molecules with important signalling functions in development and stress responses in plants and activate defense responses. Less is known about the non-enzymatically formed oxylipins. Based on their structural similarity to OPDA, signalling functions for the non-enzymatically formed phytoprostanes have been proposed. We investigated the accumulation and biological activities of phytoprostanes in Arabidopsis.

Pathogen attack leads to the generation of reactive oxygen species which triggers the formation of phytoprostanes. Levels of phytoprostanes and non-enzymatically formed hydroxy fatty acids accumulated in Arabidopsis plants after infection with P. syringae.

In order to get a comprehensive view on the biological activity of phytoprostanes, gene expression was analysed in Arabidopsis using microarrays. Phytoprostanes induced the expression of genes involved in detoxification and stress responses. In agreement with this effects, pretreatment of cell cultures with phytoprostanes lead to a protection against copper intoxication. In contrast, expression of genes involved in cell wall metabolism and cell cycle was repressed by phytoprostanes indicating a negative effect of these compounds on cell division and growth.

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Priming as a Mechanism of Systemic Acquired Resistance in Plants Gerold Beckers and Uwe Conrath Plant Biochemistry and Molecular Biology Unit, Department of Plant Physiology, RWTH Aachen University, 52056 Aachen, Germany Systemic acquired resistance (SAR) is an induced defense response of plants that confers long-lasting protection against a broad spectrum of pathogens. The induced resistance is typically associated with the ability to induce cellular defense responses more rapidly and more effectively compared to non-induced plants. The enhanced capacity to activate cellular defense responses is called priming (or sensitization) and can be induced in plant cell cultures as well as in whole plants by pretreatment with, for example, the synthetic SAR inducer benzothiadiazole (BTH). In cultured parsley cells, induction of the sensitized state was associated with the accumulation of transcripts for a mitogen activated protein kinase (MAPK). Strikingly, the encoded and accumulated MAPK protein remained inactive. It was shown that only upon treatment with even a low dose of elicitor it gained full activation, consequently inducing downstream components of the elicitor responsive signal transduction cascade more effectively compared to non-pretreated cells. We are currently exploiting the genetically accessible model system Arabidopsis thaliana to further demystify the mechanism of priming in plants.

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Poster: page (in abstract book) titel name

28 Activation of defense responses in Arabidopsis thaliana by oxylipins and yeast elicitors

Berger S. 29 Quantifying cost and benefits of Induced Systemic Resistance in a clonal plant network Gómez S. 30 Using Q-RT-PCR to determine a defence-related signal signature of Maize Danielle Karlen 31 Changes in free amino acids induced in cucumber plants by spider mites and plant growth promoting

rhizobacteria (PGPR) Tomczyk A. 32 Impact of transgenic cucumbers expressing the thaumatin II gene on the occurrence of arthropod

fauna Kielkiewicz M. 33 BABA induced resistance in grapevine

Slaughter A.R.

34 Quantification of oomycete biomass in Grapevine Dubresson R., 35 Inducible defence of Bt oilseed rape and global climate change Himanen S.J.

36 Derailed stylet mechanics during plant penetration by aphids and its potential as a mechanism of host plant resistance

Tjallingii, W.F. 37 Transgenic Plantlets of Grapevine (Vitis spec.) provide a look insight of host-pathogen interaction Seibicke T. 38 Activation of plant defence response contributes to the antiviral activity of Diocin2 from Phytolacca

dioica Faoro F

39 Different Chitinase Expression In Sugar Beet Plants After Acibenzolar-S-Methyl Applications Marinello S. 40 Possible Induction Of Systemic Acquired Resistance Against Cercospora Leaf Spot In Sugar Beet By

Trichoderma Foliar Applications Burzi P.L. 41 Involvement of phospholipid signalling system in early stages of SA perception Burketová L. 42 Breeding for inducible resistance against Potato virus disease Masoomeh Peiman 43 Effect of resistance inducing agents on seed borne Didymella lycopersici in tomato

Malathrakis N.E. 44 Early events of the signalling process leading to defence responses in grapevine Poinssot B. 45 Activation of defense responses in Arabidopsis by non enzymatically formed oxylipins

Berger S. 46 Priming as a Mechanism of Systemic Acquired Resistance in Plants Gerold Beckers