Effectors in plant–microbe interactions - New …...22nd New Phytologist Symposium E f fectors in plant–microbe interactions INRA Versailles Research Centre, Paris, France 13–16 - [PDF Document] (2024)

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• 22nd New Phytologist Symposium

  EEffffeeccttoorrss iinn ppllaanntt––mmiiccrroobbeeiinntteerraaccttiioonnss INRA Versailles Research Centre, Paris,France 13–16 September, 2009 Programme, abstracts andparticipants

  http://www.versailles.inra.fr/

• 22nd New Phytologist Symposium

  Effectors in plant–microbe interactions

  INRA Versailles Research Centre, Paris, France

  Organizing committee

  Sophien Kamoun (The Sainsbury Laboratory, JIC, UK) Marc-HenriLebrun (CNRS-Bayer Cropscience, France)

  Francis Martin (INRA-Nancy, France) Nick Talbot (University ofExeter, UK)

  Holly Slater (New Phytologist, Lancaster, UK)

  Acknowledgements

  The 22nd New Phytologist Symposium is funded by the NewPhytologist Trust.

  New Phytologist Trust

  The New Phytologist Trust is a non-profit-making organizationdedicated to the promotion of plant science, facilitating projectsfrom symposia to open access for

  our Tansley reviews. Complete information is available atwww.newphytologist.org

  Programme, abstracts and participant list compiled by JillBrooke. ‘Effectors in plant-microbe interactions’ illustration byA.P.P.S., Lancaster, U.K.

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  http://www.versailles.inra.fr/http://www.newphytologist.org/

  See Also

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• Table of Contents

  Programme........................................................................................3 Speaker Abstracts.............................................................................6 Poster Abstracts..............................................................................33 Participants......................................................................................69

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• Programme Sunday 13 September (Hotel Novotel Château deVersailles) 18:00–19:30 Registration – collect your delegate pack19:30–21:00 Welcome reception at the Hotel Novotel Château de

  Versailles Monday 14 September (INRA Versailles) 08:00–08:30Registration 08:30–08:35 Welcome and announcements Marc-HenriLebrun & Sophien Kamoun Session 1: Genome-wide analyses ofmicrobial effectors Chair: Marc-Henri Lebrun 08:35–09:15 1.1Ustilago effectors Regine Kahmann 09:15–09:55 1.2 Ralstoniasolanacearum: Molecular basis of adaptation

  to plants Stéphane Genin

  Session 2: Effector evolution Chair: Marc-Henri Lebrun09:55–10:35 2.1 The evolution of the Pseudomonas syringae HopZ

  family of type III effectors David Guttman 10:35–11:15 2.2Evolutionary and functional dynamics of Phytophthora

  infestans effector genes Sophien Kamoun 11:15–11:45 CoffeeSession 3: Microbial effector functions: virulence and avirulenceChair: Nick Talbot 11:45–12:25 3.1 Elicitation and evasion of plantimmunity by

  Pseudomonas effectors AvrPto and AvrPtoB Greg Martin

  12:25–13:05 3.2 Pseudomonas syringae type III effectors:Enzymatic activities, sites of action, and their ability tosuppress plant innate immunity

  Jim Alfano 13:05–14:00 Lunch 14:00–14:40 3.3 Flax rust Avr-Rinteractions

  Peter Dodds 14:40–15:20 3.4 Leptosphaeria maculans AVRs andSSPs

  Thierry Rouxel

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• 15:20–16:00 3.5 The pathogen effectors of the downy mildewoomycete pathogen, H. arabidopsidis, and host responses to stressJim Benyon

  16:00–16:30 Coffee 16:30–17:10 3.6 Cladosporium fulvum effectorsand functional

  hom*ologues in Dothideomycete fungi Pierre de Wit

  17:10–17:50 3.7 How Xanthom*onas type III effector proteinsmanipulate

  the plant Ulla Bonas

  17:50–19:30 Posters and reception Tuesday 15 September Session4: Effector trafficking: processing/uptake by plants Chair: FrancisMartin 08:30–09:10 4.1 Effector secretion and translocation duringrice blast

  disease Barbara Valent

  09:10–09:50 4.2 Host-selective toxins of Pyrenophoratritici-repentis,

  inside and out Lynda Ciuffetti

  Session 5: Effector trafficking: secretion/delivery by microbesChair: Francis Martin 09:50–10:30 5.1 Investigating the delivery ofeffector proteins by the rice

  blast fungus Magnaporthe oryzae Nick Talbot

  10:30–11:00 Coffee 11:00–11:40 5.2 Bacterial effector deliveryGuy Cornelis 11:40–12:20 5.3 How oomycete and fungal effectorsenter host cells Brett Tyler Session 6: Plant targets ofmicrobes/bioagressor effectors Chair: Nick Talbot 12:20–13:00 6.1Localisation and function of Phytophthora infestans

  RXLR effectors and their host targets Paul Birch 13:00–14:00Lunch 14:15 Afternoon excursion to the Palace of Versailles 18:00Bus leaves Hotel Novotel Château de Versailles for

  Conference Dinner with a cruise on the River Seine

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• Wednesday 16 September Session 6 cont.: Plant targets ofmicrobes/bioagressor effectors Chair: Nick Talbot 08:30–09:10 6.2Xanthom*onas perforans effector proteins as predictive

  indicators of durable and sustainable resistance to bacterialspot disease of Tomato Brian Staskawicz

  09:10–09:50 6.3 Small RNA pathways and their interference by

  pathogens in eukaryotes Olivier Voinnet

  09:50–10:30 6.4 Nematode effector proteins: Targets andfunctions in

  plant parasitism Dick Hussey 10:30–11:00 Coffee Session 7:Microbial effectors in symbiotic interactions Chair: Sophien Kamoun11:00–11.40 7.1 Secretome of the basidiomycete Laccaria bicolorand

  the ascomycete Tuber melanosporum reveal evolutionary insightsinto ectomycorrhizal symbiosis

  Francis Martin 11:40–12:20 7.2 Fungal signals and plant fungalperception in the

  arbuscular mycorrhizal symbiosis Natalia Requena 12:20–13:00 7.3The role of effector proteins in the legume–rhizobia

  symbiosis William Deakin

  13:00–14:00 Lunch Session 8: Emerging Effectors – nematodes,insects, metabolites Chair: Sophien Kamoun 14:00–14:40 8.1 Nematodeeffectors a genome wide survey Pierre Abad 14:40–15:20 8.2 Usingpathogen effectors to investigate host resistance

  mechanisms Jonathan Jones

  15:20–15:50 Coffee 15:50–16:30 8.3 RNAi knockdown of insectsalivary proteins Gerald Reeck 16:30–17:10 8.4 Secondarymetabolites as effectors: Fungal secondary

  metabolism is an essential component of the complex interplaybetween rice and Magnaporthe grisea

  Marc-Henri Lebrun 17:10–17:30 Closing comments 17:30 Meetingclose and depart

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• Speaker Abstracts Session 1: Genome-wide analyses of microbialeffectors Chair: Marc-Henri Lebrun 1.1 Ustilago effectorsREGINE KAHMANN, K. SCHIPPER, A. DJAMEI, T. BREFORT, G.DOEHLEMANN, F. RABE, J. WU, L. LIANG, G. BAKKEREN*, J. SCHIRAWSKIMax Planck Institute for Terrestrial Microbiology,Karl-von-Frisch-Strasse, D-35043 Marburg, Germany. *PacificAgri-Food Research Centre, 4200 Hwy 97, Summerland, BC, Canada Thebasidiomycete fungus Ustilago maydis is a biotrophic maize pathogenthat codes for a large set of novel secreted effector proteins. Asignificant percentage of the respective genes is clustered in thegenome and upregulated during pathogenic development (Kaemper etal., 2006). Many of these gene clusters have crucial roles duringdiscrete stages of biotrophic growth. We now show that U. maydis iseliciting distinct defense responses when individual clusters orindividual genes, respectively, are deleted. Maize gene expressionprofiling has allowed us to classify these defense responses andprovides leads to where the fungal effectors might interfere. Wedescribe where the crucial secreted effector molecules localize,their interaction partners and speculate how this may suppress theobserved plant responses. A comparative genomics approach in whichthe genomes of the related smut fungi Sporisorium reilianum, U.scitaminea and U. hordei were sequenced using 454-technology hasrevealed that these genomes contain paralogs of most effectorsfound in U. maydis. However, resulting from a coevolutionary armsrace between pathogen and host these effectors are highly divergentcompared to other proteins. This aids their detection andfunctional analysis.

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• 1.2 Ralstonia solanacearum: Molecular basis of adaptation toplants STÉPHANE GENIN Laboratoire Interactions PlantesMicroorganismes, INRA-CNRS, 31326 Castanet-Tolosan Cedex, FranceRalstonia solanacearum is a devastating plant pathogen with widegeographic distribution and an unusually wide host range since itis the agent of vascular wilt disease in more than 200 plantspecies. Host range is directly controlled in some cases by TypeIII effectors, either by extending or restricting the ability of R.solanacearum to infect and multiply on given hosts. Comparativegenomics can provide insights about the evolution of someavirulence gene sequences that could result in a betteradaptability of the pathogen. An experimental evolution approach bymaintaining the bacterium on fixed plant lines by serial passageexperiments for over 300 generations and aimed to identify thegenetic basis of the adaptation of the bacterium to different hostplants will also be presented.

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• Session 2: Effector evolution Chair: Marc-Henri Lebrun 2.1 Theevolution of the Pseudomonas syringae HopZ family of type IIIeffectors DAVID S. GUTTMAN, JENNIFER D. LEWIS, AMY H. LEE, PAULINEW. WANG, YUNCHEN GONG, DARRELL DESVEAUX Department of Cell &Systems Biology, Centre for the Analysis of Genome Evolution &Function, University of Toronto, 25 Willco*cks St. Toronto, OntarioM5S3B2 Canada The plant pathogen Pseudomonas syringae uses the typeIII secretion system to secrete and translocate effector proteinsinto its plant hosts. Many of these effectors suppress host defensesignaling and / or induce resistance (R) protein-mediated defenses.The YopJ / HopZ family of effectors is a common and widelydistributed class found in both animal and plant pathogenicbacteria. In previous work, we showed that the P. syringae HopZfamily includes three major allele types (one ancestral and twobrought in by horizontal gene transfer) whose diversification wasdriven by the host defense response (Ma et al., 2006), and thatvirulence and defense induction phenotypes are stronglyallele-specific (Lewis et al., 2008). We have now the R proteinresponsible for HopZ1a-dependent immunity. This previouslyuncharacterized R protein functions independently of RIN4 and allother known R proteins, and shows HopZ effector allele specificity.Further, we designed a novel, high-throughput interactor screenusing next-generation genomics technology to elucidate the HopZ-Rprotein resistance complex and host targets of all the HopZalleles. This diverse effector family beautifully illustrates hownatural genetic variation modulates host target and R proteinspecificity and influences host specific virulence and defense.

