Dear friends and participants Welcome16.00 –18.00 Session 3: Nitrogenase and regulation of...

Dear friends and participants Welcome

The Local Organising Committee is pleased to welcome you in Gent at the 8th European Nitrogen Fixation Conference. Traditionally, the biennial European Nitrogen Fixation Conferences bring together researchers from all over the world who study different aspects of biological nitrogen fixation. Also in this edition, we are happy to have over 300 participants arriving from more than 30 different countries. Biological nitrogen fixation is an essential component of the life cycle of our planet and holds the potential of providing a sustainable and environmentally friendly input of nitrogen for large-scale agriculture, as a natural and valuable alternative for industrial nitrogen fertilisers. Biological nitrogen fixation is carried out by a wide range of prokaryotes with different lifestyles and hence its study is covered by very diverse research disciplines, most of which will be represented in our Conference. These disciplines deal with many aspects of fundamental research, such as the enzymology of nitrogen fixation, the genes involved and the regulation of their expression, the diversity and evolution of the microbes, and the molecular biology of associative and symbiotic interactions. The present research focuses will be highlighted at this meeting and ample attention will be given to genomic approaches and to new tools and technologies. Several sessions will be dedicated to applied aspects of biological nitrogen fixation and address the issues of restrictions and limitations for more extensive use. We are all fully aware of the great potential of biological nitrogen fixation and wonder how we can enhance its integration in agri- and horticultural practises. The Gent conference is attended by both experienced senior scientists and many young researchers. We hope this combination will provide a fruitful mix to promote information transfer, stimulate learning, and create new collaborations. We wish you a pleasant and successful stay in Gent! Marcelle Holsters, Chair of the Local Organising Committee

Local Organising Committee Chair: Prof. Marcelle Holsters Prof. Sofie Goormachtig Dr. Danny Vereecke Dr. Martine De Cock Mr. Stephane Rombauts Department of Plant Systems Biology, VIB, and Department of Molecular Genetics, Ghent University, Technologiepark 927, 9052 Gent - Belgium Prof. Anne Willems Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, K.L. Ledeganckstraat 35, 9000 Gent - Belgium Prof. Jos Vanderleyden Department of Microbial and Molecular Systems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20 - bus 02460, 3001 Heverlee - Belgium Conference coordinator: Danny Vereecke Abstract book editors: Martine De Cock and Danny Vereecke Webmaster: Kenny Billiau Organising Secretariat: Evident European Steering Committee Julie Cullimore (France) Frans de Bruijn (France) Ray Dixon (UK) Andy Johnston (UK) György Kiss (Hungary) Adam Kondorosi (France) (Chair) José Olivares (Spain) Antonio Palomares (Spain)† Alfred Pühler (Germany) International Advisory Board Mohamed Elarbi Aouani (Tunesia) Ton Bisseling (The Netherlands) Nick Brewin (UK) Allan Downie (UK) Peter Gresshoff (Australia) Hauke Hennecke (Switzerland) Maurizio Iaccarino (Italy) Carmen Lluch (Spain) Sharon Long (USA) Yaacov Okon (Israel) Rafael Palacios (Mexico) Katharina Pawlowski (Sweden) Barry Rolfe (Australia) Tomas Ruiz-Argüeso (Spain) Rachid Serraj (India) Janet Sprent (UK) Gary Stacey (USA) Jens Stougaard (Denmark) Samba Sylla (Senegal) Igor Tikhonovich (Russia) Michael Udvardi (Germany)

Table of contents Programme of the 8th European Nitrogen Fixation Conference 1 Opening lectures 7 Session 1: Signalling in nodulation 9 Session 2: Cyanobacterial and associative nitrogen fixation 55 Session 3: Nitrogenase and regulation of free-living nitrogen fixation 61 Session 4: Diversity and evolution 79 Session 5: Highlights in plant and bacterial genomes 123 Session 6: Genetics of legume nodulation 135 Session 7: Non-legume associations: endophytic and symbiotic interactions 145 Session 8: Nodule organogenesis 161 Session 9: Ecology and sustainable agriculture 177 Session 10: Comparative and functional genomics of nitrogen-fixing bacteria 203 Session 11: New tools to study biological nitrogen fixation 227 Session 12: Nodule functioning 233 Closing Remarks 257 Workshop 1 259 Workshop 2 275 Workshop 3 281 List of Attendees 291 General Information 299 Notes 303

The 8th European Nitrogen Fixation

Conference

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Programme of the 8th European Nitrogen Fixation Conference Saturday August 30 14.00 –17.00 Registration and welcome coffee 17.00 –17.10 Opening by Prof. Van Cauwenberge, Rector of the Ghent

University 17.10 –17.30 Welcome word by Marcelle Holsters In Memoriam of Antonio Palomares by Jos Vanderleyden 17.30 –18.50 Opening lectures Chaired by Marcelle Holsters (Gent, Belgium) 17.30 –18.10 Claudine Elmerich (Paris, France): Nitrogen fixation: a glimpse into

history 18.10 –18.50 The EMBO lecturer Ton Bisseling (Wageningen, The Netherlands): The

legume-Rhizobium symbiosis: from initial recognition to symbiosome maintenance

18.50 –22.00 Welcome reception Sunday August 31 8.30 –12.00 Session 1: Signalling in nodulation Chaired by Jens Stougaard (Aarhus, Denmark) 8.30 –9.00 Simona Radutoiu (Aarhus, Denmark): Rhizobium signalling in Lotus

japonicus 9.00 –9.30 Giles Oldroyd (Norwich, UK): Coordinating epidermal and cortical

responses during the initiation of a nodule 9.30 –9.50 Thomas Ott (München, Germany): A novel protein controls rhizobial

infection and mediates post-translational regulation of symbiotic receptor proteins

9.50 –10.10 Myriam Charpentier (München, Germany): New class of cation channels essential for perinuclear calcium spiking

10.10 –10.30 Shimpei Magori (Tokyo, Japan): TOO MUCH LOVE locus is necessary for the long-distance control of nodulation in Lotus japonicus

10.30 –11.00 Coffee break 11.00 –11.20 Yiliang Ding (Norwich, UK): ABA coordinates Nod factor and cytokinin

signalling during the regulation of nodulation 11.20 –11.40 William Deakin (Geneva, Switzerland): Study and improvement of the

Lotus japonicus-Rhizobium sp. NGR234 symbiosis

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11.40 –12.40 Session 2: Cyanobacterial and associative nitrogen fixation Chaired by Jos Vanderleyden (Leuven, Belgium) 11.40 –12.10 Karl Forchhammer (Tübingen, Germany): Nitrogen storage and ammonia

stress acclimation in cyanobacteria: novel insights in similarities to plant physiology

12.10 –12.40 Yvan Moënne-Loccoz (Lyon, France): Signalling and functioning of Azospirillum in the rhizosphere

12.40 –13.30 Lunch 14.00 –15.30 Guided walk through Gent with the support of Gent Congres vzw

and Dienst Toerisme 15.30 –16.00 Coffee 16.00 –18.00 Session 3: Nitrogenase and regulation of free-living nitrogen

fixation Chaired by Ray Dixon (Norwich, UK) 16.00 –16.30 Dennis Dean (Blacksburg, USA): Nitrogenase biochemistry 16.30 –17.00 Stefan Nordlund (Stockholm, Sweden): The role of PII and AmtB proteins

in the regulation of nitrogenase activity and ammonium assimilation in Rhodospirillum rubrum

17.00 –17.20 Luis Rubio (Berkeley, USA): Complete synthesis of the iron-molybdenum cofactor of nitrogenase with purified Nif proteins

17.20 –17.40 Luciano Huergo (Curitiba, Brazil): In vitro interactions between the PII proteins and the nitrogenase regulatory enzymes in Azospirillum brasilense

17.40 –18.00 Peter Slavny (Norwich, UK): Role of the PAS2 domain of NifL in signal transduction

18.00 –20.00 Poster session 1 (even numbers) with refreshments Monday September 1 8.30 –10.30 Session 4: Diversity and evolution Chaired by Anne Willems (Gent, Belgium) 8.30 –9.00 Peter Young (York, UK): Nitrogen fixers: more diversity, more evolution 9.00 –9.30 Bernard Dreyfus (Montpellier, France): Nod factor-independent stem

nodulation in the legume genus Aeschynomene is correlated with plant molecular phylogeny

9.30 –9.50 Martin Parniske (München, Germany): Structural and functional adaptation of the receptor-like kinase gene SYMRK is exceptional among symbiosis genes, and paved the way for the evolution of root endosymbiosis with nitrogen-fixing bacteria

9.50 –10.10 Aneta Dresler-Nurmi (Helsinki, Finland): Molecular phylogeny of rhizobia isolated from Calliandra calothyrsus Meisn. trees growing in Central America, Cameroon, Kenya and New Caledonia with emphasis on biogeography and symbiosis

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10.10 –10.30 Monika Janczarek (Lublin, Poland): rosR and pssA genes involved in polysaccharide synthesis as new taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species

10.30 –11.00 Coffee break 11.00 –12.40 Session 5: Highlights in plant and bacterial genomes Chaired by Stephane Rombauts (Gent, Belgium) 11.00 –11.30 Chris Town (Rockville, USA): The Medicago truncatula genome project 11.30 –12.00 Katia Bonaldi (Montpellier, France): Interaction between Aeschynomene

and photosynthetic Bradyrhizobium: an enigmatic Nod-independent symbiotic process

12.00 –12.20 Wayne Reeve (Perth, Australia): The Sinorhizobium medicae WSM419 genome sequencing project

12.20 –12.40 Ruth Schmitz-Streit (Kiel, Germany): Screening for non-coding RNAs in the archaeal diazotroph Methanosarcina mazei

12.40 –14.30 Lunch break 14.30 –16.10 Session 6: Genetics of legume nodulation Chaired by Gabriella Endre (Szeged, Hungary) 14.30 –15.00 Makoto Hayashi (München, Germany): Infection thread mutants in Lotus

japonicus 15.00 –15.30 Pascal Ratet (Gif sur Yvette, France): Medicago truncatula insertion

mutants as tools to dissect plant-microbe interactions 15.30 –15.50 Manuel Chamber (Sevilla, Spain): A new low-nodulating isoline mutant of

chickpeas (Cicer arietinum): genetic characterisation of its progeny and detection of SNPs in several genes related to this mutation

15.50 –16.10 Nataliya Pobigaylo (Freiburg, Germany): Investigation of proteins involved in cell cycle regulation in Sinorhizobium meliloti

16.10 –16.40 Coffee break 16.40 –18.20 Session 7: Non-legume associations: endophytic and

symbiotic interactions Chaired by Katharina Pawlowski (Stockholm, Sweden) 16.40 –17.10 Philippe Normand (Villeurbanne, France): Modifications in the expression

pattern of Frankia alni in vitro and in symbiosis 17.10 –17.40 Barbara Reinhold-Hurek (Bremen, Germany): Functional genomic

analyses and signalling cascades in rice-endophyte interactions 17.40 –18.00 Akiko Tomitani (Yokosuka, Japan): Isolation and characterisation of the

genes involved in hormogonia formation from Nostoc punctiforme, a diazotrophic symbiotic cyanobacterium

18.00 –18.20 Sergio Svitoonoff (Montpellier, France): Searching for plant and bacterial signals involved in actinorhizal symbioses

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18.20 –21.00 Poster session 2 (uneven numbers) with ´Breugel tafel´ and

Belgian beers Tuesday September 2 8.30 –10.10 Session 8: Nodule organogenesis Chaired by Martin Crespi (Gif sur Yvette, France) 8.30 –9.00 Florian Frugier (Gif sur Yvette): New pathways regulating Medicago

truncatula root architecture 9.00 –9.30 Pascal Gamas (Castanet-Tolosan, France): New regulators of nodule

development in Medicago truncatula 9.30 –9.50 Dong Wang (Stanford, USA): The protein secretory pathway component

DNF1 is required for nodule function 9.50 –10.10 Virginie Mortier (Gent, Belgium): CLE peptide signalling during nodulation

on Medicago truncatula 10.10 –10.40 Coffee break 10.40 –12.40 Session 9: Ecology and sustainable agriculture Chaired by Manuel Megías (Sevilla, Spain) 10.40 –11.10 Francisco Martínez-Abarca (Granada, Spain): Bacterial group-II introns:

new mobile elements in the rhizosphere 11.10 –11.40 Mark Peoples (Black Mountain, Australia): The role of biological nitrogen

fixation in cropping systems and its environmental impact 11.40 –12.00 Ignacio D. Rodríguez Llorente (Sevilla, Spain): Genetic engineering of

the Rhizobium-legume interaction for bioremediation 12.00 –12.20 María del Carmen Villegas (Tlaxcala , Mexico): Analysis of sustainable

agriculture alternatives in Chihuahua, Mexico 12.20 –12.40 Zhongmin Dong (Halifax, Canada): Hydrogen fertilisation: is this the

benefit of crop rotation? 12.40 –14.30 Lunch break 14.30 –16.10 Session 10: Comparative and functional genomics of

nitrogen-fixing bacteria Chaired by Jacques Batut (Castanet-Tolosan, France) 14.30 –15.00 William Broughton (Genève, Switzerland): Minimal set of symbiotic

rhizobial genes 15.00 –15.30 Delphine Capela (Toulouse, France): Genome sequence of the β-

Rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia

15.30 –15.50 Hiroshi Oyaizu (Tokyo, Japan): Genome structure and symbiosis-related genes of the versatile nitrogen-fixing Azorhizobium caulinodans

15.50 –16.10 Hans-Martin Fischer (Zürich, Switzerland): Transcriptomics and functional genomics to study the Bradyrhizobium japonicum- soybean symbiosis

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16.10 –16.30 Marij Frederix (Norwich, UK): A novel mechanism of quorum sensing gene regulation in Rhizobium leguminosarum

16.30 –17.00 Coffee break 17.00 –18.00 Session 11: New tools to study BNF Chaired by Helge Küster (Bielefeld, Germany) 17.00 –17.30 Anke Becker (Bielefeld, Germany): Postgenomic strategies and

databases to explore the symbiotic lifestyle of Sinorhizobium meliloti 17.30 –18.00 Rene Geurts (Wageningen, The Netherlands): Monitoring dynamics of

transcriptional regulation of Nod factor signalling genes using an improved DsRED-E5 variant

19.30 – Banquet and party in “De Centrale” Wednesday September 3 10.00 –12.00 Session 12: Nodule functioning Chaired by Eva Kondorosi (Gif-sur-Yvette, France) 10.00 –10.30 Peter Mergaert (Gif-sur-Yvette, France): Terminal differentiation of

nitrogen fixing bacteroids induced by antimicrobial plant peptides in Medicago truncatula nodules

10.30 –11.00 Phil Poole (Reading, UK): Role of amino acids in bacteroid development and nitrogen fixation

11.00 –11.20 Jan Michiels (Leuven, Belgium): Oligopeptide transporters of Rhizobium etli fulfil an essential role during symbiotic nitrogen fixation and free-living growth under stress conditions

11.20 –11.40 Gail Ferguson (Aberdeen, UK): Investigating the role and biosynthesis of lipopolysaccharide very-long-chain-fatty-acids in the Sinorhizobium meliloti-alfalfa symbiosis

11.40 –12.00 Brent Kaiser (Adelaide, Australia): Soybean nitrogen fixation is dependent on the activity of the novel peribacteroid membrane-bound transcription factor, GmSAT1

12.00 –12.30 Closing Remarks by Peter Gresshoff (Brisbane, Australia) 12.30 – Coffee and Goodbye

Opening Session

Chaired by Marcelle Holsters Gent, Belgium

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Opening session

Nitrogen fixation: a glimpse into history Claudine Elmerich Département de Microbiologie, Institut Pasteur, 75724 Paris Cedex 15, France Discovery that legumes, bearing root nodules induced by bacteria, could use atmospheric nitrogen was established by the end of the 19th century. Soon after, rhizobia were isolated from nodules and free-living nitrogen fixers belonging to Clostridium and Azotobacter genera were obtained in pure culture. Demonstration that ammonia was the product of N2 assimilation by nitrogenase enzyme was derived from experiments using 15N as a tracer in the 1940s. However, attempts to obtain cell free extracts with nitrogenase activity were unsuccessful for more than 25 years. A method to obtain extracts of C. pasteurianum that displayed nitrogenase activity in the presence of pyruvate was reported in 1960. The role of pyruvate in supporting nitrogen fixation through the phosphoroclastic reaction and the reduction of ferredoxin was soon elucidated. By 1966, it was known that nitrogenase activity required a combination of two proteins. Purification of these proteins from different sources was achieved and the nitrogenase of Klebsiella pneumoniae was among the first ones to be fully characterized in 1972. A first report of alternative nitrogenase devoid of molybdenum in Azobacter vinelandii was published in 1980. Genetic analysis of nitrogen fixation and identification of nif genes was initiated in K. pneumoniae, using classical methods, until molecular techniques became available.

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Opening session

The legume-Rhizobium symbiosis: from initial recognition to symbiosome maintenance Ton Bisseling Laboratory of Molecular Biology:Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands The Rhizobium-legume symbiosis involves fascinating interactions between plant and microbe, from the initial recognition up to the maintenance and senescence of the nitrogen-fixing organelles. The development of legume models has provided the means to clone several genes that by forward genetic screens had been shown to play key roles in nodule formation. In addition, transcriptome analysis combined with reverse genetics turned out to be successful in identifying additional key players. These genetically identified key players have been and will be crucial tools to obtain more in-depth insight in processes, such as regulatory networks controlling entrance of rhizobia into plant cells, and the induction of organogenesis, the formation and maintenance of bacteria containing host membrane compartments, and the evolution of nodule symbiosis. The rhizobial Nod factors are most likely recognised during several steps of the interaction. This recognition controls early responses in the epidermis, start of infection thread growth in root hairs, but probably also in the nodule. Studies on signalling networks have been focused on the genetically identified regulators. Although an extension with more common signalling components will be essential, it has already become clear that positive and negative feedbacks play a role. Further, it is striking that several of the Nod factor signalling components are also essential in other intracellular symbioses. Better insight into the function of these signalling components can shed light on why these components are used in these different symbiotic interactions. Ultimately, the rhizobia are hosted in organelle-like structures. Although it has been a long lasting hypothesis that rhizobia enter plant cells by an endocytotic process, it should be kept in mind that until recently, it was questioned whether endocytosis occurred in plants. Knowledge that has now been generated in Arabidopsis thaliana can be used to study endocytosis in model legumes. This knowledge can be used to study the relationship between symbiosome formation and endocytosis. A striking observation is that plasma membrane syntaxins that have been co-opted to function in plant pathogenic fungal interaction are also used in symbiosome formation, pointing to an evolutionary relationship of pathogenecity and symbiosome formation. The evolutionary origin of the legume-Rhizobium symbiosis has also been studied by testing the functionality of the non-legume Nod factor signalling homologues and, surprisingly, these are in general partially functional in nodule formation. However, in other biological systems it has become clear that gene duplication and promoter diversification have been a major driving force in evolution and, so, these aspects need further attention.

Session 1

Signalling in nodulation

Chaired by Jens Stougaard Aarhus, Denmark

Abstracts of the oral presentations

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Session 1: Signalling in nodulation

S1-1 Rhizobium signalling in Lotus japonicus Simona Radutoiu, Lene Krusell, Lene Madsen, Niels Jørgensen, Gitte Vestergaard, Mette Wibroe Nielsen, Anna Jurkiewicz, Niels Sandal, Anne Heckmann, Svend Haaning, Kirsten Sørensen, Finn Pedersen, and Jens Stougaard Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, University of Aarhus, 8000C Aarhus, Denmark Root nodules initiate from cortical cells that dedifferentiate to stem cells, which subsequently re-differentiate and accept a new developmental fate. Lipochitooligosaccharides (Nod factors), secreted by rhizobia function as the morphogenic signals inducing root hair deformation and cell division, leading to the formation of nodule primordia. The LysM receptor kinases NFR1 and NFR5 enable the model legume Lotus japonicus to recognise the Nod factors synthesized by Mesorhizobium loti. We have shown that NFR1 and NFR5 are host-specific determinants distinguishing Nod factors synthesized by different rhizobia. Host plants also control the nodule developmental program and rhizobial infection processes. Mutants as well as natural variants impaired in their ability to bring the symbiotic process to completion were isolated in several legume species. We have used molecular and genetical approaches to characterise and dissect the early events that take place in the roots upon inoculation. High-throughput transcriptomics, and genome-sequencing programs have recently been developed for Lotus japonicus, and their concerted input for a detailed analysis of root nodule development and symbiotic nitrogen fixation will be discussed.

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S1-2 Coordinating epidermal and cortical responses during the initiation of a nodule

Giles E.D. Oldroyd, Jiyoung Kim, Sibylle Hirsch, Yiliang Ding, Alfonso Muñoz, and Allan Downie John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK Two diverse developmental programmes are necessary to generate a nitrogen fixing nodule: bacterial infection via infection threads must be initiated at the root epidermis and the cell cycle must be activated in the root cortex to form the nodule primordium. Genetics suggests that these two developmental programmes can be separated since mutants exist that initiate nodule organogenesis in the absence of bacterial infection and conversely bacterial infection can occur in the absence of nodule initiation. However, during nodulation these developmental processes are highly coordinated to ensure bacterial infection at the site of nodule formation. Nod factor is a key regulator of epidermal responses, while cytokinin is a major regulator of cortical responses. There are a number of common components between the signalling pathways downstream of Nod factor and cytokinin perception during nodule formation: dual functions for the GRAS proteins NSP1 and NSP2 and the putative transcriptional regulator NIN. NSP1 and NSP2 form a complex with CCaMK and early nodulation promoters during Nod factor signalling and an analogous function may exist for the induction of cortical specific nodulation genes. NIN is a negative regulator of Nod factor induced ENOD11 expression, but a positive regulator of ENOD11 during cortical activation of this gene by cytokinin. Furthermore, NIN is a positive regulator of other genes expressed in the cortex during the formation of the nodule. The dual requirement for NIN in both epidermal and cortical developmental programmes suggests that NIN may be a master regulator capable of coordinating these diverse processes.

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PS1-1 A novel protein controls rhizobial infection and mediates post-translational regulation of symbiotic receptor proteins

Benoit Lefebvre1, Ton Timmers1, Malick Mbengue1, Sandra Moreau1, Dörte Klaus1, Laurent Deslandes1, Sylvain Raffaele2, Sebastien Mongrand2, Julie Cullimore1, Pascal Gamas1, Andreas Niebel1, and Thomas Ott1* 1Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France; 2Laboratoire de Biogenèse Membranaire, Centre National de la Recherche Scientifique/Université Victor Segalen Bordeaux 2, UMR5200, 33076 Bordeaux Cedex, France; *present address: Genetics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany Stringent control of signal perception and transduction during the molecular dialogue between legumes and rhizobia are crucial steps to allow and control rhizobial infections. The molecular crosstalk between rhizobia and the host plant involves several essential receptor-like kinases (RLKs), of which most of them have been demonstrated to serve functions at the initial recognition of rhizobia as well as during progression of infection threads (ITs) and bacterial release into the host cells. We identified a novel legume-specific protein that was so far annotated as "unknown" function. It is a member of a so far almost uncharacterised protein family that is solely found within all clades of the plant kingdom. One particular member of this multigene family is almost exclusively expressed in root nodules while transcripts of virtually all other members can be found in all plant organs. This nodulation-specific gene is strongly induced during bacterial infection while induced expression is independent of symbiotic nitrogen fixation and nodule organogenesis per se indicating roles during bacterial infection. Using RNAi-mediated gene silencing, we show that rhizobial infections are aborted at early stages in the root cortex, resulting in the inability of the plant to develop functional nodules. The protein specifically accumulates at the plasma membrane (PM) of nodular ITs and on the symbiosome membrane. The strongest accumulation was detected at sites were bacteria were released from ITs into the plant host cytoplasm. Subcellular localisation and differential extraction of specific PM fractions indicates a presence of the protein in specific PM subdomains. Using several approaches to identify protein-protein interactions we can show that the protein oligomerises and interacts with at least two symbiotic RLKs. Results from several other experiments strongly indicate roles of this protein in spatial post-translational regulation of symbiotic RLKs:a mechanism that has never been described in plants before.

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PS1-2 New class of cation channels essential for perinuclear calcium spiking

Myriam Charpentier1, Rolf Bredemeier2*, Gerhard Wanner3, Enrico Schleiff2*, and Martin Parniske1 1Genetics, and 2Botany, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany; *present address: Cluster of Excellence Macromolecular Complexes, Department of Biosciences, Goethe University, 60439 Frankfurt, Germany Legume-rhizobia symbiosis results in the formation of a new organ, the nitrogen-fixing root nodule. A chemical communication between both partners accompanies the invasion of plant host cells by bacteria and the development of the root nodule. In response to plant-released flavonoids, rhizobia produce lipo-chitooligosaccharide signalling molecules, so-called Nod factors [1]. Early signal transduction in legumes, such as Lotus japonicus and Medicago truncatula, is associated with a succession of tightly orchestrated ion fluxes across different membrane systems of the host cell. The Nod factor perception at the plasma membrane triggers Ca2+ oscillations that are associated with the nucleus [2]. CASTOR and POLLUX are required for Ca2+ spiking. Homology modeling suggested CASTOR and POLLUX might be ion channels. However, experimental confirmation was lacking. Therefore, we performed biochemical and electrophysiological analysis to define their role. Here, we show that CASTOR and POLLUX form two independent homocomplexes in planta. We reconstituted CASTOR in planar lipid bilayers and electrophysiological measurements revealed that CASTOR is a cation channel preferentially permeable to potassium. The potassium permeability of the sequence-related POLLUX could be demonstrated through complementation of potassium uptake-deficient yeast mutants. Our data identify CASTOR and POLLUX as members of a novel class of cation channel preferentially permeable to potassium. CASTOR and POLLUX may act as counter ion channels to facilitate a rapid efflux of charge associated with the calcium efflux. Alternatively, and not mutually exclusive, they may catalyze a nuclear membrane depolarization, leading to the activation of calcium channels responsible for calcium spiking. [1] Lerouge et al. (1990). Nature 344:781-784. [2] Ehrhardt et al. (1996). Cell 85:673-681.

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PS1-3 TOO MUCH LOVE locus is necessary for the long-distance control of nodulation in Lotus japonicus

Shimpei Magori1, Erika Oka-Kira1, Satoshi Shibata2, Yousuke Umehara2, Hiroshi Kouchi2, Shusei Sato3, Satoshi Tabata3, and Masayoshi Kawaguchi1 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; 2Plant Physiology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan; 3Kazusa DNA Research Institute, Chiba 292-0818, Japan Leguminous plants develop symbiotic root nodules to confine soil bacteria, called rhizobia, which supply the hosts with ammonia produced through nitrogen fixation. Since this new organ formation is energetically expensive, the development and the number of nodules should be tightly controlled by the host plants. In Lotus japonicus, this regulation requires the functional HAR1 (CLAVATA1-like receptor kinase gene) in the shoots, suggesting that there exists a long-distance communication between the shoots and the roots. To better understand the molecular mechanism of this long-distance control of nodulation, we have characterized a hypernodulating mutant of L. japonicus named too much love (tml), which was previously produced by ion beam mutagenesis. Compared to the wild-type plants, tml mutants produce much more nodules that densely cover a wider range of the roots. This is a typical hypernodulation phenotype reminiscent of har1 mutants; however, reciprocal grafting experiments showed that tml hypernodulation is root-determined, suggesting that TML might function in the roots rather than in the shoots. Moreover, grafting a har1 shoot onto a tml root stock did not exhibit any obvious additive effect on the nodule number. This observation indicates that a shoot factor HAR1 and a root factor TML participate in the same genetic pathway, constituting the long-distance signalling of the nodule number control. To further investigate its role, map/transcript-based cloning of TML is in progress. The tml mutation was mapped between two SSLP markers, TM0805 and TM0356. Within this region of tml genome, a large deletion (>220 kb) was found and >40 ORFs are predicted in the corresponding interval of the wild-type genome. Microarray analysis revealed that one of the predicted genes is significantly downregulated in tml roots. Hypothesizing that this gene corresponds to TML, we are in the process of a complementation test using Agrobacterium rhizogenes-mediated hairy root transformation.

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PS1-4 ABA coordinates Nod factor and cytokinin signalling during the regulation of nodulation

Yiliang Ding1, Péter Kaló1,2, Craig Yendrek3, Jongho Sun1, John F. Marsh1, Jeanne Harris3, and Giles E. D. Oldroyd1 1Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK; 2Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, 6701 Szeged, Hungary; 3Department of Plant Biology, University of Vermont, Burlington, VT 05405-0086, USA Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here we characterize the nature of abscissic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signalling and impacting on the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signalling through the use of a dominant-negative allele of ABI1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, STA, that dictates the sensitivity of the plant to ABA. Mutations in sta-1 cause a reduction in nodulation and bacterial infection. Our findings suggest that the major sites of STA action for nodulation regulation are beyond the Nod factor signalling pathway. We propose that ABA functions directly on both epidermal and cortical cell programs during nodulation. ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin signalling in the cortex. As such, ABA has the capability of coordinately regulating the diverse developmental pathways associated with nodule formation and as such can intimately dictate the nature of the plant's response to the symbiotic bacteria.

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PS1-5 Study and improvement of the Lotus japonicus-Rhizobium sp. NGR234 symbiosis

Olivier Schumpp, Michèle Crèvecoeur, William J. Broughton, and William J. Deakin Laboratoire de Biologie Moléculaire des Plantes Supérieures, Université de Genève, 1211 Genève 4, Switzerland Lotus japonicus and its alternative microsymbiont, Rhizobium species NGR234 are genetically well characterised. Nodules induced by NGR234 were persistent, did not senesce prematurely, and began to have a positive effect on growth of Lotus 6 to 8 weeks after inoculation. NGR234 is however a relatively ineffective partner for Lotus. Comparisons of nodules induced by its natural microsymbiont Mesorhizobium loti and NGR234 revealed enlarged symbiosomes in the latter. Microarray analyses showed that although nodulins were induced normally after NGR234 inoculation, numerous Lotus genes had altered transcription patterns. Screening NGR234 mutants of Nod factor synthesis and substituent addition identified the fucosyl group on the reducing terminus as being essential for nodule formation. Although Nod factors lacking this fucosyl group stimulate root hair curling and entrapment of NGR234, infection thread initiation was altered. In addition to Nod factors, NGR234 utilizes numerous "secondary" signal molecules that, although not always necessary for every symbiosis, nevertheless govern its host range, sometimes negatively. The major secondary signals are various surface polysaccharides as well as a type-III protein secretion system, of which mutants were tested. One mutation, in an enzyme responsible for the processing of a surface polysaccharide dramatically enhanced the NGR234-Lotus symbiosis. A library of random NGR234 transposon mutants was also applied to Lotus plants, resulting in the identification of several NGR234 mutants with an improved symbiosis (relative to the wild-type strain) –one mutant in particular resulted in Lotus plants comparable in size to those inoculated with M. loti. This work demonstrates the usefulness of using NGR234 to investigate nodule formation in Lotus. In one theme the molecular mechanisms responsible for the inefficiency of the symbiosis with NGR234 can be studied. Secondly use of the newly identified "efficient" NGR234 strains will permit investigation of the NGR234 symbiotic signal molecules and the responses of Lotus to them.

Session 1

Signalling in nodulation

Chaired by Jens Stougaard Aarhus, Denmark

Abstracts of the posters

17


PS1-6 Legume AGP-Extensin –a unique component of the Rhizobium-legume infection process

Nicholas J. Brewin Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK AGP-extensins are a legume-specific family of plant glycoproteins, previously termed legume Root Nodule Extensins (RNEs). They are abundant in the matrix of Rhizobium-induced infection threads. Because AGPE glycoproteins apparently co-evolved with legumes, because they are structurally conserved in legumes and because they are always physically associated with rhizobial cells in the infection thread, it is suggested that they may play a role in regulating the infection process. The following structural features are considered to be of key importance: (i) AGPE is a molecular co-polymer, unique to legumes [1]; (ii) AGPE is related to Gum Arabic Glycoprotein from Acacia senegal [2]; (iii) AGPE is a component of intercellular and transcellular infection threads; (iv) AGPEs from Pisum, Medicago, Lotus, Sesbania, and all other legumes tested are recognised by rat monoclonal antibodies MAC265, MAC236, and MAC204; (v) AGPE is secreted by legume root hairs soon after inoculation with Rhizobium; (vi) AGPE attaches to the bacterial surface, presumably because of its high Lys content; (vii) AGPE is subject to cross-linking by peroxide [3], presumably because of its high Tyr content; (viii) AGPE mRNAs from pea (psRNE1) and Medicago (MtN12) have a conserved 3'-UTR sequence containing multiple UUGU motifs that are putative targets for Pumilio-family (Puf) proteins involved in polarised transport of mRNA. Polarised transport of mRNA could lead to polarized secretion of AGPE at the growing point of infection threads. In the lumen of the infection thread, it is proposed that peroxide-driven cross-linking of AGPEs (and other plant glycoproteins and polysaccharides) can modify the rigidity of the infection thread wall and the fluidity of the infection thread matrix. [1] Rathbun et al. (2002). Mol. Plant-Microbe Interact. 15:350-359. [2] Brewin, N.J. (2004). Crit. Rev. Plant Sci. 23:1-24. [3] Wisniewski et al. (2000) Mol. Plant-Microbe Interact. 13:413-420.

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PS1-7 Towards the identification of secondary messengers and calcium transporters involved in the initiation and regulation of Nod factor-induced calcium spiking

Ward Capoen, Sibylle Hirsch, and Giles Oldroyd Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK To develop a nodule, an impressive signalling network needs setting up and in recent years our understanding of the signalling events leading to this development has improved dramatically. Cloning of genes involved in Nod factor signal transduction include candidate plasma membrane Nod factor receptors and an array of nucleus-localized proteins. Also, calcium imaging has revealed a distinct nuclear calcium oscillation dependent on several of the cloned genes and apparently necessary for nodulation-specific gene expression. A major hiatus in our understanding of Nod factor signalling however is how the signals are transduced from the plasma membrane to the nucleus. Forward genetics techniques have failed to identify candidates for calcium channels and pumps or enzymes involved in the generation of specific secondary messengers. In this regard, an important question remains how the calcium oscillation signature is generated and regulated at the molecular level. Several approaches were undertaken to identify the secondary messengers involved in this and the proteins involved in the generation of the calcium oscillations. A promising candidate messenger is cyclic ADP Ribose (cADPR) as inhibition of cADPR biosynthesis blocks both calcium spiking and nodulin gene expression. This observation might help to narrow down the candidates for the enigmatic calcium channel responsible for Nod factor-induced calcium spiking. On the other side of the calcium balance, calcium ATPases are thought to be required for the resequestration of calcium after Nod factor-induced release. We have made headway in the search for the calcium ATPases involved in this process. We have identified two ATPase genes with a putative nuclear localization that are likely involved in this process. We are currently engaged in the functional analysis of this gene family and their putative roles will be discussed.

19


PS1-8 Nitrogen status and symbiotic competence, a transcriptomic analysis in Lotus japonicus

Selim Omrane1, Alberto Ferrarini2, Enrica D'Apuzzo1, Alessandra Rogato1, Massimo Delledonne2, and Maurizio Chiurazzi1 1Institute of Genetics and Biophysics "Adriano Buzzati-Traverso”, Consiglio Nazionale delle Ricerche, 80131 Napoli, Italy; 2Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, 37134 Verona, Italy Microarray analyses have been already used to identify nitrogen-responsive genes in different plants. Most of the studies have been performed on Arabidopsis to analyze the molecular basis of plant response to nitrate and were designed to test profiles of gene expression after a transient shift of plants to different nitrate conditions [1]. In the case of leguminous plants, the analysis of gene expression profiles in different nitrogen conditions is directly linked to the nitrogen fixation symbiotic performance. In fact, limitation of combined nitrogen in the soil is a prerequisite for the symbiotic interaction and addition of excessive amounts of NO3

-, NH4+, and urea depresses nodulation. The

direct effect of the presence of high concentration of combined nitrogen sources on early steps of the Nod factor-induced signalling pathway has been recently reported, revealing a differential effect of ammonium and nitrate sources on different steps of the transduction pathway [2]. However, little is known about how a different nitrogen status of the plants, as a consequence of different growth conditions prior to the rhizobia inoculation, can affect the state of competence of leguminous plants. Here, we analyze the nodulation response of Lotus plants pre-incubated on 10 mM NH4NO3 inhibitory conditions, before shifting on a nitrogen starvation regime for M. loti inoculation. This treatment affects the symbiotic performance of the Lotus plants and the interval of time needed to re-acquire a full symbiotic competence was measured. A molecular analysis of the Lotus plants grown on different nitrogen conditions, leading to the symbiotic phenotypes, was performed by a microarray analysis (COMBIMATRIX platform) to reveal gene expression profiles in roots. This analysis reveals a set of differentially expressed genes that could affect the nodulation program either through the involvement in a general nitrogen status-dependent plant response pathway or through specific signalling pathway(s) affecting the root response efficiency. [1] Scheible et al. (2004). Plant Physiol. 136:2483-2499. [2] Barbulova et al. (2007). Mol. Plant-Microbe Interact. 20:994-1003.