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• 2.2 Evolutionary and functional dynamics of Phytophthorainfestans effector genes SOPHIEN KAMOUN The Sainsbury Laboratory,Norwich, United Kingdom It is now well established that oomyceteplant pathogens secrete effectors that target the apoplast or aretranslocated inside host plant cells to enable parasitic infection.Apoplastic effectors include several types of inhibitor proteinsthat interfere with the activities of extracellular planthydrolases. Host-translocated (cytoplasmic) effectors include theRXLR and Crinkler (CRN) families, which carry conserved motifs thatare located downstream of the signal peptide and mediate deliveryinto host cells. How these effectors perturb plant processesremains poorly understood although some RXLR effectors are known tosuppress plant immunity. This presentation will report on theprogress we made in unravelling the evolutionary dynamics ofeffector genes of the potato late blight pathogen Phytophthorainfestans. More specifically, we will focus on the insightsobtained from sequencing the genomes of P. infestans and that offour closely related species, and our progress in deciphering thevirulence activities of P. infestans effectors.

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• Session 3: Microbial effector functions: virulence andavirulence Chair: Nick Talbot 3.1 Elicitation and evasion of plantimmunity by Pseudomonas type III effectors AvrPto and AvrPtoB GREGMARTIN, K. MUNKVOLD, H. NGUYEN, I. YEAM, J. MATHIEU, L. ZENG BoyceThompson Institute for Plant Research and Department of PlantPathology, Cornell University, Ithaca, New York, USA

  Pseudomonas syringae pv. tomato, which causes bacterial speckdisease of tomato, delivers ~30 type III effector proteins into thehost cell. Tomato varieties that are resistant to speck diseaseexpress the Pto kinase which physically interacts with two of theseeffectors, AvrPto or AvrPtoB, and activates a variety of hostimmune responses including localized programmed cell death. The Ptokinase is encoded by one member of a clustered, five-member genefamily. Another member of this family, Fen, recognizes certaintruncated versions of AvrPtoB. An NBARC-LRR protein, Prf, isrequired for Pto- and Fen-mediated immunity. AvrPto (18 kD) andAvrPtoB (60 kD) both make significant contributions to P. syringaevirulence and have many other similarities. Each is a modularprotein with discrete domains that have distinct activities. One ofthese domains in both proteins targets the FLS2/BAK1 complex todisrupt PAMP-triggered immunity. In each effector, this domainforms a contact surface involved in the interaction with Pto. Botheffectors have an additional unique contact with Pto and theirstructures overall are very different. Host-mediatedphosphorylation of each effector promotes its virulence activityand for AvrPto this phosphorylation-dependent virulence activitywas found to be independent of the FLS2/BAK1 disruption mechanism.Interestingly, each effector is targeted by two differentresistance proteins that recognize a structural element importantfor effector virulence activity. I will present recent dataregarding the molecular basis of the multiple virulence activitiesof AvrPto and AvrPtoB. Supported by NIH-R01GM078021 andNSF-DBI-0605059.

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• 3.2 Pseudomonas syringae type III effectors: Enzymaticactivities, sites of action, and their ability to suppress plantinnate immunity JIM R. ALFANO Center for Plant Science Innovationand the Department of Plant Pathology, University of Nebraska,Lincoln, NE 68588-0660 USA The bacterial pathogen Pseudomonassyringae is dependent on a type III protein secretion system andthe type III effector proteins (T3Es) it injects into host cells tocause disease. The enzymatic activities of T3Es and their planttargets remain largely unknown. I will discuss our progress on theDC3000 T3E HopU1, which we determined is amono-ADP-ribosyltransferase (ADP-RT). Using ADP-RT assays coupledwith mass spectrometry we identified the major HopU1 substrates inArabidopsis thaliana extracts to be several RNA-binding proteinsthat possess RNA-recognition motifs (RRMs). HopU1 ADP-ribosylatesan arginine residue in position 49 of the glycine-rich RNA-bindingprotein AtGRP7, which is within its RRM. We found thatADP-ribosylated AtGRP7 was reduced in its ability to bind RNA.Another T3E that we are currently focused on is HopG1, whichlocalizes to plant mitochondria and when expressed transgenicallyresult in plants that are infertile, dwarfed, and possess increasedbranching. HopG1 also has the ability to suppress innate immunity,which suggests that pathogens may target mitochondria as apathogenic strategy. Finally, I will also discuss recentexperiments that suggest that the majority of DC3000 type IIIeffectors can suppress plant immunity.

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• 3.3 Flax rust Avr-R interactions

  PETER DODDS1, M. RAFIQI2, P. H. P. GAN2, M. BERNOUX1, M.RAVENSDALE1, M. KOECK1, G. LAWRENCE1, B. KOBE3, D. A. JONES2 A. R.HARDHAM2, J. ELLIS1 1CSIRO-Plant Industry, GPO Box 1600, Canberra,Australia; 2Plant Cell Biology Group, Research School of BiologicalSciences, School of Biology, The Australian National University,Canberra ACT 0200 Australia; 3School of Molecular and MicrobialSciences and Institute for Molecular Bioscience, University ofQueensland, Brisbane, Australia Flax rust (Melampsora lini) is abiotrophic basidiomycete pathogen that infects flax plants (Linumusitatissimum). Nineteen different rust resistance genes have beencloned from flax, including 11 allelic variants of the L locus,which all encode cytosolic TIR-NBS-LRR proteins. Four families ofAvr genes, AvrL567, AvrM, AvrP123 and AvrP4 have been identified inflax rust and all encode small secreted proteins that are expressedin haustoria. Recognition occurs inside the plant cell andyeast-two-hybrid analyses indicate that, in at least two cases,this is based on direct interaction with the correspondingcytosolic NB-LRR R proteins. This suggests that the Avr proteinsare translocated into host cells during infection, andimmunolocalisation experiments have detected the AvrM inside hostcells during infection. Expression of various GFP-tagged AvrL567and AvrM mutants in plants suggest that these proteins are taken upinto host cells in the absence of the pathogen and that thistransport is dependent on sequences in the N terminal region.Although the LRR domain is primarily responsible for determiningrecognition specificity of the flax R proteins, y2h assays indicatethat a functional NBS domain is also required for Avr proteininteraction. The TIR domain is not required for recognition andseveral TIR mutations disrupt HR induction, without affectingrecognition. Furthermore, overexpression of the TIR domain aloneinduces HR, suggesting a primary signalling role for this domain.Direct recognition has led to strong diversifying selection in therust Avr genes to escape recognition and host resistance.

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• 3.4 Leptosphaeria maculans AVRs and SSPs I. FUDAL1, J.GRANDAUBERT1, A. DILMAGHANI1, N. GLASER1, P. BALLY1, P. WINCKER2,A. COULOUX1, B. HOWLETT3, M-H. BALESDENT1, THIERRY ROUXEL11INRA-BIOGER, Avenue Lucien Brétignières, BP 01, 78850Thiverval-Grignon France; 2CEA, DSV, IG, Genoscope, Centre Nationalde Séquençage, 2, rue Gaston Crémieux, 91057 Evry Cedex, France;3School of Botany, The University of Melbourne, VIC 3010, AustraliaThe genome of the ascomycete Leptosphaeria maculans shows theunusual characteristics to be organized in isochores, i.e., thealternating of hom*ogeneous GC% regions with abrupt changes from oneto the other. GC-equilibrated isochores (average 52% GC) aregene-rich whereas AT-rich isochores (40–43% GC) are mostly devoidof active sequences and are made up of mosaics of intermingled anddegenerated repeated elements. The three avirulence (AvrLm) genesidentified so far in this species are “lost in middle of nowhere”genes, isolated in the middle of large AT-rich isochores. Ourpostulate thus was that AT-rich isochores were specific “ecologicalniches” for avirulence genes and effectors in L. maculans. This wasfirstly validated by analysis of three genes lying in the samegenome environment (LmCys genes) and showing the samecharacteristics as AvrLm genes (low GC content, strongoverexpression at the onset of plant infection, encoding for smallsecreted proteins -SSP- often rich in cysteines). Of these, one,LmCys2, was shown to act as an effector (see I. Fudal et al.poster). A systematic search for SSP as effector candidates wasperformed using bioinformatics. 455 AT-rich isochores wereextracted from the genome data and their repeat content maskedusing the L. maculans repeated element database. Non-repeatedregions were then investigated with a pipe-line dedicated to theidentification of SSP. This provided us with three datasets: 529SSP-encoding genes in GC-equilibrated isochores, 498 non-SSP- and122 SSP-encoding genes in AT-rich isochores. Part of this latterset of genes was analyzed for their occurrence in naturalpopulations and expression data in vitro and in planta. Finally,the 122 putative AT-SSP showed structural features reminiscent ofthe AvrLm and LmCys genes and occasional RxLR-like motifs. Possiblediversification mechanisms favoured by this genome location will bediscussed.

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• 3.5 The pathogen effectors of the downy mildew oomycetepathogen, H. arabidopsidis, and host responses to stress

  JIM L. BENYON Warwick HRI, Warwick University, Wellesbourne,Warwick, CV35 9EF, UK To enable a pathogenic lifestyle manyorganisms produce a repertoire of proteins that enable them tocolonize host tissue. These proteins, effectors, are likely to betargeted to suppressing host immune mechanisms and redirectingnutritional resources to benefit the pathogen. We are studying theinteraction between the downy mildew pathogen Hyaloperonosporaarabidopsidis and Arabidopsis. In a community collaborative effortwe have just completed the sequencing and annotation of the H.arabidopsidis genome and it reveals a gene content that suggeststhat it has been adapted to a biotophic lifestyle. It has a verylarge effector content that suggests complex mechanisms ofinteraction between host and pathogen. We are analyzing the role ofindividual effector proteins in interacting with the host plantimmune system via yeast two hybrid analyses. Finally, we areanalyzing the role nature of the host plant response to biotic andabiotic stress using systems biology approaches.

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• 3.6 Cladosporium fulvum effectors and functional hom*ologues inDothideomycete fungi PIERRE J.G.M. DE WIT1, 3, H. A. VAN DEN BURG1,R. MEHRABI1, B. ÖKMEN1, G. WANG1, H. BEENEN1, G. A. KEMA2, I.STERGIOPOULOS1 1Laboratory of Phytopathology, Wageningen University& Research Centre, P.O. 8025, 3700 EE Wageningen, TheNetherlands; 2Plant Research International BV, PO Box 16, 6700 AA,Wageningen, The Netherlands; 3 Centre for BioSystems Genomics, P.O.Box 98, 6700 AB Wageningen, The Netherlands Cladosporium fulvum isa biotrophic pathogen that causes leaf mould of tomato. So far, teneffectors have been identified from this fungus includingavirulence (Avrs: Avr2, Avr4, Avr4E and Avr9) and extracellularproteins (Ecps: Ecp1, Ecp2, Ecp4, Ecp5, Ecp6 and Ecp7). All Avrsand Ecps are assumed to be virulence factors. Avr2 is an inhibitorof apoplastic plant cysteine proteases and Avr4 is a chitin-bindingprotein that protects chitin present in the cell walls of fungiagainst deleterious effects of plant chitinases during infection.Ecp6 contains chitin-binding LysM domains that are supposed to bindchitin fragments released in the apoplast during infection.Recently we have sequenced the genome of race 0 of C. fulvum thatenabled us to perform initial comparative genome analyses withother sequenced members of the plant pathogenic Dothideomycetes. Sofar, the genome of C. fulvum is most related to Mycosphaerellafijiensis the causal agent of black Sigatoka, a devastating fungaldisease of banana. We have identified functional hom*ologues of C.fulvum Avr4, Ecp2 and Ecp6 effectors in Dothideomycetes, includingM. fijiensis, M. graminicola, Cercospora nicotianae and C.beticola. We have also shown that Avr4 and Ecp2 are not onlystructural but also functional hom*ologues of the C. fulvumeffectors.

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• 3.7 How Xanthom*onas type III effector proteins manipulate theplant ULLA BONAS Department of Genetics, Martin-Luther-UniversityHalle-Wittenberg, Halle, Germany We study the interaction betweenpepper and tomato and the Gram-negative bacterium Xanthom*onascampestris pv.vesicatoria (Xcv), which causes bacterial spotdisease on pepper and tomato. Successful interactions of Xcv withthe plant depend on the type III secretion (T3S) system, amolecular syringe which injects 20-30 effector proteins (termed Avror Xop = Xanthom*onas outer protein) into the plant cell cytoplasm.One of the best understood type III effectors is AvrBs3, whichfunctions as transcription factor in the plant cell nucleus andaffects both susceptible and resistant plants. Xcv strainsexpressing AvrBs3 induce the hypersensitive reaction (HR;programmed cell death) in pepper plants carrying the resistancegene Bs3. In pepper plants lacking Bs3 and other solanaceous plantsAvrBs3 induces a hypertrophy (cell enlargement) of mesophyll cellsthat probably helps to disseminate the bacteria. AvrBs3 activitydepends on a central region of tandem repeats, its localization tothe plant cell nucleus and the presence of an acidic activationdomain. One of the direct targets of AvrBs3 is UPA20 (UPA,upregulated by AvrBs3) which encodes a transcription factor and isa key regulator of hypertrophy. New insights into AvrBs3 actionwill be presented.

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• Session 4: Effector trafficking: processing/uptake by plantsChair: Francis Martin 4.1 Effector secretion and translocationduring rice blast disease

  B. VALENT1, C.H. KHANG1, M.C. GIRALDO1, M. YI1, G. MOSQUERA1, 4,R. BERRUYER1, 5, K. CZYMMEK2, S. KANG3 1Kansas State University,Manhattan, KS, USA; 2University of Delaware, Newark, DE, USA;3Pennsylvania State University, University Park, PA, USA;4Currently: International Center for Tropical Agriculture, Cali,Colombia; 5Currently: Université d'Angers, Angers, France To causedisease, Magnaporthe oryzae sequentially invades living rice cellsusing specialized intracellular invasive hyphae (IH) that areenclosed in host-derived Extra-Invasive-Hyphal Membrane. Blast IHspecifically express numerous Biotrophy-Associated-Secreted (BAS)proteins including known effectors, AVR-Pita, PWL1, and PWL2. Weidentified a highly-localized pathogen-induced structure, theBiotrophic Interfacial Complex (BIC), which accumulatesfluorescently-labeled effectors and other BAS proteins secreted byIH. BICs contain complex lamellar membranes, and are associatedwith dynamically-shifting host cytoplasm. In successively invadedrice cells, fluorescent effectors were first secreted into BICs atthe tips of filamentous hyphae that entered the cell. FluorescentBICs then moved off the hyphal tips and remained beside the firstdifferentiated IH cells as IH continued their colonization.Fluorescent effectors that accumulated in BICs were translocated tothe cytoplasm of invaded host cells. Translocated effectors werealso observed in uninvaded neighboring cells, suggesting that thefungus sends effectors to hijack host cells before entry.

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• 4.2 Host-selective toxins of Pyrenophora tritici-repentis,inside and out LYNDA M. CIUFFETTI, V. M. MANNING, I. PANDELOVA, M.F. BETTS Department of Botany and Plant Pathology, Oregon StateUniversity, 2082 Cordley Hall, Corvallis, OR 97331, USAHost-selective toxins (HSTs) are virulence factors produced byplant pathogenic fungi. Often, host-selective toxins follow aninverse gene-for-gene interaction where a single locus in the hostis responsible for toxin sensitivity. The ability of thesevirulence factors to promote cell death by a variety of mechanismsbenefits the necrotrophic life style of the fungus. Our long-termgoal is to fully describe the molecular interactions of thehost-selective toxin producing fungus, Pyrenophoratritici-repentis, with its host plant, wheat. This includes theidentification and characterization of genes involved inpathogenicity and host specificity, the mechanisms by which thisfungus acquires these virulence factors, and the determination ofthe molecular site- and mode-of-action of these toxins. ToxA andToxB are two proteinaceous HSTs of P. tritici-repentis whichpromote virulence through distinctly different mechanisms. ToxAinduced changes occur rapidly and result in necrosis. In contrast,the plant responses to ToxB are slower and result in chlorosis.High affinity binding to a plant receptor and rapid internalizationof ToxA leads to altered Photosystem homeostasis, the accumulationof reactive oxygen species and major transcriptional reprogramming.Unlike ToxA, ToxB appears to have an extracellular site-of- actionand lacks a high affinity receptor. Additionally, plant responsesto ToxB require prolonged exposure to the toxin.

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• Session 5: Effector trafficking: secretion/delivery by microbesChair: Francis Martin 5.1 Investigating the delivery of effectorproteins by the rice blast fungus Magnaporthe oryzae

  NICHOLAS J. TALBOT, ANA-LILIA MARTÍNEZ-ROCHA, MARTIN J. EGAN,MUHAMMAD BADARUDDIN, THOMAS MENTLAK, LUIGI CIBRARIO, THOMAS A.RICHARDS School of Biosciences, University of Exeter, UnitedKingdom Plant pathogenic fungi deliver proteins directly into plantcells to facilitate tissue invasion and to suppress plant defence,but how the fungus delivers these effector proteins during plantinfection is currently unknown. We are studying the processes ofpolarised exocytosis and endocytosis during plant infection by therice blast disease-causing fungus Magnaporthe oryzae.Interestingly, our preliminary data suggests that the site ofeffector secretion may be distinct from the normal polarised tipsof fungal hyphae. We are also studying the role of the MgAPT2 genein effector delivery. MgAPT2 encodes a P-type ATPase in M. oryzae,which is required for both foliar and root infection by the fungus,and for the rapid induction of host defence responses in anincompatible reaction. We have explored the relationship betweenMgAPT2 and the yeast DRS2 gene in detail and investigated the roleof MgAPT2 in protein delivery during pathogenesis. In parallel, weare using comparative genomics to define the repertoire ofeffector-encoding genes in M. oryzae in greater detail. We are alsoinvestigating endocytosis during plant infection and the potentialrole of eisosome organelles which localise to specialised domainson the plasma membrane, where they are thought to function inmembrane remodelling, and the spatial regulation of endocytosis. Wehave functionally characterized putative eisosome components in M.oryzae andused target gene-deletion to genetically dissect the roleof eisosome-associated proteins in this important plant pathogenicfungus, and fluorescent protein fusions to demonstrate thedifferential localisation of these proteins duringinfection-related development. In spores of the rice blast fungus,a Pil1-GFP fusion protein localises to punctate patches at the cellperiphery, in a pattern consistent with that of eisosomes.Interestingly, the spatial distribution of Pil1-GFP is radicallydifferent in vegetative hyphae and invasive hyphae, which are usedby the fungus to proliferate within living plant tissue. Thissuggests that endocytic mechanisms may be distinct in these twodevelopmental stages.

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• 5.2 Bacterial effector delivery GUY R. CORNELIS InfectionBiology, Biozentrum, University of Basel, Klingelbergstrasse 50/70,Basel 4056, Switzerland The type III secretion injectisome is ananosyringe that injects bacterial effector proteins straight intothe cytosol of eukaryotic cells. It is related to the flagellum,with which it shares structural and functional similarities. Itconsists of a basal body made of several rings spanning thebacterial membranes, connected by a central tube. On top of thebasal body, comes a short stiff needle terminated with a tipstructure. Three of these rings can assemble and be functional evenwhen their subunit is fused to a fluorescent protein. Thecombination of these hybrid proteins with an array of mutations inall the injectisome components allowed to decipher the order ofassembly of the basal body by fluorescence microscopy. The basalbody is assembled sequentially by the Sec pathway. As soon as theexport apparatus itself is assembled, it takes over the assemblyprocess and exports the needle subunits (early substrates). Needleelongation is controlled by YscP, acting as a molecular ruler or atimer, released at the end of the process. YscP seems to bepartially folded and its total length approximates the length ofthe needle plus the basal body, supporting the ruler model.According to experiments carried out with partial diploids, onlyone ruler determines the length of a needle. When assembly of theneedle is complete, the C-terminal domain of YscP interacts withthe export apparatus and changes the substrate specificity, whichbecomes ready to export the needle tip protein, then pore formersand finally effectors. One protein from the export apparatusspecifically recognizes the various classes of exportsubstrates.