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PS1-9 Analysis of swarming motility and modeling of swarming behaviour Bachaspatimayum Debkumari, Kristien Braeken, Natalie Verstraeten, Maarten Fauvart, Paul Phillips, Jan Fransaer, Jan Vermant, and Jan Michiels Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium Swarming motility is considered as a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer and the phenomenon has previously been linked with biofilm formation and virulence. Quorum sensing (QS), depending on N-acylhomoserine lactone (AHL)-based LuxIR-type systems, was demonstrated to be involved in the regulation of swarming in several species including Rhizobium etli (the symbiotic partner of the common bean plant) and Pseudomonas aeruginosa. We analysed the involvement of AHLs in swarming and tried to model the swarming behaviour. Swarming was analysed using genetically modified strains, colony morphology was assessed by white-light interferometry, and colony expansion by time-lapse video microscopy. A dual role for AHL signal molecules in controlling swarming behaviour of R. etli was demonstrated. R. etli produces several QS signal molecules via the cinIR and raiIR quorum sensing systems. By analyzing the expression of cin-gusA and rai-gusA fusions and complementation analysis of defined mutants, we demonstrated that the cin system is the major swarming regulator in R. etli [1]. In general, long-chain AHLs (including the cognate AHL produced by CinI) play an important signalling role in swarmer cells. In addition, by measuring surface activities of AHLs, a second role for the long chain AHLs was disclosed as they possess an important surface activity and induce liquid flows, known as Marangoni flows. These are the result of gradients in surface tension. A tentative mathematical model for swarming was built and a simulation of swarming behaviour performed. A dual role for AHLs in swarming is demonstrated and a modeling of swarming behaviour performed. [1] Daniels et al. (2006). Proc. Natl. Acad. Sci. USA 103:14965-14970.

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PS1-10 Seven in Absentia (SINA) proteins interact with SYMRK and affect the infection process during Lotus japonicus root symbiosis

Griet Den Herder1, Satoko Yoshida1,2, and Martin Parniske1,2 1Genetics, Department of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany; 2The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK SINA E3 ligases are part of the proteasomal degradation pathway and act as dimers to ubiquitinate their target proteins. In plants, they function in regulation of lateral root formation [1] and rhizobial infection during Medicago truncatula nodulation [2]. Yeast two-hybrid analysis revealed that Lotus japonicus SINA family members interact with the SYMRK kinase, but not with other receptor kinases such as NFR1 or NFR5. These SINAs show ubiquitination activity in vitro, except for one SINA that has some special characteristics. Its interaction with SYMRK is weaker and specific for the active kinase, and it showed a different subcellular protein localization. However, a SYMRK-specific regulatory function remains possible through potential heterodimerisation with an enzymatically active SINA. The SINA genes are expressed throughout the plant, so functional diversity is probably obtained through posttranslational regulation and the level of turnover via self-ubiquitination. Therefore, dominant negative mutant forms of the SINA genes were ectopically expressed in L. japonicus plants and the consequences for symbiosis studied. This dominant negative form contains an amino acid substitution at a conserved cysteine residue in the RING finger, still allowing dimerisation, but not ubiquitination, resulting in inhibition of endogenous SINA functioning. A delayed nodulation and the formation of fewer nodules turned out to be the consequence of a defective infection thread initiation and elongation in the 35S:SINA1DN lines. These data demonstrate that SINA E3 ligases are essential for infection thread formation and, furthermore, support the hypothesis that SYMRK protein abundance and activity require a very tight control in which posttranslational modifications, such as phosphorylation increase activity [3], while SINA-mediated ubiquitination is needed for proper initiation of the signalling process by either inactivating or relocalising the target protein. [1] Xie et al. (2002). Nature 419:167-170. [2] Den Herder et al. (2008). Plant Physiol., in press. [3] Yoshida & Parniske (2005). J. Biol. Chem. 280:9203-9209.

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PS1-11 Cloning, expression and purification of the Pisum sativum symbiotic receptor kinase SYM10

Elena Dolgikh, Irina V. Leppyanen, A.V. Khodorenko, and Igor Tikhonovich All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia Legume plants are capable of forming root nodules containing symbiotic nitrogen-fixing bacteria, commonly referred as rhizobia. The initiation of the symbiotic relationship involves the recognition by the host plant of rhizobial signals, called Nod factors (NFs). NFs trigger transcriptional and developmental changes within the root, thereby providing the basis for subsequent bacteria entry, infection and nodule formation. In the model legume plants, Medicago truncatula and Lotus japonicus, genetic approaches have identified genes encoding receptor-like kinases (RLKs) possessing extracellular LysM domains, as being essential for NF perception. Positional cloning of orthologous genes in pea (Pisum sativum L.) has been carried out based on microsynteny of genetic maps of P. sativum and the corresponding model legumes. In pea, two genes Sym10 (the ortholog of MtNFP and LjNFR5) and Sym37 (the ortholog of MtLyk3 and LjNFR1) are the most probable candidates. However, concerning the putative NF receptors, experimental proofs about the involvement of these proteins in NF recognition are still lacking. The aim of our study is to purify one of the putative pea receptors, the SYM10, and elucidate its role in NF perception by pea. To address this goal, isolation of the PsSym10 gene and heterologous expression of sequence encoding its extracellular domain in Escherichia coli were carried out. Purification of the histidine-tagged protein was done by affinity chromatography and, as a result, the pea protein SYM10 became available and allowed us to obtain antiserum and to purify polyclonal antibodies. The specificity of the purified antibodies was verified by protein gel blot analysis. At present, we have initiated experiments on the localization of the SYM10 protein at different stages of the symbiosis development. This work was supported by RFBR 07-08-00700, CRDF RUXO-ST-012-00 and RFBR-NWO (06-04-89000) grants.

23


PS1-12 Plant density influence on the nodule formation capacity in Pisum sativum ssp. arvense

Carmen Dragomir, Neculai Dragomir, I. Pet, and I. Chesa Banat's University of Agricultural Sciences and Veterinary Medicine Timisoara, 300645 Timişoara, Romania The root nodule represents a perfectly integrated anatomical unit of the host plant and has the role to put together the atmospheric nitrogen for the plant's benefit. The main purpose of this research is the study of the plant density influence over the way of nodule formation in Pisum sativum ssp. arvense. The experiment was carried out on four variants with different densities: the first with 40 plants/m2 (100 kg/ha); the second with 60 plants/m2 (150 kg/ha); the third with 80 plants/m2 (200 kg/ha); and the fourth with 100 plants/m2 (250 kg/ha). The results pointed out that there was a negative correlation between the plant density and the number of nodules: the number of nodules decreases from 59.9 nodules/plant (with a density of 40 plants/m2) to 14.9 nodules/plant (with a density of 100 plants/m2). Therefore, a signifiant decrease was observed in the weight of formed nodules (between 23.6% to 77%), correlated with the plant density. If the number of formed nodules is reported to the surface unity, an increase could be noted for the density 60 plants/m2, after which a decrease with 40% was observed for the density of 100 plants/m2.

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PS1-13 Rhizobium etli HrpW is associated with the type-III secretion system and can degrade pectic substances

Maarten Fauvart, Bruno Dombrecht, Debkumari Bachaspatimayum, Maarten Vercruysse, Jos Vanderleyden, and Jan Michiels Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium Type-III secretion systems (T3SS) are essential virulence determinants of many phytopathogenic bacteria and are known to secrete at least two classes of proteins. One class consists of actual effector proteins, which are injected directly into the host cell cytoplasm via the T3SS pilus. The second class is composed of helper proteins, which are either part of the extracellular secretion apparatus or which assist in the translocation of effectors. Harpins belong to the latter category. HrpW is typical of a subclass of harpins that have a clear modular structure, much like true T3SS effectors. Its C-terminal domain shows similarity to pectate lyases. The protein was previously characterized in the phytopathogens Erwinia amylovora and Pseudomonas syringae. It was found to bind to pectate, but not to degrade it. A role in virulence was not apparent. Homologues were subsequently discovered in the genomes of other phytopathogenic bacteria as well, such as Ralstonia solanacearum and Xanthomonas spp. Recently, HrpW was shown to contribute to T3SS effector translocation in P. syringae. Surprisingly, a gene encoding a protein similar to HrpW is also present in the T3SS region of the Rhizobium etli genome. R. etli is a nitrogen-fixing microsymbiont of the common bean plant, Phaseolus vulgaris. In order to determine the function of R. etli HrpW in symbiosis, we constructed a hrpW mutant strain and determined its symbiotic phenotype. HrpW does not appear to play a major role in the interaction with P. vulgaris. We also studied the biochemical characteristics of R. etli HrpW by overexpressing and purifying the protein. Unexpectedly, HrpW was found to possess pectate lyase activity. Here, we describe its enzymatic properties and provide in silico evidence for its divergence from enzymatically inert phytopathogenic homologues. Our results suggest an additional function for HrpW-like proteins in microbe-plant interactions that is conserved in some, but not all, family members, indicative either of specialisation or redundancy.

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PS1-14 Nodulated legumes and associated rhizobia from the Indian Thar Desert

Hukam S. Gehlot1, Dheeren Panwar1, Rashmita Parihar1, Harchand R. Dagla1, N.S. Shekhawat1, and Janet I. Sprent2 1Department of Botany, Jai Narain Vyas University, Jodhpur-342001, India; 2Department of Biological Sciences. University of Dundee, Dundee DD1 5EH, UK The hot and arid North-West Indian Thar desert comprises degraded and fragile soils interspersed with saline patches, and receives an annual precipitation of 50 to 100 mm. More than 30 species of native legume belonging all to the three subfamilies of Leguminosae were found to nodulate in this arid environment. The major nodulating genera were the papilionoids Tephrosia, Indigofera, Crotalaria, Rhynchosia, and the mimosoids Acacia, Prosopis and Mimosa. These plants play an important role in providing food, fodder, shelter and medicine for the rural population of the region. Additionally, many of these native plants are good soil binders and symbiotically fix nitrogen to improve fertility of nutritionally poor soils. In natural habitats, nodulation occurs in both annual and perennial plants during the rainy season when plenty of soil moisture is available. After the monsoon, as the soil gradually tends to become hard and dry, the nodules start shrinking and eventually senesce. Newly formed nodules were internally pink due to leghemoglobin. Most of the nodules had indeterminate growth, branched, crotallaroid, mucunoid, and fan shaped having infection zone interspersed with infected and uninfected cells. Desmodioid nodules were seen in Rhynchosia, Alysicarpus and Indigofera oblongifolia. The nodulation pattern and wide-spread occurrence of nodulation at different natural sites point towards a wide availability of compatible rhizobia in the soil. To our knowledge, the presence of active nodules in Acacia jacquemontii, Rhynchosia aurea, Tephrosia leptoclada and Tephrosia falciformis is being reported for the first time. After a first time surveying and finding nodulation in a number of native legumes, rhizobia were isolated from dissected nodules of 22 species from this region. Rhizobial diversity was exhibited in the form of growth characteristics (fast-growing, medium fast, slow, and very slow-growing colonies) and other phenotypic traits of rhizobia, such as high to low acid-producing, high to low EPS producing, high temperature tolerance (42-45°C), as well as low to high NaCl tolerance (0.75% to 8% NaCl). Intrinsic antibiotic resistance properties were also investigated. Isolated rhizobia were found infective to their natural host in case of Indigofera, Rhynchosia, Alysicarpus, Acacia jacquemontii, and Mimosa spp. The results of present investigation will be discussed in relation to the occurrence of nodulation and rhizobial diversity in the arid regions of the Indian Thar Desert.

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PS1-15 Ram1 and Ram2: signalling specificity in mycorrhizal symbiosis John F. Marsh1, Enrico Gobbato1, Michael Schultze2, and Giles E.D. Oldroyd1 1Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK; 2Department of Biology, University of York, York YO10 5YW, UK Legume plants can establish symbiotic interactions with both N2-fixing bacteria and arbuscular mycorrhizal (AM) fungi. Despite dramatic differences between the two processes, common genes have been identified that are required in the early signalling events of both pathways [1]. In order to isolate novel regulatory components necessary for signalling specificity, a fast-neutron mutagenized Medicago truncatula population was screened to identify mutants impaired in AM symbiosis (Myc-), but showing wild-type responses to Sinorhizobium meliloti infection (Nod+). Here, we show the characterization of the two mutants ram1 and ram2 (reduced arbuscular mycorrhiza 1 and 2). In ram1 and ram2 plants, symbiosis with multiple AM fungi is blocked at very early stages, but the capability of inducing hyphal branching in germinated AM fungal spores [2] is retained. The absence of any obvious developmental phenotype together with a normal behaviour in response to pathogens displayed by both ram1 and ram2 plants indicate the highly specific function in symbiosis of the mutated genes. With a combination of traditional marker-based linkage mapping and transcript-based mapping [3], the deleted regions in both ram1 and ram2 mutant plants have been defined and complementation experiments with candidate genes likely to be affected by the deletions are being performed. [1] Parniske M. (2004) Curr. Opin. Plant Biol. 7:414-421. [2] Akiyama et al. (2005). Nature 435:824-827. [3] Mitra et al. (2004). Proc. Natl. Acad. Sci. USA 101:4701-4705.

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PS1-16 Calcium spiking patterns and the role of calcium/calmodulin-dependent kinase CCaMK in lateral root base nodulation in Sesbania rostrata

Ward Capoen1,2,3, Jeroen Den Herder1,2, Jongho Sun3, Christa Verplancke1,2, Annick De Keyser1,2, Riet De Rycke1,2, Sofie Goormachtig1,2, Giles Oldroyd3, and Marcelle Holsters1,2 1Department of Plant Systems Biology, Flanders Institute for Biotechnology, and 2Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium; 3Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK Nod factor (NF) signal transduction in the legume-rhizobial symbiosis involves calcium oscillations that are instrumental to elicit nodulation. To date, Ca2+ spiking has been exclusively studied in legumes with intracellular bacterial invasion via growing root hairs in zone I. However, this mechanism is not the only one by which bacteria gain entry into their legume hosts: the tropical legume Sesbania rostrata can be invaded by rhizobia at cracks caused by lateral root emergence and this process is associated with cell death for infection pocket formation. We show that Ca2+ spiking is important during this lateral root base mode of nodulation because suppression of the calcium/calmodulin-dependent protein kinase (CCaMK) abolishes nodule development, albeit without effects on the intercellular rhizobial colonization. Furthermore, root hair initials at lateral root bases responded to NFs with Ca2+ oscillations with faster and more symmetrical patterns than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca2+ spiking frequency and stimulated intracellular root hair invasion by rhizobia. These data suggest a dual pathway model downstream of NF perception at lateral root bases, with Ca2+ spiking and CCaMK required for nodule development, and the generation of secondary signals independent of CCaMK involved in infection pocket formation.

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PS1-17 Characterization of NO-inducing polysaccharides from Mesorhizobium loti

Masahito Hashimoto1, Keita Tsunemi1, Hisanori Takada2, Kenji Kajiyama2, Daishi Honda1, Yasuo Suda1, Maki Nagata3, Ken-ichi Kucho4, Mikiko Abe4, Toshiki Uchiumi4 1Department of Nanostructure and Advanced Materials, 2Department of Applied Chemistry and Chemical Engineering, 3Graduate School of Science and Technology, and 4Department of Chemistry and Bioscience, Kagoshima University, Kagoshima 890-0065, Japan Mesorhizobium loti is a member of the rhizobia and forms nitrogen-fixing symbioses with several Lotus species. Recently, it was reported that bacterial cells of M. loti and their extracts induced nitric oxide (NO) in the root of L. japonicus. However, the bacterial components responsible for the NO induction are still unclear. In this study, we separated lipopolysaccharides (LPS)/lipooligosaccharides (LOS) from M. loti and analysed their chemical structure and NO-inducing activity in plants. M. loti MAFF303099 was subjected to phenol-hot water extraction to obtain LPS and LOS fractions. Lipid A moiety, polysaccharides, and core oligosaccharide parts were prepared by weak acid hydrolysis followed by chromatographic separation. Mass spectra were obtained by MALDI-QIT-TOF or ESI-Q-TOF. NMR experiments were done on an ECA-600 spectrometer. NO induction in roots was detected by fluorescence microscopy using a DAF-FM diacetate. The LOS fraction was obtained from the aqueous extracts of M. loti, whereas the LPS fraction was recovered from the phenolic phase of the extraction, probably due to their hydrophobic property. Lipid A was composed of two glucosamine and a galacturonic acid as a backbone and five or six fatty acids. The polysaccharide parts consisted mainly of glucose and rhamnose and 6-deoxy-hexose. Both LOS and LPS fractions induced NO in the root of L. japonicus. Polysaccharides and core oligosaccharide parts also possessed the activity. These results suggest that the common structure of the saccharides is responsible for the activity.

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PS1-18 Cytokinin is an important secondary signal for root nodule development in Lotus japonicus

Anne B. Heckmann, Niels Sandal, Simona Radutoiu, Lene H. Madsen, Sabine Zitzenbacher, Leïla Tirichine, Anita Albrektsen, and Jens Stougaard Laboratory of Gene Expression, Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark We have previously isolated three mutant lines that develop spontaneous nodules (snf) in the absence of rhizobia. Further characterisation of spontaneous snf1 and snf2 nodules showed them to be similar to wild-type nodules in respect to nodule structure, up-regulation of early nodulin genes, and repression of nodule formation in the presence of nitrogen in the growth medium. Detailed phenotypic and genetic analysis showed that the snf2 mutant was caused by a gain-of-function mutation in a cytokinin receptor [1]. We have investigated the effect of exogenous application of cytokinins to the growth medium that results in formation of nodule structures and expression of the early nodulin gene Nin in the nodules formed. By testing various symbiotic mutants on plates with cytokinin it was possible to place cytokinin in the signal transduction pathway. We hypothesize that cytokinin is the direct stimulus that initiates the cell divisions shaping the nodule primordia in the root cortical cells as well as co-ordinated cortical and epidermal developmental events. To obtain further insight into the role of cytokinin during nodulation, we investigate the possible changes in cytokinin production and/or re-localisation during nodulation. We have identified several Lotus japonicus genes expected to be involved in cytokinin biosynthesis. The expression pattern of these genes have been investigated following inoculation with rhizobia. The goal of this project is to identify genes playing a role in the regulation of cytokinin levels and genes downstream of the cytokinin receptor involved in the formation of the nodule structure. [1] Tirichine et al. (2007). Science 315:104-107.

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PS1-19 Receptor kinase function in legume-rhizobia symbiosis establishment: analysis of protein partners

Christine Hervé, Csaba Seregélyes, Pablo Badin, Malick Mbengue, Dörte Klaus, Fernanda de Carvalho Niebel, Sylvie Camut, and Julie Cullimore Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France The legume-rhizobia symbiosis is characterized by the ability of bacteria to induce morphogenesis of a new plant organ, the root nodule, in which the bacteria fix atmospheric dinitrogen. This symbiosis is important not only because of its tremendous agricultural and ecological value, but also because of its interest as a model to understand the mechanisms of recognition between two organisms during plant-microbe interactions, symbiotic establishment, and plant organogenesis. The legume-rhizobia symbiosis is initiated by a specific lipochitooligosaccharide signal, the Nod Factor (NF), produced by the rhizobia, which is necessary for recognition, host specificity, infection, and initiation of the nodule primordium. Genetic analysis identified only a few genes involved in perception and transduction of NFs in the plant model Medicago truncatula. Some of these genes encode transmembrane proteins resembling receptor-like kinases (RLKs). Currently, the mechanisms of NF perception and signal transduction by these receptors are largely unknown. To identify new proteins of the NF signal transduction pathway we searched for proteins that interact with the full version of two RLKs, NFP, and DMI2 or only with their cytoplasmic domains. cDNA libraries from young nodules or from NF-treated root hairs have been used in two different yeast two hybrid systems. We could identify several candidates as interactors of DMI2 or NFP. The specificity of these interactions has been analysed in yeast. The challenge is now to demonstrate the ability of the chosen candidates to interact with these receptors in planta and to identify their function in symbiosis establishment. One part of this study is the cellular localization of the partners by using fluorescent reporter proteins. The visualization of the association of proteins inside living cells is tried by the Bimolecular Fluorescence Complementation (BiFC) method.

31


PS1-20 Mowing the GRAS - the role of NSP1 and NSP2 in nodulation Sibylle Hirsch1, Anne B. Heckmann2, Allan J. Downie2, and Giles E.D. Oldroyd1 Departments of 1Disease and Stress Biology and 2Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Nitrogen-fixing bacteria enter a symbiotic interaction with legume species such as Medicago truncatula and Lotus japonicus. During the establishment of the symbiotic relationship, rhizobia produce a lipochitooligosaccharide signal, Nod factor, which activates a specific signalling pathway in the plant that involves oscillations in the cytosolic calcium concentration of root hair cells. Several components of the signalling pathway downstream of calcium spiking have been identified in legumes. NSP1 and NSP2 encode two putative transcriptional regulators in the GRAS protein family shown to be located in the nucleus after Nod factor application. Studies in the Gal4-based yeast two-hybrid system indicate an interaction between NSP1 and NSP2, with the leucine-rich I (LHRI) domain of NSP2 as essential and sufficient. The heterodimerisation between the two GRAS proteins has been confirmed by BiFC experiments in Nicotiana benthamiana. Interestingly, the deletion of the LHRI domain of NSP2 that abolishes the interaction with NSP1 lacks the ability to complement a M. truncatula nsp2 null mutant. This suggests that the heterodimerisation between NSP1 and NSP2 is necessary for nodule formation. Consistent with this hypothesis, we show that a single point mutation in the LHRI domain affects the protein-protein interaction in yeast. This mutation causes a hypernodulation phenotype in L. japonicus that produces multiple nodules impaired in nitrogen fixation. We propose that the GRAS proteins, essential during early Nod factor signalling, also play a role during later stages of nodulation.

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PS1-21 A plasma membrane-associated DNA-binding transcription factor (MtNIN) regulates nodulation gene expression

Jiyoung Kim, Jongho Sun, and Giles E.D. Oldroyd Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK The symbiotic interaction between rhizobia and legumes leads to the induction of the nitrogen-fixing root nodule. NIN is known to function in initiation and maintenance of infection threads in the root epidermis as well as reinitiation of cell division in the root cortex, suggesting that it is a key regulator mediating these processes. Even though NIN plays important roles in nodulation, the molecular mechanism of its action remains unknown. Here, we report that NIN is originally plasma membrane (PM)-localized and appears to relocalize to the nucleus by proteolytic cleavage in response to Nod factors. NIN binds directly to both promoters of early and late nodulation genes through a novel cis-acting element, the Rhizobium Response Element that is very similar to the previously characterized "organ-specific element", known to be critical for late nodulation gene expression. NIN acts as a bifunctional transcription factor having both features of a repressor and an activator in regulation of nodulation genes. This work reveals the first known PM-associated DNA-binding transcription factor and provides evidence that NIN can coordinate early and late nodulation processes.

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PS1-22 Receptor activation studies of the intracellular parts of NFP and LYK3

Dörte Klaus1, Benoit Lefebvre1, Sylvie Camut1, Malick Mbengue1, Christine Hervé1, Michel Rossignol2, Carole Pichereaux2, and Julie Cullimore1 1Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, and 2Plateforme Protéomique, Pôle de Biotechnologie Végétale, Centre National de la Recherche Scientifique, IFR40, 31326 Castanet-Tolosan Cedex, France NFP and LYK3 are two lysine motif-receptor-like kinases (LysM-RLKs).They play essential roles in Nod factor (NF) perception and infection in Medicago truncatula. These proteins are hypothesised to recognise NFs (rhizobial lipochitooligosaccharidic signals) produced by Sinorhizobium meliloti and transduce the NF signal into the cell. The aim of this work is to determine the mode of action of these two receptors in transduction of the NF signal. RLKs reside in plasma membranes and are proteins with a ligand binding and a signalling domain in one molecule. NFP and LYK3 are composed of three extracellular LysM domains, a transmembrane domain, and an intracellular region resembling a serine-threonine kinase. Specifically, this work focuses on the examination of the intracellular domains of NFP and LYK3, in particular how their mode of action is modulated due to the recognition of NFs. In vitro studies have shown that the intracellular part of LYK3 consists of an active kinase, whereas that of NFP showed no kinase activity in vitro and may thus resemble a "Dead kinase". Here, we present data of a more detailed in vitro and in vivo approach to specifically study the mode of action of the LYK3 kinase. This approach comprises mutagenesis of catalytic motifs and predicted phosphorylation sites within the kinase domain. Testing these mutations in phosphorylation and complementation studies will illustrate their biological relevance for the function/action of the LYK3 kinase. Furthermore, we are facing the challenge to purify these proteins from plant material. This will give us the possibility to analyse posttranslational modifications of these receptors due to the application of NFs and/or bacteria by MS/MS. This work is supported by the EU-funded research training network "NODPERCEPTION".

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PS1-23 Characterizing receptors and their ligands involved in development and regulation of legume-Rhizobium symbiosis

Lene Krusell, Svend Haaning, Christina Grossmann, Thomas Boesen, Mick Blaise, Esben Quistgaard, and Jens Stougaard Centre for Carbohydrate Recognition and Signalling. Department of Molecular Biology, University of Aarhus, Aarhus, Denmark. Symbiotic interaction between legumes and rhizobia results in the development of a new organ, the root nodule. The development and restriction of nodule number involves a complex exchange of signals, not only between the two symbiotic partners, but also over long distances, such as between the root and shoot of the plant. To date, several different receptor molecules involved in these processes have been identified (e.g. LjNFR1, LjNFR5, and LjHAR1). Elucidation of how these receptors function can give us an understanding of how symbiotic interactions are established and controlled, which is important for development of a sustainable agriculture in the future. This project focuses on the establishment of a platform for in vitro expression and purification of putative receptors. Both bacterial, yeast and plant expression systems are applied for the production of affinity-tagged proteins that can be purified using affinity chromatography. The Nicotiana benthamiana heterologous protein expression system will be used to investigate the cellular localisation of fluorescence-tagged receptor proteins. Determination of proteins interacting in a receptor complex followed by detection of receptor-ligand interaction will be investigated with the Isothermal Titration Calorimetry and the BiaCore technology.

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PS1-24 Mutation in rosR gene affects surface polysaccharides in Rhizobium leguminosarum bv. trifolii

Jolanta Kutkowska, Monika Janczarek, Anna Skorupska, and Teresa Urbanik-Sypniewska Department of Genetics and Microbiology, M. Curie-Skłodowska University, 20-033 Lublin, Poland Rhizobium leguminosarum bv. trifolii produces various polysaccharides, such as exopolysaccharides (EPSs), capsular polysaccharides, lipopolysaccharides (LPSs), and cyclic β-(1,2)-glucans, that are necessary for establishing a nitrogen-fixing symbiosis with clover. Recently, a rosR gene encoding a transcriptional regulator involved in EPS production has been identified in R. leguminosarum bv. trifolii 24.2 and TA1 strains [1]. Rhizobium leguminosarum bv. trifolii RosR possesses a characteristic Cys2-His2-type zinc-finger motif in the C-terminal domain. This protein shares significant identity with the RosR of Rhizobium etli, which affects the expression of diverse genes, as revealed by genome-wide genetic screening [2]. The rosR frame-shift mutant of R. leguminosarum bv. trifolii formed dry, wrinkled colonies and induced ineffective nodules on clover. Regulatory mutants in rosR of the 24.2 strain formed morphologically changed colonies. These mutants exhibited increased susceptibility to detergents and ethanol, had a different antibiotic sensitivity pattern, and highly reduced the amount of EPS released into the medium. EPSs of rosR mutants showed changed the ratio of the high-molecular-weight to the low-molecular-weight fraction. Moreover, various LPS migration patterns were detected in these rosR mutants. LPS electrophoretic profiles of rosR mutants indicated disturbances in the ratios and mobility of LPSII/LPSIII. [1] Janczarek & Skorupska (2007). Mol. Plant-Microbe Interact. 20:867-881 [2] Bittinger & Handelsman (2000). J. Bacteriol. 182:1706-1713.

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PS1-25 Nod factor signalling during Rhizobium infection Alessandra Lillo, Andreas Untergasser, Ton Bisseling, and René Geurts Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands Mutual recognition of legume plant hosts and Rhizobium bacteria is essential to establish a symbiotic relation. The exchange of biochemical signals appears to be essential for the formation of the root nodular organ as well as bacterial infection, and so forms a basis for host range determination. The underlying mechanisms for recognition of the rhizobial signalling molecules, named Nod factors, and the underlining signalling cascade has been unravelled genetically. In order to define the factors that determine host specificity between rhizobia and legumes we have studied, in Medicago truncatula, the molecular mechanisms involved in Nod factor perception in relation to polar attachment by the bacteria on root hairs. We focused on the process by which Rhizobium provides positional information to the root hair cell that it will use to penetrate the plant root. This is essential because to enable infection the root hair should grow around a single bacterium that subsequently becomes entrapped in the pocket of the curl. From there penetration starts. To be able to provide positional information it is essential that the bacterium attaches firmly to the plant cell wall. We have found evidence that Rhizobium firm polar attachment requires secretion of a plant component of unknown nature, which is Nod factor signalling driven. Furthermore, we demonstrate that this component is not a flavonoid. Characterization of this component is the aim of this research project.

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PS1-26 NodI and NodJ genes of Ensifer fredii HH103 play a role in Nod factor secretion efficiency

Carmen García Trigueros1, Marta S. Dardanelli2, Hamid Manyani1, and Manuel Megías1 1Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; 2Universidad Nacional de Rio Cuarto, Rio Cuarto, Córdoba, Argentina Soybean is actually one of the most important and extensively grown crops in the world. It accounts for almost 30% of the world's processed vegetable oil and it is a rich source of vegetable protein for food and feed. Ensifer fredii HH103 is a fast-growing soybean symbiont able to nodulate Asiatic and American soybean cultivars as well as a great variety of legumes forming determinate and indeterminate nodules [1]. Successful legume nodulation by rhizobia results from a series of complex plant-microbe interactions. Plant flavonoid compounds exuded from roots can induce the expression of nodulation (Nod) genes in compatible rhizobia. Nod gene expression leads to the synthesis and secretion of lipochitooligosaccharide molecules, called Nod factors. These molecules are secreted to the extracellular medium through a type-I secretion system consisting of NodIJ proteins in which NodI is an ABC-ATPase and NodJ is a transmembrane protein [2]. The main purpose of this work was to identify and characterize Nod genes involved in Nod factor secretion by genetic and symbiotic analysis of E. fredii HH103 nodI and nodJ mutants. In order to set the individual role of NodI and NodJ genes in Nod factors secretion, we have performed individual non-polar mutagenesis of both genes and the double nodIJ non-polar mutant in E. fredii HH103 genetic background. We have studied Nod factors production and secretion in E. fredii HH103 nodI, nodJ and nodIJ mutants. We have also determined the effect of the absence of NodI and NodJ genes on nodulation in Glycine max. Results obtained in this work demonstrate that while both nodI and nodJ play an important role in the efficiency of Nod factors secretion, only the NodJ gene is essential in the establishment of effective symbiosis between E. fredii HH103 and G. max. [1] Dowdle & Bohlool (1985). Appl. Environ. Microbiol. 50:1171-1176. [2] Spaink et al. (1995). J. Bacteriol. 177:6276-6281.

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PS1-27 Role of nodD1 gene of Rhizobium tropici CIAT899 in symbiosis establishment in abiotic stress conditions

Esau Megías1, J. Estevez1, Beatriz Guasch2, I. del Castillo1, M. Camacho4, M. Albareda4, M.E. Soria3, Miguel A. Rodríguez-Carvajal2, and Hamid Manyani1 Departments of 1Microbiology and Parasitology, 2Microbiology, and 3Organic Chemistry, University of Sevilla, 41012 Sevilla, Spain; 4Centro de Investigación y Formación Agraria, Instituto de Investigación y Formación Agraria y Pesquera, Centro Las Torres-Tomejil, 41200 Sevilla, Spain Grain legumes are able to establish symbiotic relationships with a group of soil microorganisms, collectively called rhizobia. The Rhizobia-legume symbiotic interaction is a complex process that requires a sequence of highly regulated events, involving the coordinated expression of both plant and bacterial genes. Nod factors are produced and secreted by rhizobia in response to plant exudates. Nod factors perception by the plant induces several morphological and physiological changes in the roots, which are essential for successful nodulation. Rhizobium tropici CIAT899 is a bean symbiont with at least four copies of nodD regulatory gene. In this work, we have mutagenized the nodD1 gene to determine its role on Nod factor production and symbiosis establishment. TLC analysis of Nod factor production, indicated the lack of LCO synthesis in the nodD1 mutant. However, this mutant was able to nodulate Phaseolus vulgaris and Leucaena leucocephala plants, though less efficiently than the parental strain. The structural analysis of Nod factors by mass spectrometry showed the presence of LCO molecules that could be responsible for nodulation on common bean. TLC analysis of Nod factors, performed under abiotic stress conditions without flavonoid induction, indicated that while the LCO biosynthesis is independent of nodD1 under salt stress conditions, it is nodD1-dependent under acid stress conditions. Divergences observed in Nod factors induction under different abiotic stresses suggest the existence of different response pathways. This work has been supported by Junta de Andalucía Excellency Project number 18.10.03.27.01-2006/596.

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PS1-28 Is Nod factor-induced calcium flux required for the infection of legumes by rhizobia?

Giulia Morieri, Giles E.D. Oldroyd, and J. Allan Downie John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK In the rhizobia/legume symbiosis bacterial infection results in the formation of nitrogen-fixing root nodules. The establishment of this symbiosis requires the plant to perceive a bacterially derived molecule, called Nod factor. Low concentrations of Nod factor can induce many events associated with nodulation, including periodic calcium oscillations, termed calcium spiking. However, with the application of higher concentrations of Nod factor an additional calcium flux precedes calcium spiking. It has been proposed that such high concentrations of Nod factor may be found when the bacteria become entrapped in a curled root hair and may play a role in infection thread formation. In order to test this hypothesis, structurally modified Nod factors released by bacterial mutants (NodFL and NodL), which are compromised for infection, were analysed for induction of calcium flux. Nod factor-induced calcium flux was also analysed in hcl mutants of Medicago truncatula that are defective for infection thread growth. In this work, we show that the calcium flux requires structural specificity of Nod factor and the HCL gene. NodFL and NodL Nod factors were unable to induce calcium flux and the hcl mutant was also compromised for calcium flux. HCL have been proposed to recognise structural specificity of Nod factor; therefore, we tested the induction of the calcium flux induced by NodL Nod factor in hcl mutants. We found that the hcl mutant is unable to discriminate between wild-type and NodL Nod factor for induction of calcium flux. Taken together, our results suggest that the induction of calcium flux correlates with the genetic compounds in the plant and the bacteria that are associated with bacterial infection.

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PS1-29 Identification of plant signals that induce symbiotic changes in the Nod-independent bacterium Bradyrhizobium ORS278

Nico Nouwen, Adeline Rénier, and Eric Giraud Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France Photosynthetic rhizobia from the genus Bradyrhizobium are capable of forming nitrogen-fixing nodules on the roots and stems of Aeschynomene species (a tropical aquatic legume). In the classical Nod-dependent Rhizobium-legume interactions, establishment of the symbiosis depends on the continuous exchange of molecular signals between the plant and the bacteria. Initially, molecular signals from the plant (in most cases flavonoids) induce the synthesis and secretion of lipochitooligosaccharides (LCOs or Nod factors) by the rhizobia. In turn, the Nod factors trigger the formation of a nodule meristem in the plant, whereas at later stages, additional bacterial signals, such as surface polysaccharides or secreted proteins further add to the efficient nodulation of the plant. Recently, the genomes of two photosynthetic Bradyrhizobium strains, Bradyrhizobium BTAi1 and Bradyrhizobium sp. ORS278 have been sequenced. Remarkably, the analysis of the sequencing data showed that both genomes do not contain genes encoding the NodA, NodB, and NodC proteins [1]. As these three proteins play a pivotal role in the synthesis of the LCO backbone structure, this observation implies that these two photosynthetic Bradyrhizobium strains use novel signalling molecules for communication with the plant. As so far nothing is known about the exchange of molecular signals between Aeschynomene indica and Bradyrhizobium ORS278, we decided to identify the plant signals that induce physiological changes in Bradyrhizobium ORS278 and are related to symbiosis in other rhizobia. For this purpose, we are currently identifying flavonoids present in the root exudate of Aeschynomene indica and testing if these and other pure flavonoids alter the LCO and (exo)polysaccharide pattern of Bradyrhizobium ORS278. Recent results will be presented. [1] Giraud et al. (2007). Science 316:1307-1312.

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PS1-30 Is a fast and transient intracellular ROS response a specific signature of the legume-rhizobia symbiosis?

Luis Cárdenas, Adán Martínez, Olivia Santana, Noreide Nava, Federico Sánchez, and Carmen Quinto Departamento de Biología Molecular de Plantas. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, Mexico The production of reactive oxygen species (ROS) in root-nodule development and metabolism has been studied to a great extent. Nevertheless, there is limited evidence showing ROS changes during the earliest steps of the legume-rhizobia interaction. Herein, using ratio-imaging analysis, transient ROS levels were detected at the tip of actively growing root hair cells within seconds after addition of Nod factors (NFs). This transient response (which lasted up to 3 minutes) was NF-specific because chitin oligomers (pentamers) failed to induce a similar response. When chitosan, a fungal elicitor, or ATP was used instead, a sustained signal that continuously increased was found. Since ROS levels are transiently elevated after NF addition, we propose that this ROS response is a characteristic of the symbiotic interaction. Furthermore, we discuss the remarkable spatial and temporal coincidences between ROS and transiently increased calcium levels observed at the tip of root hair cells immediately after NF perception.