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• 5.3 How oomycete and fungal effectors enter host cells BRETT M.TYLER1, SHIV D. KALE1, BIAO GU1,2, DANIEL G. S. CAPELLUTO3, DAOLONGDOU1,4, FELIPE D. ARREDONDO1, CHRISTOPHER B. LAWRENCE1, WEIXINGSHAN2 1Virginia Bioinformatics Institute, Virginia PolytechnicInstitute and State University, Blacksburg, VA 24061, USA; 2Collegeof Plant Protection and Shaanxi Key Laboratory of Molecular Biologyfor Agriculture, Northwest A & F University, Yangling, Shaanxi712100,China; 3Department of Biological Sciences,VirginiaPolytechnic Institute and State University, Blacksburg,VA24061,USA; 4Current address: Department of Plant Pathology, NanjingAgricultural University,Nanjing 210095, China Pathogens of bothplants and animals produce effectors and/or toxins that act withinthe cytoplasm of host cells to suppress host defenses and causedisease. Effector proteins of oomycete plant pathogens utilize anN-terminal motif, RXLR-dEER, to enter host cells, and a similarmotif, Pexel (RxLxE/D/Q), is used by Plasmodium effectors to entererythrocytes. Host cell entry by oomycete effectors does notrequire the presence of any pathogen encoded machinery. We havefound that oomycete RXLR-dEER motifs, as well as the PlasmodiumPexel motifs, are responsible for binding of the effectors tophosphatidyl-inositol-3-phosphate (PI-3-P) and/orphosphatidyl-inositol-4-phosphate (PI-4-P). Stimulation of hostcell entry by PI-4-P, and inhibition by inositol 1,4 diphosphate orby the phosphoinositide-binding domain of the protein VAM7, supportthe hypothesis that phosphoinositide binding mediates cell entry.Effectors of fungal plant pathogens were found to contain variantsof the RXLR-dEER motif which can enable cell entry. While somefungal effectors bound phosphoinositides, many others bounddifferent phospholipids, consistent with independent convergentevolution of this entry mechanism. Effectors from all threekingdoms could also enter human cells, suggesting thatphospholipid-mediated effector entry may be very widespread inplant, animal and human pathogenesis. Inhibition of both plant andhuman cell entry by the endocytosis inhibitor tyrphostin A23 andthe localization of effector-GFP fusions to endosomes in humancells support the hypothesis that entry occurs by receptor-mediatedendocytosis.

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• Session 6: Plant targets of microbes/bioagressor effectorsChair: Nick Talbot 6.1 Localisation and function of Phytophthorainfestans RXLR effectors and their host targets PAUL R.J. BIRCH1,3, JIB BOS4, M.R. ARMSTRONG3, E.M. GILROY1, R.M. TAYOR1, 5, I.HEIN2, P.C. BOEVINK1, S. BREEN1, L. PRITCHARD1, A. SADANANDOM5, S.KAMOUN4, S.C. WHISSON1 1Plant Pathology Programme; 2GeneticsProgramme; 3Division of Plant Science, University of Dundee,Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK;4The Sainsbury Laboratory, John Innes Centre, Colney, Norwich, NR47UH, UK; 5Biomedical and Life Sciences Department, University ofGlasgow, Glasgow G12 8QQ, United Kingdom. Eukaryotic plantpathogens, like their better-characterised prokaryoticcounterparts, secrete an array of effector proteins that manipulatehost innate immunity to establish infection. Deciphering thebiochemical activities of effectors to understand how pathogenssuccessfully colonize and reproduce on their host plants has becomea driving focus of research in the fields of fungal and oomycetepathology. AVR3a, the first effector characterized from theoomycete pathogen of potato and tomato, Phytophthora infestans, wasfound to contain N-terminal RxLR and dEER motifs required for itstranslocation across the host plasma membrane. Genomic resourceshave allowed large-scale computational screening for this conservedmotif to reveal >450 P. infestans RXLR-EER effectors. We arecloning RXLR effector genes to investigate their roles invirulence, their localisation in plant cells and to determinewhether they are recognised by host resistance proteins.Yeast-2-hybrid and bimolecular fluorescence complementation arebeing used to investigate effector-target protein interactions, andto localize these during infection. I will present our progress inthe investigation of pathogenicity functions of selected RXLReffectors, including the consequences of stable silencing of theseeffectors in the pathogen, and data showing the host proteins withwhich they interact. A range of approaches, including virus-inducedgene silencing, are being used to determine the roles of hosttargets in defence.

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  http://www.nature.com/nature/journal/v450/n7166/abs/nature06203.html#a1#a1http://www.nature.com/nature/journal/v450/n7166/abs/nature06203.html#a1#a1http://www.nature.com/nature/journal/v450/n7166/abs/nature06203.html#a1#a1http://www.nature.com/nature/journal/v450/n7166/abs/nature06203.html#a1#a1http://www.nature.com/nature/journal/v450/n7166/abs/nature06203.html#a1#a1http://www.nature.com/nature/journal/v450/n7166/abs/nature06203.html#a1#a1

• 6.2 Xanthom*onas perforans effector proteins as predictiveindicators of durable and sustainable resistance to bacterial spotdisease of Tomato BRIAN J. STASKAWICZ Department of Plant andMicrobial Biology, University of California, Berkeley, CA 94720USA

  Data will be presented on the identification andcharacterization of bacterial (TTSS) effector proteins that occurin several naturally occurring field isolates of X. perforans. Itis generally accepted that the genetic complement of effectorproteins delivered to the host via the bacterial TTSS allows thepathogen to suppress or manipulate the host innate immune system,resulting in pathogen multiplication and disease in susceptiblehosts. A comprehensive understanding of effector function in X.perforans will ultimately reveal the molecular mechanismscontrolling virulence in the bacterium and resistance interactionsthat occur between this bacterium and its host tomato. The abilityto rapidly and inexpensively determine the genome sequence ofnatural field isolates of X. perforans from infected tomatoes willprovide novel insights into the evolution of pathogen virulence andthe allelic diversity of effector genes in natural populations. Theknowledge gained from these studies will also provide acomprehensive understanding of the role effector proteins play inbacterial pathogenicity in X. perforans and provide the foundationto identify novel sources of resistant germplasm in wild species ofSolanum to control this disease in a durable and environmentallysustainable manner. Our progress towards these goals will bepresented.

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• 6.3 Small RNA pathways and their interference by pathogens ineukaryotes OLIVIER VOINNET Institut de Biologie Moléculaire desPlantes, Strausbourg, France RNA silencing is a pan-eukaryotic generegulation process whereby small interfering (si)RNAs and micro(mi)RNAs produced by Dicer-like enzymes repress gene expressionthrough partial or complete base-pairing to target DNA or RNA.Besides their roles in developmental patterning and maintenance ofgenome integrity, small RNAs are also integral components ofeukaryotic responses to adverse environmental conditions, includingbiotic stress. Until recently, antiviral RNA silencing wasconsidered a paradigm of the interactions linking RNA silencing topathogens: Virus-derived sRNAs silence viral gene expression and,accordingly, viruses produce suppressor proteins that target thesilencing mechanism. However, increasing evidence shows thatendogenous, rather than pathogen-derived, sRNAs also have broadfunctions in regulating plant responses to various microbes. Inturn, microbes have evolved ways to inhibit, avoid, or usurpcellular silencing pathways, thereby prompting the deployment ofcountercounterdefensive measures by plants, a compellingillustration of the never ending molecular arms race between hostsand parasites. Several original illustrations of these variousaspects will be provided, using examples from ongoing studies inour laboratory.

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• 6.4 Nematode effector proteins: Targets and functions in plantparasitism DICK S HUSSEY1, M. G. MITCHUM2, T. J. BAUM3, E. L.DAVIS4 1Departments of Plant Pathology, University of Georgia,Athens, GA 30602; 2University of Missouri, Columbia, MO 65211;3Iowa State University, Ames, IA 50011; 4N. C. State University,Raleigh, NC 27695, USA Parasitism genes developmentally-expressedin three enlarged secretory gland cells of sedentary endoparasiticnematodes encode multiple effector proteins that are secretedthrough the nematode’s stylet (oral spear) to facilitate the worm’smigration within plant roots and mediate the transformation ofselected root cells into elaborate permanent feeding cells. Somenematode parasitism genes encode effectors with similarity to knownproteins that are involved in cell-wall degradation, peptidesignaling, altering cellular metabolism, protein degradation, andnuclear localization, but the majority (>70%) of the predictedeffector proteins are unique to these microscopic obligatebiotrophs. Examples include a novel nematode effector peptide thatinteracts with plant SCARECROW-like transcription factors anddramatically increases root growth, a cellulose-binding proteinthat interacts with a plant pectin methylesterase to condition hostcell walls for parasitism, and a functional mimic of plantCLAVATA3/ESR-like peptides that appears to interact in signalingpathways that effect plant cell differentiation. The identificationand functional analysis of the effector proteins is revealing thecomplex nature of the secretions that make a nematode a plantparasite.

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• Session 7: Microbial effectors in symbiotic interactions Chair:Sophien Kamoun 7.1 Secretome of the basidiomycete Laccaria bicolorand the ascomycete Tuber melanosporum reveal evolutionary insightsinto ectomycorrhizal symbiosis FRANCIS MARTIN, J. PLETT, M.KEMPPAINEN, J. GIBON, A. KOHLER, V. PEREDA, V. LEGUE, A. PARDO UMR‘Tree-Microbe Interactions’ Department, INRA-Nancy Université, INRACenter, 54280 Champenoux, France Plants gained their ancestraltoehold on dry land with considerable help from their mycorrhizalfungal symbionts. The genetic mechanism of this kind of symbiosiscontributes to the delicate ecological balance in healthy forests.The genomic sequence for two representative of symbiotic fungi, theBasidiomycota Laccaria bicolor and the Ascomycota Tubermelanosporum, have been released. We bioinformatically identify>200 candidate genes coding for effector-like secreted smallsecreted proteins (SSPs) in each of the genomes of L. bicolor andT. melanosporum, several of which are only expressed in symbiotictissues and/or fruiting body. Both symbionts thus secreteeffector-like molecules that may facilitate the colonisation oftheir hosts, but expression of several of the secreted smallsecreted proteins is also upregulated during fruiting bodydevelopment suggesting a complex interplay between SSPs. In L.bicolor, the most highly expressed secreted protein MiSSP7accumulates in the Hartig net hyphae colonizing the host apoplast.RNAi-inactivation of MiSSP7 showed that this gene has a decisiverole in the establishment of the symbiosis. Whether some of theseSSPs are similar to those found in other fungi during hyphal fusionand homing, and aggregation of hyphae leading to the formation ofsexual organs, remains to be investigated. The unravelling of thesesecretomes provides tantalizing hints about differences betweensymbiotic fungi and their saprotrophic and pathogenicrelatives.

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• 7.2 Fungal signals and plant fungal perception in the arbuscularmycorrhizal symbiosis NATALIA REQUENA Plant-Fungal InteractionsGroup, Botanical Institute, University of Karlsruhe and KarlsruheInstitute of Technology, Hertzstrasse 16, D-76187 Karlsruhe,Germany Arbuscular mycorrhizal (AM) fungi form a mutualisticsymbiosis with the root of most vascular plants. This mycorrhizaassociation is evolutionarily dated as one of the oldestfungal-plant symbiosis on earth reflecting the success of thisinteraction. Amazingly, it is perhaps one of the most obscureassociations due to the genetic intractability of the fungalpartner. Thus, while enormous advances on the knowledge about plantperception and accommodation of AM fungi has been achieved in thelast years, not so much is known about the details governing thelife cycle of the fungus. In our group we are interested inunderstanding how AM fungi talk to plants and persuade them oftheir good intentions. With a combination of several molecularapproaches we are aiming to identify the chemical signals thattrigger plant fungal recognition during the AM symbiosis. We haveidentified a family of putative effector proteins that are secretedand able to enter the plant and travel to the nucleus. Expressionof these proteins in planta appears to increase the susceptibilityto mycorrhiza formation, indicating that they might play a role insilencing the immune response of the plant. We are currentlyinvestigating how the plant perceives these and other fungalsignals leading to the activation of the symbiotic program.

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• 7.3 The role of effector proteins in the legume-rhizobiasymbiosis WILLIAM J. DEAKIN, SILVIA ARDISSONE, KUMIKO KAMBARA,FABIA KESSAS, PATRICIA LARIGUET, OLIVIER SCHUMPP, WILLIAM J.BROUGHTON LBMPS, Dépt de Biologie Végétale, Université de Genève,Sciences III, 30 Quai Ernest Ansermet, CH-1211 GENEVE 4,Switzerland Rhizobia form symbiotic associations with leguminousplants. The symbiosis begins with an exchange of molecular signalsbetween the two organisms. Flavonoids exuded by plant roots inducethe synthesis of Nod factors (NFs) by rhizobia. NFs induce theformation of new plant organs (nodules), into which rhizobia arereleased. Within nodules rhizobia reduce atmospheric nitrogen toammonia, which is taken up by the host plant in return forphotosynthates. Although Nod factors are essential for noduleformation, there are other determinants, such as secreted proteins,that influence the extent of the symbiosis. Certain rhizobiapossess protein secretion systems generally associated withpathogenic bacteria. Pathogens use these systems to inject“effector” proteins into the cytoplasm of their eukaryotic hosts.Rhizobium species NGR234 has a functional type III secretion system(T3SS) that is induced by flavonoids and translocates effectorsinto legume cells. Depending on the legume host, the T3SS canimprove or reduce the symbiotic ability of NGR234. NGR234translocates several effectors; some are members of families ofeffector proteins found in pathogenic bacteria of both animals andplants, and generally have negative effects on nodulation byNGR234. Molecular characterisation has shown they have similarproperties to their pathogenic hom*ologues. Whereas other NGR234effectors are specific to T3SS-possessing rhizobia and generallyhave positive effects on symbiosis. Our working hypothesis is thatNGR234 may have acquired a T3SS and adapted it to aid the processof nodulation through the development of specific “rhizobialeffectors”. Although relics of the original system may betrayNGR234 as a potential pathogen to some plant species, initiating aplant defence response that blocks the symbiotic interaction.

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• Session 8: Emerging Effectors – nematodes, insects, metabolitesChair: Sophien Kamoun 8.1 Nematode effectors a genome wide surveyPIERRE ABAD INRA 1301-UNSA-CNRS 6243 - Interactions Biotiques etSanté Végétale, Sophia Antipolis, France The Root Knot Nematode(RKN) Meloidogyne incognita is a widespread and polyphagousobligate asexual endoparasite of plants that causes serious andgrowing problems to agriculture. This lifestyle implies dramaticalchanges of plant cells into complex feeding sites, which areaccomplished by effector molecules secreted by the nematode,so-called parasitism proteins. An integrated approach of moleculartechniques has been used to functionally characterize nematodeparasitism proteins. Very recently, the complete genome sequence ofthis nematode has been achieved. The assembled sequence of M.incognita spans 86 Mbp, and mostly consists of hom*ologous butdivergent segment pairs that might represent former alleles in thisspecies. A combination of different processes could explain thispeculiar genome structure in M. incognita, including polyploidy,polysomy, aneuploidy and hybridization, all features that arefrequently associated with asexual reproduction. Anotherinteresting feature of the genome is the spectacular presence of anextensive set of plant cell wall-degrading enzymes in thisnematode, which has no equivalent in any animal studied to date.This suite of enzymes likely modify and subvert the hostenvironment to support nematode growth. Initial analyses show thatthese enzymes are not found in other metazoan animals and theirclosest hom*ologs are bacterial, suggesting that these genes wereacquired by multiple horizontal gene transfer events.

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• 8.2 Using pathogen effectors to investigate host resistancemechanisms JONATHAN D.G. JONES, ERIC KEMEN, KEE-HOON SOHN, GEORGINAFABRO, JORGE BADEL, SOPHIE PIQUEREZ Sainsbury Lab, Norwich, UKPlant pathogens use small molecules and also proteins to rendertheir hosts susceptible. Many bacteria and other pathogens use aspecialized secretion system to deliver proteins into host cellsthat interfere with host defence. We have taken advantage of thebacterial type III secretion system (T3SS) to investigate effectorsfrom filamentous pathogens such as oomycetes. We are using T3SSdelivery of oomycete effectors from Pseudomonas sp to investigatethe effector complement of the downy mildew pathogenHyaloperonospora parasitica (Hpa). I will report recent data on Hpaeffector functions and on the use of the Solexa/Illumina sequencinginstrument to advance our understanding of Hpa pathogenicity. Weare using Illumina paired read sequencing and Velvet software(Zerbino and Birney, Genome Research, 2008) to assemble sequencesof multiple races of another oomycete pathogen, Albugo candida,which is particularly effective at shutting down host defence. Theanalysis of its effectors is likely to provide very interesting newinsights into host defence mechanisms. In addition, we are usingT3SS delivery of oomycete effectors to investigate the molecularbasis of pathogen/host specificity and non-host resistance. Anupdate on recent progress will be presented.

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• 8.3 RNAi knockdown of insect salivary proteins GERALD REECKBiochemistry, University of Kansas, 61 Chalmers Hall, Manhattan,Kansas 66506, USA Speaking only somewhat in jest, an aphid salivarygland can be thought of as a plant pathogen with legs. That is, itis proteins and enzymes of aphid saliva that, on the insect side,determine whether an interaction with a plant is successful or not,participating at every stage of interaction and continuingthroughout feeding. The importance of proteins and enzymes of aphidsaliva has been fully recognized for many years, but it is only inthe past several years that we have begun to enumerate and know, inany detail, the components of aphid saliva, much less begun toevaluate individual proteins/enzymes as effectors. I will summarizework from several laboratories, including mine, on current, ongoingefforts to catalog the components of aphid saliva (focusing on peaaphid, the model system) using both transcriptomic and proteomicapproaches. Then I will turn to our work on Protein C002, the firstsalivary component for which there is direct evidence in support ofits role as an effector (Mutti et al. PNAS 105: 9965 (2008)), andtwo other recombinantly expressed proteins of saliva that we arecurrently working with.

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• 8.4 Secondary metabolites as effectors: Fungal secondarymetabolism is an essential component of the complex interplaybetween rice and Magnaporthe grisea JEROME COLLEMARE1, RAHADATIABDOU1, MARIE-JOSE GAGEY1, ZHONGSHU SONG2, WALID BAKEER2, RUSSELLCOX2, ELSA BALLINI3, DIDIER THARREAU3, MARC-HENRI LEBRUN1 1UMR5240CNRS-UCB-INSA-BCS, CRLD Bayer Cropscience, 69263 Lyon Cedex 09,France; 2School of Chemistry, Bldg 77, University of Bristol,Bristol BS8 1TS, UK; 3UMR BGPI, CIRAD-INRA-SupAgro, Baillarguet TA41/K, 34398 Montpellier cedex 5, France Functional analyses offungal genomes are expanding our view of the metabolic pathwaysinvolved in the production of secondary metabolites. These genomescontains a significant number of genes encoding key biosyntheticenzymes such as polyketides synthases (PKS), non-ribosomal peptidesynthases (NRPS) and their hybrids (PKS-NRPS), as well as terpenesynthases (TS). Magnaporthe grisea has the highest number of suchkey enzymes (22 PKS, 8 NRPS, 10 PKS-NRPS, and 5 TS) among fungalplant pathogens, suggesting that this fungal species produce alarge number of secondary metabolites. In particular, it has 10hybrid PKS-NRPS that likely produce polyketides containing a singlean amino-acid. Three of them (ACE1, SYN2 and SYN8) have the sameexpression pattern that is specific of early stages of infection(appressorium-mediated penetration), suggesting that thecorresponding metabolites are delivered to the first infectedcells. M. grisea mutants deleted for ACE1 or SYN2 by targeted genereplacement are as pathogenic as wild type Guy11 isolate onsusceptible rice cultivars. Such a negative result could resultfrom a functional redundancy between these pathways. However, ACE1null mutants become specifically pathogenic on resistant ricecultivars carrying the Pi33 resistance gene compared to wild typeGuy11 isolate that is unable to infect such rice cultivars.Introduction of a Guy11 wild type ACE1 allele in Pi33 virulent M.grisea isolates restore their avirulence on Pi33 resistant ricecultivars, showing that ACE1 behaves as a classical avirulence gene(AVR). ACE1 differs from other fungal AVR genes (proteins secretedinto host tissues during infection) as it likely controls theproduction of a secondary metabolite specifically recognized byresistant rice cultivars. Arguments toward this hypothesis involvethe fact that the protein Ace1 is only detected in the cytoplasm ofappressoria and is not translocated into infectious hyphae insideepidermal cells. Furthermore, Ace1-ks0, an ACE1 allele obtained bysite-directed mutagenesis of a single amino acid essential for theenzymatic activity of Ace1, is unable to confer avirulence.According to this hypothesis, resistant rice plants carrying Pi33are able to recognize its fungal pathogen M. grisea through theperception of one fungal secondary metabolite produced duringinfection. The map based cloning of the Pi33 rice gene wasinitiated and this gene maps at a locus rich in classical NBS-LRRresistance genes. Further work is ongoing to identify which gene isPi33. In order to characterize the secondary metabolite produced byACE1, this gene was expressed in a heterologous fungal host such asAspergillus oryzae under the control of an inducible promoter. Theremoval of the three introns of ACE1 allowed the expression of theenzyme in A. oryzae. Characterization of the novel metaboliteproduced by Ace1 is in progress.