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PS1-31 Sinorhizobium fredii HH103 ndvB mutants Juan Carlos Crespo1, Ángeles Hidalgo1, Isabel Margaret1, Ana Buendía-Clavería1, Antonio Gil2, Javier Lloret3, Javier Ollero1, María Reguera3, Miguel Ángel Rodríguez2, Dulce N. Rodríguez4, José E. Ruiz-Sainz1, and José M. Vinardell1 Departments of 1Microbiology and 2Organic Chemistry, University of Seville, 41012 Sevilla, Spain; 3Department of Biology, Universidad Autónoma de Madrid, 28049 Madrid, Spain; 4Instituto de Investigación y Formación Agraria y Pesquera, Centro Las Torres-Tomejil, 41200 Sevilla, Spain Sinorhizobium fredii HH103 produces neutral and anionic cyclic glucans (CGs) of 18 to 24 glucose residues in which sn-1-phosphoglycerol is the only substituent. The S. fredii HH103 ndvB gene was sequenced and mutated by inserting the lacZ-gentamicin-resistant cassette. The mutant SVQ562 did not produce CGs, was immobile, and grew more slowly in the hypoosmotic GYM medium. However, the survival of SVQ562 in distilled water was not different from that of its parental strain HH103 RifR. Lipopolysaccharides (LPSs), K-antigens (KPSs), and outer nodulation proteins (ONPs) produced by SVQ562 did not show any detectable alteration. SVQ562 showed increased exopolysaccharide (EPS) production and real time-RT-PCR experiments indicated that the exoA gene was transcribed at higher levels. In GYM medium, part of the EPS produced by SVQ562 had a much higher molecular mass (2000 kDa or more) than that produced by the parental strain HH103 RifR. NMR experiments indicated that the EPS produced by SVQ562 either in the hypoosmotic GYM medium or in GYM supplemented with 100 mM NaCl contains higher levels of substituents (pyruvate and acetate) than that of HH103 RifR. These differences were not observed when bacteria were grown in YMB medium. The expression of the SVQ562 ndvB::lacZ fusion was influenced by the pH and the osmolarity of the growth medium. S. fredii ndvB mutants SVQ561 (ndvB::Ω cassette) and SVQ562 only formed pseudonodules in Glycine max (determinate nodules) and Glycyrrhiza uralensis (indeterminate nodules). Macroscopic root responses were not observed in Vigna unguiculata (determinate nodules) roots inoculated with HH103 RifR ndvB mutants, although the bacteria produced nodulation factors and induced root nodule primordia. It is concluded that the nodulation process induced by S. fredii ndvB mutants is aborted at earlier stages in V. unguiculata than in G. max. This work was supported by grant BIO2005-08691-CO2-01/02.

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PS1-32 Symbiotic properties of Sinorhizobium fredii HH103 mutants unable to produce KPS

Isabel Margaret1, Ángeles Hidalgo1, Juan Carlos Crespo1, Miguel Ángel Rodríguez2, Antonio Gil2, Javier Moreno3, Ildefonso Bonilla4, Javier Lloret4, María Reguera4, Ana Buendía-Clavería1, Javier Ollero1, Maribel Parada1, José M. Vinardell1, and José E. Ruiz-Sainz1 Departments of 1Microbiology, 2Organic Chemistry, and 3Cell Biology, University of Seville, 41012 Sevilla, Spain; 4Department of Biology, Universidad Autónoma de Madrid, 28049 Madrid, Spain Capsular polysaccharides (KPSs) are involved in the symbiotic capacity of Sinorhizobium fredii HH103 because Glycine max (soybean) and Cajanus cajan plants inoculated with HH103 rkpG or rkpH mutants showed reduced nodulation and severe symptoms of nitrogen starvation [1]. In S. meliloti AK631, genes involved in KPS transport are clustered in the rkp-1 and rkp-3 regions while those responsible for the biosynthesis of the KPS repeating unit are mainly located in the rkp-3 region. Different genes of the S. fredii HH103 rkp-1 (rkpA, rkpI, rkpJ, and rkpU) and rkp-3 (rkpM) regions have been mutated. RMN experiments showed that none of the mutants produced the wild-type KPS (a homopolymer of a pseudaminic acid derivative). All the rkp-1 region mutants tested showed reduced nodulation and nitrogen-fixation capacity with soybean cv. Williams and Glycyrrhiza uralensis. In Vigna unguiculata (cowpea), the three rkp-1 mutants tested (rkpA, rkpJ, and rkpU) were as effective as the parental strain. Thus, KPS appears to be important for the symbiosis with soybean but not with cowpea. The mutant affected in rkpM only formed pseudonodules with soybean and small ineffective nodules with cowpea plants (both legumes form determinate nodules). In G. uralensis (indeterminate nodules), the rkpM mutant was still able to form some nitrogen-fixing nodules. LPS electrophoretic profiles of rkp-1 mutants were unaffected while that of that of the rkpM mutant was clearly altered. We conclude that the symbiotic impairment showed by the rkpM mutant, which is more severe than that shown by mutants in the rkp-1 region, could be due to alterations in the LPSs rather than to the bacterial incapacity to produce KPSs. This work was supported by grant BIO2005-08691-CO2-01/02. [1] Parada et al. (2006). Mol. Plant-Microbe Interact. 19:43-52.

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PS1-33 Role of CCaMK in nodulation of a non-"infection thread" legume Arachis hypogea

Senjuti Sinharoy and Maitrayee DasGupta Department of Biochemistry, University of Calcutta, Kolkata 700019, India Arachis hypogea is a Papilionoid legume in which aeschynomenoid nodules are formed following rhizobial invasion through "cracks" that develop with lateral root emergence. "Crack invasion" bypasses the complex processes involved in epidermal infection via root hairs and is considered to be primitive. Transcellular infection threads are never formed in aeschynomenoids. In "infection thread" (IT) legumes, formation of nodule primordia in the cortex and bacterial invasion in the epidermis is uncoupled. In aeschynomenoid nodules, infected cells are not interspersed with uninfected ones, suggesting the process of bacterial infection and cortical cell division to be coupled for giving rise to the characteristic uniformly infected central tissue. The calcium/calmodulin-dependent protein kinase, CCaMK, functions immediately downstream of calcium spiking in the nodulation pathway. In IT legumes, autoactivated CCaMK has been found to induce nodule organogenesis in the cortex in the absence of bacterial or Nod-factor elicitation. Considering the critical importance of CCaMK in nodulation signalling, our objective was to investigate the role of this kinase in a non-IT legume, such as Arachis hypogea. We have cloned CCaMK (AhCCaMK) from Arachis hypogea roots (Genbank accession no EU395429). The expressed full length kinase shows calcium independent autophosphorylation and calcium/calmodulin-dependent substrate phoshorylation activity. A partial reduction of CCaMK in transgenic roots developed nodules with unusual ultrastructural features and drastically reduced the number of nodules per plant, indicating that CCaMK is functional in Arachis and is required for its nodule formation. But in contrast to IT legumes, overexpression of autoactivated AhCCaMK in Arachis transgenic roots failed to generate empty nodules, though the kinase was active in vivo, and was localized in the nucleus. Our results indicate that in primitive legumes, such as Arachis, activation of CCaMK needs to be coupled with other activation principles for initiating nodule organogenesis. The implication of the results would be discussed.

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PS1-34 Indole-3-acetic acid-regulated genes in Rhizobium etli CNPAF512 Stijn Spaepen, Frederik Das, Ellen Luyten, Jan Michiels, and Jos Vanderleyden Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium Many rhizobial strains are capable of producing the auxin indole-3-acetic acid (IAA). However, the exact role of IAA for the bacterial partner in the legume-Rhizobium symbiosis is not known yet. To identify the effect of IAA on bacterial gene expression, a transposon (mTn5gusA-oriV) mutant library of Rhizobium etli, pre-screened for differential gene expression in conditions that mimic symbiotic conditions, was screened for altered gene expression upon IAA addition. Four mutants showed altered gene expression when tested in presence and absence of IAA. These mutants corresponded to four different gene fusions. DNA sequence analysis revealed that these genes are involved in plant signal processing, motility and attachment to plant roots, clearly demonstrating a distinct role for IAA in the bacterial partner of the legume-Rhizobium interactions.

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PS1-35 Differential and chaotic calcium signatures in the symbiosis signalling pathway of legumes

Jongho Sun1, Sonja Kosuta1, Saul Hazeldine2, Hiroki Miwa3, Richard J. Morris2, J. Allan Downie3, and Giles E.D. Oldroyd1 1Departments of Disease and Stress Biology, 2Computation and Systems Biology, and 3Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Legumes fulfill a significant amount of their nutritional needs through symbiotic interactions with both nitrogen-fixing rhizobial bacteria and mycorrhizal fungi. The establishment of these symbioses involves a molecular dialogue between the plant and the microorganisms, with the diffusible signals the Nod and Myc factor(s) activating respectively rhizobial and mycorrhizal responses in the plant. The perception of Nod and Myc factors by the plant involves a conserved symbiosis (Sym) signalling pathway. Oscillations in calcium, termed calcium spiking, act as a secondary messenger in the plants' response to Nod factor and the recognition of this calcium signal is the function of a calcium and calmodulin dependent protein kinase (CCaMK), a component of the Sym pathway. Here we show that mycorrhizal fungi also activate an oscillatory calcium response and this appears to be the function of a diffusible Myc factor. The structure of this calcium response is very different to Nod factor induced calcium spiking. However, spatial resolution of Myc factor induced calcium oscillations are similar to Nod factor induced calcium spiking which is mostly restricted to the cytosol associated with the nucleus. Nod factor and Myc factor induced calcium oscillations show features of chaotic behaviour. Chaotic systems are inherently more flexible than stable linear systems, and thus this chaotic nature should allow a pliant signal transduction pathway with the flexibility to differentially respond to these two symbiotic signals. This work provides a mechanisms by which CCaMK can be differentially activated by these divergent calcium signatures, and this may define specificity in this signalling pathway.

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PS1-36 Expression of a class 1 hemoglobin gene and production of nitric oxide in response to rhizobia and plant pathogens in Lotus japonicus

Toshiki Uchiumi1, Maki Nagata2, Ei-ichi Murakami2, Yoshikazu Shimoda3, Fuyuko Shimoda-Sasakura4, Ken-ichi Kucho1, Masahito Hashimoto5, Akihiro Suzuki6, Shiro Higashi1, and Mikiko Abe1 1Department of Chemistry and Bioscience, and 2Graduate School of Science and Technology, Kagoshima University, Kagoshima 890-0065, Japan; 3National Institute of Agrobiological Sciences, Chiba 292-0818, Japan; 4Frontier Science Research Center, Kagoshima University, Kogoshima, Kagoshima 890-8520 Japan; 5Department of Nanostructure and Advanced Materials, Kagoshima University, Kagoshima 890-0065, Japan; 6Department of Agricultural Sciences, Saga University, Saga 840-8502, Japan Rhizobium-legume symbiosis is the important biological system in the global nitrogen cycle, and some molecular aspects during the early stage of host-symbiont recognition have been revealed. However, it is not be well understood whether the plant immune system functions during symbiotic process between rhizobium and its host plant. To understand the responses of a host plant against various bacteria, we examined the expression of a class 1 hemoglobin gene LjHb1 and the production of nitric oxide (NO) in Lotus japonicus after inoculation with rhizobia or plant pathogens. NO induces plant defense system and class 1 hemoglobins control various physiological processes of plants by modulating NO level [1, 2]. When the symbiotic rhizobium Mesorhizobium loti was inoculated, expression of LjHb1 and NO production were induced transiently in the roots at 4 h after inoculation [3]. In contrast, inoculation with the nonsymbiotic rhizobia Sinorhizobium meliloti and Bradyrhizobium japonicum induced neither expression of LjHb1 nor NO production. When L. japonicus was inoculated with plant pathogens (Ralstonia solanacearum or Pseudomonas syringae), continuous NO production was observed in roots, but induction of LjHb1 did not occur. These results suggest that modulation of NO levels and expression of class 1 hemoglobin are involved in the establishment of the symbiosis. [1] Seregélyes et al. (2003). Plant Sci. 165:541-550. [2] Seregélyes et al. (2004). FEBS Lett. 571:61-66. [3] Shimoda et al. (2005). Plant Cell Physiol. 46:99-107.

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PS1-37 Transferring eight key regulators required for NOD factor signalling to non-legumes

Andreas Untergasser, Marijke Hartog, Ton Bisseling, and René Geurts Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University and Research Centre, 6700 ET, Wageningen, The Netherlands Since the beginning of legume research, scientists are fascinated by the vision to transfer the ability to fix nitrogen to non-legumes. The intensive research in the past years identified key genes involved in this process. Interestingly, a homologous set of genes was found in mutant screens in Medicago, Lotus, and pea, ranging from receptors on the membrane over calcium calmodulin kinases up to transcription factors in the nucleus. The high overlap in the results of these screenings suggested that they are close to saturation. But do these genes contain all the information needed to enable plants for symbiosis? To answer this question we cloned the receptors Lyk3 and NFP, the three DMI genes, and the transcription factors NSP1 and NSP2 in their genetic context. This resulted in a 100-kb plasmid that was transferred into the non-legumes tobacco, poplar, and strawberry. We will show the method of cloning a complete signalling pathway, the transfer of these artificial BAC into different plant species, and the initial results.

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PS1-38 Characterisation and phylogenetic analysis of LysM receptor-like kinase genes in Lotus japonicus

Gitte Vestergaard, Mette Wibroe Nielsen, Y. Shimoda, Satoshi Tabata, Shusei Sato, Simona Radutoiu, Lene H. Madsen, and Jens Stougaard Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark In Lotus japonicus, two LysM receptor kinases LjNFR1 and LjNFR5 have been identified as Nod factor receptors initiating a signal cascade leading to symbiotic nodule development in response to inoculation with rhizobia. Similar genes have also been identified in other legumes: Medicago truncatula, Glycine max, and Pisum sativum. In Arabidopsis thaliana, five LysM-RLK genes are present, one of which, AtCerk1, has been identified as the chitin receptor. This study presents the identification of 13 additional LysM receptor-like kinase (LysM-RLK) genes in L. japonicus based on a search in the L. japonicus genome database. Their location on the L. japonicus genome has been determined as well as their gene structure based on cDNA genomic sequence alignment. A detailed phylogenetic analysis of the newly identified LysM-RLK genes will be presented.

50


PS1-39 Functional characterisation of LysM receptor-like kinase genes in Lotus japonicus

Mette Wibroe Nielsen, Gitte Vestergaard, S. Bucholdt, Kirsten Sørensen, Jesper B. Christensen, Y. Shimoda, Satoshi Tabata, Shusei Sato, Lene H. Madsen, Simona Radutoiu, and Jens Stougaard Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark LjNFR1 and LjNFR5 are LysM receptor kinases mediating Nod-factor perception and initiation of a downstream signalling cascade that leads to nodule development in Lotus japonicus. A search in the L. japonicus genome sequence database followed by in silico analysis has revealed the presence of at least 13 LysM receptor-like kinase (LysM-RLK) genes in addition to LjNfr1 and LjNfr5. The role of the L. japonicus LjNfr1/LjNfr5 homologous genes is so far unknown. This study presents a detailed expresssion analysis of the new LysM-RLK genes using Affymetrix array and quantitative RT-PCR. The spatio-temporal expression of selected homologs has been analysed by promoter GUS fusion in transformed hairy roots. A possible involvement of different L. japonicus LysM-RLK genes in the response to chitin and mycorrhiza is under investigation. In order to get more insight into their function, a reverse genetic approach employing RNAi and TILLING has been initiated for a selected subset of genes. The TILLING and RNAi lines obtained are currently being analysed for their ability to form symbiosis with rhizobia and mycorrhizal fungi.

51


PS1-40 A novel polar attachment factor in Rhizobium leguminosarum 3841 induced by pea root exudates

Fang Xie, Alan Williams, and J. Allan Downie Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Attachment to root hairs is an early step for rhizobia to infect and nodulate legume plants. In addition to attaching to legume roots, rhizobia can attach to inert surfaces and form biofilms. Rhizobium leguminosarum bv. viciae forms a three-dimensional biofilm when grown in minimal medium static culture. The biofilm forms over a period of 4 days, begins with single cells attaching laterally to glass, and then the attached cells begin to form microcolonies by division and by attachment of planktonic cells. When pea root exudate was added to the minimal growth medium, it induced a switch from lateral attachment to glass to polar attachment. Tests with root exudates from others legume and non-legume plants revealed that the polar attachment was present, even in Arabidopsis thaliana root exudates. Currently, we are trying to purify and identify the polar attachment factor. To identify the R. leguminosarum genes required for polar attachment, we have constructed several mutants. These include mutants that affected the production of exopolysaccharides and surface proteins including flagellar and chemotaxis components. Mutations affected the synthesis of cellulose, glucomannan, gel-forming exopolysaccharide, and type-I secretion system did not affect root exudate-induced polar attachment. Mutations affecting LPS and the acidic EPS alter the biofilm such that it was difficult to know whether the polar attachment occurs in response to root exudates.

52


PS1-41 Daidzein transforms Sinorhizobium fredii USDA191 NodD1 into its activated mode with enhanced solubility and stability

Ken-ichi Yoshida1, Yohei Takada1, Hari B. Krishnan2, and Hitoshi Ashida1 1Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan; 2Plant Genetics Research Unit, Department of Agronomy, University of Missouri, Columbia, MO 65211, USA Sinorhizobium fredii USDA191 forms nitrogen-fixing nodules on soybean. During the early stages of the plant-microbe symbiosis, nodulation inducers such as flavonoids released from the plant root activate the bacterial transcription factors, NodD1 and NodD2, to induce the expression of bacterial genes required for nodulation, including nodABC. Daidzein, a potent inducer of nodulation genes, is known to complex with NodD1. NodD1 binds to the nod-box promoter sequences located upstream of nod genes and activates their transcription. When USDA191 cells were stimulated by daidzein, NodD1 was able to bind to the nodABC promoter region. DNA binding of NodD1 was not affected by the addition of daidzein in vitro, suggesting that activation of NodD1 could require some cellular function triggered by daidzein. A specific antibody was raised against the recombinant His-tagged NodD1 produced in Escherichia coli, and used for immunobolt analysis of the subcellular distribution of NodD1. USDA191 cells grown in the presence and absence of daidzein were disrupted by the treatment with lysozyme and sonication, and then separated by centrifugation into soluble and precipitated cell debris fractions. NodD1 mostly accumulated in the precipitated cell debris in the absence of daidzein; however, in its presence almost twice more NodD1 was distributed in the soluble fraction, suggesting that daizdein might enhance solubility of NodD1. Addition of chloramphenicol to the culture media to arrest protein synthesis resulted in higher amounts of NodD1 in the soluble fraction, indicating that NodD1 with the enhanced solubility was more stable against proteolysis. Taken together, our data suggest that daidzein may stimulate the unidentified cellular function to transform NodD1 into its activated mode through enhanced solubility and stability.

53


PS1-42 Expression analysis of the type-III secretion system of Bradyrhizobium japonicum

Susanne Zehner, Grit Schober, Mandy Wenzel, and Michael Göttfert Institut für Genetik, Technische Universität Dresden, 01069 Dresden, Germany Bradyrhizobium japonicum is able to establish symbiosis with different legumes, such as soybean, Vigna unguiculata and Macroptilium atropurpureum. Symbiosis is influenced by a type-III secretion system (T3SS) [1]. To test whether the T3SS is expressed in early or late stages of symbiosis, we used lacZ reporter gene fusions with various genes coding for secreted proteins or the secretion machinery. We found reporter activity in the rhizosphere as well as in fully developed nodules. Interestingly, genes of the T3SS are much more strongly expressed in M. atropurpureum than in soybean and V. unguiculata. The expression of tts genes in nodules depended on TtsI, a two-component response regulator. A conserved sequence motif, the tts box, was identified in the promoter region of genes encoding secreted proteins and the secretion machinery [1]. In order to analyse the function of the tts box, we mutated the tts box upstream of nopB and measured transcriptional activity after flavonoid induction. A deletion of the 30-bp conserved motif abolished transcription. Modification of two or more conserved nucleotides strongly reduced activity. These data demonstrate that the tts box is an essential promoter element [2]. [1] Krause et al. (2002). Mol. Plant-Microbe Interact. 15:1228-1235. [2] Zehner et al. (2008). Mol. Plant-Microbe Interact. 21:1087-1093.

54


PS1-43 Two soybean kinase-associated protein phosphatases are phosphorylated by nodule autoregulation receptor kinase in vitro

Akira Miyahara1,4, Tripty A. Hirani1, Marie Oakes3, Attila Kereszt1, Bostjan Kobe2, Giles Oldroyd4, Michael A. Djordjevic3, and Peter M. Gresshoff1

1Australian Research Council Centre of Excellence for Integrative Legume Research, and 2School of Microbial and Molecular Sciences and Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072, Australia; 3Australian Research Council Centre of Excellence for Integrative Legume Research, Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia; 4Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK A number of legume genes control the systemic regulation of nodulation (AON). Nodule number and nodule distribution in seedlings are AON controlled, involving at least two signals, a root-derived activation signal (termed Q) and a leaf-derived nodule progression inhibitor (SDI). AON-deficient mutants show a supernodulation or hypernodulation phenotype as well as yet unexplained pleiotropic responses, such as altered root growth. The Nodule Autoregulation Receptor Kinase (NARK) gene (equivalent to MtSUNN and LjHAR1), which regulates proliferation of nodule primordia in several legumes, encodes a receptor kinase that is composed of an extracellular leucine-rich repeat and an intracellular serine/threonine protein kinase domain. The putative catalytic domain of GmNARK was expressed and purified as a maltose-binding or a glutathione S-transferase fusion protein in Escherichia coli. The recombinant GmNARK proteins showed autophosphorylation activity in vitro. The kinase-inactive protein K724E failed to autophosphorylate, as did three other proteins corresponding to phenotypically detected mutants defective in whole-plant AON. A wild-type GmNARK fusion protein transphosphorylates a kinase-inactive mutant fusion protein, suggesting that it is capable of intermolecular autophophorylation in vitro. In addition, two closely related soybean kinase-associated protein phosphatases (GmKAPP1/2) were isolated as putative interacting components of GmNARK. Transphosphorylation experiments showed that recombinant GmKAPP proteins are substrates of GmNARK in vitro. Furthermore, autophosphorylated GmNARK was, in turn, dephosphorylated by both GmKAPP1 and GmKAPP2. These results suggest a model for the signal transduction pathway involving GmNARK in nodule development control.

Session 2

Cyanobacterial and associative nitrogen

fixation

Chaired by Jos Vanderleyden Leuven, Belgium


55

Session 2: Cyanobacterial and associative nitrogen fixation

S2-1 Nitrogen storage and ammonia stress acclimation in cyanobacteria: novel insights in similarities to plant physiology

Karl Forchhammer, Sabine Beez, Christina Herrmann, and Miriam Drath Institut für Mikrobiologie der Eberhad-Karls-Universität Tübingen, 72076 Tübingen, Germany Acclimation of cyanobacteria to various nitrogen conditions is pivotal for their survival in natural habitats. The ability to buffer excess combined nitrogen is a selective advantage under fluctuating nitrogen regimes. During nitrogen excess conditions (such as ammonia surplus) and during nitrogen fixation, cyanobacteria can store nitrogen as arginine within the polymer cyanophycin (multi-L-arginyl-poly-L-aspartic acid). The synthesis of cyanophycin follows an increase in the arginine level, which itself is controlled by the PII signal transduction protein [1]. PII regulates the activity of the controlling enzyme, N-acetyl glutamate kinase (NAGK) through protein-protein interaction, which causes catalytic activitation and relief from arginine inhibition. A similar interaction between PII and NAGK has been demonstrated in chloroplasts of plants. Similar to cyanobacteria, plants can use arginine as nitrogen storage. Recently, the crystal structures of PII-NAGK complexes from the cyanobacterium Synechococcus elongatus [2] and from Arabidopsis thaliana could be resolved. Despite a high overall similarity, the complexes show several distinguishing features. Functionally, differences were proposed with respect to the role of 2-oxoglutarate (2-OG) and the catalytic activation of NAGK by PII. The present investigation was carried out to clarify the previous conflicting results on the role of 2-OG and to compare PII-mediated NAGK regulation in plants and cyanobacteria, with the question in mind, if plant and cyanobacterial PII and NAGK proteins are mutually exchangeable across the domains of life. Special attention was given to aspects, where the NAGK-PII system in cyanobacteria differs from that in plants, in particular, the effect of arginine and PII on NAGK activity. If present in excess, ammonia can be detrimental to plants and cyanobacteria. Here, we show that ammonia may be harmful to the cells by enhancing photodamage to PSII. A novel model for ammonia toxicity in cyanobacteria is presented [3]. [1] Maheswaran et al. (2006). J. Bacteriol. 188:2730-2734. [2] Llacer et al. (2007). Proc. Natl. Acad. Sci. USA. 104:17644-17649. [3] Drath et al. (2008). Plant Physiol. 147:206-215.

56


S2-2 Signalling and functioning of Azospirillum in the rhizosphere Yvan Moënne-Loccoz, Emeline Combes-Meynet, Vincent Walker, Joël Pothier, Olivier Couillerot, Floriant Bellvert, Cédric Bertrand, Gilles Comte, Florence Wisniewski-Dyé, and Claire Prigent-Combaret Laboratoire d'Ecologie Microbienne, Centre National de la Recherche Scientifique, UMR5557, Université Lyon 1, 69622 Villeurbanne Cedex, France The rhizosphere is a biologically active soil compartment, where microorganisms are involved in a myriad of plant-microbe and microbe-microbe interactions. These interactions are often mediated by molecular signals. Associative nitrogen fixers, such as Azospirillum play an important role in this network of interactions, because nitrogen fixation is a key step of the nitrogen cycle and these bacteria have the potential to promote plant growth. Indeed, Azospirillum PGPR are used as biofertilizers for certain crops. However, the molecular interactions of Azospirillum PGPR with their plant and microbial partners are only partially understood, which limits our knowledge of the conditions in which Azospirillum can function in the rhizosphere. First, screening of an Azospirillum promoter library by differential fluorescence induction coupled to flow cytometry was used to identify genes responding to seed exudates. One of them was the nitrite reductase gene nirK, which is upregulated when Azospirillum colonizes the root. Besides Azospirillum, other PGPR may also be present in the rhizosphere, for instance antagonistic fluorescent Pseudomonas strains that protect roots from fungal pathogens by producing the biocontrol metabolite 2,4-diacetylphloroglucinol (DAPG). Therefore, differential fluorescence induction and flow cytometry were also used to assess the effect of DAPG on Azospirillum. It appears that DAPG can act as a signal on Azospirillum, leading to upregulation of numerous genes, e.g. genes encoding putative sensors or ABC transporters. Second, a global metabolomic approach was followed to characterise the physiological response of the plant to Azospirillum. Inoculation of seeds with Azospirillum strains resulted noticeably in changes in the production of root secondary metabolites such as phenolic compounds. The results illustrate the importance of signalling in the functioning of Azospirillum interactions in the rhizosphere.

Session 2

Cyanobacterial and associative nitrogen

fixation

Chaired by Jos Vanderleyden Leuven, Belgium


57


PS2-1 Nitrogen fixation by Antarctic diazotrophic cyanobacteria Rajan Kumar Gupta Algal Research Laboratory, Department of Botany, Government Post Graduate College (affiliated to HNB Garhwal University), Rishikesh 249201 (Dehradun), Uttarankhand, India The cyanobacteria growing in all the ecosystem, i.e. free living, in aquatic systems, on soil, porus rocks, and as epiphytes on mosses are of ecological significance in the Antarctic ecosystem as contributor of new nitrogen via biological nitrogen fixation. Nostoc commune, the dominant and important constituent of the microbial flora in the Schirmacher oasis (Antarctica) was isolated and established in auxenic state. The organism prefers to grow in the medium devoid of combined inorganic nitrogen sources. An attempt was made to estimate the contribution of N2 fixation potentiality during the 11th and 14th Indian Scientific Expeditions to Antarctica. The N2 fixation was measured at different temperatures and temperature optima of Antarctic cyanobactera for nitrogenase activity was determined. At temperature close to 10°C or in low light intensity, Nitrogenase activity (C2H2 reduction) of Nostoc commune was compared with Nostoc muscorum, N. calcicola, Anabaena doliolum, and Gloeocapsa sp. The temperature profile of acetylene reduction (5-30°C) for Nostoc commune revealed that the rate was highest at 25°C, although it was low in comparison to N. muscorum, but the activity continued at lower temperatures, which was not evident in other cyanobacteria. Although the temperature influences the activity, the Antarctic Nostoc commune is well adapted to the lower temperature in terms of nitrogen fixation. Data analysis revealed that variation in temperature was statistically significant at a 10% probability level. Also two strains differed significantly (p<0.0025) in nitrogenase activity with respect to the temperature profile (F2,15=9.4). The activation energy for N. muscorum and N. commune were also determined and a Arrhenius plot for the N. muscorum showed that the energy of activation was 108 KJ/mol (temperature range from 15-25°C), on the other hand N. commune exhibited two levels of activation energy, one at a temperature range of 5°C (59 KJ/mol), the other at 15-25°C (33 KJ/mol).

58


PS2-2 Phenotype characterization of Azospirillum brasilense sp7 ABC transporter (wzm) mutant

Anat Lerner, Saul Burdman, and Yaacov Okon Department of Plant Pathology and Microbiology, and The Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel Azospirillum, a free-living nitrogen fixer, belongs to the plant growth-promoting rhizobacteria (PGPR), living in close association with plant roots. These bacteria are able to exert beneficial effects on plant growth and yield of many crops of agronomic importance under a variety of environmental and soil conditions. These positive effects are mainly the result of morphological and physiological changes in the inoculated plant roots, with enhanced water and mineral uptake. Plant growth-promoting substances produced by the bacteria seems to be at least partially responsible for these effects. Azospirillum cells are surrounded by a thick, dense, and tightly cell-bound layer of capsular polysaccharide (CPS) and by an outer lighter exopolysaccharide (EPS) layer bound to the cell. The EPS is involved in the attachment of Azospirillum to the plant root. Several genes involved in the Azospirillum brasilense-plant root interaction are carried on a 900-MDa plasmid, called p90. p90 carries also genes involved in motility, adsorption to roots, colony morphology and genes belonging to the glycosyl or mannosyl transferase, sugar dehydratase families, and genes involved in the ABC transporter-dependent pathway (wzm and wzt). These ATP-binding cassette (ABC) superfamily transporters (or traffic ATPases) are frequently involved in the translocation of complex carbohydrates across the cytoplasmic membrane. An A. brasilense wzm mutant was generated and its phenotype in comparison with the wild-type strain Sp7 was evaluated. The wzm mutant was more resistant to heat, osmotic shock, osmotic pressure, desiccation, and starvation, but was more sensitive to elevated levels on NaCl, UV radiation, and hydrogen peroxide. Differences in sensitivity to antibiotics and growth on different carbon sources were observed between the two strains. The wzm mutants also exhibited changes in cell morphology and motility.

59


PS2-3 Role of SipA, the NblS histidine kinase-binding factor, in acclimation to stress and photosynthesis regulation in Synechococcus sp. PCC7942

Paloma Salinas1, Javier Espinosa1, Karl Forchhammer2, and Asunción Contreras1 1Departamento Fisiología Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, 03080 Alicante, Spain; 2Department of Microbiology/Organismic Interactions, Faculty of Biology, University of Tübingen, 72076 Tübingen, Germany In Synechococcus sp. PCC7942, different stress conditions result in photosynthesis down-regulation and phycobilisome degradation. The small protein NblA is a key effector of this process of chlorosis or bleaching. Transcription of the nblA gene is subjected to a complex control in response to a variety of signals and regulatory factors. Amongst them, the histidine kinase NblS and the response regulator NblR that belong to different signalling systems and have, respectively, negative and positive effects on nblA expression. Yeast two-hybrid approaches led to the identification of SipA (NblS interacting protein A), a protein that binds to the ATP-binding domain of NblS. Constitutive expression of the sipA gene from an ectopic promoter resulted in a strong non-bleaching phenotype, a result supporting SipA cooperation with NblS in down-regulation of nblA. The lethality of the NblR mutant under nitrogen deprivation and high light stress was significantly suppressed by inactivation of sipA, suggesting that SipA impairs recovery from chlorosis in the absence of a functional nblR gene. Inactivation of the sipA gene had a very small effect on pigment content, nblA expression, and phycobilisome degradation induced by nitrogen deprivation or high light stress. With the aim of discovering the "sipA phenotype", we have investigated photosynthesis performance and acclimation to a series of stress conditions. In particular, we have monitored chlorophyll fluorescence and oxygen evolution activities of photosystem II in different genetic backgrounds and environmental conditions, and tested the sensitivity to and recovery from additional types of stresses. As a result, a pleiotropic phenotype was unravelled for the SipA strain, which seems to be specifically impaired in acclimation to particular types of stress.

60


Session 3

Nitrogenase and regulation of free-living

nitrogen fixation

Chaired by Ray Dixon Norwich, UK


61

Session 3: Nitrogenase and regulation of free-living nitrogen fixation

S3-1 Nitrogenase biochemistry Dennis R. Dean Department of Biochemistry and Institute for Biomedical and Life Sciences, Virginia Tech, Blacksburg, VA 24061, USA One aspect of the biochemical analysis of biological nitrogen fixation has focused on where and how substrates interact with the nitrogenase active site, how electrons and protons are delivered to the substrate, and how nucleotide hydrolysis is coupled to this process [1]. Biochemical, genetic, and structural studies have provided considerable insight into these processes, but some fundamental questions still remain. A second avenue of very active research in the field of biological nitrogen fixation –with substantial progress in the last two years [2, 3]– has involved a molecular analysis of the mechanism for the assembly of the simple and complex metalloclusters required for the activation of nitrogenase. Such work has also provided a paradigm for the formation of metalloclusters required to sustain other essential life processes, such as photosynthesis and respiration. This lecture will briefly summarize our current understanding of nitrogenase catalysis and will focus on recent progress in understanding the mobilization of iron and sulphur required for the formation of simple and complex metalloclusters. [1] Dos Santos et al. (2005) Accounts Chem. Res. 38:208-214. [2] Rubio & Ludden (2008). Annu. Rev. Microbiol. 62:93-111. [3] Hu et al. (2008). Biochemistry, in press.

62


S3-2 The role of PII and AmtB proteins in the regulation of nitrogenase activity and ammonium assimilation in Rhodospirillum rubrum

He Wang1, Pedro Teixeira1, Anders Jonsson1, Simina Vintila2, Agneta Norén1, and Stefan Nordlund1 Departments of 1Biochemistry and Biophysics and 2Botany, Stockholm University, 106 91 Stockholm, Sweden In the phototroph Rhodospirillum rubrum nitrogenase activity is regulated by reversible ADP-ribosylation of the Fe-protein. DRAT catalyzes the addition of the ADP-ribose moiety from NAD+ and DRAG catalyzes the removal of the modifying group and thereby the activity is restored. One of the major questions has been the identity of the signal(s) to this system and some years ago, we proposed that the binding of DRAG to the chromatophore membrane plays a central role in the regulatory mechanism. Since then, we and a number of other groups have provided evidence supporting this model and also demonstrated the involvement of AmtB1 and PII proteins in this regulation. There are three PII paralogues in R. rubrum, GlnB, GlnJ, and GlnK. GlnB and GlnJ have both been shown to have specific functions in the regulation of nitrogen metabolism, whereas no specific function has been identifíed for GlnK. We have now studied the role of PII proteins in the regulation of nitrogenase activity with respect to the interaction with AmtB1 in the chromatophore membrane. We have also analyzed the expression of the glnBglnA, glnJamtB1, and glnKamtB2 operons under different growth conditions with respect to nitrogen source. All together, our studies aim at establishing a model explaining the communication within the cell, leading to a concerted regulation of nitrogen assimilation and nitrogen fixation.

63


PS3-1 Complete synthesis of the iron-molybdenum cofactor of nitrogenase with purified Nif proteins

Leonardo Curatti, José A. Hernandez, Robert Y. Igarashi, Basem Soboh, Dehua Zhao, and Luis M. Rubio Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA The iron-molybdenum cofactor (FeMo-co) found in the molybdenum nitrogenase is among the most complex iron-sulphur prosthetic moieties in biology. This cofactor, which is composed of 7Fe, 9S, Mo, R-homocitrate and an additional atom X of unknown identity, is required by the NifDK component of nitrogenase to catalyze the reductive lysis of dinitrogen. The synthesis of FeMo-co requires a specialized cellular machinery, involving multiple gene products of the nitrogen fixation (nif) gene cluster [1]. The products of the nifD and nifK genes do not seem to be required for the biosynthesis of FeMo-co. It is accepted that FeMo-co is assembled elsewhere in the cells and then incorporated into a FeMo-co- deficient apo-NifDK protein. The proteins involved in FeMo-co biosynthesis can be functionally divided into three classes: scaffold proteins, metallocluster carrier proteins, and enzymes that provide substrates in chemical forms that are suitable for FeMo-co synthesis. We have achieved the purification of most of these proteins and have developed an assay that drives the synthesis of FeMo-co and the formation of active nitrogenase in vitro. This assay demonstrates that NifB, NifEN, and NifH are sufficient to perform all essential reactions to synthesize FeMo-co in vitro from its basic components (molybdenum, iron, sulphur, and homocitrate) [2]. Importantly, FeMo-co assembly involves radical chemistry in the reaction catalyzed by NifB, which provides the link between the biosyntheses of simple iron-sulphur clusters and that of the structurally complex FeMo-co [3]. [1] Rubio & Ludden (2005). J. Bacteriol. 187:405-414. [2] Curatti et al. (2007). Proc. Nat. Acad. Sci. USA 104:17626-17631. [3] Curatti et al. (2006). Proc. Nat. Acad. Sci. USA 103: 5297-5301.

64


PS3-2 In vitro interactions between the PII proteins and the nitrogenase regulatory enzymes in Azospirillum brasilense

Luciano Fernandes Huergo1, Rose Adele Monteiro1, Mike Merrick2, Leda Satie Chubatsu1, Fabio Oliveira Pedrosa1, and Emanuel Maltempi Souza1 1Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Curitiba, PR, 80060-000, Brazil; 2Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Nitrogen metabolism in prokaryotes is coordinated by the signal transduction proteins from the PII family. In Gram-negative bacteria, PII proteins can be modified by uridylylation according to the cellular nitrogen status; these proteins can also bind small effectors, such as ATP and 2-oxoglutarate. Recent data indicate that PII proteins can also bind ADP, thus acting as "energy charge sensors". PII proteins control the activity of transcriptional regulators, enzymes, and transporters through direct protein-protein interaction, which, in turn, are regulated by the PII uridylylation status and by the effectors bound. Nitrogenase activity in the plant-associative nitrogen-fixing bacterium Azospirillum brasilense is reversibly inactivated by NH4

+ through ADP-ribosylation of the Fe-protein. This process is catalysed by DraT and is reversed, upon NH4

+ exhaustion, by DraG. The activities of both DraT and DraG are regulated accordingly to the external levels of ammonium in a PII-dependent manner, A. brasilense codes for two PII-like proteins, namely GlnB and GlnZ. Our previous data have suggested a model for the regulation of DraT and DraG activities. According to the model, upon an ammonium shock, DraT is activated through interaction with the de-uridylylated GlnB and DraG is negatively regulated by membrane sequestration through the formation of a AmtB-GlnZ-DraG ternary complex. Here, we have used pull-down assays and gel filtration to further characterise the influence of the PII uridylylation status and the PII small effectors on the DraT-GlnB and DraG-GlnZ in vitro complex formation. We observed that both interactions are maximised when PII proteins are de-uridylylated and when ADP is present. The physiological significance of these findings will be discussed. Gel filtration analysis of the DraT-GlnB complex indicates a 1:1 stoichiometry (DraT monomer:GlnB trimer) and suggested a major conformational change in DraT upon complex formation that presumably triggers the DraT activation. Financial support: CNPq and Fundação Araucária

65


PS3-3 Role of the PAS2 domain of NifL in signal transduction Peter Slavny1, Richard Little1, Paloma Salinas2, and Ray Dixon1 1Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK; 2División de Genética, Universidad de Alicante, 03080 Alicante, Spain The NifL-NifA system regulates the transcription of genes required for nitrogen fixation in Azotobacter vinelandii. The activity of the transcriptional activator, NifA, is controlled by its partner protein NifL, in response to changes in redox potential and fixed nitrogen status. The ability of NifL to inhibit transcriptional activation by NifA is mediated solely through the formation of the NifL-NifA protein-protein complex. NifL contains two N-terminal PAS domains and a C-terminal region similar to histidine protein kinases. The first PAS domain, PAS1, contains a FAD co-factor and is responsible for redox sensing, whereas the second PAS domain, PAS2, has no known co-factor and its function remains unclear. We have identified two classes of mutation in the PAS2 domain of NifL. The first class results in a "locked-on" form of NifL that constitutively inhibits NifA, irrespective of environmental conditions. The second class of mutation results in failure to transduce the redox signal so that the mutant proteins fail to inhibit NifA under oxidising conditions, but retain the capacity to respond to the nitrogen status. These results suggest that PAS2 plays a pivotal role in transducing the redox signal from PAS1 to the C-terminal domains of NifL. Using the bacterial adenylate cyclase two-hybrid (BACTH) system, we have detected an interaction between subunits of the isolated PAS2 domain, suggesting that this domain is a multimer. Biochemical studies demonstrate that the isolated PAS2 domain is dimeric in solution. The PAS2-PAS2 interaction detected by BACTH is maintained in PAS2 mutations that give rise to a defective redox response, but is perturbed by mutations in PAS2 that result in a "locked-on" phenotype. This suggests a model for signal transduction in NifL whereby redox-dependent conformational changes in PAS1 are relayed to the C-terminal domains of NifL via changes in the quartenary stucture of the PAS2 domain.

66


Session 3

Nitrogenase and regulation of free-living

nitrogen fixation

Chaired by Ray Dixon Norwich, UK


67


PS3-4 The uridylylation of Herbaspirillum seropedicae PII proteins (GlnB and GlnK) responds differently to the ATP/ADP ratio

Ana C. Bonatto, Emanuel M. Souza, Rose A. Monteiro, Luciano F. Huergo, and Fábio O. Pedrosa Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Curitiba, PR, 80060-000, Brazil Herbaspirillum seropedicae is an endophytic diazotroph that associates with important crops, such as wheat, sugarcane, and rice. In this organism, several proteins involved in nitrogen metabolism were identified, including two PII proteins, GlnB and GlnK. PII proteins are signal transducers that integrate metabolic signals and transmit this information to proteins involved in nitrogen metabolism, such as NtrB, GlnE, and NifA (directly or through NifL). In many proteobacteria, PII proteins are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. Usually, uridylylation and de-uridylylation reactions require the effectors ATP and 2-oxoglutarate, which bind directly to PII proteins. In Escherichia coli, the PII proteins also bind ADP and the ATP/ADP ratio influences PII activities. In other organisms, such as Azospirillum brasilense, ADP is also involved in regulating the interactions of PII with their targets. In this work, the PII interaction with the N-terminal domain of NifA was evaluated by limited proteolysis assays and uridylylation of purified H. seropedicae GlnB and GlnK by GlnD was analysed under different ATP/ADP ratios. The proteolysis assays indicated that the protection of PII from tryptic digestion by the N-terminal domain of NifA is modulated by the presence of ATP or ADP. The GlnB uridylylation rate was higher in the presence of ATP when compared with ADP and AMP, while GlnK uridylylation in the presence of either ATP or ADP was similar. At low concentrations of 2-oxoglutarate (100 µM), uridylylation of GlnB decreased with increasing ADP/ATP ratio, whereas uridylylation of GlnK remained constant under these conditions. The results suggest that in addition to the nitrogen levels, H. seropedicae GlnK uridylylation responds only to fluctuations in the carbon levels, while GlnB responds to both carbon and energy levels in vitro. Supported by CNPq/Instituto do Milênio

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PS3-5 In vitro characterisation of the FixK2 regulatory protein of Bradyrhizobium japonicum

Mariette Bonnet, Hans-Martin Fischer, Hauke Hennecke, and Socorro Mesa Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zürich, 8093 Zürich, Switzerland In the soybean endosymbiont Bradyrhizobium japonicum, two linked cascades are involved in the regulation of its different lifestyles. In the FixLJ-FixK2 cascade, the FixLJ two-component regulatory system activates expression of the transcription factor gene fixK2 at a concentration of approximately 5% of O2 in the gas phase. FixK2 is a member of the CRP/FNR superfamily, but unlike its homologs, it neither possesses the CRP-specific residues involved in cAMP binding nor contains the FNR-specific cysteine residues necessary to bind [4Fe-4S]2+ clusters. Previous experiments showed that FixK2 acts as homodimer without any cofactor [1]. Recently, we have noticed that FixK2 can be inactivated by the formation of an intermolecular disulphide bridge via C183 in the DNA-binding domain [unpublished data]. Replacement of C183 by a serine residue led to a non-redox-responsive protein derivative that is even more active than the wild-type protein. In addition to this type of regulation, we observed that a truncated FixK2 derivative is always co-purified together with the full-length protein from FixK2-overproducing Escherichia coli cells. Mass spectrometry analysis revealed that this secondary form is a C-terminally cleaved FixK2 derivative that lacks the last 12 amino acids (FixK2 1-220). Different strategies have been applied to get a more homogenous population and to biochemically characterize FixK2. Especially, the construction of a C-terminal His-tagged derivative allowed us to purify a full-length active FixK2. In order to get insights into the molecular mechanism of FixK2, we will focus on two goals: (i) crystallization trials with the (C183S) FixK2-His protein variant and (ii) deciphering whether or not C-terminal cleavage of FixK2 is an additional way of regulating FixK2 in vivo activity. [1] Mesa et al. (2005). J. Bacteriol. 187:3229-3388.

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PS3-6 Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae

Jens Glöer, Robert Thummer, Heike Ullrich, and Ruth A. Schmitz Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, 24118 Kiel, Germany In the diazotroph Klebsiella pneumoniae, the nitrogen sensory protein GlnK mediates the cellular nitrogen status towards the NifL/NifA regulatory system that regulates transcription of the nitrogen fixation genes in response to ammonium and molecular oxygen. In order to identify the amino acids of GlnK essential for this direct protein-protein interaction, we performed a random point mutagenesis by PCR amplification under conditions of reduced Taq polymerase fidelity and developed an in vivo screen for change-of-function mutations resulting in a Nif- phenotype. Of three independent pools, 3,200 mutants were screened for mutants that would no longer complement a K. pneumoniae glnK mutant strain for growth under nitrogen-fixing conditions. Twenty-four glnK mutants resulting in a Nif- phenotype were identified, which showed up to 11 amino acids changes. Based on those, 20 single amino acid mutations were introduced into glnK by site-directed mutagenesis, and the respective effects on NifA-mediated nif gene induction were monitored in K. pneumoniae using a nifH-lacZ fusion. The results obtained will be presented and a working model for signal transduction will be discussed.

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PS3-7 Characterisation of the DraT/DraG-system involved in nitrogenase Fe-protein modification in the endophytic bacterium Azoarcus sp. BH72

Janina Oetjen1, Agneta Norén2, and Barbara Reinhold-Hurek1 1Laboratory of General Microbiology, Faculty of Biology and Chemistry, University of Bremen, 28334 Bremen, Germany; 2Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 106 91 Stockholm, Sweden Reversible covalent modification of nitrogenase Fe protein is a common strategy in bacteria to regulate nitrogenase activity posttranslationally. Fe-protein modification by ADP-ribosylation of a specific arginine residue has been demonstrated in case of the α-proteobacteria Rhodospirillum rubrum and Rhodobacter capsulatus. ADP-ribosylation of one subunit of nitrogenase Fe-protein leads to the so called "switch-off" response. Functional inactivation of nitrogenase Fe-protein by ADP-ribosylation was also proposed for Azospirillum brasilense and A. lipoferum. Dinitrogenase reductase ADP-ribosyltransferase (DraT) was shown to catalyze the ADP-ribosylation after addition of an external nitrogen source to nitrogen-fixing bacteria or by energy depletion. The reversibility of this process is accomplished by the action of DraG, acting as the dinitrogenase reductase-activating glycohydrolase. A protein of increased electrophoretic mobility has been shown during Western blot analysis indicating Fe-protein modification in the endophytic β-Proteobacterium Azoarcus sp. BH72, as well. This bacterium, with its ability to infect rice and expressing the nifH gene in rice apoplast, possesses a draT ortholog and two draG copies in its genome. Azoarcus draT deletion mutants were unable to modify the nitrogenase Fe-protein, but performed a physiological switch-off response. draG mutants were disabled in their ability to demodify nitrogenase Fe-protein and to reactivate the enzyme after anaerobiosis shifts. Cells responding to ammonia addition indicated that nitrogenase turn-over in Azoarcus sp. BH72 is very fast. Activity was abolished during nitrogenase in vitro assays of draG mutant strains after ammonia addition and anaerobiosis shifts, whereas unmodified control strains (draT mutants) showed activity. This is the first characterisation of a DraT/DraG system in a β-Proteobacterium. Furthermore, we would like to discuss the type of Fe-protein modification in this endophytic bacterium.

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PS3-8 Role of GlnK in the control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1501

Sheng He1, Ming Chen1, Zhihong Xie1, Yongliang Yan1, Hongquan Li1, Ying Fan1, Shuzhen Ping1, Claudine Elmerich2, and Min Lin1 1Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R. China; 2Département de Microbiologie, Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Institut Pasteur, 75724 Paris Cedex 15, France Pseudomonas stutzeri A1501 (Chinese Culture Collection, CGMCC 0351) was isolated from rice paddy soils and has been widely used as a crop inoculant in China. The strain is capable of fixing nitrogen under microoxic conditions. Genetic information specific to synthesis and functioning of nitrogenase is clustered in a 49-kb island, suggesting that this property was acquired by lateral gene transfer. GlnK, a protein belonging to the PII family, is a signal protein playing a major role in overall regulation of nitrogen metabolism. P. stutzeri A1501 carries a single gene encoding glnK, which is co-transcribed with two distantly related copies of the amtB genes, encoding putative ammonium channels. Inactivation of glnK led to a mutant strain devoid of nitrogenase activity and auxotrophic for glutamine, while inactivation of amtB led to a prototrophic and Nif+ mutant strain. Transcription analysis showed that nifA expression was abolished in the glnK mutant consistent with the Nif phenotype. In contrast, glnA remained transcribed and glutamine auxotrophy resulted from inability to deadenylylate glutamine synthetase. Introduction of a plasmid that expressed nifA from a constitutive promoter restored nitrogen fixation to the glnK mutant and nitrogenase activity was observed even in the presence of ammonia. GlnK signalling appears to be a key regulatory element in the control of ammonia assimilation, of nifA expression, and in modulation of NifA activity by NifL.

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PS3-9 Expression of Fe protein from a heterologous promoter can enhance nitrogen fixation activity of Azotobacter vinelandii

Papri Nag, and Subrata Pal Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700 032, India The consequences of overexpressing the nifH gene (encoding dinitrogenase reductase I) in a rec+ Azotobacter vinelandii were assessed. The rationale for overexpressing the nifH gene was the multiple functions carried out by the NifH protein in the nitrogenase enzyme system. The nifH gene was cloned in a broad host range plasmid (pJB654) known to survive in A. vinelandii. This plasmid contained the Pm promoter from the TOL plasmid found in Pseudomonas putida. The high recombination frequency in rec+ A. vinelandii caused the plasmid to integrate into the chromosomal DNA. A nifH:vnfH hybrid was selected for further studies. This was done because the open reading frames present in this region are organised into two transcriptional units. One contains vnfH (encoding dinitrogenase reductase II) and a ferredoxin-like open reading frame (Fd) and the second one includes vnfD (encoding the subunit of dinitrogenase II), vnfG (encoding a product similar to dinitrogenase II from A. chroococcum), and vnfK (encoding the three subunit of dinitrogenase 2). Thus, freeing the nifH gene from the NifA/NifL or VnfA control, it could be overexpressed. VnfH and NifH are very similar proteins and can substitute each other in function. The increase in the NifH:VnfH protein (3-fold over the wild type) as determined by native protein gel blotting resulted in the increase in acetylene reduction capacity (1.7 fold over the wild type). All the experiments were carried out in the presence of iron and molybdenum for assessing the effect on the nitrogenase I system only. Concomitant changes were observed also in the cellular levels of siderophores and iron superoxide dismutase (FeSOD) and catalase. The level of Fe protein in the cell thus appeared to be at least one of the rate-limiting factors for Azotobacter nitrogenase activity.

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PS3-10 A new regulatory role of the Rnf system of Azoarcus sp. strain BH72 Abhijit Sarkar, and Barbara Reinhold-Hurek Laboratory of General Microbiology, University of Bremen, 28334 Bremen, Germany Supply of reducing power to nitrogenase is one of the limiting factors of nitrogen fixation. However, the electron pathway(s) to mobile electron carrier(s) reducing nitrogenase is poorly understood in most bacteria. Rnf proteins are proposed to form trans-membrane–peripheral protein complexes coupling the energy of ion transport to reduce ferredoxins at very low redox potentials. In this work, the role of rnf gene clusters has been investigated for the diazotrophic endophytic β-Proteobacterium Azoarcus sp. strain BH72. Among the two clusters of rnf-like genes present in its genome, the expression of the rnf1 cluster was found to be specifically induced by 15-fold under nitrogen fixation and strongly repressed by ammonium. Its expression was found to be modulated by transcription factors, such as NtrC and NifA from an upstream σ54-dependent promoter consensus. The diazotrophic growth was partially impaired by an in-frame deletion of the rnf1 cluster. Although the "switch off" response to ammonium was completely abolished in absence of the Rnf1 complex, posttranslational modification of nitrogenase Fe protein was still partially retained. Although the Rnf2 complex was constitutively expressed (albeit at low levels), it could not complement the Rnf1 complex. Spectrophotometric time course measurements by ferricyanide reduction supported an Rnf1-mediated direct electron transport involved in the nitrogen fixation of strain BH72. A deeper insight into the signalling process interestingly revealed a novel specific membrane sequestration of the signal transmitter protein, GlnK, by the Rnf1 complex. A working model will be proposed on the basis of this study.

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PS3-11 Insights into the NrpR regulon in Methanosarcina mazei Gö1 Katrin Weidenbach1, Claudia Ehlers1, Jutta Kock1, Armin Ehrenreich2, and Ruth Schmitz-Streit1 1Department of General Microbiology, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany; 2Department of Mircobiology and Genetic, University of Göttingen, 37077 Göttingen, Germany The methanogenic archaeon Methanosarcina mazei strain Gö1 contains two homologues of NrpR, the transcriptional repressor of nitrogen assimilation genes recently discovered and characterized in Methanococcus maripaludis [1]. Insertion of a puromycin resistance-conferring cassette into MM1085, encoding a single NrpR domain with an N-terminal helix-turn-helix domain (NrpRI) led to a significant reduction of the lag-phase after a shift from nitrogen sufficiency to nitrogen limitation. Consistent with this finding, loss of NrpRI resulted in significantly increased transcript levels of genes involved in nitrogen fixation or nitrogen assimilation, though growing under nitrogen sufficiency as demonstrated by qTR-PCR analysis. Genome-wide analysis using DNA microarrays confirmed that transcript levels of 27 open reading frames were significantly elevated in the M. mazei MM1085::pac mutant under nitrogen sufficiency, including genes known to be up-regulated under nitrogen limitation (e.g., nifH, glnA1, and glnK1), and 17 additional genes involved in metabolism (4), encoding a flagella-related protein (1), and genes encoding hypothetical proteins (12). Using cell extracts of Escherichia coli expressing MM1085 fused to the maltose-binding protein (MBP-NrpRI) and employing promoter binding studies by DNA affinity chromatography demonstrated that MBP-NrpRI binds specifically to the nifH promoter. Deletion of various bases in the promoter region of nifH confirmed that the regulatory element ACC-N7-GGT is required for specific binding of NrpRI to the promoter. [1] Lie & Leigh (2003). Mol. Microbiol. 47:235-246.

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PS3-12 Protein expression profiles of Rhodospirillum rubrum under different nitrogen availability conditions

Tiago Toscano Selão, Stefan Nordlund, and Agneta Norén Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden Rhodospirillum rubrum is a free-living, photosynthetic, purple, non-sulphur, nitrogen-fixing bacterium, which has been extensively used as a model organism for metabolic and regulatory studies regarding nitrogen fixation. In order to understand the metabolic effects when changing between different nitrogen sources, we have studied both the soluble and the chromatophore membrane-associated proteome of R. rubrum, using a combination of electrophoretic techniques and mass spectrometry analysis. The analysis of the soluble fraction, using two-dimensional PAGE and MALDI-TOF, revealed the intricate relation between nitrogen and carbon fixation pathways. This interplay allows a regulation of both the carbon/nitrogen ratio and the redox balance in the cell, because nitrogen fixation induces a down regulation of carbon fixation pathways [1]. We have also analyzed the chromatophore membrane protein complexes using two-dimensional Blue Native/SDS-PAGE and identified many of the main components, expressed under different nitrogen availability conditions. [1] Selão et al. (2008). J. Proteome Res. 7:3267-3275.

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PS3-13 The PII signal transduction proteins as sensors of cellular carbon, nitrogen and energy status in Rhodospirillum rubrum

Pedro Filipe Teixeira, and Stefan Nordlund Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden The PII family of signal transduction proteins is widespread amongst the three domains of life and its members have a fundamental role in the general control of nitrogen metabolism [1]. These proteins exert their regulatory role by direct protein-protein interaction with a multitude of cellular targets, modulated by the binding of metabolites, such as ATP, ADP, 2-oxoglutarate, and also by reversible post-translational modification. In the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum, three PII paralogues were identified and termed GlnB, GlnJ, and GlnK. In this work, we have analysed the interaction of GlnJ with several cellular targets, such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD [2, 3]. Our results show that the interaction of GlnJ with cellular targets is regulated by the concentration of manganese, 2-oxoglutarate, and the ADP/ATP ratio. 2-Oxoglutarate abolishes the interaction of GlnJ with AmtB1 and GlnE, while favouring the interaction with GlnD. Conversely, ADP acts by relieving the 2-oxoglutarate signal, indicating an additional mechanism of regulation. This study provides further evidence that PII proteins are sensitive to the carbon, nitrogen, and energy status in the cell. This work was supported by grants from the Swedish Research Council to SN and by Fundação para a Ciência e a Tecnologia (Portugal) through the PhD fellowship SFRH/BD/22162/2005 to PFT. [1] Forchhammer (2008). Trends Microbiol. 16:65-72. [2] Jonsson et al. (2007). FEBS J. 274:2449-2460. [3] Teixeira et al. (2008). Microbiology 154:2336-2347.

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PS3-14 Nitrogenase regulation in Nodularia spumigena strain AV1 in response to darkness and ammonium supplementation

Simina Vintila1, Tiago T. Selao2, Agneta Norén2, and Rehab El-Shehawy1 Departments of 1Botany, and 2Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden Nodularia spumigena is a brackish-water, filamentous, heterocystous cyanobacterium that dominates the annual, toxic cyanobacterial blooms in the Baltic Sea. Nitrogen-fixing cyanobacteria are contributing significantly to the annual nitrogen input into the Baltic Sea and it is, therefore, crucial to understand the regulation of nitrogen fixation in these organisms. The focus of this study was to characterize the nitrogen fixation behaviour in N. spumigena strain AV1 in response to ammonium supplementation and darkness. In Rhodospirillum rubrum and some other species of nitrogen-fixing bacteria, nitrogenase activity is regulated by reversible ADP-ribosylation leading to a "switch-off" effect in darkness or when ammonium is supplemented [1]. A post-translational regulation of nitrogenase exists also in cyanobacteria although it has not yet been characterized [2]. Western blot of two-dimensional gel electrophoresis shows that a modification of NifH occurs in N. spumigena strain AV1 in response to darkness, but no switch-off effect could be seen after ammonium supplementation. In addition, a second form of NifH, which is very sensitive to oxidation, can also be seen on Western blots. Mass spectometry data will be presented. Taken together, our data suggest that there might be several mechanisms for regulation of nitrogenase expression and activity in N. spumigena strain AV1. [1] Ekman et al. (2008). J. Exp. Bot. 59:1023-1034. [2] Nordlund, S., and Ludden, P.W. (2004). In Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria, Vol. 2, W.

Klipp, B. Masepohl, J.R. Gallon, and W.E. Newton (Eds.). Dordrecht, Kluwer, pp. 175-196.

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PS3-15 Structural studies of key enzyme involved in the post-translational regulation of nitrogenase in Rhodospirillum rubrum

He Wang, Catrine Berthold, Martin Hogbom, Agneta Norén, and Stefan Nordlund Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is regulated by reversible modification in response to nitrogen availability and energy status. DRAG (Dinitrogenase Reductase-Activating Glycohydrolase) and DRAT (Dinitrogenase Reductase ADP-ribosyl Transferase) are the key enzymes responsible for this post-translational regulation in a number of nitrogen-fixing bacteria. We have recently solved the three-dimensional structure of DRAG using X-ray crystallography. The crystal structure reveals manganese ion-binding site in the cavity of the active site, which confirms the physiological relevance of Mn2+-dependent activation. In addition to earlier biochemical and biophysical data from studies on R. rubrum mutant strains, our crystal structure of DRAG contributes to the understanding of the mechanism of catalysis and to elucidate the metabolic pathways involved.

Session 4

Diversity and evolution

Chaired by Anne Willems Gent, Belgium


79

Session 4: Diversity and evolution

S4-1 Nitrogen fixers: more diversity, more evolution Cyril Bontemps1, Rebecca Lawton1, Geoffrey Elliott2, Euan James2, Janet Sprent2, Gehong Wei3, Weimin Chen1,3, Elisa Giuntini1, Xavier Bailly1, Jasper Green1, Ryan Lower1, Peter Harrison1, and J. Peter W. Young1 1Department of Biology, University of York, York YO10 5YW, UK; 2College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK; 2College of Life Science, Northwest A&F University, Shaanxi, P. R. China Nitrogen fixation is widespread in prokaryotes, but seen only in a minority of species. Complete genome sequences reveal the potential in some unsuspected species, but also demonstrate categorically that most organisms do not have nitrogenase. The sporadic distribution and irregular phylogeny of nitrogenase are typical of accessory gene systems, and nodulation is another accessory system with a separate but intertwined history. Horizontal transfer of nodulation genes into different genetic backgrounds has been the common theme of most rhizobial diversity studies in recent years, and appears to have occurred throughout the history of the legume-rhizobia symbiosis. In examples from Europe and Asia, we see host specificity determinants passed from species to species within Mesorhizobium. In Brazil, we have recently completed an extensive survey of indigenous Mimosa species. Virtually all the symbionts are Burkholderia, apparently the result of an ancient transfer of nod genes from α- to β-proteobacteria. Curiously, though, there is little evidence for further transfer within Burkholderia. Returning to a smaller scale in a smaller country, we have been exploring the population genomics of Sinorhizobium medicae nodulating Medicago lupulina on a British roadside. High-throughput sequencing using the Roche 454 FLX reveals the shared and unique features of strains within a population. These different studies illustrate an important general principle of bacterial genome evolution and adaptation. Common housekeeping functions are carried out by a set of core genes that mostly share a common evolutionary history, but adaptations to a particular environment are conferred by a distinct set of accessory genes that are often transferred between species and could be said to belong to the niche rather than to any particular species.

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S4-2 Nod factor-independent stem nodulation in the legume genus Aeschynomene is correlated with plant molecular phylogeny

Bernard Dreyfus1, Clémence Chaintreuil1, Gilles Béna1, Benoît Mallen1, Mame Samba Mbaye1, José-Antonio Munive2, María del Carmen Villegas2, José Luis Contreras-Jiménez2, Lucie Miché1, Marc Boursot1, Eric Giraud1, and Lionel Moulin1 1Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France; 2Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico Approximately 150 species are presently ascribed to the genus Aeschynomene, one half of them from the new world, mainly South and Central America, the other half found across the tropical regions of Africa, South-East Asia, Australia, and the Pacific Islands. The genus Aeschynomene includes both herbaceous and shrubby species, annuals and perennials, some of them growing up to 8 m height and 0.5 m base width (such as A. elaphroxylon). Half of the species are hydrophytes growing in marshes, temporary or permanent ponds, rice fields, waterlogged meadows, and along streams, and river banks. The other half is more xeric and is found in savannas or dry forests. Although many Aeschynomene species only form root nodules in association with classical bradyrhizobia, most aquatic species have the additional capacity to form stem-nodules in symbiosis with specific photosynthetic bradyrhizobia. These stem-nodulating bacteria, which contain a cluster of photosynthesis-related genes, are unique because they do not produce the common Nod factors synthesized by all other rhizobia. In order to determine the relationships between the character stem nodulation and plant phylogeny, we conducted a molecular taxonomy analysis of 56 different Aeschynomene species or accessions originated from both the New and Old World. This phylogenetic study was investigated with sequences from both the chloroplast DNA trnL intron and the nuclear ribosomal DNA intergenic spacer/5.8S region. A parsimony and Bayesian analysis of individual and combined data resolved five distinct phylogenetic groups of Aeschynomene. Simultaneously, we tested the stem-nodulation capacity of each Aeschynomene species by inoculation with different rhizobial strains including the non-Nod factor-producing strain ORS278. We found that all Aeschynomene species nodulated by ORS278 belonged to the same phylogenetic group. We thus demonstrated that the atypical Nod factor-independent stem nodulation is an important plant taxonomic character in the genus Aeschynomene.

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PS4-1 Structural and functional adaptation of the receptor-like kinase gene SYMRK is exceptional among symbiosis genes, and paved the way for the evolution of root endosymbiosis with nitrogen-fixing bacteria

Katharina Markmann1,2, Gábor Giczey2,3, Judith Müller1, Akio Miyao4, Hirohiko Hirochika4, and Martin Parniske1,2 1Genetics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany, Germany; 2The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK; 3Quintiles, 1124 Budapest, Hungary; 4Molecular Genetics Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan Intracellular root symbiosis with nitrogen-fixing bacteria (RNS) is restricted to a few angiosperm orders, which likely form a monophyletic group within the rosid clade. This is in contrast to arbuscular mycorrhiza (AM) with glomeromycotan fungi, which is widespread among land plants. In the legume Lotus japonicus, both types of symbiosis share at least seven "common symbiosis" genes that are necessary for intracellular accommodation of the respective microbial symbionts. This genetic overlap indicates that these functions may have been recruited from AM during RNS evolution. Among the common symbiosis genes, the receptor-like kinase gene SYMRK acts near or at a point of conversion of AM and RNS signalling circuits. We found that at least three distinct structural versions of SYMRK exist in different angiosperm lineages, and tested their functional capabilities in root endosymbiosis. The longest version was found within the rosids clade, which contains all RNS-forming species, and is the only version of the three that can support functional RNS. In contrast, the two shorter versions from the asterid tomato and the monocot rice, both exhibiting a reduced predicted extracellular extension, can only support AM. This divergence contrasts to the structural and functional conservation of other common symbiosis components, such as the nuclear protein CYCLOPS. This protein is required for AM formation in rice, where a putative ortholog with identical exon-intron structure exists, confirming the hypothesis of a conserved AM genetic program in monocot and dicot angiosperm lineages. If introduced in legume mutants, rice CYCLOPS can restore both AM and RNS. The combined findings suggest that SYMRK played an essential role in RNS evolution, by recruiting the AM genetic program for intracellular accommodation of microbes. This step likely formed a basis for intracellular uptake of bacteria, a key capability of RNS-forming plants.

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PS4-2 Molecular phylogeny of rhizobia isolated from Calliandra calothyrsus Meisn. trees growing in Central America, Cameroon, Kenya, and New Caledonia with emphasis on biogeography and symbiosis

Aneta Dresler-Nurmi1, Pablo Vinuesa2, Didier Lesueur3, Lars Paulin4, Kristina Lindström1, and Leena A. Räsänen1 1Department of Applied Chemistry and Microbiology, 00014 University of Helsinki, Finland; 2Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, Mexico; 3CIRAD/Tropical Soil Biology and Fertility Institute/CIAT c/o World Agroforestry Centre, 00100 Nairobi, Kenya; 4DNA Sequencing Laboratory, Institute of Biotechnology, 00014 University of Helsinki, Finland The molecular diversity of 192 strains nodulating Callinadra calothyrsus Meisn trees from native sites of Central America and non-native areas of Cameroon, Kenya, and New Caledonia was assayed by genotypic and phenotypic traits. Genotypic semi-automated one-gene 16S rDNA-RFLP approach, automated whole genome AFLP method and analyses of partial sequences of "core" housekeeping 16S rDNA, glnII, recA, and "accessory" symbiotic nodA and nifH genes were used. The 16S rDNA-RFLP analysis together with 30 reference strains revealed eight genotypes. Phylogenetic studies of 16S rDNA, glnII, and recA sequences confirmed that Calliandra isolates represent Rhizobium tropici (116 strains), R. etli (6), Sinorhizobium meliloti (44), S. saheli (5), S. terangae (3), Agrobacterium (18), Zooglea (1), and Ralstonia (1). The housekeeping 16S rDNA gene correctly delineated Calliandra isolates at genus level; however, the resolution at the species level was considerably higher in strongly supported concatenated partitions of housekeeping glnII and recA. The diversity at the strain level was evaluated by fluorescently labelled AFLP [1]. According to all genotypic approaches, the diversity of Calliandra rhizobia, both at species and strain levels was higher in the gene center of C. calothyrus (Central America) than in non-native areas, where the tree had been introduced. Phylogenetic analysis of available symbiotic nodA genes revealed that Calliandra sequences cluster together with strains isolated from Leucaena, Prosopis, Mimosa, and Phaseolus. According to plant tests, Calliandra isolates induce effective nodules with Phaseolus and Prosopis plants. The type strains used in AFLP and NodA trees were clustered separately from our Calliandra strains what illustrates how important in modern rhizobiology are biogeoraphical data are such as site of isolation as well as original host plant. Based on phylogenetic analyses of symbiotic nodA and nifH sequences and host-range experiments, the biovariety mimosae for Calliandra R. etli is possible. Origin of species nodulating Calliandra trees in non-native areas is still unclear. [1] Dresler-Nurmi et al. (2000). J. Microbiol. Methods 41:161-172.

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PS4-3 rosR and pssA genes involved in polysaccharide synthesis as new taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species

Monika Janczarek, Michał Kalita, and Anna Skorupska Department of Genetics and Microbiology, M. Curie-Skłodowska University, 20-033 Lublin, Poland Rhizobia are widely occurring soil bacteria belonging to a Proteobacteria that are able to establish a nitrogen-fixing symbiosis with legumes. Acidic, species-specific exopolysaccharides (EPSs) secreted in large amounts by these microorganisms play an important role in the establishment of an effective symbiosis with host plants that form indeterminate-type nodules. Using highly conserved genes involved in the polysaccharide synthesis, we developed and tested a reliable PCR-based method for identification and subsequent discrimination between Rhizobium leguminosarum field isolates. The chromosomal genes used for identification of these strains were, accordingly, rosR, encoding the transcriptional regulator of EPS synthesis [1]; pssA, encoding the first glucosyl-IP-transferase in EPS-repeating-unit synthesis [2]; and the pssY gene with homology to Sinorhizobium meliloti exoY, encoding galactosyltransferase that transfers UDP-galactose-1-phosphate to an isoprenylphosphate (IP) lipid carrier. For identification of R. leguminosarum, three sets of primers (I to III), complementary to the sequences of the rosR, pssA, and pssY genes, were established. Further sets of primers (IV to VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a broad range of R. leguminosarum strains isolated from root nodules of clover, vetch, pea, and bean, originating from different regions of Poland. The results demonstrate a high discriminating power of the primer sets I to III, which allow us to distinguish R. leguminosarum and two closely related species, i.e., R. etli and R. gallicum. This new approach is applicable when identifying and differentiating, in a short time, R. leguminosarum strains originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of the rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA genes might be useful markers to study relationships among R. leguminosarum strains. [1] Janczarek & Skorupska (2007). Mol. Plant-Microbe Interact. 20:867-881. [2] Janczarek & Skorupska (2003). Res. Microbiol. 154:433-442.

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Session 4

Diversity and evolution

Chaired by Anne Willems Gent, Belgium


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PS4-4 dnaJ is a useful phylogenetic marker for α-proteobacteria Ana Alexandre1, Marta Laranjo1, J. Peter W. Young2, and Solange Oliveira1 1Instituto de Ciências Agrárias Mediterrânicas, Universidade de Évora, 7004-516 Évora, Portugal; 2Department of Biology, University of York, York YO10 5YW, UK In the past, bacterial phylogeny relied almost exclusively on 16S rRNA gene sequence analysis. More recently, multilocus sequences have been used to infer organismal phylogenies. In this study, the dnaJ chaperone gene was investigated as a marker for phylogeny studies in α-proteobacteria. dnaJ-based phylogenies were analysed at three taxonomic levels: proteobacteria, α-proteobacteria, and the genus Mesorhizobium. Dendrograms based on DnaJ and 16S rRNA gene sequences revealed the same topology already described for proteobacteria, indicating that the DnaJ phylogenetic signal is able to reproduce the accepted relationships among the proteobacteria classes. At a lower taxonomic level, using 20 α-proteobacteria species, the topology of the DnaJ tree is consistent with broader phylogenies from recent studies based on concatenated alignments of multiple core genes, contrary to the one based on 16S rRNA gene. For example, the DnaJ tree shows the two Rhizobiales clusters closely related, as expected, while the 16S rRNA gene-based phylogeny shows them distantly related. In order to evaluate the phylogenetic performance of dnaJ at the genus level, a multilocus analysis based on five housekeeping genes (atpD, gapA, gyrB, recA, and rplB) was performed for 10 Mesorhizobium species. This analysis clarified the phylogenetic relationships among Mesorhizobium species, namely the proximity of M. ciceri, M. loti, and M. huakuii. In contrast to the 16S rRNA gene, the DnaJ sequence analysis generated a tree similar to the multilocus dendrogram. For identification of chickpea Mesorhizobium isolates, a dnaJ nucleotide-based tree was used. Despite different topologies, 16S rRNA gene- and dnaJ-based trees led to the same species identification. This study suggests that the dnaJ gene is a good phylogenetic marker, particularly for the α-proteobacteria class, because its phylogeny is consistent with phylogenies based on multilocus approaches [1]. Funding: FCT, EU-FEDER (POCTI/BME/44140/2002) and fellowship to A.A. (BD/18162/2004) and to M.L. (SFRH/BPD/27008/2006). [1] Alexandre et al. (2008). Int. J. Syst. Evol. Microbiol., in press.

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PS4-5 Evolution of Nod factor signalling Gerben Bijl, Marijke Hartog, Carolien Franken van Butselaar, Ton Bisseling, and René Geurts Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University and Research Centre, 6700 ET, Wageningen, The Netherlands Over the past decade, genetic screens in multiple legume species have resulted in the identification of a number of key regulators essential in early Nod factor signalling (NFS). Seven of these key regulators are constitutively expressed in roots prior to Nod factor perception. Analysis of the genome sequences of Arabidopsis thaliana, Oryza sativa, Vitis vinifera, and Populus trichocarpa has revealed that homologues of these genes can be found in plants outside the legume family. Of these plant species, P. trichocarpa is phylogenetically most related to legumes as both belong to the clade "Eurosids I". The presence of NFS gene homologues in non-legumes implies the presence of common ancestral genes at the moment of divergence. The question remains to what extent these genes have evolved specifically in the legume lineage to enable Nod factor perception and signalling. To answer this, we quantify to what extent homologous genes from poplar can functionally complement NFS mutants in the legume Medicago. We discriminate between evolution of coding sequences and cis-regulatory elements. By conducting these trans-complementation studies, we discovered that the evolutionary forces that shaped Nod factor signalling genes in the Leguminosae lineage differ from gene to gene. We hypothesise that the differences in evolutionary diversification between these genes are due to differences, on the one hand, in selective pressure on certain key amino acids and/or regulatory elements and, on the other hand, in the constraints imposed by the non-symbiotic function(s) of these genes.