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• Poster Abstracts Listed alphabetically by first author,presenting author is underlined 1. Towards the characterization ofa quantitative resistance to downy mildew in cultivated Sunflower,Helianthus annuus

  F. AS-SADI1, N. POUILLY1, M-C. BONIFACE1, A. BORDAT1, A.BELLEC2, N. HELMSTETTER2, S. VAUTRIN2, H. BERGES2, D. TOURVIEILLEde LABROUHE3, F. VEAR3, P. VINCOURT1, L. GODIARD1 1Laboratoire desInteractions Plantes Micro-organismes (LIPM), INRA-CNRS, Castanet-Tolosan, France ; 2Centre National de Ressources GénomiquesVégétales (CNRGV), INRA, Castanet-Tolosan, France; 3UMR 1095INRA-Université Blaise Pascal, Domaine de Crouelle,Clermont-Ferrand, France

  Quantitative resistance to sunflower Downy Mildew caused by theoomycete Plasmopara halstedii was studied on a population ofrecombinant inbred lines (RIL) not carrying efficient majorresistance gene, in fields naturally infested by one race of thepathogen (703 or 710). The major quantitative trait locus (QTL)localized on linkage group 10 explains almost 40% of variation, andis not linked to any of the known race-specific resistance genescalled Pl genes. This QTL confers resistance to at least 2different downy mildew races and its support interval is 5 cm long.We constructed and screened a BAC library of the RIL parent (XRQ)having the QTL with the closest genetic markers in order to build aBAC contig in the QTL region, a first step towards the positionalcloning strategy. The polymorphic BAC ends are currently being usedas new genetic markers on the RIL population. We also screened anF2 population of 3500 plants in order to increase the number ofplants presenting a recombination event between the closest QTLmarkers. The evaluation of the resistant phenotypes of suchrecombinant plants may help restricting the QTL support interval.In order to characterize the expressed genes during the interactionfrom both partners, plant and oomycete, we initiated a cDNAsequencing approach of infected sunflower plantlets using the 454®sequencing method. 2. Identification of genes putatively involvedin manipulating plant defense responses during rice blast infectionMUHAMMAD BADARUDDIN, DARREN M. SOANES, NICHOLAS J. TALBOT School ofBiosciences, Geoffrey Pope Building, University of Exeter, Exeter,EX4 4QD, UK Phytopathogenic fungi have evolved different strategiesto proliferate and cause disease. One of the mechanisms used bythese pathogens involves the delivery of a battery of effectorproteins into the host cell which act to subdue defense responsesby interacting with host proteins. In this investigation, threegenomic sequences of M. oryzae were analysed, 70-15, Y34 and 131 inorder to identify genes putatively undergoing diversifyingselection. The dN/dS ratio was estimated for each pairwisecomparison using Codeml and the twenty five best candidateeffectors were identified with a dN/dS > 1. Five genes which hada dN/dS>1 were selected for functional characterization. In asecond approach, comparative secretome analysis was carried out toidentify proteins specifically present only in ascomycetephytopathogenic fungi. An isochorismatase motif was found in thesecretome of five different species of phytopathogens.Isochorismate is a precursor of salicylic acid which is activelyinvolved in plant defense, so it is worth speculating that secretedisochorismatase could be a virulence factor employed by the fungusto reduce salicylic acid. Here we report on the functionalcharacterization of an isochorismatase encoding gene ISM1 in M.oryzae.

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• 3. Arabidopsis downy mildew avirulence locus ATR5 containssingle or multi copy, highly polymorphic non-RXLR effectors amongpathogen isolates

  KATE BAILEY, VOLKAN ÇEVIK, NICK HOLTON, ERIC HOLUB, MAHMUT TÖRWarwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF,UK The intricate genetic dance between plant pathogens and theirhosts involves the pathogen attack, host defence and thepathogen-counter attack with the use of secreted molecules.Pathogen effector molecules perform inter- and intracellular tasksas adaptation factors and manipulators of the defence network. TheArabidopsis-Hyaloperonospora pathosystem has been playing asignificant role in uncovering major complementaryeffector-receptor genes. Common conserved regions including RXLRand EER motifs in the secreted effectors have been identified fromseveral oomycete pathogens, and have been under detailedinvestigation. Arabidopsis La-er accession carries RPP5, whichrecognizes ATR5 from Noks1/Noco2 and Emoy2 isolates. We have mappedATR5 using F2 mapping populations derived from different crossesbetween isolates of H. arabidopsidis. A genetic interval for ATR5has been established and a physical map of ATR5 in the Emoy2 genomewas constructed using the publicly available genomic and BAC-endsequences, as well as the BAC contig data. The ATR5 gene has beenplaced on a single BAC clone. Fine mapping has put the gene to a25kb interval. There is segmental gene duplication in the Emoy2genome at the ATR5 locus. Bioinformatic studies supported byexpression analysis revealed the presence of five genes, three ofwhich have the characteristics of polymorphic effector molecules inisolate Emoy2. Interestingly, none of these candidates have an RXLRmotif. Transient expression studies using bombardment assays haveidentified the ATR5Emoy2 that gives an RPP5 dependent defenceresponse. The other polymorphic effectors, ATR5L1Emoy2 andATR5L2Emoy2, are not recognized by RPP5. We made a cosmid libraryfrom isolate Noks1 and identified the clone that covers the ATR5locus. Sequence information shows no gene duplication at the locusand only one copy of the putative non-RXLR effector is present.However, this Noks1 copy does not trigger an RPP5-dependant defenceresponse. Analysis of the Cala2 genome using RACE PCR suggested 5copies of this family of effectors. Recent work on the function,evolution and further analysis will be presented. 4. Fungal cellwall and secreted proteins: insights from the Tuber melanosporumgenome R. BALESTRINI1, F. SILLO1, A. KOHELER2, P. WINCKER3, F.MARTIN2, P. BONFANTE1 1Instituto per la Protezione delle Piante -CNR and Dipartimento di Biologia Vegetale – UniTO, Italy; 2UMRINRA-UHP 1136 Interactions Arbres/Micro-organismes, Centre INRA deNancy, 54280 Champenoux, France; 3Genoscope, Evry, France Fungalcell wall is a dynamic structure that plays crucial roles inmaintaining cell morphology, protecting mycelia from environmentalstresses, and allowing interactions with substrates and the otherliving organisms. Formation and remodelling of the fungal cell wallinvolves numerous pathways and the concerted actions of manyproteins within the fungal cell. Starting from the genomesequencing project of Tuber melanosporum, an ectomycorrhizalfungus, we performed an in silico analysis mostly focusing oncell-wall related genes. The results gave us a glimpse on cellwall-related and secreted proteins, allowing the identification ofthe several members in some gene families (CHSs, chitinases,hydrophobins). Interestingly, arrays expression data suggest thattwo chitinase genes could be involved in the ectomycorrhizalformation. These proteins could have a role in the cell wallremodelling during the switch from free-living to symbiotic status,but we could also hypothesize a role in the formation ofchitin-derived elicitors. A set of genes coding for secretedenzymes involved in a subtle degradation of plant cell wallpolysaccharides have been also identified. Future work will befocused to understand whether multiple members of the same genefamily have specialized roles

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• in one or more biological processes (e.g. ectomycorrhizae,fruiting bodies development). The Tuber genome sequencing projectis a collaborative effort involving the Génoscope-CEA (coordinator:P. Wincker) and the Tuber Genome Consortium (coordinator: F.Martin). 5. Unraveling the mode- and site-of-action of thehost-selective toxin Ptr ToxB MELANIA F. BETTS, VIOLA A. MANNING,IOVANNA PANDELOVA, KARA MILES-ROCKENFIELD, LYNDA M. CIUFFETTIDepartment of Botany and Plant Pathology, Oregon State University,Corvallis, Oregon, 97331 USA Pyrenophora tritici-repentis is anecrotrophic ascomycete and the causal agent of the disease tanspot of wheat. Ptr ToxB (ToxB) is one of the proteinaceoushost-selective toxins produced by this pathogen and is responsiblefor the development of chlorotic symptoms in susceptible cultivars.ToxB is encoded by a multicopy gene, ToxB (261 bp), whoseexpression results in an 87 amino acid (aa) pre-protein. Thispre-protein contains a signal peptide of 23 aa and the remaining 64aa encode the mature form of the toxin (6.5 KDa). There are nocharacterized motifs within ToxB to give clues to its functionsite. An allele of ToxB, toxb, is found in non-pathogenic isolatesand encodes an inactive protein. Construction of chimeric proteinscontaining ToxB and toxb coding regions, and site-directedmutagenesis based on the aa sequence differences have providedinformation on the structural requirements for ToxB activity. AProteinase protection assay using heterologously expressed ToxBindicated that ToxB must be present in the apoplastic space for 8hours to induce maximum symptom development. Barley Stripe MosaicVirus-mediated transient systemic expression of ToxB and toxb isconsistent with the hypothesis that ToxB acts as an apoplasticeffector. 6. A functional genomics approach to elucidate the roleof aphid salivary gland proteins in plant infestation JORUNN I.B.BOS1, DAVID PRINCE1, MARCO PITINO, JOE WIN2, SASKIA A. HOGENHOUT11Department of Disease and Stress Biology, The John Innes Centre,Norwich, NR4 7UH, United Kingdom; 2The Sainsbury Laboratory,Norwich, NR4 7UH, United Kingdom Aphids are amongst the mostdevastating hemipteran sap-feeding insects of plants. They induceextensive feeding damage and vector the majority of described plantviruses worldwide. Myzus persicae (green peach aphid) is considereda generalist, with host plants in over 40 plant families. The M.persicae salivary glands probably produce effector proteins thatare secreted into the plant host during aphid feeding and modulateplant cell processes. Our aim is to identify and characterize theseproteins and to elucidate the molecular mechanisms underlying theirfunctions. Genomics resources recently became available offeringunprecedented opportunities for investigating aphids and theperturbations they cause in plants. We applied a data miningstrategy combined with functional assays to identify andfunctionally characterize secreted salivary gland proteins from M.persicae. We identified 115 proteins from a salivary gland ESTdatabase (3233 ESTs) that are predicted to be secreted. Currently,we are screening this set of proteins for effects on aphidsurvival, fitness and host range specificity using in plantaover-expression followed by aphid challenge as well as RNAi inaphids. In addition, we are using transient over-expression assaysin Nicotiana benthamiana to investigate whether these proteinsaffect plant cell processes, especially those involved indefense.