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PS4-6 Stem nodulation in the legume genus Aeschynomene is a plant taxonomy character

Clémence Chaintreuil1, Gilles Béna1, Benoît Mallen1, Antonio Munive1, Maria Carmen Villegas2, José Luis Contreras3, Lucie Miché1, Marc Boursot1, Eric Giraud1, Bernard Dreyfus1, and Lionel Moulin1 1Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France; 2Centro de Investigación en Biotecnología Aplicada, Instituto de Politécnico Nacional, Tlaxcala, Tlax. 90700, Mexico; 3Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico Approximately 150 species are presently ascribed to the genus Aeschynomene, one half of them from the new world, mainly South and Central America, the other half found across the tropical regions of Africa, South-East Asia, Australia, and the Pacific Islands. The genus Aeschynomene includes both herbaceous and shrubby species, annuals and perennials, some of them growing up to 8 m height and 0.5 m base width (for instance, A. elaphroxylon). Half of the species are hydrophytes growing in marshes, temporary or permanent ponds, rice fields, waterlogged meadows, and along stream and river banks. The other half is more xeric and is found in savannas or dry forests. Although many Aeschynomene species only form root nodules in association with classical bradyrhizobiam, most aquatic species have the additional capacity to form stem nodules in symbiosis with specific photosynthetic Bradyrhizobium sp. These stem-nodulating bacteria, which contain a cluster of photosynthesis-related genes, are unique because they do not produce the common Nod factors synthesized by all other rhizobia. In order to determine the relationships between the character stem nodulation and plant phylogeny, we conducted a molecular taxonomy analysis of 56 different Aeschynomene species or accessions originated from both New and Old World. This phylogenetic study was investigated with sequences from both the chloroplast DNA trnL intron and the nuclear ribosomal DNA ITS/5.8S region. A parsimony and Bayesian analysis of individual and combined data resolved five distinct phylogenetic groups of Aeschynomene. Simultaneously, we tested the stem-nodulation capacity of each Aeschynomene species by inoculation with different rhizobial strains including the non-Nod Factor producing strain ORS278. We found that all Aeschynomene species nodulated by ORS278 belonged to the same phylogenetic group. We thus demonstrated that the atypical Nod factor-independent stem nodulation is an important plant taxonomic character in the genus Aeschynomene.

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PS4-7 Genetic diversity of rhizobia associated with alfalfa in Serbian soils Olivera Stajković1, Sofie De Meyer2, Bogić Miličić1, Dušica Delić1* and Anne Willems2* 1Institute of Soil Science, Belgrade, Serbia; 2Laboratory of Microbiology, Ghent University, 9000 Gent, Belgium; *equal contribution. We have evaluated the genetic diversity and phylogeny of alfalfa rhizobia isolated from different types of soils in Serbia and their ability to establish an effective symbiosis with alfalfa. Approximately 80 strains isolated from nodules of Medicago sp. were characterised by rep-PCR analyses, partial and complete 16S rDNA gene sequencing and recA gene sequencing. Symbiotic effectiveness (nodulation and shoot dry weight) of rhizobia was evaluated in glasshouse experiments. The results of the sequence analyses revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules. One strain was identified as S. medicae, while three other strains were identified as Rhizobium sp. According to current data, these Rhizobium sp. strains can be recognized as a new species; however, further analyses are needed to clarify the taxonomic identity of these strains. Despite the fact that the majority of the strains were identified as S. meliloti, a high genetic diversity at strain level was detected. Almost all isolates shared the ability to nodulate and fix nitrogen with Medicago sp., except 12 of them that were not capable of fixing nitrogen with this plant. This was the first systematic study of rhizobia isolated from Serbian soil.

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PS4-8 Preliminary evaluation of symbiotic nitrogen fixation of common bean from northwest of Spain

Antonio M. De Ron1, María De la Fuente1, and Jean-Jacques Drevon2 1Plant Genetic Resources Department, Misión Biológica de Galicia, Consejo Superior de Investigaciones Científicas, 36080 Pontevedra, Spain; 2Institut National de la Recherche Agronomique-Montpellier-SupAgro, UMR1222, Rhizosphère et Symbiose, 34060 Montpellier Cedex, France Common bean is a traditional and important crop grown by farmers in southern Europe during the warm season. Symbiotic nitrogen fixation (SNF) potential in common bean is considered to be low in comparison with other grain legumes. Common bean is often grown in marginal lands limited in available soil N, and with minimal fertilization. Thus, nitrogen availability is often a problem where N fertilizers are not available or are limited for environmental purposes. Improvement in the ability of this crop to fix atmospheric nitrogen symbiotically would reduce N depletion in such soils, improve grain yield, and, in some cases, enhance protein content. In this study, we screened a broad diversity represented by different accession of common bean representative of the European market classes from the germplasm collection at the Misión Biológica de Galicia that were chosen from previous research. Common bean plants were grown in aerated nutrient solution in a glasshouse and the seeds were inoculated with Rhizobium tropici CIAT899. It seems that there is an important genotypic variability associated with SNF potential and quantity of N2 fixation. This variability emphasizes the need to explore the potential of indigenous rhizobial strains for improving the symbiotic performance of P. vulgaris. The existence of genetic variation in symbiotic N2 fixation among bean landraces opens a real possibility for enhancing N2 fixation through selection and breeding. This work can help to reduce the costs of production and preserving the environmental quality, and to promote the agriculture in less favoured regions and under difficult environmental conditions.

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PS4-9 Genetic diversity of Bradyrhizobia from Aeschynomene plants in Mexico

Elizabeth Delgado-Lopez1, Esmeralda Parra-Basilio1, Clémence Chaintreuil2, José Luis Contreras-Jiménez3, María del Carmen Villegas4, Bernard Dreyfus2, and José-Antonio Munive1 1Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico; 2Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France; 3Herbario-Jardín Botánico, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico; 4Centro de Investigación en Biotecnología Aplicada, Instituto de Politécnico Nacional, Tlaxcala, Tlax. 90700, Mexico Nitrogen-fixing symbioses between legumes and soil bacteria occur almost exclusively in nodular tissues on plant roots. Notable exception has been reported in Neptunia, Sesbania, and Aeschynomene, in which rhizobia-induced nodules also develop on stems. Bacteria capable of inducing stem nodules in Aeschynomene plants belong to the genus Bradyrhizobium. These bacteria are of great significance because of their ability to produce the photosynthetic pigment bacteriochlorophyll a. This pigment is not produced by any other rhizobia that enter into plant symbioses. These stem-nodulating endophytes are of special significance for improvements in the symbiotic nitrogen fixation for crop production. Several of the Aeschynomene species naturally occur in flooded areas and have been investigated as potential green manure crops for use in lowland rice production. Previous work has shown that in bradyrhizobia, the partial sequence of the 16S-23S rDNA intergenic spacer (the region between the tRNA-Ile and 23S rDNA genes) reflects the whole DNA homology among strains and can be used as a screening method to recognise genospecies among Bradyrhizobium isolates. Given that these bacteria occupy a rather specialized ecological niche and share a unique combination of physiological characteristics, the aim of this project was to analyse the genetic diversity of the Bradyrhizobium-Aeschynomene mutualistic association. The project involved primarily the isolation of strains from root and stem nodules of natural populations of Aeschynomene sp. in Mexico. Emphasis was placed on including representative photosynthetic isolates obtained form different Aeschynomene species grown in widely separated geographical regions. In this study we obtained 136 bacterial isolates from root and stem nodules from Aeschynomene species from Veracruz, Morelos, and Guerrero. As expected, ITS sequences exhibited considerable variation both in the length and the sequence. 16S-23S rDNA intergenic sequence analyses have given a first snapshot of the bacterial population diversity and provided information useful for deducing relationships between these nitrogen-fixing photosynthetic symbiotic microorganisms.

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PS4-10 Twenty-five years of rhizobial population genetics: a retrospective Bertrand D. Eardly The Penn State University, Berks College, Reading, PA 19610, USA The primary goal of population genetics is to explain how and why the genetic composition of a population may change over time. Factors that influence allele frequencies in a population include mutation, recombination, migration, population size, and selection. Beginning with the pioneering studies [1] in the 1980', there have been over 45 published studies that have examined the population genetics of rhizobia. Several important conclusions that have been drawn from these studies will be highlighted. For example, it was initially assumed that, like other Gram-negative bacteria, rhizobia would possess a clonal population structure. Collective results from 11 studies examining six species in three genera have shown that this may not be the case, and that the degree of clonality observed in a rhizobial population depends on several factors, including the species under consideration, its geographic distribution, the sampling strategy, the genomic element being characterized, and the unit of statistical analysis (such as the individual strain versus the multilocus genotype). More recent studies have also focused on the role of selection in shaping the genetic composition of natural populations. These studies show that different forms of selection appear to be operating on different loci within the rhizobial genome [2]. These results are important because they provide insight on how certain agriculturally important traits, such as symbiotic host range, may have evolved as a consequence of interactions between rhizobia and their legume hosts. [1] Young (1985). J. Gen. Microbiol. 131:2399-2408. [2] Bailly et al. (2006). Mol. Ecol. 15:2719-2734.

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PS4-11 Population structure of Vicia faba-nodulating rhizobia in Chinese soils

Chang Fu Tian1,4, En Tao Wang2, Li Juan Wu1, Tian Xu Han1, Samih M. Tamimi3, and Wen Xin Chen1 1Key Laboratory of Agro-Microbial Resource and Application, Ministry of Agriculture/College of Biological Sciences, China Agricultural University, 100094 Beijing, China; 2Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 11340 México D.F., Mexico; 3Department of Biology, Faculty of Science, University of Jordan, Amman, Jordan; 4Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France Vicia faba, domesticated approximately 7000 years ago in the Middle East and North East Africa, was introduced into China approximately 4000-5000 years ago. With a polyphasic approach, we revealed that most rhizobia isolated from root nodules of V. faba grown in China were Rhizobium leguminosarum, and six strains were defined as R. fabae sp. nov. Three endemic clusters identified among these rhizobia by genomic fingerprinting and nodulation gene analyses corresponded respectively to three eco-regions: the autumn-sowing region where the winter ecotype of V. faba was cultivated, the spring-sowing region where the spring ecotype was grown, and the Yunnan province where the intermediate ecotype was sown. In the nodulation test, the representative strain for the cluster relating to the spring-sowing region dominated nodules of spring ecotype of V. faba, but it was less effective on the winter ecotype. In the comparative analyses of atpD, recA, and glnII sequences, R. fabae sp. nov. strains were genetically differentiated from the faba bean-R. leguminosarum strains isolated from China, Jordan, Spain, Canada, and Peru, while apparent gene flow events were identified among R. leguminosarum strains isolated from different origins. The nodD sequence analysis showed that the dominating nodD genotype of UK and France documented earlier was the main nodD genotype in Spain and in spring sowing region of China, and it was also found in Canada and Jordan. Several rare nodD genotypes in spring-sowing region and those dominating in the autumn-sowing region of China were also found in Jordan. Strains with different housekeeping genotypes could have the same nodD genotype. The most published nodD genotypes could be found in strains isolated from Jordan, which is near the domestication origin of V. faba. Therefore, we suggested that rhizobia were co-transferred with V. faba and nodD gene is strongly selected by V. faba.

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PS4-12 Study on the diversity of Frankia from environmental samples by applying DGGE fingerprinting and band sequencing

Onofrio Gallina, Angel Valverde, María González Tirante, Ignacio Santa-Regina, and José M. Igual Departamento de Desarrollo Sostenible de Sistemas Agroforestales y Ganaderos, Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas, 37071 Salamanca, Spain Due to their great importance for humankind and worldwide distribution, the most important N2-fixing mutualistic symbioses are those established between rhizobia and legumes and the actinorhizal symbiosis, the later including more than 200 woody dicotyledonous plant species symbiotically associated with the actinomycete Frankia. The great ubiquity of Frankia, which is found in inhospitable latitudes and in soils devoid of actinorhizal plants, indicates that it can live as a saprophyte and compete with soil microorganisms. It is known that Frankia has a great genetic diversity, but studies on biodiversity face the difficult isolation and slow growth in vitro of this actinomycete. Therefore, molecular techniques with high resolution power that, without the need of isolation, allow analyzing the microbial diversity in environmental samples can be extremely useful in the case of Frankia. The aim of this work was, therefore, to adapt the DGGE technique to study the diversity of this microsymbiont. The methodology followed includes the amplification by nested PCR of a hypervariable fragment of the 16S rRNA gene with, first, the specific primers for Frankia FN1F and FN1R; and, later, with the primers FNF and GC-X5R, by which fragments of 400 bp appropriate for separation through DGGE are obtained. This methodology has been successful with different environmental samples.

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PS4-13 Identification and symbiotic effectiveness of rhizobia isolated from Phaseolus vulgaris L. nodules in mountainous agro-ecosystems from Dominican Republic

César Díaz-Alcántara1, Daniel Mulas2, Encarna Velázquez3, and Fernando González-Andrés2 1Facultad de Ciencias Agronómicas y Veterinarias, Universidad Autónoma Santo Domingo, República Dominicana; 2Departamento de Ingeniería y Ciencias Agrarias, Universidad de León, 24071 Léon, Spain; 3Departamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, Spain Sixty isolates were extracted from Phaseolus vulgaris L. root nodules in 14 soils distributed homogenously in three provinces of the central-southern mountainous areas of the Dominican Republic: Elías Piña, San José de Ocoa, and La Vega. Thirty-two isolates reinfected the plants inoculated, and their symbiotic effectiveness was determined in vitro, by growing inoculated plants in N2-free hydroponic solution. Two of the infective isolates showed no significant N2-fixing rate, whereas the plants inoculated with 18 of them did not differ in biomass production and N2 content from the control fertilized with N2. The isolates were grouped by means of their TP-RAPD patterns obtained with the primers 879F and 1522R targeting 16S rRNA gene [1]. According to the results obtained, they were distributed into five groups plus other 10 isolates that were not grouped; from each one a representative strain was identified by 16S-23S intergenic spacer (ITS) sequencing. The ITS regions have phylogenetically divergent sequences in species from the same genus and also they differ among strains from the same species [2]. Therefore, the ITS region sequence analysis constitutes a good tool for the identification of rhizobial isolates. All groups found in this study belong to the phylogenetic group of R. leguminosarum, nevertheless some of them presented divergent sequences with respect to the species currently accepted within this group and we are currently analysing several housekeeping genes to establish the taxonomic status of these groups. [1] Rivas et al. (2002). Eur. J. Plant Pathol. 108:179-184. [2] Kwon et al. (2005). Int. J. Syst. Evol. Microbiol. 55:263-270.

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PS4-14 Conservation and divergence in genome structure in Rhizobium etli Víctor González, José Luis Acosta, Rosa Isela Santamaría, Patricia Bustos, José Luis Fernández, Ismael Luis Hernández, Rafael Díaz, Margarita Flores, Jaime Mora, Rafael Palacios y Guillermo Dávila Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, Mexico Strains of the same bacterial species regularly show an ample genomic variation. To examine the extent of this variation in Rhizobium etli, we determined the complete genome sequence of R. etli CIAT652 and the partial sequence of the genomes of six other R. etli strains from different geographical origins. The DNA and protein content of these strains were compared among them and with the genome of R. etli CFN42 already reported. We found that 70% of the DNA is common among the strains and the rest corresponds to DNA particular to each strain. This extra DNA represents approximately 500-1000 kb or the fifth or sixth portion of the genome. It encodes accessory functions as transport of sugars and amino acids, secondary metabolism, mobile elements, and others that could be related to adaptation. Sequences corresponding to the symbiotic plasmids show high DNA identities (approximately 99%) whereas chromosomal sequences and those with matches in other plasmids show the identities of approximately 90-95% on average. These data support the notion of an extended genome structure in R. etli, variable from strain to strain. Although all R. etli strains are able to nodulate and fix nitrogen in bean plants, the extra DNA might provide a means for adaptation to particular soil conditions.

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PS4-15 Loci determining specificity at steps post nodule induction in Lotus Jasmin A. Gossmann1, Laura Rose2, and Martin Parniske1 1Genetics, and 2Evolutionary Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany The root nodule symbiosis between legume species and their rhizobial partners shows specificity at successive steps of the nodulation process. We are aiming to identify loci conferring specificity at steps post nodule induction in the Lotus genome by exploiting natural allelic variation. The root nodule symbiosis is a tight and relatively specific symbiotic interaction including the exchange and specific recognition of signals between the symbiotic partners. The specificity of the signals exchanged determines inefficient vs. efficient symbiosis. Such selection and adaptation processes drive the co-evolution between legumes and rhizobia. We screened Lotus species and ecotypes for naturally occurring polymorphisms in nodulation-specificity. Our screen consisted of three steps: (i) the collection of Lotus corniculatus and Lotus uliginosus, totaling 61 accessions from throughout Europe, (ii) the isolation of the corresponding nodulating rhizobia, and (iii) a nodulation assay, where the diploid Lotus species Lotus burttii, Lotus filicaulis, and three cultivars of Lotus japonicus ('Miyakojima' MG-20, 'B-129' Gifu, and Nepal) were cross-inoculated with 10 rhizobia isolates from the genera Mesorhizobium, Rhizobium, Bradyrhizobium, Rhodopseudomonas, and Burkholderia. Known and novel combinations displaying incompatibility at different time points of the nodulation process were identified.

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PS4-16 A remarkable evolutionary conservation of RNA structures and expansion domains encoded by Enod40 genes from various plants

Alexander P. Gultyaev1, and Andreas Roussis2 1Leiden Institute of Biology, Leiden University, 2311 GP Leiden, The Netherlands; 2Department of Botany, Faculty of Biology, National and Kapodistrian University of Athens, 157 84 Athens, Greece Plant enod40 genes are associated with the earliest phases of the developmental program that control root nodule organogenesis and are keyregulators of the symbiotic interaction between leguminous plants and bacteria or fungi. They are also involved in non-symbiotic cell development, while the details of the molecular mechanisms of the enod40 action still remain an enigma. The enod40 mRNAs encode rather small peptides and the RNAs contain highly-ordered structures, that is, two possible functional properties that may, in parallel, determine the enod40 function. Based on predictions of conserved enod40 RNA structures in different species, we undertook a search for potential enod40 sequences stored in nucleotide databases. This search revealed new enod40 genes in various angiosperm plant families [1] and comparative analysis of predicted enod40 folds allowed us to establish the most conserved domains of the enod40 structure and assign certain evolutionary features. Insterestingly, the enod40 structures turn out to contain stable domains of variable length, located at homologous positions, exhibiting structure and evolution reminiscent of the so-called expansion domains observed in structural RNAs, such as rRNAs or other ribozymes. Enod40 RNA structures seem to be much more conserved than enod40 peptides: while a number of enod40 homologues do not encode for conserved peptides, the core enod40 RNA structure is universally conserved in all retrieved sequences. [1] Gultyaev & Roussis (2007). Nucleic Acids Res. 35:3144-3152.

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PS4-17 The host ranges of the promiscuous β-rhizobia Burkholderia phymatum STM815 and Cupriavidus taiwanensis LMG19424 extend throughout the subfamily Mimosoideae, but only B. phymatum STM815 forms effective symbioses outside the genus Mimosa

Euan K. James1, Geoffrey N. Elliott1, Colin Hughes2, Sergio M. de Faria3, Wen-Ming Chen4, Cyril Bontemps5, Marcelo F. Simon2, J. Peter W. Young5, and Janet I. Sprent1 1College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK; 2Department of Plant Science, University of Oxford, Oxford OX1 3RB, UK; 3EMBRAPA Agrobiologia, Seropédica, R.J., 23851-970, Brazil; 4Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung City 811, Taiwan; 5Department of Biology 3, University of York, York YO10 5YW, UK Two strains of β-rhizobia, Burkholderia phymatum STM815 and Cupriavidus taiwanensis LMG19424, which have previously been shown to nodulate several species in the large genus Mimosa [1], were tested for their ability to nodulate 68 species across the legume subfamily Mimosoideae, including species in all three largest tribes, the Acacieae, Ingeae, and Mimoseae. Cupriavidus taiwanensis LMG19424 failed to nodulate any species effectively, but could ineffectively nodulate a wide range of species (29 in total) across all three tribes, whereas B. phymatum STM815 effectively nodulated 12 species, particularly those closely related to Mimosa in the tribe Mimoseae, such as Anadenanthera peregrina and three species in the polyphyletic genus Piptadenia (P. gonoacantha, P. stipulacea, and the former P. obliqua, now Pityrocarpa obliqua). It also effectively nodulated more distant species in the Mimoseae, such as Leucaena leucocephala (but not the 10 other Leucaena spp. tested), Prosopis juliflora (but not the other seven Prosopis species tested), as well as Acacia pennatula and A. seyal, which are both in the subgenus Acacia, which is phylogenetically close to the Mimoseae. Interestingly, it failed to effectively nodulate any species in either of the other Acacia subgenera, Aculeiferum and Heterophyllum, which are both distant from the Mimoseae. In general, B. phymatum STM815 nodulated only a few of the tested species in the Ingeae, such as Calliandra calothyrsus, C. juzepzuckii, C. trinervia var. arborea, and Pithecellobium dulce. It is concluded that C. taiwanensis LMG19424 can be a highly promiscuous and opportunistic colonizer of Mimosoid legumes, but may not be capable of forming a symbiotic relationship outside the genus Mimosa. In contrast, B. phymatum STM815 can form symbioses with a range of Mimosoid legumes, and has the ability to nodulate legumes that cannot be nodulated by other broad host range rhizobia, such as Sinorhizobium sp. NGR234. [1] Elliott et al. (2007). New Phytol. 173:168-180.

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PS4-18 Phylogenetic analysis of Bradyrhizobium sp. (Genista tinctoria) strains

Michał Kalita, and Wanda Malek Department of Genetics and Microbiology, M. Curie-Skłodowska University, 20-033 Lublin, Poland From 40 Genista tinctoria nodule isolates six strains representing three separate AFLP genomic and phenotypic groups were used to obtain phylogenetic relationships to known rhizobial species. Phylogeny was determined by comparative nucleotide sequence analysis of the 16S rDNA, atpD, and dnaK genes using neighbour-joining (NJ) and maximum likelihood methods. All gene trees, independently of the construction method, support grouping of rhizobia specific for G. tinctoria together with Bradyrhizobium spp., although the studied strains do not form homogeneous group and are dispersed on phylograms among other strains of the genus Bradyrhizobium. In the majority of the cases, the analysed isolates and B. japonicum bv. genistearum gather together into one common cluster. The sequence similarity of the 16S rDNA, atpD and dnaK genes of six G. tinctoria nodule isolates was 98%-100%, 99%-100%, and 97%-100%, respectively, and between these sequences and the 16S rDNA, atpD and dnaK ones of other bradyrhizobia ranged from 93% to 98%, 92% to 99%, and 90% to 97%, respectively. G. tinctoria microsymbionts indicated the highest sequence similarities of the studied genes to those of B. japonicum bv. genistearum. This relationship was supported by DNA-DNA hybridisation analysis in microdilution wells. Polish isolates showed over 70% DNA-DNA similarity with all analysed strains belonging to the species B. japonicum, whereas Ukrainian and English strains showed over 60% similarity only with B. japonicum bv. genistearum. Our previous report on the phylogeny of nodulation genes and the symbiotic properties of G. tinctoria microsymbionts together with the currently presented results allow us to place the analysed strain within the species B. japonicum bv. genistearum.

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PS4-19 Co-evolution in the Rhizobium-legume symbioses Kristina Lindström1, Elena Chizhevskaya1,2, David Fewer1, and Evgeny Andronov1,2 1Department of Applied Chemistry and Microbiology, 00014 University of Helsinki, Finland; 2All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia The taxonomic relationship of bacteria symbiotically interacting with legumes is not congruent with that of their host plants, as determined by the phylogeny of key core genes. We have studied a wide range of symbiotic associates with the aim at understanding how rhizobia track their legume hosts in evolution. Using the Rhizobium galegae-Galega sp. system, we have found that rhizobial Nod genes in this very specific symbiosis evolved under host constraint. The diversity of the bacterial biovar orientalis strains collected in the Caucasus, the center of origin for G. orientalis, was greater than that of the Caucasian biovar officinalis isolates. Morphological and genomic diversity of the host plants was greater for the G. orientalis accessions than for the G. officinalis, confirming the gene center theory. Partial sequencing of SymRK and NFR5 gene sequences from selected plant accessions displayed diversity within the Galega genus. The Galega sequences were distinct from other sequenced plant genes and belonged to the same clade as other galegoid plants. There was evidence for positive selection within NFR5 and a negative selection within SymRK sequences. The possible co-evolution of the plant LysM-type receptors and Nod factor structure will be discussed, and the very specific Galega symbiosis will be compared with the more promiscuous symbiosis between the legume Calliandra calothyrsus and its associated bacteria.

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PS4-20 Nitrogen-fixing bacteria from two wild legumes (Retama raetam and Genista saharae) in Tunisia

Mosbah Mahdhi1, Angèle N'Zoué2, Philippe de Lajudie2, and Mohamed Mars1 1Laboratoire de Biotechnologies Végétales Appliquées à l'Amélioration des Cultures, Faculté des Sciences de Gabès, 6072 Gabès, Tunisia; 2Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France Sixty-three isolates from nodules of Genista saharae and Retama raetam growing in arid regions of Tunisia were characterized and compared to reference strains representing different rhizobial species. A polyphasic approach, which included a phenotypic study, RFLP of PCR-amplified 16S rRNA genes, and 16S rRNA gene sequencing, was used in the comparative analysis. The isolates varied in their phenotypic and genetic characteristics. The majority of the isolates tolerate 3% NaCl and can grow up to 40°C. It was found that new isolates are diverse and most of them affiliated to Ensifer and Rhizobium. Furthermore, our results support the presence of non-nodulating commensal strains (Phyllobacterium and Agrobacterium) in legume nodules. Except for Agrobacterium and Phyllobacterium isolates, all strains are able to nodulate their host plant of origin, but the number of nodules and nitrogen fixation vary among them.

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PS4-21 Sinorhizobium meliloti populations in soil, nodules and plant tissues: what is their ecological meaning?

Francesco Pini, Emanuele G. Biondi, Lorenzo Ferri, Marco Bazzicalupo, and Alessio Mengoni Dipartimento di Biologia Evoluzionistica, Università degli Studi di Firenze, 50125 Firenze, Italy Strains of the species Sinorhizobium meliloti are ubiquitous in soils and they specifically form symbiotic nitrogen-fixing nodules on the roots of leguminous plants, such as alfalfa (Medicago spp.). S. meliloti is a model species to study symbiotic nitrogen fixation and it has been investigated as a model system also for bacterial population genetics. So far, most of the studies on S. meliloti ecology have been performed on bacteria isolated from nodules of different Medicago species (plant trapping). Actually, the current model for the lifestyle of S. meliloti is based on the alternation of free life in soil and symbiosis with host plant species. In this classical model, due to the biased bacterial sampling (only strains from nodules are isolated), the role and evolutionary significance of free-living and nodule-forming strains in a given S. meliloti population cannot be satisfactorily clarified. Actually, almost nothing is known about the presence and, especially, the genetic diversity of S. meliloti populations free living in soil and endophytically colonizing plants others than legumes [1]. To begin to answer these fundamental questions in the ecology of S. meliloti, we assayed the endophytic ability of wild-type and mutant strains on M. truncatula plants and we developed new PCR tools, based on qPCR and T-RFLP of S. meliloti-specific gene variants, for enumeration of S. meliloti cells, and analysis of their genetic polymorphism in environmental samples. Populations of S. meliloti present in the DNA extracted from soil, nodules, and leaves of leguminous (alfalfa) and grass species, were analysed and the relationships between the populations detected in the different habitats were investigated. [1] Chi et al. (2005). Appl. Environ. Microbiol. 71:7271–7278.

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PS4-22 Genotypic diversity of Sinorhizobium meliloti strains isolated from different legumes

Bacem Mnasri1, Philippe de Lajudie2, and Ridha Mhamdi1 1Laboratoire Interactions Légumineuses-Microorganismes, Centre de Biotechnologie à la Technopole Borj-Cédria, Hammam-lif 2050, Tunisia; 2Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France Sinorhizobium meliloti was first known as a specific micosymbiont of Medicago. Recently, two new biovars, meliloti and medicaginis were proposed to distinguish strains exhibiting different host specificities towards Medicago species. The biovar meliloti encompasses the long known S. meliloti strains represented by the strains ATCC9930T and RCR2011, efficient on M. sativa and M. truncatula, and forming no or ineffective nodules on M. laciniata. The biovar medicaginis groups S. meliloti strains showing nitrogen fixation-specificity with M. laciniata and M. sauvagei, but forms ineffective nodules on M. sativa. Two other biovars were also proposed for S. meliloti strains isolated from Acacia tortilis (bv. acaciae) and Phaseolus vulgaris (bv. mediterranense). These isolates did not nodulate Medicago, but were effective in nitrogen fixation with their respective original host. More S. meliloti strains were isolated from various other legumes, but their symbiotic status remains unclear. Here, we studied a collection of 47 S. meliloti strains isolated from six Medicago species and from 11 other legumes, including Acacia tortilis, Argyrolobium uniflorum, Cicer arietinum, Genista saharae, Hedysarum carnosum, Hippocrepis bicontorta, Lotus creticus, Lotus roudairei, Ononis natrix, Phaseolus vulgaris, and Retama raetam. The chromosomal diversity was assessed by rep-PCR, PCR-RFLP of 16S-23S IGS and 16S rDNA sequencing. The symbiotic diversity was assessed by PCR-RFLP of nifH and nifDK regions and nodA gene sequencing. The diversity of the symbiotic plasmids and their distribution among the chromosomal backgrounds will be discussed.

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PS4-23 Diversity and population genomics of Aeschynomene photosynthetic symbionts

Lucie Miché, Lionel Moulin, Clémence Chaintreuil, and Gilles Béna Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France Tropical aquatic legumes of the genus Aeschynomene are nodulated by Bradyrhizobium spp. that possess several peculiarities compared to more "classical" rhizobia interacting with legumes: (i) Aeschynomene symbionts are photosynthetic, a trait that is unique among rhizobia and nodulation efficiency is highly dependent on photosynthetic activity [1]; (ii) besides root nodulation, photosynthetic bradyrhizobia can also induce nodulation on the stem of their hosts; (iii) whole-genome analysis of two reference strains (ORS278 and BTAi1) highlighted the absence of canonical nodABC genes in these bacteria [2], a surprising result that raises the question of the mechanisms involved in Aeschynomene/photosynthetic bradyrhizobia interactions. A large sampling campaign was done to study the diversity of photosynthetic bradyrhizobia nodulating several species of Aeschynomene in Africa and Central America. Phylogenetic analyses based on both recA marker and whole genome AFLP studies confirmed that photosynthetic strains form a separate cluster among bradyrhizobia. Moreover, a new cluster of strains that are not photosynthetic but are able to stem-nodulate A. indica was found. Six representative strains were fully sequenced by the new Solexa (Illumena) pyrosequencing technology that generates millions of 36-bp oligomers. Data were assembled and comparative genomics carried out in order to study the core genome and accessory genes involved in the photosynthetic Bradyrhizobium spp. adaptation to their peculiar ecological niches. [1] Giraud & Fleischman (2004). Photosynth. Res. 82:115-130. [2] Giraud et al. (2007). Science 316:1307-1312.

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PS4-24 Biodiversity of Robinia pseudoacacia microsymbionts Bożena Mierzwa, Sylwia Wdowiak-Wróbel, Barbara Łotocka, Małgorzata Szlachetka, Elwira Lelito, Michał Bartosik, and Wanda Małek Department of Genetics and Microbiology, University of Maria Curie-Skłodowska, 20-033 Lublin, Poland Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia grown in Japan and Poland, were characterized for genomic diversity by the AFLP technique (Amplified Fragment Length Polymorphism), genomic DNA G+C content, phage sensitivity, plasmid presence, plant host specificity, and structure of black locust nodules induced by these rhizobia. R. pseudoacacia isolates are genomically heterogeneous as indicated by the AFLP method. The DNA patterns were found to be highly specific for nearly each strain, although DNA bands specific for most nodule isolates were also noted. The phage sensitivity test was employed to differentiate R. pseudoacacia nodulators. Black locust tree soil served as a source of the phages used for the test. Extrachromosomal DNAs were found in all strains. Nitrogenase structural genes were located on 771-961 kb megaplasmids. R. pseudoacacia rhizobia formed an effective symbiosis not only with the native host but also with Amorpha fruticosa and Amorpha caliphornica. They were more effective in N2 fixation with black locust than with desert false indigo and California false indigo. The R. pseudoacacia root nodules were designated as indeterminate with apical meristem. In the proximal part adjacent to the root of 13-week-old nodules, saprophytic bacteria, probably released from the infection threads were observed in degenerated bacteroidal tissue.

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PS4-25 Rhizobium leguminosarum is the predominant species found among rhizobia nodulating common bean (Phaseolus vulgaris L.) in León (Spain)

Daniel Mulas1, Martha Helena Ramírez-Bahena2, Paula García-Fraile2, Encarna Velázquez2, and Fernando González Andrés1 1Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de León, 24007 León, Spain; 2Departamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, Spain Effective root nodules were collected in 2006 in order to assess the diversity of rhizobia nodulating common bean (Phaseolus vulgaris L.) in NW Castilla y León fields where it has been traditionally grown for decades. Several strains were isolated and experiments leading to their taxonomic identification were performed. Molecular techniques based on the PCR procedure (TP-RAPD) [1] and the sequence of two housekeeping genes (recA and atpD) [2] were used to identify the isolated rhizobial strains. Furthermore, the strains were used in an in vitro experiment aiming at confirming their ability to induce root nodulation in common bean plants as well as to evaluate the differences among them in terms of N2 fixation efficiency by means of statistical analysis. The strains were inoculated in common bean plants grown under sterile and controlled conditions and N2-free solution was added to their irrigation. Despite the finding of two different rhizobia species in these soils, most of the strains were included in the phylogenetic group of Rhizobium leguminosarum, whereas only two of them were identified as R. giardinii. The results showed that all strains induced nodulation in common bean roots within 21 days after inoculation and the most efficient strains were those related to R. leguminosarum, while those of R. giardinii had a lower N2 fixation efficiency, as previously observed [3]. [1] Rivas et al. (2001). Electrophoresis 22, 1086-1089. [2] Gaunt et al. (2001). Int. J. Syst. Evol. Microbiol. 51, 2037-2048. [3] Armarger et al. (1997). Int. J. Syst. Bacteriol. 47, 996-1006.

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PS4-26 Presence of Gluconacetobacter diazotrophicus in tropical legume species associated to coffee crops in Mexico

Teresita Jiménez-Salgado1, T. Dolores Castañeda-Antonio1, E. Flores-Rivera1, P. Román1, José Luis Contreras-Jiménez2, Armando Tapia-Hernández1, and José-Antonio Munive1 1Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, and 2Herbario, Jardín Botánico, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico A variety of diazotrophic bacteria have been isolated from rhizosphere and roots of sugarcane plants. In 1988, isolates of the acid-tolerant nitrogen-fixing bacterium Acetobacer diazotrophicus were reported from sugarcane plants. These strains were related to the contribution on N nutrition to sugarcane crops. These bacteria were later classified as Gluconacetobacter diazotrophicus, which have been isolated from other plants, such as Cameroon grass, sweet potato, tea, banana, ragi, rice, pineapple, and even from insects that infest sugarcane. The presence of G. diazotrophicus has also been associated to coffee plants. In this work, we report the isolation of several strains belonging to the Acetobacteriacea family, including strains belonging to the genus Gluconacetobacter, from two native tropical legume trees commonly associated to coffee crops in Mexico. These strains were isolated from inner tissues of surface-sterilized roots and stems of Leucaena and Inga plants, showing phenotypical characteristics related to the Acetobacteria group, some of which have been related to the Gluconacetobacter genus. Polyphasic taxonomy has got great importance in taxonomic studies; that is why physiological tests, such as nitrogen fixation and biochemical tests, have been essayed to complete genetic studies. The taxonomical status of the strains was corroborated by 16S rDNA analysis.

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PS4-27 Local bradyrhizobia promote plant growth on both maize and legume

Angèle N'Zoué1, Lionel Moulin1, Yves Prin1, Anne Willems2, Gisèle Laguerre1, Adako Moudiongui3, Christophe Kouamé3, Antoine Galiana1, Bernard Dreyfus1, and Philippe de Lajudie1 1Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France; 2Laboratorium voor Microbiologie, Universiteit Gent, 9000 Gent, Belgium; 3Centre National de la Recherche Agronomique, Abidjan, Ivory Coast In Ivory Coast (West Africa) food crops are widely produced through a legume-cereal-tuber intermixed culture system. To assess the role and genetic diversity of rhizobial bacteria in this agronomic system, we isolated 74 rhizobial strains from root nodules of groundnut, soybean, and cowpea plants grown as intermixed cultures in fields all around the country. They all belonged to the Bradyrhizobium species (16S rDNA sequencing) with a large genetic diversity (16S-23S rDNA intertranscribed spacer [ITS] sequencing). They formed 17 clusters, some corresponding to B. japonicum, B. elkanii, B. yuanmingense, some to Bradyrhizobium spp. reference genospecies Ia, IV, V, VIII, XI [1], and six separate groups. We further performed MultiLocus Sequence analysis on five housekeeping genes (gnlII, recA, dnaK, rpoB, and atpD), using several phylogenetic methods (MP, ML, Bayesian), and obtained a consensus phylogenetic tree. Greenhouse tests were performed to screen the new strains for (i) their nitrogen-fixing potential on groundnut, soybean, and cowpea, and (ii) their plant growth promotion capacity on maize. Eight weeks after inoculation, plants were surveyed for their nodule numbers and dry weights, aerial part and root dry weight, leaf color, and ARA. Statistical analysis show that all strains (100%) are efficient on cowpea, 26% on groundnut (separate genospecies clusters 3, 8, 9, and 10), and 27% on soybean (B. japonicum cl. I, Ia, B. elkanii, cl. II, B. yuanmingense). Six strains (STM3040, STM3080, STM3078, STM3088, STM3070, and STM3079) were selected for their growth promotion on both legumes and cereal. Biochemicals tests showed ammonium production in STM3078, siderophores production in STM3080, STM3078, and STM3088. No AIA production nor phosphate solubilization were detected. Further investigations for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, ACC gene sequencing, and endophytic functions are in progress and will be discussed. [1] Willems et al. (2003). Syst. Appl. Microbiol. 26:203-210.

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PS4-28 A survey of chickpea rhizobia in Portugal shows high species diversity

Ana Alexandre, Clarisse Brígido, Marta Laranjo, Sérgio Rodrigues, and Solange Oliveira Instituto de Ciências Agrárias Mediterrânicas, Departamento de Biologia, Universidade de Évora, 7002-554 Évora, Portugal Chickpea is one of the most important legumes worldwide, being cultivated for its seeds, which are a major protein source. Besides the two specific microsymbionts, Mesorhizobium ciceri and M. mediterraneum, strains close to M. amorphae, M. loti, and M. tianshanense are able to induce effective nodules in this legume [1, 2]. The aim of this study was to evaluate the genetic diversity and symbiotic effectiveness of native chickpea rhizobia from Portugal. With trap plants using soils from the 13 provinces of Portugal, including Madeira and the Azores islands, 110 isolates were obtained. Isolates are highly diverse and group with almost all Mesorhizobium-type strains, generating four main clusters (A to D), according to the 16S rRNA gene-based phylogeny. Interestingly, only 37% of the isolates grouped with M. ciceri (cluster B) and M. mediterraneum (cluster D). Most isolates are included in cluster A, that also includes M. huakuii and M. amorphae, but probably belong to new species. The association found between cluster and origin site of the isolates suggests a geographical distribution of species in Portugal. Most isolates from the North, Centre and South belong to cluster B, cluster A, and cluster D, respectively. Of the rhizobia isolates, 40% showed a symbiotic effectiveness (SE) above 50%. Approximately 10% of the isolates were found to be highly effective (SE>75%). To our knowledge this is one of the few surveys on chickpea rhizobia and the first survey of indigenous rhizobia in Portugal. Although chickpea has been traditionally described as a restrictive host, the present work shows that several Mesorhizobium species, including putative new species, are able to effectively nodulate this legume. Funding: FCT project POCTI/BME/44140/2002, co-financed by EU-FEDER; FCT grants to M. L. (SFRH/BD/6772/2001, SFRH/BPD/27008/2006), A. A. (SFRH/BD/18162/2004) and C.B. (SFRH/BD/30680/2006). [1] Laranjo et al. (2004). FEMS Microbiol. Ecol. 48:101-107. [2] Rivas et al. (2007). Lett. Appl. Microbiol. 44:412-418.

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PS4-29 Assessing the biodiversity of indigenous cowpea-nodulating bradyrhizobia using nine genotypes in three African countries

Flora Pule-Meulenberg1, Tatiana Krasova-Wade2, and Felix D. Dakora1 1Tshwane University of Technology, Pretoria 0001, South Africa; 2Laboratoire Commun de Microbiologie, IRD/ISRA/UCAD, Dakar, Senegal Nine cowpea genotypes (Omondaw, Brown eye, Bostswana White, Glenda, Mamlaka, Fahari, Apagbaala, ITH98-46, and IT82D-889), originating from Ghana, Botswana, South Africa, Tanzania, and IITA in Nigeria, were used as trap hosts to assess the biodiversity of cowpea bradyrhizobia in three African countries, namely Ghana, Botswana, and South Africa. Nine hundred root nodules collected from the nine cowpea varieties grown in Botswana, Ghana and South Africa, were used to directly extract the DNA of indigenous bradyrhizobia from soils of the three countries, followed by PCR-RFLP analysis of the IGS region using MSpI and HaeIII restriction enzymes. The bacterial DNA isolated from the 900 nodules grouped into 19 IGS types (I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX). In terms of country of origin, 16 out of the 19 IGS types (i.e. I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XIII, XIV, XVI, XVII, and XVIII) came from root nodules of the nine cowpea genotypes planted in South Africa, five (IV, V, VI, XV and XIX) from nodules of the same nine genotypes grown in Botswana, while another five IGS types (II, V, VII, VIII, and XII) came from root nodules of the nine cowpea genotypes planted in Ghana. Of the 19 IGS types found in nodules of cowpea from South Africa, 10 were unique to that country, two IGS types were also unique to Botswana, while only one was exclusively restricted to Ghana. In terms of geographic distribution, only IGS type V was found in soils of all three countries, while IGS types III and VI were only found in soils of Botswana and South Africa, but not Ghana. IGS types II and VIII were also only found in soils of Ghana and South Africa, but not Botswana. No IGS type was common to Ghana and Botswana. Of the nine cowpea genotypes used as trap hosts for indigenous cowpea bradyrhizobia in Botswana, Ghana, and South Africa, the variety Fahari from Tanzania (which ranked number one out of 27 genotypes for superior symbiotic traits) was nodulated by the highest number of indigenous bradyrhizobia, with nodule occupancy by eight out of the 19 IGS types. Bradyrhizobia belonging to IGS types V and VIII were able to nodulate all the nine cowpea genotypes, except for varieties Omondaw and Glenda, respectively, with cv. Mamlaka and Botswana White showing the highest nodule occupancy of 78.9% and 57.9%, respectively. This study has provided some insights into the diversity of indigenous bradyrhizobia nodulating cowpea in African soils with some implications for cowpea production continentally.

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PS4-30 Genetic diversity and inter-strain relatedness of bradyrhizobia isolated from peanut and wild legume nodules in China

Leena A. Räsänen1, Iiris Mattila1, Qiang Chen2, Xiaoping Zhang2, and Kristina Lindström1 1Department of Applied Chemistry and Microbiology, Biocenter 1, FIN-00014 University of Helsinki, Finland; 2Department of Applied Microbiology, Sichuan Agricultural University, 625000 Sichuan, P. R. China Peanut or groundnut (Arachis hypogaea L.) is the world's fourth most important source for edible oil and the third most important source of vegetable protein. Developing countries account for 96% of the global peanut area and 92% of the global production [1]. China is nowadays the major peanut producer, being responsible for 40% of world production of 36.4 metric million tons in 2004 [2]. The modern peanut originates from South America. In China, it has been introduced in different places at different times, the earliest records of its cultivation date from 1368 [3]. N2-fixing symbionts of peanuts, bradyrhizobia, form in general a very diverse group of bacteria and can nodulate a large variety of different legume species. At the phylogenetic level, bradyrhizobia are more related to many other β-proteobacteria than to nodulating genera of Rhizobium, Mesorhizobium, and Sinorhizobium. We compared AFLP fingerprints and RFLP patterns of amplified housekeeping genes (ITS region, recA, and glnII) and symbiotic genes (nifH and nodC) of 17 peanut bradyrhizobia to those obtained from bacteria isolated from a perennial twining vine, kudzu (Pueraria lobata (9 strains), other wild legumes (5 strains), and reference (14) strains to find out which kind of bradyrhizobia are nodulating cultivated and wild legumes in China. Moreover, our aim was to study how different genes group strains and whether the groupings reflect bradyrhizobial species, host plant species, or geographic origin. According to cluster analyses of AFLP fingerprints and RFLP patterns, the genetic diversity of peanut and kudzu bradyrhizobia was great. Chinese strains formed three major croups: (i) bradyrhizobia nodulating peanut, (ii) B. japonicum-like strains nodulating kudzu, and (iii) B. elkanii-like strains nodulating kudzu. International type and reference bradyrhizobia also showed a great heterogeneity. The origin of bradyrhizobia being able to nodulate peanuts in China will be discussed. [1] http://www.icrisat.org/GroundNut/GroundNut.htm. [2] FAO Statistical Yearbook 2005-2006, Rome, Food and Agriculture Organization. [3] Yao (2004). Peanut in Local and Global Food Systems Series, Report no. 4, R.E. Rhoades, and V. Nazarea (Eds.), Athens, University of Georgia (http://www.lanra.uga.edu/peanut/download/china.pdf).

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PS4-31 Rhizobia-bean association in the northwest of Spain Ana Paula Rodiño1, Marlene Pérez-Barbeito1, Jean-Jacques Drevon2, and Marta Santalla1 1Plant Genetic Resources Department, Misión Biológica de Galicia, Consejo Superior de Investigaciones Científicas, 36080 Pontevedra, Spain; 2Institut National de la Recherche Agronomique-Montpellier-SupAgro, UMR1222, Rhizosphère et Symbiose, 34060 Montpellier Cedex, France Common bean (Phaseolus vulgaris L.) is a traditional crop of American origin with two major areas of domestication in South and Middle America. The Iberian Peninsula is considered as a secondary center of diversification because of the exceptional gene exchange between beans originated from both pools, by contrast with the rest of Europe, where the Andean origin dominates, and America, where such gene flow did not occur. The microorganisms associated with the common bean plant for its symbiotic nitrogen fixation may exhibit a similar arrangement of genetic diversity in Mesoamerican and Andean gene pools. In Europe, rhizobia strains that nodulate common bean have a narrow genetic diversity that was correlative to beans being an introduced crop. Crop legumes as the common bean have been used extensively in agriculture over the past century, mainly for maintaining soil fertility. The low fertility can also have a negative impact on the legume-rhizobia symbiotic relationship reducing the ability of rhizobia to form nodules with optimal N2-fixing capacity. The symbiotic fixation of nitrogen provides an ecologically acceptable alternative to the high applications of nitrogenous fertilizers, especially in Europe, and an economic alternative to the limited access to these fertilizers of the developing countries. Inoculation is required to increase yield through N2 fixation and to reduce the external inputs. The objective of this work was to study the rhizobia-bean association and the ability of rhizobia to form nodules when introducing common bean into different soil environments. There are the differences in the interaction nodulation-bean in different environments and that could be exploited in breeding programs for enhanced nodulation and N2 fixation in each bean market class.

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PS4-32 The evolution of symbiotic pathway Natalya Saveljeva1, Gerben Bijl2, René Geurts2, Ludmila Lutova1, and Ton Bisseling2 1St. Petersburg State University, St. Petersburg 199034, Russia; 2Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands The evolution of plant symbiotic interactions is one of the interesting scientific questions. An evolutionary queue frequently occurring is based on changes in gene expression, a process named regulatory evolution. Gene expression may evolve through changes in either the activity or the deployment of the proteins (primarily transcription factors) that govern gene expression, or in the cis-regulatory sequences that modulate the expression of individual genes. Also in the case of legumes, changes in expression could be a prerequisite for symbiosis with Rhizobium. The focus of this work is to identify specific cis-regulatory elements (CREs) that govern symbiotic legume genes and are absent in orthologous counterparts of non-legumes plant species. To study this we used two model plants: Medicago truncatula (legume) and Poplar trichocarpa (non-legume). Mutants in the legume Medicago affected in symbiotic signalling where trans-complemented using promoter constructs of the non-legume P. trichocarpa. This revealed that at least for one gene, encoding a calcium/calmodulin kinase (CCaMK), specific cis-regulatory elements are lacking in the poplar construct. To compare both CCaMK promoter regions, reporter constructs were used. Experiments with GUS as reporter showed that both promoters are active in root tissues. To study the temporal and spatial regulation of both promoters, we used the DsRED-E5 reporter construct. DsRED-E5 has a prolonged maturation time, by which it has a bright green fluorescence for a period of 24 h, which subsequently shifts to red. By quantifying the ratio between green and red fluorescence, promoter regulation can be studied at the cellular level. Preliminary results using this reporter showed a strong up-regulation of the Medicago CCaMK promoter in roots of Medicago during symbiotic infection. Next, the regulation of the poplar promoter will be studied.

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PS4-33 Genetic diversity of indigenous alfalfa (Medicago sativa) rhizobia in different soil types of Istrian peninsula

Sanja Sikora1, Marco Bazzicalupo2, Katarina Huić Babić1, Majid Talebi3, and Mihaela Blažinkov1 1Department of Microbiology, Faculty of Agriculture, University of Zagreb, 10000 Zagreb, Croatia; 2Dipartimento di Biologia Evoluzionistica, Università di Firenze, 50125 Firenze, Italy; 3Department of Plant Breeding and Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran Composition and characteristics of rhizobial field population are of great importance in the alfalfa production, particularly when introducing commercial inoculant strains under natural conditions. The main aim of the present investigation was to identify and to characterize the indigenous alfalfa rhizobia isolated from different field sites in the inland of the Istrian peninsula. The actual composition and genetic diversity of natural field population was studied by two PCR fingerprinting methods. PCR-RFLP of 16S rDNA clearly showed that all isolates can be determined as Sinorhizobium meliloti. Cluster analysis of RFLP patterns obtained with RsaI, showed that none of the isolates was identical with S. medicae-type strains. However, several isolates produced slightly different RFLP patterns from S. meliloti-type strains and other isolates. Dendrogram derived from AFLP profiles revealed considerable genetic diversity among S. meliloti isolates. Only a few strains were identical or nearly identical to each other. Most of the isolates were grouped within one major cluster while the other major cluster was formed from the same seven isolates that were characterized by slightly different PCR-RFLP profiles in comparison with all other S. meliloti strains. AFLP analysis was also performed with some S. meliloti strains of different origin. Dendrograms derived from combined AFLP profiles showed that the Istrian strains were clearly separated from the other ones. Analysis of molecular variance (AMOVA) allowed relating the genetic structure of the symbiotic population to various factors, including location, soil type, vegetation, and chemical properties of soil samples. Location (14.44%), soil pH (10.85%), amount of total soil nitrogen (9.42%), and soil type (6.91%) significantly influenced the distribution of the genetic variability of the population, while the effects of vegetation and amount of potassium in the soil were not found to be significant.

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PS4-34 Leguminosae nodulating Burkholderia isolated from soils under different land use systems in the Amazon region

Krisle da Silva, Alice de Souza Cassetari, Adriana Silva Lima, and Fátima Maria de Souza Moreira Department of Soil Science, Federal University of Lavras, Lavras, MG 37200-000, Brazil The Amazon region represents a great potential as a source of new species of Nitrogen-Fixing Leguminosae-nodulating bacteria (NFLNB). The genus Burkholderia comprises approximately 45 species, but only four are able to nodulate Leguminosae. Previous work isolated 44 Burkholderia strains belonging to NFLNB communities occurring in Amazonian soils under different land use systems: forest, young secondary forest, old secondary forest, agroforestry, crops, and pasture. These strains were isolated by using siratro (Macroptilium atropurpureum) as trap species [1]. Their 16S rRNA sequences have similarity levels lower than 98% with Burkholderia species when compared to the GenBank database and they can be divided into three groups. The strains were evaluated through cultural characteristics on 79 medium [2], BOX-PCR profiles, solubilization of calcium, aluminum and iron phosphates, acetylene reduction assay on free-nitrogen LO medium [3] with three carbon sources (lactate, mannitol, and fructose), and symbiotic efficiency. All tests included type strains of 11 nitrogen-fixing, nodulating, and associative Burkholderia species. The Amazonian strains had just a few differences in cultural characteristics on 79 medium and presented among them distinct BOX-PCR patterns that differ also significantly from those of known Burkholderia species type strains. All Amazonian and type strains did not solubilize Al and Fe-phosphates, but in Ca-phosphate medium, 40 strains and four type strains presented this characteristic. Most of the strains grew on semi solid nitrogen-free media and presented acetylene reduction mainly when fructose was used as carbon source, followed by lactate and mannitol. Some of these strains were able to nodule siratro efficiently. These results corroborate the diversity of Burkholderia as NFLNB as well as their potential to contribute for plant growth both by symbiotic and associative relationships and by other biochemical processes. [1] Lima (2007) PhD thesis, Federal University of Lavras, Lavras, Brazil. [2] Fred & Waksman (1928) Laboratory Manual of General Microbiology. New York, McGraw-Hill. [3] Dreyfus et al. (1983). Int. J. Syst. Bacteriol. 38:89-98.

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PS4-35 Evaluation of genetic diversity of rhizobial strains nodulating soybean (Glycine max L.) isolated from Serbian fields

Olivera Stajković1, Sofie De Meyer2, Bogić Miličić1, Dušica Delić1*, and Anne Willems2* 1Institute of Soil Science, 11000 Belgrado, Serbia; 2Laboratory of Microbiology, Ghent University, 9000 Gent, Belgium; * equal contributions Among various legumes in Serbia, soybean (Glycine max L.) is widely grown and plays a central role as food and forage crop. However, the diversity of native rizobial strains in Serbia that can nodulate soybean is poorly understood. Diversity and phylogeny of 50, mainly slow-growing; strains, isolated from root nodules of soybean in different geographical regions of Serbia, were studied using rep-PCR, 16S rDNA, and recA gene sequencing. On the basis of 16S rRNA gene sequences, the majority of the rhizobia were affiliated to the genus Bradyrhizobium, with B. japonicum as the closest related species. Phylogenetic analysis of recA sequences revealed a high diversity of strains nodulating soybean. These isolates according to recA sequences clustered into at least six distinct groups within the genus Bradyrhizobium. One group of isolates was identified as B. japonicum and one strain as B. liaoningense (100% similarity). The four other Bradyrhizobium groups may represent new species. Additionally, two strains isolated from soybean belong to the genus Rhizobium, showing low similarity (89.9%) to R. rhizogenes and they may form new Rhizobium species, too.

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PS4-36 Identification of microsymbionts associated to the leguminous tree Parapiptadenia rigida

Cecilia Taulé, M. Zabaleta, Raúl Platero, and Elena Fabiano Laboratorio de Ecología Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Universitad de la República, Montevideo 11600, Uruguay Among Uruguayan leguminous trees, Parapiptadenia rigida comes out as one of the most promising species for agroforestry. In addition to its use for re-vegetation and erosion control, this specie produces quality fuel wood, has a long-lasting hardwood, and is attractive for works of refined carpentry. In the process that leads to tree plantations, the stage of nursery is from great importance, because the vitality of the seedlings will determine the success of the forest. Unfortunately, there is no knowledge about the behaviour of Uruguayan native leguminous trees on the stage of nursery for massive purpose. Our aim is to take advantage of symbiotic nitrogen-fixing bacteria associated to P. rigida to inoculate this species under nursery conditions in order to achieve post-planting success. Our approach was to isolate microsymbionts from nodules obtained from young plants present in natural forests. We also employed a trap plant method by using soil samples collected from different regions. To identify probable diazotrophic bacteria, the presence of the nifH gene was evaluated by PCR. Out of 92 isolates, 56 gave positive results. In order to distinguish the selected isolates, PCR of consensual repetitive extragenic region and PCR-RFLP were performed. According to 16S rRNA sequencing, 13 isolates belong to Burkholderia genera and two to Rhizobium genera. Symbiotic nitrogen-fixing Burkholderia have recently been named β-rhizobia in analogy to the well know α-rhizobia species. The capacity of the isolates to establish symbiotic associations with P. rigida was corroborated by single-strain inoculation of plant seedlings. This work was supported by INIA-FPTA 216 and PDT/C/OP/67/03.

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PS4-37 Transfer of Mesorhizobium loti symbiotic genes into the Bradyrhizobiaceae chromosome

Rujirek Noisangiam1, Clive Ronson2, Joshua P. Ramsay2, John T. Sullivan2, Nantakorn Boonkerd1, Achara Nuntagij3, and Neung Teaumroong1 1School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; 2Department of Microbiology, University of Otago, Dunedin, New Zealand; 3Soil Microbiology Group, Soil Science Division, Department of Agriculture, Bangkok 10900, Thailand The evolutionary history among rhizobial groups, Rhizobium palustris, soybean-symbiotic, and non-symbiotic bradyrhizobial isolates from Thailand was elucidated. The phylogenetic analysis of the housekeeping genes atpD, glnII, recA, and 16S rRNA gene together with the biochemical analysis using API 20NE revealed the close relationship among bradyrhizobia, R. palustris and the non-symbiotic isolates. It was hypothesized that lateral gene transfer of the symbiotic system might play a significant role for their evolution. Symbiosis island transfer in Mesorhizobium loti is a case of rapid evolution and diversification for the rhizobial group related to host-plant interaction. The ability of the M. loti symbiosis island to transfer by conjugation to symbiotic and non-symbiotic Bradyrhizobia was determined. A mini-island, comprising a suicide plasmid containing the attP region and intS, could integrate into chromosome of bradyrhizobia and R. palustris via site-specific integration at PhetRNA. A putative staggered cleavage event is proposed. Surprisingly, we also demonstrated the ability of symbiosis islands to integrate into the chromosome of a symbiotic isolate as B. yuanmingense in which some nodulation genes were not detected. However, some genes were thought to be deleted after recombination. The mechanism is under investigation.

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PS4-38 Pyruvate carboxylase encoded by midK is required for efficient metabolism of mimosine by Rhizobium sp. strain TAL1145

Panlada Tittabutr1, Jonathan D. Awaya2, Qing X. Li2, and Dulal Borthakur2 1School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; 2Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA The midK gene, which encodes a protein similar to pyruvate carboxylase, is located downstream of midR in the cluster of genes for mimosine degradation in Rhizobium sp. strain TAL1145. The transcription of midK is also regulated by MidR, like other Mid genes in the presence of mimosine. To determine the role of MidK in mimosine degradation, the midK mutants of TAL1145 were constructed by Tn3Hogus mutagenesis, and found that the midK mutants degraded mimosine more slowly than the wild type. However, these mutants could utilize pyruvate as a source of carbon, indicating that there is another pyruvate carboxylase (pyc) gene in TAL1145. Two classes of clones were isolated from the library of TAL1145 by complementing a pyc mutant of Rhizobium etli: one class contained midK, while the other carried pyc. Both midK and pyc of TAL1145 complemented the midK mutant for mimosine degradation, and also the R. etli pyc mutant for pyruvate utilization. A phylogenetic tree, comprising midK of TAL1145 and pyc from different rhizobia and other Gram-negative bacteria, shows that midK is closely related to pyc of Sinorhizobium meliloti and S. medicae, while pyc of TAL1145 is closer to those of R. etli, R. leguminosarum bv viciae, and R. leguminosarum bv trifolii. It is likely that midK evolved through a duplication of pyc that got inserted into the Mid gene cluster during evolution and is required for an efficient conversion of mimosine into 3-hydroxy-4-pyridone (HP).

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PS4-39 Biogeography and evolutionary genetics of five Bradyrhizobium species that nodulate wild and cultivated legumes across the globe, assessed by multilocus sequence analysis

Pablo Vinuesa1, Iraís Figueroa-Palacios1, Agustín Avila1, Enrique Zozaya1, Bruno Contreras-Moreira2, and Dietrich Werner3 1Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, 62210 Morelos, Mexico; 2Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, 50059 Zaragoza, Spain; 3Fachbereich Biologie, Philipps-Universität Marburg, 35032 Marburg, Germany A highly supported maximum likelihood species phylogeny was inferred from partial atpD, recA, glnII, and rpoB sequences corresponding to 109 Bradyrhizobium strains, including 76 novel isolates from the nodules of Glycine max (soybean) trap plants inoculated with soil samples from Myanmar, India, Nepal, and Vietnam, as well as 40 other isolates from diverse geographic origins and host tribes. Seventy five of the Asiatic soybean isolates could be classified as B. japonicum type Ia (USDA110/USDA122-like), B. liaoningense, B. yuanmingense, or B. elkanii, whereas one represented a novel Bradyrhizobium lineage. This is the first report showing that B. yuanmingense nodulates soybeans. Most Nepalese B. japonicum Ia isolates belong to a highly epidemic clone closely related to strain USDA110. Significant phylogenetic evidence was found against the monophyly of the B. japonicum I and Ia lineages. Analysis of their DNA polymorphisms revealed high population distances, significant genetic differentiation, and contrasting population genetic structures, suggesting that the strains in the Ia lineage should not be classified as B. japonicum. All species showed broad geographic and environmental distribution ranges. For example, B. yuanmingense is distributed across both hemispheres, colonizing habitats with climates that range from hot arid or semiarid to warm or cold humid with marked seasonal variations in water availability. Bradyrhizobium japonicum, B. liaoningense, and B. elkanii were found to be preferentially distributed in areas with different types of humid climates. The DNA polymorphism patterns of all species conformed to the expectations of the neutral mutation and population equilibrium models and, excluding the B. japonicum Ia lineage, were consistent with intermediate recombination levels. All species displayed epidemic clones.

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PS4-40 Phylogeny of nodule bacteria deriving from root nodules of the legume plant Astragalus cicer

Sylwia Wdowiak-Wróbel, Wanda Malek, and Małgorzata Szlachetka Institute of Microbiology and Biotechnology, Department of Genetic and Microbiology, Maria Curie Skłodowska University, 20-033 Lublin, Poland A multilocus phylogenetic approach was applied to elucidate the phylogeny of Astragalus cicer rhizobia derived from Poland, Ukraine, and Canada. The strains selected represented three main geographically different phenons of these bacteria. One of the basic criteria in bacterial taxonomy used to delineate bacterial genus and species is the 16S rRNA gene analysis. It is a molecular marker that reveals the evolutionary history. Also the other conservative genes as atpD, glnII, dnaK, and recA have been used in taxonomic and phylogenetic studies of nodule bacteria. Phylogenetic analyses of A. cicer microsymbionts was performed with three chromosomal core loci (16S rDNA, atpD, and glnII) and four symbiotic genes located on a plasmid (nodA, nodB, nodC, and nifH). A "core" and "auxiliary" gene tree revealed that A. cicer nodule isolates were intermingled with strains of the Mesorhizobium species pointing that they are descendents of the same ancestor as mesorhizobia and fall into Mesorhizobium genus. By both kinds of genes (except 16S rRNA) A. cicer rhizobia are distinguished from currently known Mesorhizobium species. Whether they constitute a new rhizobial species in this genus needs further confirmation. The noted congruence of the housekeeping and symbiotic gene phylogenies of A. cicer microsymbionts indicates that Sym loci are spread and transferred through vertical transmission without a significant participation of the intergeneric horizontal gene transfer. The phylogeographic patterns of the sym genes of intercontinental strains point to their relatively long, separate, evolutionary history.

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Session 5

Highlights in plant and bacterial genomes

Chaired by Stephane Rombauts

Gent, Belgium


123

Session 5: Highlights in plant and bacterial genomes

S5-1 The Medicago truncatula Genome Project Chris Town (for the Medicago Genome Sequencing Consortium) The J. Craig Venter Institute, Rockville, MD 20850, USA An international consortium from the USA and EU is sequencing the "euchromatic gene space" of Medicago truncatula A17 using a bacterial artificial chromosome (BAC-by-BAC) strategy, with a goal of completion by the end of 2008. Release Mt2.0 was assembled following a data freeze in July 2007, with 2111 BACs being incorporated into approximately 215 Mb of pseudomolecule sequence and most centromeric boundaries and telomeres defined by BAC fluorescence in situ hybridisation (BAC-FISH). EST capture rate suggests that this corresponds to approximately 65% of the gene space. Annotation of Mt2.0 by the International Medicago Genome Annotation Group (IMGAG) revealed approximately 39,000 gene models, including approximately 13,000 lacking experimental support. Most unsupported gene models were short (<100 amino acids), although several lines of evidence (RT-PCR, Affymetrix chip data, and 454 and Solexa transcriptome sequencing) indicate that many are transcribed. Chromosome 6 and the top of chromosome 3 are notably different from other chromosomes, with lower gene densities, elevated numbers of transposons, few synteny blocks, and a high number of disease-resistance genes (NBS-LRRs). Genome duplications appear less widespread than in other sequenced plants, although this may be due partly to the incomplete state of the M. truncatula assembly. Medicago displays extensive synteny with both Lotus and the draft sequence of the soybean genome, as well as many shorter synteny blocks with poplar and Arabidopsis. Synteny with the soybean assemblies is being used both to orient some sequenced contigs and to position unanchored, potentially gene-rich FPC contigs in gaps in the current pseudomolecules. A third set of pseudomolecule sequences and annotation will be released towards the end of 2008 to be followed by a genome publication in early 2009. Supported by the EU FP6 Grain Legumes Project, INRA, the French Ministère de la recherche, the UK BBSRC, the Noble Foundation, and the US National Science Foundation.

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S5-2 Interaction between Aeschynomene and photosynthetic Bradyrhizobium: an enigmatic Nod-independent symbiotic process

Katia Bonaldi, Fabienne Cartieaux, Joel Fardoux, Laure Hannibal, G. Bastien, Lionel Moulin, Nico Nouwen, Yves Prin, Adeline Rénier, and Eric Giraud Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France Nodule formation in legume plants was assumed to be exclusively initiated by the binding of bacterial host-specific lipochitooligosaccharidic Nod factors to kinase-like receptors of the plant. Recently, sequence analysis of the genomes of two photosynthetic Bradyrhizobium strains (ORS278 and BTAi1), symbiotic of some Aeschynomene species, overturned this dogma because these bacteria lack the canonical nodulation genes (nodABC) involved in the synthesis of Nod factors [1]. This indicates that certain rhizobia use another mechanism to trigger nodule organogenesis in legumes. The most relevant characteristic of the photosynthetic Bradyrhizobium genomes will be presented. To identify the bacterial genes involved during this symbiosis, we individually screened a large Tn5 mutant library (20,000 mutants) of Bradyrhizobium ORS278 strain for their inability to induce nodules on Aeschynomene or to fix nitrogen. Results of this screening strategy will also be discussed. [1] Giraud et al. (2007). Science 316:1307-1312.

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PS5-1 The Sinorhizobium medicae WSM419 genome sequencing project Wayne Reeve1, Patrick Chain2, Ravi Tiwari1, Graham O'Hara1, John Howieson1, and Lambert Bräu1 1Centre for Rhizobium Studies, Murdoch University, Murdoch, WA 6150, Australia; 2Lawrence Livermore National Laboratory, Livermore, CA 94551, USA Sinorhizobium medicae is capable of fixing nitrogen with Medicago arabica, M. murex, M. polymorpha, M. truncatula, and M. sativa, the last two of which are also hosts for Sinorhizobium meliloti Sm1021. S. medicae WSM419 is saprophytically competent in moderately acidic soils (>pH 4.9) that are challenging to other sinorhizobia; a feature that enabled pasture production to be extended in southern Australia by a further 1 million ha. We now report on the complete genome sequence of S. medicae WSM419. For the sequencing strategy, a shotgun assembly approach was adopted using four libraries; one of which was constructed in a functional genomics vector (pTH1522) [1]. Double-ended plasmid sequencing reactions were then performed at the US Joint Genome Institute. Approximately 92,100 sequencing reads were assembled, producing an average of 12.9-fold coverage across the genome. Processing of sequence traces, base calling, assessment of data quality and assembly were performed with the PHRED/PHRAP/CONSED package. The initial assembly consisted of 30 contigs with at least 20 reads per contig. Gaps in the sequence were closed by primer walking on gap-spanning library clones or genomic DNA-amplified PCR products. Sequence finishing and polishing added 638 reads. Automated gene prediction was completed by assessing congruence of gene call results from Critica, Generation, and Glimmer, and by comparing the translations to GenBank's nonredundant database. Analysis of the genome (6 817 576 bp) reveals a multipartite structure consisting of a chromosome (3 781 904 bp) and three plasmids (pSMed01, 1 570 951 bp; pSmed02, 1 245 408 bp; and pSMed03, 219 313 bp) with a GC content 61.15%. In total, 6523 protein encoding ORFs could be identified of which 4646 (70.53%) could be assigned a putative function. This presentation will give an update on the current state of comparative analyses and future research directions. This project has been made entirely possible by funding from the Joint Genome Institute Community Sequencing Program (Department of Energy; USA), the Australian Research Council, and Murdoch University. [1] Cowie et al. (2006). Appl. Environ. Microbiol. 72:7156-7167.

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PS5-2 Screening for non-coding RNAs in the archaeal diazotroph Methanosarcina mazei

Dominik Jäger1, Jens Thomsen1, Claudia Ehlers1, Cynthia Sharma2, Jörg Vogel2, Heiko Liesegang3, Wolfgang Hess4, and Ruth A. Schmitz-Streit1 1Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, 24118 Kiel, Germany; 2RNA Biologie, Max-Planck-Institut für Infektionsbiologie Berlin, 10117 Berlin, Germany; 3Laboratorium für Genomanalyse, Georg-August-Universität Göttingen, 37077 Göttingen, Germany; 4Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany Small non-coding RNAs (sRNAs) are an emerging field of research as their global impact in regulatory processes becomes more and more obvious. Non-coding RNAs have been identified in all three domains of life. In Eukarya and Bacteria functions have been assigned for many sRNAs; however still little is known about sRNAs in Archaea. To get an insight into potential regulatory roles of sRNAs in Archaea, we chose the methanogenic archaeon Methanosarcina mazei strain Gö1 as model system, which is able to fix molecular nitrogen. It is a perfect candidate because, due to its high ecological importance in biogenic methane production, many aspects of the organism's adaptation to different stress situations are currently under investigation (e.g., carbon stress [1], nitrogen limitation [2] and osmotic stress [3]). The goal of this work is to identify non-coding RNAs in M. mazei using two different approaches: (i) computational screens, based on comparative analysis of the three Methanosarcina genomes (M. acetivorans, M. barkeri, and M. mazei) and (ii) direct sequencing of M. mazei cDNA populations by massive parallel sequencing technology. In silico screens predicted more than 600 potential sRNAs in the 4.1-Mb genome of M. mazei. Direct sequencing of M. mazei cDNA populations using the 454-sequencing system resulted in 44,000 reads in total, approximately 14,000 reads of which exclusively correspond to intergenic regions. Results from both approaches will be compared and discussed. [1] Hovey et al. (2005). Mol. Genet. Genomics 273:225-239. [2] Veit et al. (2006). Mol. Genet. Genomics 276:41-55. [3] Pflüger et al. (2007). FEMS Microbiol. Lett. 277:79-89.

Session 5

Highlights in plant and bacterial genomes

Chaired by Stephane Rombauts

Gent, Belgium


127


PS5-3 Motility and exopolysaccharide production in the symbiotic soil bacterium, Sinorhizobium meliloti 1021: gene expression and regulatory circuits

Melanie J. Barnett, and Sharon R. Long Department of Biology, Stanford University, Stanford, CA 94305, USA Sinorhizobium meliloti engages in a nitrogen-fixing symbiosis with leguminous plants of the genera Medicago, Melilotus, and Trigonella. In order to understand the molecular processes of this intimate and complex interaction, we constructed a dual-genome Affymetrix Symbiosis Chip for coordinate study of gene expression in the S. meliloti-Medicago truncatula symbiosis [1]. Data from our bacterial gene expression analyses suggested that upregulation of S. meliloti genes for exopolysaccharide (EPS) biosynthesis correlates with down-regulation of genes encoding motility and chemotaxis functions, and with altered expression of additional previously uncharacterised genes. We and others have noted an inverse correlation between EPS production and motility [2] and suggest that this is part of a complex multitrait adaptation that responds to environmental cues. Because EPS is required for invasion of the plant host, and because motility is presumably not required within the plant nodule, understanding this regulatory circuit may provide insight into important developmental checkpoints during the transition from free-living bacterium to nitrogen-fixing bacteroid. To further understand the relationship between motility and EPS production, we mutagenized S. meliloti 1021 and screened for mutants with a mucoid colonial morphology. Our screen differed from previous screens in that we used a transposon, Tn5-110 [3], that confers constitutive expression from both Tn5 ends. We found many novel mutants, most of which overproduce EPS-I (succinoglycan). Surprisingly, we isolated many more mucoid mutants that were motile than expected. Therefore, the relationship between EPS production and motility is more complex than previously suggested. [1] Barnett et al. (2004). Proc. Nat. Acad. Sci. USA 101:16636-16641. [2] Barnett & Fisher (2006). Symbiosis 42:1-24. [3] Griffitts & Long (2008). Mol. Microbiol. 67:1292-1306.