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• 7. Structure/function studies of Phytophthora effectors using amedium-throughput approach LAURENCE S. BOUTEMY1, RICHARD K.HUGHES1, ALICE C. MAXWELL1, SOPHIEN KAMOUN2, MARK J. BANFIELD1 1Department of Biological Chemistry, John Innes Centre, NorwichResearch Park, Colney, Norwich, Norfolk, NR4 7UH, United Kingdom;2The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich,Norfolk, NR4 7UH, United Kingdom Phytophthora species are plantpathogens responsible for massive economic and agricultural lossesas they infect crops such as potatoes and tomatoes (Phytophthorainfestans) or soybeans (Phytophthora sojae). They manipulate hostcell structure and function through delivery of an array ofeffector proteins. The RxLR and the CRN (Crinkler) proteins formthe two main families of Phytophthora effectors. Although many ofthese effectors can be associated with a virulence and/oravirulence function and phenotype in the host plant, decipheringtheir biological function remains difficult as they do not shareany sequence similarity with proteins of known function.Determining the three-dimensional structure of these proteins wouldprovide significant insights into their function and help directfurther biological studies. Here we describe a medium-throughputapproach that was used for the design, cloning, expression andpurification of a set of Phytophthora effectors. This set containsRxLR and CRN proteins whose expression is known to be induced oninteraction with the host plant and have a demonstratablephenotype. The purified effectors will ultimately be used forstructure determination by X-ray crystallography as well asbiochemical and biophysical assays. 8. Identification of oomycteeffector targets using in planta co-immunoprecipitation TOLGA O.BOZKURT, JOE WIN, ALEX JONES, SOPHIEN KAMOUN The SainsburyLaboratory, Norwich Research Park, Colney, Norwich NR4 7UH, UKOomycete pathogens deliver a variety of effector proteins intoplant host cells to suppress defense responses and enablesuccessful colonization. In this study, we aimed to find targetproteins of the 52 validated oomycete effectors including severalwith avirulence activity (RXLR family). We used an in vivoco-immunoprecipitation (co-IP) assay to identify the targets of oureffectors. We made expression constructs by replacing the secretionsignals with the Flag tag and cloning into pJL-TRBO, a binaryplasmid derived from a modified Tobacco mosaic virus. We deliveredeffector constructs into the leaves of Nicotiana benthamiana andtransiently overexpressed them by agroinfiltration. We thenharvested the leaves 2-3 days after infiltration and extractedtotal proteins. Effector proteins and their interactors from theplant were co-IPed with anti-FLAG resins. Eluted proteins were thenrun on SDS-PAGE, gel slices were excised, digested with trypsin,and identified by LC-MS/MS. Accepted proteins were required to haveMascot scores of more than 50 and two or more unique peptidesidentified. We are using reverse co-IP and split YFP assay and toconfirm interactions. We will report and discuss identifiedeffector target proteins and any alterations in plant immunityresulting from overexpression or virus-induced gene silencing ofthese targets.

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• 9. Role of the TtsI protein of Bradyrhizobium elkanii in T3SS(type three secretion system)-mediated protein secretion andsoybean nodulation S. B. CAMPOS1, L. M. P. PASSAGLIA1, W. J.BROUGHTON2, W. J. DEAKIN2 1Department of Genetics, FederalUniversity of Rio Grande do Sul, Av. Bento Gonçalves, 9500, PortoAlegre, RS, CEP 91501-970 Brazil; 2Sciences III, 30 QuaiErnest-Ansermet, CH-1211 GENEVE 4,Switzerland Bradyrhizobiumelkanii is an important soil bacterium which fixes nitrogen andinduces nodule formation in soybean (Glycine max). Theplant-rhizobia interaction is fundamental for the establishment ofthe symbiosis. Several bacteria release factors that act aselicitors for this interaction, such as Nops (nodulation outerproteins) secreted by type three protein secretion systems (T3SS).TtsI is the transcriptional activator of the system, recognizingconsensus sequences (tts-box) in the promoter regions of the T3SSgenes. To study the B. elkanii TtsI protein an omega cassette wasused to disrupt the ttsI gene, generating a B. elkanii ttsI mutant.The mutant and wild type bacteria were used in soybean nodulationand protein secretion assays. Two soybean cultivars were used: inthe cultivar Peking a nodulation delay was observed for the mutantstrain, while no difference between the wild type and mutantstrains was observed for the cultivar McCall. Using Genistein as aninductor for the T3SS, no protein was secreted by the mutantstrain. In contrast, the wild type showed a positive western-blotsignal against NopA and NopL antibodies. To date, this is the firstrecord of activation of the T3SS in B. elkanii by a specificflavonoid. 10. The Ralstonia solanacearum GMI1000 effectome A-C.CAZALÉ, N. PEETERS, C. BOUCHER, S. GENIN Laboratoire desInteractions Plantes Micro-organismes (LIPM), UMR CNRS-INRA2594/441, F-31320 Castanet Tolosan, France Ralstonia solanacearum,the causal agent of bacterial wilt disease, targets more than twohundred species including economical important crops. The type IIIsecretion system plays a major role in its pathogenicity. Seventyfour type III effectors have been identified in strain GMI1000. Todate, 48 have been experimentally validated either through in vitrosecretion assays or demonstration of translocation into the plantcell. We suspect substantial functional overlap among thisrepertoire since only two single disruption mutants were found tobe slightly altered in pathogenicity on Arabidopsis or tomatoplants. In order to get insights into the functions of this largeset of effectors, we have defined a systematic functional approachusing in planta transient expression assays to test the responsesinduced at the macroscopic level and the localization in the plantcell. The first results show that eight out of the 30 effectorstested induce the development of a necrosis in Nicotianae. Most ofthe effectors-RFP fusions are observed in both the nucleus and thecytoplasm except for three that are exclusively nuclear-localized.For the most promising candidates, plant interacting partnerproteins will be searched. 11. Mechanism of cell-death suppressionby the Phytophthora infestans RXLR effector protein AVR3a ANGELACHAPARRO-GARCIA, JORUNN BOS, SOPHIEN KAMOUN The SainsburyLaboratory, Colney Lane, Norwich, NR4 7UH, UK Phytophthorainfestans effector protein AVR3a belongs to the RXLR class ofcytoplasmic effectors. AVR3a induces R3a-mediated hypersensitivityand suppresses the cell death induced by P. infestans INF1elicitin, a protein with features of pathogen-associated molecularpatterns (PAMPs). AVR3a mutants that activate R3a but do notsuppress cell death were identified suggesting that distinct aminoacids condition the effector activities. One example isAVR3aY147del mutant, which lacks cell death

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• suppression activity but retains R3a activation. These datapoint to a model in which AVR3a interacts with one or more hostproteins. To identify candidate virulence targets of AVR3a, ourcollaborators in the Birch and Michelmore labs found that AVR3ainteracts with the E3 ligase CMPG1 in yeast-two-hybrid assays.Interestingly, AVR3a stabilizes CMPG1 in planta whereasAVR3aY147del does not. CMPG1 is required for INF1-induced celldeath suggesting that it could mediate the virulence activity ofAVR3a. Our goals are to characterize the AVR3a-CMPG1 interactionand to determine its contribution to INF1 cell death suppression.Currently, we are mapping the interaction sites between AVR3a andCMPG1 and testing the extent to which AVR3a suppresses differentaspects of the PAMP-triggered immunity elicited by INF1. 12.Identification of Phytophthora cactorum genes expressed duringinfection of strawberry XIAOREN CHEN, SONJA SLETNER KLEMSDAL, MAYBENTE BRURBERG Bioforsk, Plant Health and Plant ProtectionDivision, Department of Genetics and Biotechnology, Høgskoleveien7, 1432 Ås, Norway The oomycete Phytophthora cactorum causes crownrot disease in strawberry, resulting in big economic losses. Tounravel the molecular mechanisms that are involved in thepathogenicity of P. cactorum on strawberry, two strategies werefollowed, SSH cDNA library and effector specific differentialdisplay. Two cDNA libraries were made, enriched for P. cactorumgenes upregulated during infection of strawberry or genes expressedin in vitro germinating cysts (a developmental stage essential forinfection). Recent characterization of oomycete AVR/effector genesrevealed that they encode proteins with conserved RxLR-dEER motifsrequired for translocating these effectors into host cells. Thepresence of such a conserved “tag” has provided a tool fordiscovering the otherwise structurally diverse effector genes. Toselect RxLR effector genes from P. cactorum differential displaywas performed on eight cDNA populations, including fourdevelopmental stages (mycelium, sporangium, zoospore andgerminating cyst) as well as four time points during infection (0,3, 5, 7 days post-inoculation), using the RxLR and EER motifdegenerate primers. Using these strategies several genespotentially relevant for pathogenicity, including several putativeeffector genes were discovered, and their differential expressionconfirmed using real-time quantitative PCR. 13. Secretion of fungaleffectors: a comparative view between a symbiotic and a pathogenicfungus C. COMMUN, S. DUPLESSIS, F. MARTIN, A. BRUN, C.VENEAULT-FOURREY UMR INRA-UHP 1136 " InteractionsArbres/Microorganismes", IFR 110 Génomique, Ecophysiologie etEcologie Fonctionnelles 54280 Champenoux, France In a forestecosystem, trees are in continuous interaction with differentmicroorganisms including fungi. Among these fungi, some of them aresymbiotic as Laccaria bicolor while others are pathogenic asMelampsora larici-populina. These two organisms are biotrophicfungi. Development of a functional biotrophic interface betweenfungus and poplar Populus trichocarpa needs active secretion offungal effectors. The recent sequencing of both L. bicolor and M.larici-populina genomes combined with “transcriptomic” analysesallowed us to establish a genomic view of secretion pathway(s)within both fungi. In addition, we started functional analysis ofP4-ATPases family in both fungi as members of this family areinvolved in endo/exocytosis (1) and secretion of one avirulencegene in Magnaporthe grisea (2).Preliminary results will bepresented (1). Graham TR. 2004. Trends in Cell Biology 14: 670–677.(2). Gilbert et al., 2006. Nature 440: 535–539.