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PS5-4 Genome features of an annual and a perennial clover microsymbiont Lambert Bräu1, Lynn A. Goodwin2, Graham O'Hara1, Ravi Tiwari1, John Howieson1, Ron Yates3, and Wayne Reeve1 1Centre for Rhizobium Studies, Murdoch University, Murdoch, WA 6150, Australia; 2US Department of Energy, Joint Genome Institute, Walnut Creek, CA 94598, USA; 3Department of Agriculture and Food, Western Australia, South Perth, WA 6151, Australia Rhizobium leguminosarum bv. trifolii is one of the most utilized species of root nodule bacteria, required for symbioses with annual (eg. Trifolium subterrraneum) and perennial (e.g., T. pretense, T. repens, and T. polymorphum) clover species. Most clover rhizobial inoculants only form effective nitrogen-fixing symbioses with either annual or perennial species (and very few with both). This constraint provides a considerable barrier to agricultural productivity, because background populations of R. leguminosarum bv. trifolii may nodulate with an incompatible host and then fix nitrogen only poorly [1]. Knowledge at the genetic level is essential to develop an understanding of this incompatibility and progress in this pursuit will be greatly enhanced by complete genome sequence information. Thus, the genomes of two R. leguminosarum bv. trifolii strains were sequenced by the US Joint Genome Institute Community Sequencing Program; the Mediterranean Trifolium spp. isolate WSM1325 and the south American Trifolium polymorphum isolate WSM2304. Strain WSM1325 is compatible with Mediterranean perennial clovers (i.e., T. pratense) but not with American or African perennial clovers, while the reverse is true for strain WSM2304. To sequence the genomes, a shotgun assembly approach was adopted to assemble a draft genome of each organism. Three libraries were constructed for each strain, including one library for each strain in the functional genomics vector pTH1522 [2]. Assembly of the sequence data is in the draft stage and reveals that the genome of WSM2304 is 6.8 Mb in size (27 contigs of 20 reads or greater), contains a G+C content of 61.1%, and encodes 6487 candidate protein-encoding genes. In comparison, the WSM1325 genome is 7.8 Mb in size (143 contigs of 20 reads or greater), contains a G+C content of 60.7%, and encodes 7528 candidate protein-encoding genes. We will discuss comparative studies performed on the genome sequences of these two strains. [1] Yates et al. (2008). Soil Biol. Biochem. 40:822-833. [2] Cowie et al. (2006). Appl. Environ. Microbiol. 72:7156-7167.

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PS5-5 The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis

Ana M. Cosme1, Anke Becker2,3, Mário R. Santos1, Larissa A. Sharypova2, Pedro M. Santos1, and Leonilde M. Moreira1 1Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisboa, Portugal; 2Institute for Genome Research and Systems Biology, Center for Biotechnology, Bielefeld University, 33501 Bielefeld, Germany; 3Institute of Biology III, University of Freiburg, 79104 Freiburg, Germany Sinorhizobium meliloti is a soil bacterium capable of establishing a symbiotic nitrogen fixation relationship with the leguminous plant Medicago sativa. Throughout this process, it must cope with diverse environments, having evolved different types of transport systems that help its propagation in the plant roots. The TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In particular, the S. meliloti tolC mutant induced a reduced number of non-fixing nitrogen nodules in Medicago sativa roots. We have characterized the protein TolC from S. meliloti 2011. The insertional mutation in the tolC gene strongly affected resistance to antimicrobial agents, and it induced higher susceptibility to osmotic and oxidative stress. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type vs. tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of ORF SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by TolC-dependent secretion system. In the absence of TolC, neither of the two exopolysaccharides involved in the symbiosis that are produced by this bacterium, succinoglycan and galactoglucan, were detected in the culture supernatant. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

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PS5-6 Regulation and function of motility genes in Rhizobium leguminosarum

Michael F. Hynes, Dinah D. Tambalo, and Denise Bustard Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada Motility is an important property of rhizobia that allows them to find optimal growth conditions in soil and on plant roots. The genome of Rhizobium leguminosarum 3841 contains seven flagellin genes (flaA to flaG) and four motility genes (motA, motB, motC, and motD). Four flagellin genes (flaABCD) are located within the main flagellar gene cluster of R. leguminosarum; flaE is on plasmid pRL11JI, and the other two flagellin genes (flaF and flaG) are located outside the main flagellar regulon. FlaA, FlaB, FlaC, FlaE, and FlaG are highly homologous and the amino and carboxy-terminal ends are highly conserved. Individual mutations in each of the seven Fla genes, motA, and motB were constructed by insertional mutagenesis in strains VF39 and 3841. None of the fla mutants was completely impaired in swimming and swarming motility but a major role of FlaA, and important roles of FlaB and FlaC were established. A flaABCD deletion mutant was non-motile. Mutation of motA and motB, which code for the stator protein of the flagellum, resulted in paralysis. Gene regulation studies were done using gusA transcriptional fusions. The highest transcription activities were observed for flaA, flaC, and flaD, while flaB, flaE, flaF, and flaG exhibited minimal transcription values. These results suggest that FlaE, FlaF, and FlaG are at most minor components of the flagellar filament. The role of three known regulatory genes (visN, visR, and rem) in the expression of the flagellin and motility genes was also determined using gusA gene fusions in wild-type and regulatory mutant backgrounds. The flagellin genes flaABCD and the motility gene motB are regulated by visNR and rem while the other flagellin genes (flaEFG), which are located outside the main flagellar regulon, are not. All flagellar and motility genes, as well as visNR and rem, are down-regulated during symbiosis.

131


PS5-7 Toxin-antitoxin-like module in Bradyrhizobium japonicum Sebastian Paul Miclea1, Ibolya Horváth2, and Ilona Dusha1 1Institute of Genetics, and 2Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, 6726 Szeged, Hungary Toxin-antitoxin (TA) modules composed of two adjacent genes are ubiquitous in bacteria. They are thought to help the adjustment of metabolic processes to varying conditions. Based on similar organization, size and sequence homology, a TA-like module (designated as bat/bto operon) was identified in Bradyrhizobium japonicum. Deletion of the bat/bto operon resulted in the alteration of various metabolic pathways in the mutant bacteria. The generation time of the mutant was considerably decreased in media containing complex sources of carbon and nitrogen, but the mutant cells were unable to grow in minimal medium. Different imaging techniques (AFM and LSM) revealed the altered (shorter and wider) shape of mutant cells. The lipopolysaccharide production of the mutant was four-fold lower than that of the wild-type cells, and resulted in the synthesis of mainly incomplete molecules. By determining the fatty acid and phospholipid components, remarkable differences were observed in the membrane composition of the wild-type and mutant strains, which may explain the observed phenotypic alterations of the mutant bacteria. In the wild-type strain, 80% of the total fatty acids were represented by oleic acid. In contrast, a variety of fatty acids were present in the mutant membranes. The analysis of phospholipid content revealed the absence of phosphatidylcholine, and the increased amount of cardiolipin and phosphatidylethanolamine in mutant membranes. The higher amount of cardiolipin domains may greatly contribute to the increased division rate. Changes in the division rate and cell shape may also be provoked by the altered elongation of the bacterial cell wall and membrane composition. Our results suggest that the bat/bto system is an important regulator of main metabolic pathways and cell division in B. japonicum. These data represent new aspects concerning the role of TA systems in bacteria.

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PS5-8 The potential role of CLE-signalling peptides in the nodulation of legumes

Karsten Oelkers1,2, Tancred Frickey1,3, Nicolas Goffard1,3, Georg F. Weiller1,3, Peter M. Gresshoff1,4, and Ulrike Mathesius1,2 1Australian Research Council Centre of Excellence for Integrative Legume Research, University of Queensland, Brisbane, QLD 4072, Australia; 2School of Biochemistry and Molecular Biology, and 3Research School of Biological Sciences, Australian National University, Canberra, ACT 0200, Australia; 4University of Queensland, Brisbane, QLD 4072, Australia Genome analysis predicted that leucine-rich repeat receptor-like kinases (LRR-RLKs) are the predominant receptor family of plants. Legumes encode several LRR-RLKs linked to the process of root nodule formation, the ligands of which are unknown. CLE signalling peptides have been shown to be ligands of LRR-RLKs. Little is known about the detailed receptor interaction and specificity of the CLE family. We are testing the hypothesis that CLE-signalling peptides are ligands of the autoregulation of nodulation receptor-like kinase identified from various legumes. We used biological sequence analysis of plant genomes and EST libraries and identified 114 novel CLE-signalling peptides from a variety of plant species. Sequence analysis of the CLE family using CLuster ANalysis of Sequences (CLANS) revealed a grouping of CLE peptide sequences analogous to previously established gain-of-function phenotype classifications and phylogenetic analyses [1]. Furthermore, we identified motif extensions as well as an additional motif upstream of the primary CLE motif. We identified conserved and invariant residues in the 12-amino-acid region forming the active signalling peptide. These residues could mediate receptor interactions or even determine receptor specificity. We found deviant representatives of the CLE family that we predict to contain several active signalling peptides in the protein precursor and to be expressed in vivo. Finally, newly identified CLE-signalling peptides were tested for biological activity in peptide assays using Medicago truncatula and the sequence-specific termination of plant root growth could be confirmed. Using peptide assays we are now identifying CLE-signalling peptides that are involved in the regulation of the number of root nodules. Our analysis provides an identification and classification of a large number of novel CLE-signalling peptides. The additional motifs we found could lead to the future discovery of recognition sites for processing peptidases as well as predictions for receptor-binding specificity. [1] Oelkers et al. (2008). BMC Plant Biol. 8:1.

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PS5-9 Prediction and characterisation of small non-coding RNAs in Sinorhizobium meliloti

Omar Torres-Quesada1, Coral del Val2, Elena Rivas3, Nicolás Toro1, and José I. Jiménez-Zurdo1 1Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain; 2Escuela Técnica Superior de Ingenierías, Informática, Universidad de Granada, 18071 Granada, Spain; 3Howard Hughes Medical Institute, Ashburn, VA 20147, USA Computational and functional analyses of both eukaryotic and prokaryotic genomes have revealed the existence of a great number of untranslated RNA species that fulfil diverse cellular functions. In bacteria, most of these molecules are small sized transcripts of 50-400 nucleotides (small non-coding RNAs or sRNAs), which mainly act as post-transcriptional regulators of gene expression in response to different environmental signals. Although the vast majority of the sRNAs currently annotated in databases have been identified and characterised in the model bacterium Escherichia coli, in the last few years the post-genomic research has made possible the identification of new sRNAs in other bacterial species, mainly animal pathogens [1]. We have conducted the first search for sRNA-encoding genes in the nitrogen-fixing legume endosymbiont Sinorhizobium meliloti, performed by a genome-wide computational comparative genomics approach [2]. The unannotated intergenic sequences were used as queries to interrogate nine related α-proteobacterial genomes and the generated pairwise alignments were individually scanned with the programs QRNA and RNAz as complementary predictive algorithms to identify putative conserved and thermodynamically stable secondary structures likely corresponding to sRNAs. DNA gel blot hybridisation and 5'-RACE mapping experiments led to the identification of eight loci, expressing small transcripts and organized in autonomous transcription units with predictable promoter and termination signatures (smr genes, for S. meliloti RNA). Seven of the smr genes are differentially regulated in free-living and symbiotic conditions, which predict a riboregulatory role for their encoded transcripts. Their expression profiles and conservation patterns render the identified S. meliloti sRNAs as novel molecular markers to unravel common adaptive responses of α-proteobacteria to infect and survive in eukaryotic cells. [1] Livny & Waldor (2007). Curr. Opin. Microbiol. 10:96-101. [2] del Val et al. (2007). Mol. Microbiol. 66:1080-1091.

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PS5-10 Mutagenesis reveals new insights into the function of the cell envelope in Rhizobium leguminosarum

Elizabeth M. Vanderlinde, Dallas L. Foreman, Samantha A. Magnus, and Christopher K. Yost Biology Department, University of Regina, Regina, SK S4S 0A2, Canada In Gram-negative bacteria, such as Rhizobium leguminosarum, the cell envelope is comprised of an inner membrane, cell wall, outer membrane, and secreted polysaccharides. The envelope represents the interface between the bacterium and its external environment. Consequently, it plays a critical role as a protective barrier against the entry of harmful substances and is also important in host-bacterial interactions, because it is the contact point between the host cell and bacterial cell. We have taken a genetics approach to identify genes and their functions, which are critical to the proper functioning of the cell envelope. From this research, several notable trends have emerged that highlight the importance of the cell envelope to both free-living and symbiotic cells. Mutations affecting exopolysaccharide (EPS) production and LPS structure result in increased sensitivity to desiccation. This is the first mutational evidence of a role for either the EPS or LPS in the desiccation tolerance mechanisms of rhizobia. All of the mutants affected in cell envelope components are sensitive to detergents and antimicrobial peptides. Plants produce peptide-like molecules, called defensins, as part of their immune response. The results we have gathered so far would suggest that the cell envelope contributes significantly to providing protection against defensins and other membrane-disrupting molecules. Several of the mutants we have studied are altered in their ability to form effective symbioses, indicating the cell envelope is important in all stages of nodulation. In conclusion, our mutagenesis approach has allowed us to identify new genes important in construction of the cell envelope and identify new functions for the cell envelope that are important to both free-living and symbiotic growth of Rhizobium leguminosarum.

Session 6

Genetics of legume nodulation

Chaired by Gabriella Endre

Szeged, Hungary


135

Session 6: Genetics of legume nodulation

S6-1 Infection thread mutants in Lotus japonicus Makoto Hayashi National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan The infection thread (IT) is a plant-derived structure that is developed upon infection of root nodule bacteria. The majority of the "higher" legumes, such as soybean, common bean, alfalfa, and pea, adopt this mechanism for effective nodulation. After entrapping bacteria in the curled root hair, ITs are initiated and develop towards the base of the epidermis, and then penetrate into the cortical layers where nodules develop. There are several Lotus japonicus mutants in which IT development is aborted [1, 2] and we focus on identifying and characterizing genes from these mutants. Among them, CYCLOPS shows root hair curling and bacterial colonization, but IT initiation is mostly aborted. Concomitantly, nodule development is suppressed at the bump stage. In this mutant, Nod factor- or infection-dependent NIN expression is severely compromised. CYCLOPS encodes a nuclear protein, with two putative NLS motifs and a coiled-coil motif at the C terminus. CYCLOPS interacts with CCaMK, another nuclear protein that is crucial for decoding the calcium spiking. Interestingly, spontaneous nodule formation triggered by a gain-of-function CCaMK does not require CYCLOPS, because the size of spontaneous nodules in cyclops is not significantly different to that in the wild type. We hypothesize that the primary function of CYCLOPS is to initiate ITs in the epidermis, and this induces further development of nodules in the cortex. Another example is alb1. In this mutant, ITs are initiated, but never penetrate into the cortical layers. In some cases, abnormal ITs can be observed in a developed (Type-II) nodule, which aberrantly releases bacteria into infected cells. We cloned the gene encoding LRR-RK. Expression of Alb1 is induced by infection and confined as a patchy pattern in epidermis. We further characterise ALB1 in order to know the mechanism for IT development in the epidermis. [1] Yano et al. (2006). Mol. Plant-Microbe Interact. 19:801-810. [2] Lombardo et al. (2006) Mol. Plant-Microbe Interact. 19:1444-1450.

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S6-2 Medicago truncatula insertion mutants as tools to dissect plant-microbe interactions

Pascal Ratet1, Peter Mergaert1, Millon Tadege2, Jeremy Murray2, Jiangqi Wen2, Michael Udvardi2, and Kiran Mysore2 1Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, Cedex, France; 2Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA Interactions with soil bacteria are very important for plant harmonious development. Soil bacteria are present in the plant rhizosphere but can also colonise roots as endophytes. These plant associated bacteria can promote plant growth and protect their host from pathogens. Understanding the mechanisms controlling these interactions is important for future use in sustainable agriculture. We have been using the leguminous model plant Medicago truncatula to study plant microbe interactions and more precisely the establishment of symbiosis between legumes and rhizobia. By making use of a very efficient regeneration and transformation protocol developed for M. truncatula, we initiated an insertion mutagenesis program in this species using the LTR retrotransposon Tnt1, an autonomous 5.3-kb-long copia-like LTR element as mutagen. We demonstrated that Tnt1 transposes actively during in vitro transformation, generating from 4 to 40 insertions per plant. These insertions are stable during the life cycle of M. truncatula and most of them are genetically independent and can be separated by recombination. Sequencing of a large number of Tnt1 insertion sites indicated the lack of insertion site specificity for this element in Medicago but preferential insertion in genes. We have used these properties to construct large mutant collections in this plant (www.eugrainlegumes.org; www.noble.org). Part of the collection constructed at the Noble Foundation was screened for the presence of symbiotic mutants. Among theses nod-, nod++, and fix- mutants were isolated and mutants in the Nod factor early signalling genes were identified among them. We are currently studying more carefully the fix- mutants in order to identify new M. truncatula symbiotic genes.

137


PS6-1 A new low-nodulating isoline mutant of chickpeas (Cicer arietinum): genetic characterisation of its progeny and detection of SNPs in several genes related to this mutation

Manuel Angel Chamber Perez, Ana Aguado Puig, Antonio Daza Ortega, and Eloísa Pajuelo Domínguez 1Departamento de Agricultura Ecológica, Centro de Investigación y Formación Agraria, Centro Las Torres-Tomejil, 41200 Sevilla, Spain; 2Instituto de Investigación y Formación Agraria y Pesquera, Consejería de Innovación, Ciencias y Empresas, 41200 Sevilla, Spain An EMS mutagenesis programme has been developed in chickpea (Cicer arietinum bv. F46C) in order to isolate symbiotic mutants of this grain legume. A non-nodulating mutant of chickpea obtained by EMS mutagenesis has been analysed at the phenotypical and microscopical levels. The mutant plant shows neither nodules nor primordia when inoculated with different Mesorhizobium ciceri strains. All plants of the F1 progeny from crosses of the mutant plant with the parental showed the mutant phenotype, indicative of a dominant mutation. F2 progeny of these crosses segregated 3:1 (mutant: wild type plants). A microscopical study of the first stages on nodule development has been carried out using a M. ciceri strain carrying a lacZ reporter gene. Although there are some rhizobial infections in the mutant plant (approximately 1/10th compared to wild type), they do no form infection threads inside the root hair. Instead, Mesorhizobium invades the apical zone of the root hair sometimes forming a ballon-like structure, but never an infection thread. Occasionally, the mutant plant shows one single nodule at a frequency of one in 10 or 20 plants. The nodule structure is apparently normal, pink and functional. However, no infection thread is detected in the neighbourhood of the nodule. Addition of several phytohormones (e.g., auxins, auxin transport inhibitors, cytokinins, ethylene precursors, and inhibitors) failed in restoring the mutant phenotype. However, a significant improvement in nodulation and shoot dry weight can be observed in the presence of Ag+ ions and ACC, suggesting a possible role of ethylene in the mutation. Several nodulin genes that could be affected in this mutant are being PCR-amplified in order to analyse the expression pattern of these genes in the early stages of nodulation. Pollen germination has been affected in the mutant, but mycorhizal infections are similar to the parent plant. Studies on mycorhyzal infections showed that mutant plants were not qualitatively affected. Work is underway to detect SNPs by DGGE in nodulation genes (Nin, Enod2, Enod8, Enod40, MTN6, SYMRK, Ca2+ spiking, NORK, etc.), among those already described in the two model legumes.

138


PS6-2 Investigation of proteins involved in cell cycle regulation in Sinorhizobium meliloti

Nataliya Pobigaylo, and Anke Becker Center for Biological Systems Analysis, University of Freiburg, 79104 Freiburg, Germany Regulation of cell cycle in bacteria is a highly organized and tightly controlled process. In the indeterminate nodules, induced by Sinorhizobium meliloti on its plant hosts, a deregulation of a normal cell cycle takes place. Here, bacteria, as well as the infected plant cells become enlarged and polyploid through several cycles of endoreduplication of the genome uncoupled from cell division [1, 2]. The exact mechanisms of such deregulation of the cell cycle are not elucidated yet. We have started a systems biology project to investigate the role of regulators of the cell cycle in S. meliloti in free-living as well as in symbiotic conditions. In order to create a model of cell cycle regulation, a three-step approach was chosen. At the first step, the homologues of the known division regulator proteins have been fused to fluorescent proteins to investigate their subcellular localization throughout the cell cycle. At the second step, the unknown regulators will be identified based on the preliminary model and experimental work, and their function will be identified. At the third step, the comprehensive model of the cell cycle in S. meliloti will be created. At the end, the knowledge about the division of the S. meliloti free-living cells will be used to establish a model of division deregulation in the symbiotic conditions. [1] Bisseling et al. (1977). J. Gen. Microbiol. 101:79-84. [2] Mergaert et al. (2006). Proc. Natl. Acad. Sci. USA 103:5230-5235.

Session 6

Genetics of legume nodulation

Chaired by Gabriella Endre

Szeged, Hungary


139


PS6-3 Extracellular protein PssO of Rhizobium leguminosarum bv. trifolii TA1–possible involvement in exopolysaccharide export

Małgorzata Marczak, Andrzej Mazur, and Anna Skorupska Department of Genetics and Microbiology, Maria Curie-Skłodowska University, 20-033 Lublin, Poland Synthesis and secretion of polysaccharides by Gram-negative bacteria is the result of a concerted action of proteins localised in different compartments of the cell. We characterized the PssO protein of the symbiotic bacterium Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) as a possible component of the complex engaged in exopolysaccharide (EPS) synthesis and export. PssO is encoded within the chromosomal EPS biosynthesis region PssI. By examining a non-polar pssO mutant RtO112, we demonstrated that the pssO gene is required for EPS synthesis and/or secretion in RtTA1. The RtO112 strain infected nodule cells and was more proficient in nodule invasion than other EPS-deficient mutants of RtTA1, but was unable to fix nitrogen. RtO112 was characterised by a significant increase in sensitivity to membrane-disrupting agents. These perturbances in the outer membrane (OM) permeability and the observed non-mucoid phenotype of RtO112 could have originated from the altered spatial distribution of OM components and a disturbed organisation of the EPS transport system. Mature PssO has an extensive α-helical content (32%) and little β-sheet structure (12%). Subcellular fractionation analyses demonstrated that PssO was present in the fraction of extracellular proteins and appendages sheared from RtTA1 cells. In silico analyses indicated similarity of PssO to the type-1 fimbrial subunit protein of Klebsiella pneumoniae. The hypothesis of PssO being a part of such extracellular structures in RtTA1 cells was assessed by immunogold electron microscopy. As type-1 fimbriae have been shown to mediate adhesion of associative nitrogen fixers to grass roots, the possible engagement of PssO in RtTA1 cell adhesion was assessed in plant tests. No difference in RtTA1 and RtO112 strain abilities to adhere to root hairs was observed. Although we determined a number of PssO important features, some data remain puzzling and the precise role the protein plays in EPS surface expression is still an open question.

140


PS6-4 Role of Rhizobium cellulase CelC2 in bacterial cellulose biosynthesis

Marta Robledo1, L. Rivera1, José I. Jiménez-Zurdo2, Raúl Rivas1, Encarna Velázquez1, Frank Dazzo3, Eustoquio Martínez-Molina1, and Pedro F. Mateos1 1Centro Hispano Luso de Investigaciones Agrarias, Universidad de Salamanca, 37007 Salamanca, Spain; 2Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain; 3Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots [1]. CelC2 cellulase is a 1,4-β-D-endoglucanase belonging to the glycosyl hydrolase family 8 with high substrate specificity for non-crystalline cellulose. Orthologues to the CelC endoglucanase-coding gene are found in a diversity of eubacteria that make cellulose (Rhizobium, Agrobacterium, Gluconacetobacter, Escherichia, Salmonella, and others). In all these cellulose-producing eubacteria, the celC genes are located near putative cellulose synthase genes localized in a region of the chromosome (celABC or bcsABZ) involved in bacterial cellulose biosynthesis. Microscopy analysis of Rhizobium leguminosarum bv. trifolii ANU843 and E11 wild-type parents and its derivative strains (CelC2 knockout mutants, and recombinant strains that overproduce CelC2) indicated that this enzyme fulfils a very significant role in Rhizobium cellulose biosynthesis. The cellulose biosynthesis pattern in free-living bacteria differs from those found during colonization of its legume host root hair and root surface. [1] Robledo et al. (2008). Proc. Natl. Acad. Sci. USA 105:7064-7069.

141


PS6-5 Replication region of Rhizobium leguminosarum bv. trifolii TA1 non-symbiotic plasmid

Andrzej Mazur, Agnieszka Woloszyn, Jerzy Wielbo, Monika Marek-Kozaczuk, Małgorzata Marczak, and Anna Skorupska Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, University of Maria Curie Skłodowska, 20-033 Lublin, Poland Rhizobial extrachromosomal replicons are usually equipped with repABC genes that control their replication and maintenance in a cell population. The genome of Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) consists of five replicons: a chromosome and four plasmids, with the smallest, pRtTA1a, recognized as pSym [1]. In RtTA1, we identified a putative replication system consisting of three genes repA, repB, and repC in the same relative order. Despite their high DNA sequence identity with the respective genes of the pRL10 symbiotic plasmid of R. leguminosarum bv. viciae 3841, the identified system proved to belong to the 653-kb pRtTA1c non-symbiotic plasmid of RtTA1. In silico promoter prediction suggested an operon organization of putative genes that is very common for repABC-type systems. The hypothetical RepA and RepB have sequence similarities with proteins involved in active segregation of plasmids, while RepC seems to be a potential initiator protein. In the intergenic sequence between repB and repC, a putative incompatibility region igs was found with a well-conserved promoter comprising -10 and -35 boxes. It is located in the complementary DNA strand of the repABC and presumably drives transcription of small antisense RNA. According to the plasmid-partitioning system classification, the one detected for pRtTA1c belongs to the Type-Ia group [2], in which RepA with its N-terminal DNA-binding domain constitutes a member of the ParA superfamily. The consensus 16-bp palindromic putative parS sequence (ATTgtCaGCtGacAAT) for RepB binding is located upstream of repA gene, while another potential parS site located immediately downstream of repB is not fully palindromic. Hybridisation analyses showed that pRtTA1c repC (but not repAB) homologous sequences could also be found in the RtTA1 pSym, demonstrating replication gene diversity and RepAB co-evolution partially independent of RepC. [1] Krol et al. (2008). Mol. Genet. Genomics 279:107-121. [2] Cevallos et al. (2008). Plasmid 60:19-37.

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PS6-6 Towards synteny-based cloning of four pea (Pisum sativum L.) symbiotic genes Sym31, Sym32, Sym41, and Sym42 controlling symbiosome formation

Evgenia S. Ovchinnikova1,2, Erik Limpens2, Alexey Y. Borisov1, Ton Bisseling2, and Igor A. Tikhonovich1 1All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia; 2Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands A key step in the Rhizobium-legume interaction is the formation of nitrogen-fixing symbiosomes; however, the molecular mechanism underlying this intracellular accommodation of rhizobia is largely unknown. Pea (Pisum sativum L.) currently has the most extensive and best characterised set of mutants that are impaired in symbiosome development. We will focus on four pea mutants that are altered in the early stages of symbiosome formation and development, Sym31, Sym32, Sym41, and Sym42. The model legume Medicago truncatula will be used as intergenomic cloning vehicle to facilitate the positional cloning of these genes. We aim to develop approximately 60 cross-species markers (approximately four markers/chromosome arm) that can be used to identify the syntenic regions in M. truncatula, using the information of Aubert et al. [1]. We currently developed 30 markers that can be used as co-dominant cross-species markers in our segregating populations. These markers will be used to map the mutants in pea and to identify the syntenic region in M. truncatula, after which the extensive genomic data of M. truncatula will be used to develop new cross-species markers to fine-map the mutant loci. To date, Sym31 has been localized on chromosome III and new cross-species markers are being developed from the corresponding region in M. truncatula to allow fine mapping. The identification of the mutated genes should give valuable insight into the mechanism of accommodating the bacteria as new organelles in plant cells. This work is supported by the NWO grant 047.018.001, grants of RFBR (06-04-89000, 07-04-01171, 07-04-01558, 07-04-13566) and Russian Government grants (5399.2008.4, 02.512.11.2182). [1] Aubert et al. (2006). Theor. Appl. Genet. 112:1024-1041.

143


PS6-7 Investigating the role of MsbA-like and BacA proteins in the Sinorhizobium meliloti-alfalfa symbiosis

Silvia Wehmeier, Victoria L. Marlow, Andreas F. Haag, and Gail P. Ferguson School of Medicine, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK Sinorhizobium meliloti forms a symbiosis with the legume alfalfa. During this interaction, S. meliloti enters into the plant cell via plant-derived infection threads and differentiates into nitrogen-fixing bacteroids. We determined that the MsbA2 protein plays a critical role in ensuring the release of S. meliloti from infection threads [1]. Consequently, in the absence of MsbA2, S. meliloti accumulates in abnormally swollen infection threads and induces a heightened plant defence response. In Escherichia coli, the MsbA protein is necessary for the inner membrane transport of lipopolysaccharide (LPS) across the inner membrane. Our preliminary studies suggest that the S. meliloti msbA2 mutant has a polysaccharide alteration, but LPS transport across the inner membrane was unaffected. Since the S. meliloti Rm1021 genome encodes multiple MsbA-like proteins, we are currently investigating the function of these other proteins in the alfalfa symbiosis and LPS transport. Once inside the plant cell, the BacA protein is essential for S. meliloti bacteroid development. In their free-living state, S. meliloti bacA mutants display an increased resistance towards the glycopeptide bleomycin [2]. Since the BacA protein is functionally interchangeable with the E. coli SbmA protein, which has recently been shown to be involved in the intracellular accumulation of the proline-rich peptide, Bac7, this led to the hypothesis that the BacA protein may also affect the intracellular accumulation of bleomycin. Using fluorescently labelled bleomycin (F-BLM), we found that the S. meliloti bacA mutant accumulated less F-BLM and had a substantially lower amount of bleomycin-induced DNA degradation relative to the parent strain. These findings are consistent with a role for BacA in the uptake of modified peptides and this ability could be critical for S. meliloti bacteroid development. [1] Beck et al. (2008). Microbiology 154:1258-1270. [2] Ferguson et al. (2006). J. Bacteriol. 188:3143-3148.

144


Session 7

Non-legume associations:

endophytic and symbiotic interactions

Chaired by Katharina Pawlowski

Stockholm, Sweden


145

Session 7: Non-legume associations: endophytic and symbiotic interactions

S7-1 Modifications in the expression pattern of Frankia alni in vitro and in symbiosis

Nicole Alloisio, Clothilde Queiroux, Pascale Fournier, Petar Pujic, and Philippe Normand Laboratoire d'Ecologie Microbienne, Centre National de la Recherche Scientifique, UMR5557, Université Lyon 1, 69622 Villeurbanne Cedex, France The known nitrogen-fixing symbionts have developed different physiological strategies to trigger host plant symbiotic programmes. Such strategies have been studied and are now very well known as is the case for Rhizobium and Sinorhizobium, while they remain uncharacterised in other cases, such as Bradyrhizobium or Frankia. Furthermore, no genetics is so far possible in Frankia; therefore, alternative approaches were explored. Three Frankia spp. genomes were recently published [1] and found to contain no canonical nod genes homologues. Several nodC homologues have been detected as well as a nodB, but no nodA homologue. A transcriptomics approach was thus undertaken to identify up- or down-regulated genes under in vitro nitrogen-fixing conditions and in symbiotic Alnus nodules. Hundreds of genes were found to be up- or down-regulated in the two physiological conditions assayed; these were found to fall into various categories, such as ammonium assimilation, tricarboxylic acid cycle, etc. This approach opens up perspectives in understanding the molecular bases of interactions in the actinorhizal symbiosis. [1] Normand et al. (2007). Genome Res. 17:7-15.

146


S7-2 Functional genomic analyses and signalling cascades in rice-endophyte interactions

Barbara Reinhold-Hurek, Melanie Böhm, Anna Buschart, Sabrina Gemmer, Lena Hauberg, Julia Herglotz, Thomas Hurek, Andrea Krause, and Abhijit Sarkar Laboratory of General Microbiology, University of Bremen, 28334 Bremen, Germany Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agro-biotechnological interest because it supplies fixed nitrogen to its host and colonises plants in remarkably high numbers without eliciting disease symptoms. This raises the question of mechanisms of compatibility between host and bacterium. Recently, the complete genome was sequenced [1], allowing application of functional genomic analyses. A proteome reference map was established for pure cultures of strain BH72. Moreover, several determinants for endophytic interactions were characterized, such as ethanol dehydrogenases. Different types of motility appear to be important. The presence of type-IV pili in strain BH72 is known to be involved in the attachment to and colonization of rice roots. Analysis of a PilT mutant showed that twitching motility mediated by type-IV pili is crucial for endophytic colonization, however not for root surface colonization. Also flagella-mediated motility is required for endophytic colonization. Interestingly, flagella apparently do not have negative effects as PAMPS inducing defence reactions. Another unusual feature of this plant-associated bacterium is the apparent lack of a homoserine-lactone based quorum sensing system. Mutational analyses showed that quorum-sensing like responses of gene regulation in strain BH72 were independent of putative candidate genes related to the production of known autoinducers. For symbiotic interactions such with rhizobia and arbuscular mykorrhiza (AM), plant signalling cascades are now well characterized. The rice genome contains several orthologs of genes known to be important in legumes, that have partially been shown to be required for the establishment of AM in rice. The results of the analysis of differential gene expression in Azoarcus-infected rice roots will be presented, as well as the effect of mutations in these genes on endophytic colonization. [1] Krause et al. (2006). Nat. Biotechnol. 24:1385-1391.

147


PS7-1 Isolation and characterisation of the genes involved in hormogonia formation from Nostoc punctiforme, a diazotrophic symbiotic cyanobacterium

Akiko Tomitani1, Paula S. Duggan2, and David G. Adams2 1Institute for Research on Earth Evolution, Japan Agency for Marine-Earth Science and Technology, Yokosuka 237-0061, Japan; 2Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK Cyanobacteria are oxygenic photosynthetic bacteria, many of which have the ability to fix nitrogen. Nostoc punctiforme is a filamentous, diazotrophic, symbiotically-competent cyanobacterium, whose vegetative cells can differentiate into three types of cells, i.e. heterocysts (specialized for nitrogen fixation), akinetes (resting cells), and hormogonia, depending on the environmental growth conditions. Hormogonia are transiently differentiated small-celled filaments often capable of gliding and/or buoyant motility. Their function is to provide immotile strains with a means of dispersal in response to environmental triggers. They are also known to play an important role as infective units in the establishment of symbiotic associations with various host plants, where the cyanobionts supply fixed nitrogen to the partners. In order to identify genes involved in hormogonia differentiation, we screened transposon mutants of Nostoc punctiforme ATCC29133. Five strains were isolated as clones that seldom produce hormogonia nor infect the host bryophyte Blasia pusilla. The transposon and flanking DNAs were recovered and sequenced. Predicted proteins encoded by the genes include membrane proteins, sugar transport, and signalling pathways. The mutants were reconstructed by site-directed mutagenesis, confirming that two of them reproduced the phenotype of no hormogonia formation and no infection. Continued study of the identified genes will provide a clue to understand cyanobacterial multicellularity as well as cyanobacteria-plant symbiosis.

148


PS7-2 Searching for plant and bacterial signals involved in actinorhizal symbioses

Sergio Svistoonoff1, Louis Tisa2, Patrick Doumas1, Laurent Laplaze1, Florence Auguy1, Claudine Franche1, and Didier Bogusz1 1Equipe Rhizogenèse, Institut de Recherche pour le Développement/Institut National de la Recherche Agronomique/Université Montpellier II, UMR DIAPC, 34394 Montpellier Cedex 5, France; 2Department of Microbiology, University of New Hampshire, Durham, NH 03824-2617, USA We study the interaction between the tropical tree Casuarina glauca and the actinomycete Frankia that leads to the formation of actinorhizal nodules. We recently reported the existence of a signalling pathway shared between fungal and bacterial root endosymbioses in the actinorhizal plant Casuarina and in legumes [1]. However, the nature of the chemical signals exchanged between the two partners of actinorhizal symbioses is still unknown due to the lack of genetic tools in Frankia and of specific molecular markers of the symbiotic interaction. We exploited the results of the recent sequencing of three Frankia genomes to search marker genes of the symbiotic interaction in order to identify symbiotic signals emitted by C. glauca roots. We will report the characterisation of a molecule present in root exudates from nitrogen-starved C. glauca plants. This compound induces molecular and physiological changes in Frankia CcI3. In parallel, we use plant genes that are specifically expressed at early stages of the interaction as molecular markers to try to isolate the Frankia nodulation factors. These genes include Cg12 [2], a subtilase specifically expressed in Frankia-infected cells, CgAUX1 [3], an auxin influx carrier, and MtEnod11, a legume gene widely used as a symbiotic marker. From these converging strategies, we will propose a model for nodule organogenesis events. [1] Gherbi et al. (2008). Proc. Natl. Acad. Sci. USA 105:4928-4932. [2] Svistoonoff et al. (2004). Plant Physiol. 136:3191-3197. [3] Péret et al. (2007). Plant Physiol. 144: 1852-1862.

Session 7

Non-legume associations:

endophytic and symbiotic interactions

Chaired by Katharina Pawlowski

Stockholm, Sweden


149


PS7-3 Identification of an iron transport operon in Gluconacetobacter diazotrophicus

A.P. Vázquez-Candanedo, S. Soriano-Suárez, A. Sánchez-Espíndola, C. Ãvila Ramírez, and Beatriz Eugenia Baca Centro de Investigaciones Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico Gluconactebacter diazotrophicus, a nitrogen-fixing bacterium, is an endophyte of sugarcane. To clone components of the iron acquisition system in G. diazotrophicus, we have designed degenerated oligonucleotides to amplify by PCR a conserved region of a specific permease for the iron transport. The 400-bp amplicon obtained was used, in turn, to isolate a plasmid clone from a library of the bacterium. The sequence analysis of this clone revealed three ORFs, named feuA, feuB, and feuC, organised in an operon, encoding proteins of 314 (34 kDa), 342 (34.2 kDa), and 240 (25.4 kDa) amino acids, respectively. The deduced translation proteins showed similarity with a periplasmic binding protein, a permease, and an ATPase involved in iron transport, designated FeuA, FeuB, and FeuC respectively, for iron-regulated (Fe) uptake transporters, deposited under GeneBank accession number DQ080039. In order to determine if the feuABC operon was functional, we constructed a mutant by homologous recombination of a Km resistance cassette. The role of G. diazotrophicus in survival under iron depletion was evaluated by comparing the ability of the feuB-km mutant and the wild-type strains to grow in media, without Fe supplement and in the presence of the iron chelator 2'di-pyridyl (DPI). The growth was not affected in the medium without Fe, indicating that traces of Fe were sufficient. This also suggests the existence of an alternate system different of the FeuABC to transport iron. In contrast, when the iron chelator was added this created iron-deficient conditions and the mutant strain showed a growth defect. A transcriptional fusion was constructed with the gusA gene. The feuABC operon of G. diazotrophicus is expressed at higher levels in cells that are depleted for iron than in those that are replete for the metal. The expression of the fusion in association with sugarcane is under study to establish if the feuABC operon is required for the endophytic colonisation of the host plant. Further analysis will be also performed to determine the role of this iron acquisition system for nitrogen fixation both at the free-living state and in planta. The work was partially funded by a grant of VIEP-SEP.