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• 14. Insights into the Pseudomonas syringae pv. tomato DC3000type III effector repertoire gained through combinatorial deletionsof effector genes and identification of interacting tomato proteinsSÉBASTIEN CUNNAC, BRIAN KVITKO, ALISTAIR RUSSELL, DAVID SCHNEIDER,GREGORY MARTIN, ALAN COLLMER Department of Plant Pathology andPlant-Microbe Biology, Cornell University, Ithaca, NY 14850, USA;United States Department of Agriculture-Agricultural ResearchService, Robert W Holley Center for Agriculture and Health, Ithaca,NY 14853, USA; Boyce Thompson Institute for Plant Research, Ithaca,NY 14850, USA Pto DC3000 uses the type III secretion system toinject ca. 28 Avr/Hop effector proteins into plants, which enablesthe bacterium to grow from low inoculum levels to produce bacterialspeck symptoms in Arabidopsis thaliana and the Solanaceae species,tomato (Solanum lycopersicum) and (when lacking hopQ1-1) Nicotianabenthamiana. The effectors are collectively essential butindividually dispensable for pathogenesis. To understand the basisfor this redundancy and the potential function of the effectorrepertoire as a system, we have been constructing and analyzingDC3000 mutants with combinatorial effector gene deletions and usingyeast two hybrid screens to comprehensively identify tomatoproteins that interact with DC3000 effectors. Combinatorialdeletions involving the 18 effector genes occurring in clusters andtwo of the remaining effector genes revealed a redundancy-basedstructure in the effector repertoire, such that some deletionsdiminished growth in N. benthamiana only in combination with otherdeletions. Much of the ability of DC3000 to grow in N. benthamianawas found to be due to five effectors in two redundant-effectorgroups (REGs), which appear to separately target two high-levelprocesses in plant defense: perception of external pathogen signals(AvrPto and AvrPtoB) and deployment of antimicrobial factors (AvrE,HopM1, HopR1). Similarly, analysis of tomato proteins in theeffector interactome has revealed multiple cases of commoninteractors for two or more effectors. Deletions of complete genesets for various type III substrates (translocators, lytictransglycosylases, and effectors) in combination with coronatinebiosynthesis genes, is revealing the redundancy groups and minimalrequirements for each stage in type III effector-mediatedpathogenesis. 15. Role of the bacterial Type III Secretion System(T3SS) in the interactions between bacteria and ectomycorrhizalfungi A.M. CUSANO1, A. DEVEAU1, 2, P. BURLINSON4, B. PALIN1, S.UROZ1, A. SARNIGUET3, D. HOGAN2, G. PRESTON4 , P. FREY-KLETT11INRA, UMR1136 INRA-Nancy “Interaction Arbres/Micro-organismes”,Centre de Nancy, 54280 Champenoux , FRANCE; 2 Department ofMicrobiology and Immunology, Darthmouth Medical School, Hanover, NH03755, USA; 3INRA, UMR1099 “Biologie des Organismes et desPopulations appliquée à la Protection des Plantes ”, 35 653 Le RheuCedex, FRANCE; 4Department of Plant Sciences, University of Oxford,Oxford OX1 3RB, UK In forest ecosystems, exists a mixedfungal-bacterial continuum at the interface between soil and treeroots, called the ectomycorrhizal complex which controls planthealth and nutrition. In order to develop new strategies for asustainable management of forest ecosystems within the forestmicrobial communities, a better understanding of the interactionmechanisms between bacteria and ectomycorrhizal fungi in theectomycorrhizal complex is necessary. We hypothesize that the TypeThree Secretion System (T3SS), a key secretion-translocationapparatus used by gram-negative bacteria to colonize animal andplant hosts, mediates protein translocation between bacterial andfungal cells and modulates the functioning of the ectomycorrhizalcomplex. Up to now, T3SS studies have largely focused on bacterialpathogens of animal and plants. However there is increasingevidence to suggest that plant and animal pathogenesis is not theprimary function of the T3SS and that the ecological functions ofT3SS are more diverse than expected1. We recently demonstrated thatthe Mycorrhiza Helper Bacterial

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• strain Pseudomonas fluorescens BBc6R8, isolated from a sporocarpof the ectomycorrhizal fungus Laccaria bicolor S238N, harbours aT3SS gene cluster. Experiences are running to study thefunctionality of the Pseudomonas fluorescens BBc6R8 T3SS and toanalyse its role of in the interactions with L. bicolor. 16.LecRK79, a putative virulence target of the RXLR effector IPI-O isinvolved in cell wall-plasma membrane adhesions and PAMP-triggeredimmunity M. DE SAIN1, K. BOUWMEESTER1, R. WEIDE1, H. CANUT2, F.GOVERS1 1Laboratory of Phytopathology, Wageningen University,Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands; 2UMR5546 CNRS-Université Paul Sabatier, BP17, 31326 Castanet Tolosan,France The potato late blight pathogen Phytophthora infestanssecretes many RXLR effectors, one of which is IPI-O. In IPI-O theRXLR motif overlaps with the tripeptide RGD (RSLRGD), a typicalcell adhesion motif present in extracellular metazoan proteins thatplay a role in cell-cell interactions. A phage display aimed atselecting proteins that potentially interact with the RGD motif inIPI-O resulted in the identification of Arabidopsis lectin receptorkinase LecRK79 (Gouget et al. 2006, Plant Physiology 140, 81-90).We postulate that LecRK79 is a virulence target of IPI-O. Forfunctional characterization we generated Arabidopsis linesoverexpressing LecRK79 (OE lines) or lacking LecRK79 (knock-outlines lecrk79). Infection assays revealed changes in phenotype uponinfection with Phytophthora brassicae in both the OE and lecrk79lines and demonstrate that LecRK79 has a role in Phytophthoradisease resistance. To unravel the mechanisms underlying thisresistance we analysed callose deposition upon PAMP treatment andinvestigated the strength of cell wall-plasma membrane adhesions byinducing plasmolysis. The results indicate that LecRK79 plays animportant role in the continuum between cell wall and plasmamembrane and suggest that this lectin receptor kinase is involvedin PAMP-triggered immunity. 17. Seven in Absentia (SINA) E3 ligasesaffect SYMRK protein stability and function in rhizobial entryduring Lotus japonicus root nodule symbiosis G. DEN HERDER1, S.YOSHIDA1,2, M. ANOTLIN-LLOVERA1, M. PARNISKE1,2 1Genetics,Biozentrum, University of Munich (LMU), Munich, Germany; 2TheSainsbury Laboratory, Norwich, UK SINA E3 ligase proteins are partof the proteasomal degradation pathway, acting as dimers tospecifically ubiquitinate their substrates. In M. truncatula, theyfunction in regulation of lateral root formation and rhizobialinfection (1). Symbiosis Receptor Kinase (SYMRK) activity andphosphorylation is required during root symbiosis to allowinternalisation of the microsymbionts. Yeast two-hybrid analysisrevealed an interaction of the SYMRK kinase domain with a smallfamily of L. japonicus SINA family members, a specific interactionthat was confirmed in planta. The SINA genes are expressedthroughout the plant, and regulated through posttranslationalmodification and turnover via self-ubiquitination. Proteinstability of SYMRK was reduced upon transient co-expression ofSINA4 and SYMRK in N. benthamiana leaves, indicating thatproteasomal degradation of SYMRK is triggered via SINA4. Ectopicexpression of a dominant negative mutant form (SINADN) in L.japonicus transgenic plants inhibits SINA function, and theseplants showed impaired nodulation on the level of the infectionprocess. Our data provide evidence for a role of SINA in rhizobialentry via regulation of SYMRK, which probably involvesubiquitin-mediated internalization and subsequent proteasomaldegradation. (1) Den Herder, et al., 2008. Plant Physiology 148:369–382.

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• 18. Identity, host range, vector and spread of a phytoplasmacausing tomato stolbur disease in Turkey S. EROGLU1, F. SAHIN1, N.OZDEMIR2, Y. KARSAVURAN2 1Yeditepe University, Istanbul, Turkey 2Ege University, Izmir, Turkey This study aimed to identify thepathogen and determine host range, vector and spread of the causalagent of stolbur like disease on tomato in Turkey. Between 2004 and2008, plant and pest samples collected from diseased tomato fieldsin Bursa and Canakkale were examined by nested PCR usingphytoplasma-universal 16S rDNA based primer sets (P1/P7). Aunique1.4 kb PCR amplified rDNA band from all parts of diseasedtomato plants, except seed, were demonstrated that the phytoplasm(PLO) was the causal agent of stolbur disease in tomato. The datashowed that stolbur disease was causing epidemic in tomatoproduction areas in the provinces of Bursa (Karacabey andYenisehir) and Canakkale (Biga) in Turkey. Cuscuta campestris,Orobance ramose, Datura stramonium, Polygonum persicaria, Setariaspp., Chenopodium album and Amaranthus albus were determined asalternative hosts of tomato PLO. Only Tyhlocyba quercus among the22 insect species was found to be potential vector of tomato PLO.All the tomato cultivars/genotypes were found to be susceptible totomato PLO. RFLP analysis of the PCR-amplified 16S rDNA indicatedthat all samples contained a closely related phytoplasmas.Phylogenetic analysis of 16S rDNA sequences (1.4 kb) clusteredtomato phyoplasmas into a distinct phylogenetic lineages. 19.Hyaloperonospora arabidopsidis (Hpa) effector’s are able tosuppress PTI in A. thaliana GEORGINA FABRO, EVELYN KOERNER, DAVIDSTUDHOLME, JONATHAN D. G. JONES The Sainsbury Laboratory, JohnInnes Centre, Colney Lane, Norwich, NR4 7UH; Collaborators from theERA-PG Effectoromics consortium* We investigate how this obligateoomycete (Hpa) is able to manipulate its host, A. thaliana toestablish a successful infection. We are characterizing Hpaeffector proteins in collaboration with the ERAPG Hpa Effectoromicsconsortium. These effectors are small secreted proteins containingsignal peptide and RxLR motifs. Through bioinformatic analysis ofthe Hpa Emoy2 race genome we identified ≈ 140 potential effectorsof which 102 have been cloned. We t

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