150


PS7-4 The study of nitrogen-fixing activity on wheat roots Abbas Biabani Agricultural Faculty, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran In this study, isolated strains of nitrogen-fixing bacteria and pectinolytic bacteria that can able to create paranodule on wheat seedling were selected from soil. The best nitrogen-fixing bacteria and bacteria with high enzyme production were chosen. 2,4-Dichlorophenoxyacetic acid was used as abiological agent (Aa) and the best pectinolytic bacteria as biological agent (Ba) (Bacillus polymyxa strain 43) for study formation paranodules on wheat seedlings and as diazotroph bacteria for inoculation. Nitrogen-fixing activity on wheat roots was measured dynamically on the 8th, 10th, 14th, and 22nd day of plant growth. Nine different experiments were carried out: (1) wheat without any treatment (control); (2) wheat + Aa; (3) wheat + Ba; (4) wheat inoculated + mixed cultures of diazotrophic bacteria (Arthrobacter sp. + Xanthomonas sp.); (5) wheat + Bacillus polymyxa strain 42; (6) treatment #4 + Aa; (7) treatment #5 + Aa; (8) yreatment 4 + Ba; and (9) treatment #5 + Ba. The formation of paranodules on wheat roots was observed in the 4-5th day of plant growth. Their diameter was approximately 0.5-1 mm. The significant differences in the number of paranodules and also the rate of nitrogen-fixing activity on wheat roots was shown to depend on the treatments. The maximum nitrogen-fixing level was recorded after wheat plant inoculation with a mixed culture (Arthrobacter sp. + Xanthomonas sp.) on day 14 of plant growth.

151


PS7-5 Contribution of model legumes to the knowledge of the actinorhizal symbiosis Casuarina-Frankia

Valérie Hocher, Sergio Svistoonoff, Hassen Gherbi, Florence Auguy, Laurent Laplaze, Didier Bogusz, and Claudine Franche Equipe Rhizogenèse, Institut de Recherche pour le Développement/Institut National de la Recherche Agronomique/Université Montpellier II, UMR DIAPC, 34394 Montpellier Cedex 5, France Actinorhizal root nodules result from the interaction between the nitrogen-fixing actinomycete Frankia and roots of plants belonging to eight dicot families and 25 genera. The basic knowledge of the symbiotic interaction between Frankia and actinorhizal plants is still poorly understood, although it offers striking differences with the Rhizobium-legume symbiosis [1]. By comparing the symbiotic process involving Frankia to the one involving Rhizobium, we expect a deeper understanding of the molecular basis underlying host plant predisposition for nodulation shared by members of the Fabid clade. To investigate the similarities between legumes and actinorhizal plants, we have genetically transformed the tropical actinorhizal tree Casuarina glauca with reporter genes driven by promoters of symbiotic genes from legumes. Data obtained with the promoters ENOD40, ENOD12, and ENOD11 will be presented. Similarly, the expression patterns conferred by Casuarina symbiotic promoters have been studied in transgenic legumes. A genomic platform has also been developed to study the molecular mechanisms leading to actinorhizal nodule development and functioning [2]. Genes of primary metabolism, protein synthesis, cell division, and defence were found to be highly represented in nodules. A subtractive hybridisation library (SSH) was also constructed with roots sampled 4 days after infection, revealing that a majority of ESTs were implicated in defence, cell wall structure, and gene expression. This EST-based analysis provides the first picture of the set of genes expressed during actinorhizal symbiosis, allowing a broad comparison with the ESTs involved in the symbiotic process with Rhizobium. All these data establish that common plant genes and functions are involved in the rhizobial and actinorhizal symbioses, suggesting that all endosymbioses resulting from the interaction with Rhizobium, Frankia, and arbuscular mycorrhizal fungi are closely related [3]. [1] Laplaze et al. (2008). In Nitrogen-Fixing Actinorhizal Symbioses, Nitrogen Fixation Research: Origins and Progress, vol. 6,

K. Pawlowski, and W.E. Newton (Eds), Berlin, Springer, pp. 235-259. [2] Hocher et al. (2006). New Phytol. 169: 681-688. [3] Gherbi et al. (2008). Proc. Natl. Acad. Sci. USA 105:4928-4932.

152


PS7-6 Study of the microbial community structure and functional diazotrophic diversity in Colophospermum mopane

Claudia Sofía Burbano, Thomas Hurek, and Barbara Reinhold-Hurek Laboratory of General Microbiology, Faculty of Biology and Chemistry, University of Bremen, 28334 Bremen, Germany The tree Mopane (Colophospermum mopane) is a legume capable of growing in hot and dry areas of Southern Africa, where it is a component of the woodland vegetation, and sometimes the predominant tree. Mopane is an important food source for animals and people and it is also commonly used as firewood and construction material. Despite its economical importance in the region, little is known about its ecology and, particularly, about the nodulation status or the root-associated bacterial community. Given the fact that Mopane is found in mostly nitrogen-poor soils, endophytic diazotrophic bacteria might be involved in its growth and development. The aim of the presented work is to assess the diversity and activity of endophytic diazotrophic bacteria in roots of Mopane samples from different locations in Namibia. Root nodules were not detected. However, preliminary results showed that RNA extracts from roots of all plants contain nifH mRNA at levels detectable by RT-PCR. Phylogenetic analysis of a clone library constructed for nifH-expressed fragments showed that the sequences have high similarities to nitrogenase reductases from α-, δ-, γ-proteobacteria, Firmicutes, and to unknown nitrogen-fixing bacteria. 16S rRNA gene analysis will also be presented in order to assess the microbial community structure. Here, we report for the first time that Mopane plants contain diazotrophic bacteria transcribing nifH in non-nodulated roots.

153


PS7-7 Performance of common bean and Faba bean-rhizobial combinations under water stress east and west of the Nile delta

Youssef Garas Yanni1, Mohamed Zidan2, Carmen Vargas3, and Frank Dazzo4 Departments of 1Microbiology and 2Plant Nutrition, Sakha Agricultural Research Station, Kafr El-Sheikh, Egypt; 3Department of Microbiology and Parasitology, University of Seville, 41012 Sevilla, Spain; and 4Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA The symbiotic performance of some common and Faba bean rhizobial strains collected from slightly to highly saline and dry soils of the eastern and western Nile delta were tested in 80 field experiments during the Faba bean seasons 2004/2005, 2005/2006, and 2006/2007 and the common bean seasons 2005, 2006, and 2007. We tested eight registered Faba bean cultivars and a lot of non-identified ones (produced by farmers and stored to be used in subsequent seasons) against nine strains of Rhizobium leguminosarum bv. vicea, and two registered common bean cultivars and non-identified ones against nine Rhizobium phaseoli strains. The used salt-tolerant strains were identified using the "Capillary T-RFLP analysis of the 16S rDNA", then re-tested for authenticity and efficiency as symbiotic micropartners in greenhouse before use in field experimentation. Field soil textures ranged from calcareous, sandy-loam in the west to sandy, sandy-loam, and clay-loamy in the east, either salt-affected and/or often suffer moderate to severe shortage in irrigation water. Soil water contents were maintained at 100% or 50-60% of their field capacities all over the experimental course (main-plot treatments); three ascending doses of N2-fertilizer (split-plot treatments), and inoculation or not (split-split plot treatments). The isolates expressed high symbiotic performances that varied according to strain/cultivar combinations, as increased plant growth, seed yield, straw, harvest index (% seed yield/seed + straw), and the agronomic N fertilizer use efficiency (kg seed yield/kg fertiliser N). Superiority of the researcher package of recommendations over the conventional agro-economical management of the cooperator farmers was found attributable to the optimized crop management practices containing, in addition to inoculation, proper field stand densities, optimized NPK fertilization, integrated pest management, use of highly responsive varieties, and efficiency of researchers to disseminate knowledge to both the extension specialists and the cooperating farmers through a fluent feed/feedback information system.

154


PS7-8 Growth promotion of maize seedlings by Bacillus sp N. Martínez-Montiel1, R. Bustillos-Cristales1, J. Muñoz-Rojas1, Miguel A. Mascarúa-Esparza1, A. Sánchez-Saavedra1, G. Téllez-Torres2, and Luis E. Fuentes-Ramírez1 1Instituto de Ciencias, and 2Escuola de Biologia, Benemérita Universidad Autonoma de Puebla, Puebla, Pue. 72570, Mexico Inoculation of crops with beneficial plant-associated bacteria produces phenotypes, such as growth increase, resistance to adverse environmental conditions, reduction in occurrence of diseases, and lastly rising of yields. The microorganisms that induce these phenotypes are known as plant growth-promoting bacteria (PGPB). Possibly, some of the most studied microorganisms include species in the genera Azospirillum, Bacillus, Pseudomonas, Azoarcus, and Burkholderia. Nevertheless, the spectrum of PGPB is extremely wide and increases continually as more interactions are explored. Other authors have observed plant growth promotion of maize inoculated with Pseudomonas corrugate, P. fluorescens, P. syringae, P. putida, E. aerogenes, A. lipoferum, A. brasilense, K. pneumoniae, Pantoea agglomerans, G. diazotrophicus, H. seropedicae, R. leguminosarum, and R. etli. Several isolates obtained previously from Pseudomitrocereus fulviceps (Cactaceae) were tested in a "criollo" maize cv. These isolates were preliminary identified by 16S DNA sequencing. Inoculated sprouts of maize with some of the strains were higher and showed a larger diameter than non-inoculated plantlets. These effects were observed with an isolate of Bacillus sp. This work was partially funded by VIEP and ICUAP.

155


PS7-9 Molecular characterisation of a novel quorum sensing system in the diazotrophic grass endophyte Azoarcus sp. strain BH72

Lena Hauberg1, Melanie Böhm1, Christian Scharf2, and Barbara Reinhold-Hurek1 1Laboratory of General Microbiology, University of Bremen, 28334 Bremen, Germany; 2Functional Genomics, Greifswald University, 17487 Greifswald, Germany Quorum sensing is a regulatory mechanism operating in response to cell density. This cell-to-cell communication system involves the production and detection of autoinducers followed by transcriptional gene regulation. In proteobacteria, N-acyl-homoserine lactones (AHLs) are widely used as such signal molecules. The β-Proteobacterium Azoarcus sp. BH72 lacks an AHL-based quorum sensing system. The autoinducer in this novel quorum sensing system is not known so far, but based on its hydrophilicity it was termed HSF (hydrophilic signal factor). In former studies, it has been shown that the pilAB gene expression in Azoarcus sp. BH72 is quorum sensing regulated. Therefore, these structural genes, which are important for the formation of type-IV pili, can be used as reporter genes for quorum sensing experiments. The influence of cell-free supernatants from different Proteobacteria on the pilAB gene expression was detected. Supernatants from Azoarcus communis strain SwuB3 and Azospira oryzae strain 6a3 can induce the pilAB gene expression from Azoarcus sp. strain BH72. This observation leads to the question whether the cell-free supernatants contain the same or a similar signal molecule. Proteomics can be used as a standard method to study diverse cellular functions and regulatory processes. A whole cell proteome reference map from the exponential growth phase from Azoarcus sp. strain BH72 has been constructed to allow comparative proteomic studies. For the proteome map, several protein parameters were analysed and these theoretical data were compared to the experimental results from two-dimensional gels and mass spectrometry. Comparative proteomic analyses revealed that 25% of the detected proteins from Azoarcus sp. BH72 were regulated under quorum sensing conditions, 20% were found to be down-regulated, and 5% up-regulated. This observation demonstrates that quorum sensing is a global regulatory mechanism in this strain.

156


PS7-10 Transmembrane signalling and genomics: role of TonB-dependent receptors in the endophytic lifestyle of Azoarcus sp. BH72

Andrea Krause, Federico Battistoni, Anna Klindworth, and Barbara Reinhold-Hurek Laboratory of General Microbiology, University of Bremen, 28334 Bremen, Germany TonB-dependent receptors (TBDRs) are outer membrane proteins that are mainly responsible for the active uptake of iron-siderophore complexes. Additionally, they are important for perception of environmental signals, including the plant-pathogen interaction. Examination of the genome sequence of the nitrogen-fixing endophyte Azoarcus sp. BH72, which is capable of colonising the interior of grass roots without causing symptoms of plant diseases, revealed that 22 genes of the 3992 predicted protein-coding sequences encode proteins related to TBDRs. In contrast, experimental and genomic studies have shown that Azoarcus is unable to synthesise siderophores (iron carriers) which are important for the iron uptake. Two of these 22 TBDRs have an N-terminal extension that is known to participate in the iron-related signalling cascade. Computer-based investigations identified in the upstream regions of these two tbdr genes, a DNA motif resembling a Fur box, a regulatory element found in promoter regions of iron-responsive genes. Further analyses revealed that such a Fur box could not be found in the upstream region of every tbdr gene, indicating that not all of them are involved in iron uptake. To investigate the function of the 22 TBDRs in the endophytic lifestyle of Azoarcus, a plasmid integration mutagenesis system was established. Since the plasmid carries the gfp and gusA marker genes, the mutant strains can be additionally used to analyse the expression profile in culture and in interaction with plants. Functional and expressional analyses of some of the tbdr genes have been started. Preliminary results of the first few mutant strains did not show any influence of iron on the lifestyle of Azoarcus, which is in good agreement with the computer-based investigations that only few, but not all, of the TBRs are involved in iron uptake. Further characterisations will be presented.

157


PS7-11 A sugarcane bacterial inoculant Veronica Massena Reis1, Willian Pereira2, Guilherme de Souza Hipólito2, Fábio Lopes Olivares3, José Ivo Baldani1, Robert Michael Boddey1, and Segundo Urquiaga1 1EMBRAPA Agrobiologia, Seropédica, RJ, 23851-970, Brazil; 2Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, 23890-000 Brazil; 3Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campo dos Goytacazes, RJ, 28013-600, Brazil Brazil has a biofuel programme mainly based on sugarcane. It is one of the graminaceous species that can obtain most of its nitrogen from biological N2 fixation (BNF). A new product, based on the use of five diazotrophic bacterial strains has been selected for inoculation on sugarcane. This product is applied on stem pieces that are submerged in the bacterial solution for 60 min. This procedure guarantees that each piece contains approximately 106 bacterial cells per g fresh tissue. Greenhouse experiments, testing this mixture and other strains applied separately, confirm that biomass accumulation is higher in the mixture treatment in two varieties tested, RB72545 and RB867515. In the field experiments, these two varieties were also inoculated at the micropropagation stage and again at planting. These experiments done at three different sites and the inoculated treatment produced 14 to 22 Mg more cane compared to the control without nitrogen. The in vitro inoculation resulted in a yield 16 Mg higher than the control without nitrogen fertiliser, and 3 Mg over the control with 80 kg of added nitrogen. The measurements of the contribution of BNF using ∆15N analysis did not show any contribution of the biological process in this first year.

158


PS7-12 Transcriptomic analysis of Frankia alni ACN14a Clothilde Queiroux, Pascale Fournier, Nicole Alloisio, Petar Pujic, and Philippe Normand Laboratoire d'Ecologie Microbienne, Centre National de la Recherche Scientifique, UMR5557, Université Lyon 1, 69622 Villeurbanne Cedex, France Frankia are actinobacteria in nitrogen-fixing symbiosis with actinorhizal plants. Frankia alni ACN14a can form nodules on Alnus roots. It is not known how the bacteria can enter in symbiosis with the plant because of the lack of a genetic tool for Frankia. However, since 2005, the whole genome sequence of three Frankia strains have been published [1]. These data showed that there are no canonical nod genes in Frankia, but there are nodC and nodB-like genes. These data allowed us to conceive new approaches. In particular, we employed the transcriptomic microarray technique to find genes up- and down-regulated during symbiosis. Several conditions were tested: nitrogen depletion, nitrogen abundance, and Alnus nodules. We also work on a second approach targeted on a "Nod factor"-like molecule. The presence of a molecule, able to deform root hairs, has been shown [2]. This molecule was heat-stable, resistant to some chitinases, and hydrophilic, whereas Rhizobium Nod factors are heat-stable, chitinase sensitive, and amphiphilic. New experiments have been undertaken showing that the root hair-deforming factor was sensitive to some chitinases. [1] Normand et al. (2007). Genome Res. 17:7-15. [2] Cérémonie et al. (1999). Can. J. Bot. 77:1293-1301.

159


PS7-13 Response of upland cultivated rice (Oryza sativa L.) to microbial inoculation with α- and β-rhizobia

Manoj K. Singh1, A.K. Saxena2, Cyril Bontemps3, J. Peter W. Young3, and R.K. Singh1 1Microbial Genetic Laboratory, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005, India; 2National Bureau of Agriculturally Important Microorganisms, Kusmaur, Mau Nath Bhanjan-275101, India; 3Department of Biology, University of York, York YO10 5YW, UK We have isolated two rhizobia (RREM25 and RREM36) from surface-sterilized roots of naturally grown cultivated rice (Oryza sativa L.). They nodulate mimosa (Mimosa pudica) and French bean (Phaseolus vulgaris), respectively, under controlled laboratory conditions. Molecular analysis of 16S rDNA through PCR-RFLP and sequencing revealed that strain RREM25 has close affinity with the Burkholderia cepacia complex, whereas RREM36 is closely related to Rhizobium undicola, a species that has not previously been reported as an endophyte of rice. Endophytic effectiveness of both RREM25 and RREM36 on rice plants was tested under gnotobiotic and glasshouse conditions. A significant increase in plant height, root growth, leaf area, dry weight, chlorophyll content, and total nitrogen content of rice plants inoculated with these bacteria was observed. Various biochemical tests, such as phosphate solubilization, IAA production, and cellulase and pectinase activities were made to investigate the mechanisms for this plant growth promotion ability. It was concluded that the observed plant growth promotion due to bacterial inoculation is attained through more than one mechanism.

160


PS7-14 A quorum-quenching approach to identify quorum-sensing-regulated functions in Azospirillum lipoferum

Florence Wisniewski-Dyé, Mickaël Boyer, and René Bally Laboratoire d'Ecologie Microbienne, Centre National de la Recherche Scientifique, UMR5557, Université Lyon 1, 69622 Villeurbanne Cedex, France Many bacteria rely on cell-cell communication to modulate gene expression and behaviour within a microbial community, a process known as quorum sensing (QS). Such communication is mediated by small diffusible signalling molecules, such as the N-acyl homoserine lactones (AHLs) used by many Gram-negative bacteria. A quorum-quenching approach was exploited in order to identify QS-regulated functions in the plant growth-promoting bacterium Azospirillum lipoferum. The AttM lactonase from Agrobacterium tumefaciens was shown to enzymatically inactivate AHLs produced by two A. lipoferum strains. The targeted analysis of several phenotypes revealed that in strain B518, a rice endophyte, AHL inactivation abolished pectinase activity, increased the synthesis of siderophores, and reduced indole acetic acid production (in stationary phase), but no effect was observed on cellulase activity and on swimming and swarming motilities. None of the tested phenotypes appeared to be under QS regulation in strain TVV3 isolated from the rice rhizosphere. Moreover, AHL inactivation had no deleterious effect on the phytostimulatory effect of the two A. lipoferum in vitro. A global proteomic approach was then undertaken. Whereas little modification of the protein patterns was detected when comparing attM-expressing TVV3 and the wild-type strain, numerous proteins appeared to be up-regulated or down-regulated by the AHL-mediated QS system in strain B518. Several proteins identified by MS-MS analysis appeared to be implicated in transport (such as OmaA) and chemotaxis (ChvE). Altogether, results indicate that in A. lipoferum QS regulation is strain specific and is dedicated to regulate functions linked to rhizosphere competence and adaptation of A. lipoferum to the plant root.

Session 8

Nodule organogenesis

Chaired by Martin Crespi Gif sur Yvette, France


161

Session 8: Nodule organogenesis

S8-1 New pathways regulating Medicago truncatula root architecture Florian Frugier1, Julie Plet1, Federico Ariel3, Adnane Boualem1, Carole Laffont1, Philippe Laporte1, Mariana Jovanovic1, Loreto Naya1, Raquel Chan3, Erika Sallet2, Jérôme Gouzy2, Caroline Hartmann, Christine Lelandais1, and Martin Crespi1 1 Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France; 2Laboratoire des Interactions Plantes Micro-organismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France; 3Laboratorio de Biotecnología Vegetal, Universidad Nacional del Litoral, Santa Fe, Argentina Legumes are characterized by their ability to develop two lateral organs from primary roots depending on environmental conditions, lateral roots (common to all plants) and nitrogen-fixing nodules. Physiological and genetic data indicate that development of these organs is coordinated and involves common regulatory pathways, including phytohormonal controls. Recently, cytokinin signalling mediated by the receptor MtCRE1 has been identified as crucial for both organogeneses in the Medicago truncatula model legume [1]. We characterised symbiotic and non-symbiotic root phenotypes of M. truncatula mutants affected in cytokinin signalling genes, obtained through the TILLING platform developed in the frame of the Grain Legumes EEC project (GLIP; C. LeSignor and R. Thompson, INRA-Dijon; D. Baker and J. Clarke, JIC-Norwich). This coupled with transcriptomic approaches allowed us the identification of new cross-talk elements between nodulation and cytokinin signalling pathways. Regulation of transcript stability mediated by microRNAs has been associated to various plant developmental processes. We notably showed that MtmiR166 regulation, which targets type-III HD-ZIP transcription factors, is crucial for patterning of root vascular bundles, and formation of both lateral roots and nodules. Parallel analysis of small RNA libraries from M. truncatula roots and nodules is being done to extend our knowledge on the role of miRNA-mediated riboregulation in symbiotic interactions. Altogether, we characterized several new pathways integrating symbiotic and non-symbiotic root architectures in legumes. [1] Gonzalez-Rizzo et al. (2006). Plant Cell 18:2680-2693.

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S8-2 New regulators of nodule development in Medicago truncatula Tatiana Vernié1, Thomas Ott1,2, Jean-Philippe Combier1, Sandra Moreau1, Benoit Lefebvre1, Susana Rivas1, Françoise de Billy1, Ton Timmers1, Florian Frugier3, Giles Oldroyd4, Françoise Jardinaud5, Fabienne Vailleau5, Agnès Lepage1, Laurence Godiard1, Andreas Niebel1, and Pascal Gamas1 1Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France; 2Genetics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany; 3Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France; 4Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK; 5Laboratoire de Symbiose et Pathologie des Plantes, Institut National de Polytechnique-Ecole Nationale Supérieure Agronomique de Toulouse, 31326 Castanet-Tolosan Cedex, France We identified by transcriptomics a large set of new potential regulators of nodule development [1, 2]. The characterisation of four of them showed their involvement in the control of early and late stages of nodulation, and led to the discovery of new post-transcriptional mechanisms for the control of gene expression. MtHAP2.1 belongs to the family of CCAAT-box binding proteins. We found a new mechanism that restricts its expression to the nodule meristem, in a complementary way to miR169. It consists in differential splicing of MtHAP2-1 transcripts and trans inhibition of MtHAP2-1 expression by an intron-encoded peptide [3]. A null hap2-1-1 mutant confirmed that MtHAP2.1 is necessary for proper Nod factor induction of MtENOD11, Sinorhizobium meliloti infection and release, and nodule meristem maintenance. EFD belongs to the same subgroup of Ethylene Response Factors as ERN1-3. The characterisation of a null deletion mutant, overexpressor, or RNAi lines suggested that EFD is a negative regulator of nodulation, triggered by nodule primordium formation. EFD is also expressed in nodule zone II and required for zone II and III differentiation. The fact that the response regulator gene MtRR4 is activated by EFD leads us to propose that EFD acts by inhibiting the cytokinin signalling pathway [unpublished]. MtnodREM and MtbHLH1 are expressed in nodule zone III: MtbHLH-1 encodes a putative transcription factor expressed in non-infected cells and vascular bundles, whereas MtnodREM, a lipid raft-associated remorin, is expressed in infected cells. In addition, MtnodREM and MtbHLH1 are induced in the root at early symbiotic stages. RNAi nodREM roots are defective for Rhizobium infection, while altering bHLH1 leads to modifications in nodule vascularisation and defects in zone-III development. The possible function of these genes will be discussed. [1] El Yahyaoui et al. (2004). Plant Physiol. 136, 3159-3176. [2] Godiard et al. (2007). Mol. Plant-Microbe Interact. 20:321-332. [3] Combier et al. (2008). Genes Dev. in press.

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PS8-1 The protein secretory pathway component DNF1 is required for nodule function

Dong Wang1, Joel Griffitts1, Colby Starker1, and Sharon Long1 Department of Biology, Stanford University, Stanford, CA 94305. USA In the symbiosis between legumes and nitrogen-fixing bacteria, many host factors are necessary for the function of a nodule. The intricate molecular machinery essential for successful nodule organogenesis remains poorly understood. Recent advances in the molecular genetics of model legumes offer opportunities to dissect this complex process. We performed a genetic screen in a fast neutron-irradiated Medicago truncatula population for plants affected in the nodulation process. Several mutants with nodules defective in nitrogen fixation were discovered and were named dnf mutants. After inoculation with Sinorhizobium meliloti Rm1021, these dnf mutants form white nodules accompanied by a loss of nitrogenase activity [1]. Here, we describe the DNF1 gene, which is represented by two alleles from our screen. In both alleles of dnf1, a transcript-based approach identified deletions in a gene encoding a putative member of the protein secretory pathway. Complementation tests in planta confirmed that this gene is DNF1. The protein secretory pathway processes proteins targeted to the extracellular space or certain organelles. The cloning of DNF1 suggests that in legume-Rhizobium symbiosis, protein secretion from the host plays a crucial role. We describe efforts to characterise DNF1's function in protein secretion both in vitro and in vivo. Consistent with its mutant phenotype, the DNF1 gene is strongly expressed in nodules. Furthermore, DNF1 expression inside nodules is restricted to regions of active growth, indicating that the DNF1 function precedes the formation of fully differentiated bacteroids. We hypothesise that the substrates of DNF1 may be proteins important for the maturation of symbiosome. [1] Starker et al. (2006). Plant Physiol. 140:671-680.

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PS8-2 CLE peptide signalling during nodulation on Medicago truncatula Virginie Mortier1,2, Griet Den Herder3, Willem Van de Velde4, Ryan Whitford1,2, Marcelle Holsters1,2, and Sofie Goormachtig1,2 1Department of Plant Systems Biology, Flanders Institute for Biotechnology, and 2Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium; 3Genetics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany; 4Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France The classical hormones ethylene, cytokinin, and auxin are involved in the initiation and coordination of the nodulation process. We suspect that a new class of hormones, the peptide hormones, and more specifically, the CLE peptides, also have an important function during Medicago truncatula nodulation. Until now, CLE peptides were only assigned a role in shoot, flower, and root meristem maintenance, in vascular development, and in nematode feeding cell formation. By sequence analysis of the M. truncatula genome and MtEST libraries, we identified 25 MtCLE peptide genes. qPCR analysis revealed that three of them, MtCLE4, MtCLE12, and MtCLE13 are up-regulated from the early stages of nodulation on. Promoter-GUS analysis of the three MtCLE peptide genes indicated that each of them is activated in the nodule meristem. Moreover, MtCLE13 is also expressed very early in the nodule primordia. RNAi analysis of MtCLE13 by use of Agrobacterium rhizogenes transformation, resulted in a retarded and diminished nodulation. No major RNAi phenotypes could be observed for the other two CLE peptide genes. This could be the result of redundancy, because this has often been observed for CLE peptides. In parallel to the RNAi, the ectopic expression of the three CLE peptide genes was performed. A Nod- phenotype was generated when overexpressing MtCLE12 and MtCLE13. The results gained until now suggest a role for at least one of the nodule-specific MtCLE peptide genes in nodule primordium and meristem formation.

Session 8

Nodule organogenesis

Chaired by Martin Crespi Gif sur Yvette, France


165


PS8-3 Boron deficiency in the legume-rhizobia interaction: symbiosis or pathogenesis?

María Reguera1, José E. Ruiz-Sainz2, Luis Bolaños1, Nicholas J. Brewin3, and Ildefonso Bonilla1 1Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid; 2Departamento de Microbiología, Facultad de Biológicas, Universidad de Sevilla, 41012 Sevilla, Spain; 3Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK The rhizobia-legume symbiosis is driven by cellular and molecular plant-microbe interactions that are successful only insofar as they avoid triggering host plant defence reactions. Failure in cell or molecular components implicated in the interaction often results in pathogenic rather than symbiotic responses. Boron (B), a micronutrient essential for plant growth, seems to modulate signalling during nodule development and, apparently, helps to suppress the host defence response. Boron deficiency alters early preinfection and invasion events, resulting in an incompatible interaction between the legume and its host rhizobia, and inducing some plant defence mechanisms, including morphological barriers and synthesis of pathogenesis-related (PR) proteins. During infection thread growth, the host legume secretes glycoproteins, mainly legume-specific root nodule extensins (RNEs), otherwise known as AGP-Extensins (AGPEs). It has been shown in vitro that these glycoproteins are able to attach to the rhizobial cell surface. In the infection thread, the bacterial cell seems to escape from this "glycoprotein trap": cells are not ensnared by RNE peroxide-driven cross-linking, as has been demonstrated in vitro. However, under B deficiency, bacterial progress is arrested, presumably because of a higher degree of entrapment by RNE and peroxide. Moreover, B deficiency leads to induction of at least two PR proteins of the PR10 family, PR10.1 and ABR17. Members of the PR10 family are ribonuclease-like proteins that in legumes are induced by pathogens, but repressed during rhizobial symbiosis. Moreover, ABR17, which is a pea protein constitutively expressed in roots, is induced by abiotic ABA-signalised stresses. The use of anti-ABR17 antiserum, in Western blots and tissue-print assays indicates that the ABR17 protein occurrence is restricted at a low level to the cortical tissues of B-sufficient nodules, but it increased during B-deficient development in all nodule tissues. Financed by Ministerio de Educación y Ciencia (BIO2005-08691-C02-01) and Comunidad de Madrid (Microambiente).

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PS8-4 Cross-talk between the cell cycle progression and infection thread formation at early stages of symbiotic root nodule development on pea

Kirill N. Demchenko1, Nikolai P. Demchenko1, Alexey Y. Borisov2, Katharina Pawlowski3, and Viktor E. Tsyganov2 1Komarov Botanical Institute, Russian Academy of Sciences, 197376 St.-Petersburg, Russia; 2All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia 3Department of Botany, Stockholm University, 10691 Stockholm, Sweden In search for the molecular mechanism of rhizobial nodule morphogenesis, we aimed to identify the connection between the position of a plant cell in the cell cycle with its ability to form infection threads, using a genetic approach. Using confocal microscopy, a refined analysis of early developmental steps of nodule morphogenesis was carried out for the non-fixing-nitrogen Pisum sativum mutants RisFixA (Sym41), SGEFix--2 (Sym33), RBT3 (Sym33, Sym40), and the corresponding wild-type lines. Cell proliferation was estimated by 5-bromo-deoxyuridine immunolocalization (labelling cells in the S-phase) and determination of the mitotic index. The pattern of infection thread formation was analysed using immunovisualisation of rhizobial cell walls. After the resumption of cell proliferation in the root pericycle and cortex, nodule primordium formation occurs and an incipient nodule meristem develops. All cortical cell layers, except for the four outer ones, are involved in primordium formation prior to the formation of infection threads in primordium cells. In this stage, all nodule primordium cells are in the mitotic cycle, i.e. they synthesise DNA and divide. This leads to the development of a persistent mature nodule meristem at the nodule apex as a consequence of sustained mitotic activity of a part of the incipient nodule meristem. Cells in the incipient nodule meristem containing infection threads were still able to divide. Based on the observed sequence of changes in nodule structure, we propose that the ability of plant cells to form infection threads, while undergoing the mitotic cell cycle, is a prerequisite for incipient nodule meristem formation, and that at least two pea genes (Sym41 and Sym33) are required for this ability. Based on these results and data reported previously, a scheme for the sequential functioning of pea symbiotic genes in the pathway of nodule development is suggested. This work was supported by the Russian Foundation for Basic Research (08-04-01710).

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PS8-5 The structure of the nodules and localisation of symbiosome membrane (SM) identity markers in defective for nitrogen fixation (DNF) mutants of Medicago truncatula

Elena Fedorova1,2, Sergey Ivanov1,2, Erik Limpens1, and Ton Bisseling1 1Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands; 2K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Science, Moscow 127276, Russia In infected nodule cells, rhizobia are maintained in membrane compartments formed by the host, so-called symbiosomes, which grow and mature to be able to perform a nitrogen fixation. We hypothesise that development of symbiosomes may depend on the presence on SM of a selected set of membrane markers involved in membrane fusion, such as SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). To obtain insight into symbiosome formation, we searched for mutants with symbiosomes arrested in different developmental stages. We analysed several fix- mutants among others the six Medicago mutants dnf [1]. Two of these mutants, namely DNF1 and DNF2, form symbiosomes that are maintained at a very early stage of development for 6-8 cell layers. In DNF2 nodules, symbiosomes with unusually big peribacteroid spaces completely fill the host cells that do not contain any vacuoles. In contrast, DNF1 symbiosomes have small peribacteroid spaces and vacuoles were formed in infected cells. In the DNF1 mutant, we also found a lot of autolytical bodies containing part of the host cytoplasm, even in young infected cells. On the wild-type nodules, symbiosomes are characterized by the occurrence of specific identity markers (SNAREs and small GTPases of the Rab family) at different stages of development [2, 3]. The occurrence of these identity markers in wild-type and DNF1/DNF2 mutants will be compared and will be combined with detailed ultrastructural analyses. [1] Mitra & Long (2004). Plant Physiol. 134:595-604. [2] Limpens et al. (2008). This volume (WS3, S1-1). [3] Ivanov et al. (2008). This volume (PS8-7).

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PS8-6 New approaches to study nodule development in pea, an older model plant

Scott R. Clemow, and Frédérique C. Guinel Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, N2L 3C5, Canada Recently, great advances in our knowledge of the nodulation process have been made. Molecular tools, new model plants, and improved microscopy techniques have led to a detailed picture of the pre-infection and penetration stages of the legume-Rhizobium symbiosis. The time has arrived to revisit the pea symbiosis, as recalcitrance of pea to transformation impeded its use and past studies did not account for pre-infection events. Our study had two objectives: (i) to produce composite plants, i.e. plants with transformed roots but non-transformed shoot, and make them nodulate; and (ii) to develop a spot-inoculation technique to visualize changes occurring to the roots in response to rhizobia. Towards goal 1, a protocol was modified [1]. Cut stems, inserted into Fibrgro cubes imbibed previously with Agrobacterium rhizogenes carrying a GUS reporter gene, were placed in trays covered with domes. The plants, upon transfer to pots, were inoculated with Rhizobium leguminosarum. Three weeks later, individual roots were assayed for GUS and GUS-positive roots had their nodules counted. To achieve goal 2, seeds were pre-germinated for 48 h, transferred to germination pouches, and inoculated with R. leguminosarum carrying a lacZ reporter. The plants were harvested at different time points and their roots assayed for lacZ and embedded in agar; vibratome sections were observed for signs of infection. Nodules formed on transformed roots of composite plants and the transformation efficiency was comparable to that of other legumes. Spot-inoculation was successful with early infection events caught at the site of inoculation. We foresee the use of these techniques for the study of our mutant collection. Transformation will be useful as we plan to modify the cytokinin levels of the plant via the insertion of cytokinin metabolic genes, while spot-inoculation would permit the observation of the effects these alterations have on nodule organogenesis. [1] Collier et al. (2005). Plant J. 43:449-457.

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PS8-7 SNAREs on the symbiosome membrane Sergey Ivanov1,2, Erik Limpens1, Elena Fedorova1,2, and Ton Bisseling1 1Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands; 2K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Science, Moscow 127276, Russia Rhizobium bacteria are hosted inside root nodule cells as transitory organelles, called symbiosomes (SBs), where they convert atmospheric nitrogen to ammonia. In the model legume Medicago truncatula (Medicago), the bacteria are internalised into the plant cell by an endocytosis-like process, and they become individually enclosed by a membrane of plant origin, the symbiosome membrane (SM). After maturing to their nitrogen-fixing form, the symbiosomes are maintained for some time as individual units, but, eventually, they start to fuse and form lytic compartments in a process called senescence. We hypothesise that SB development and maintenance depends on the presence on the SM of specific sets of integral membrane proteins that control vesicle fusion, the N-ethylmalemide sensitive factor attachment protein receptors (SNAREs). Such SNARE proteins are key membrane identity markers of the different endomembrane compartments. With the aim to specify the SB identity, we have analysed the localisation of SNAREs proteins during symbiosome development. Therefore GFP-fusions of Medicago SNARE proteins were localised in both roots and nodules. We analysed SNAREs that control the fusion to the plasma membrane (SYP132, SYP121, VAMP721/722 and VAMP724), and SNAREs that control vacuole formation (SYP21/22, SYP51/52, VTI11/12, VAMP711, and VAMP714). Symbiosomes appear to have plasma membrane identity as they retain plasma membrane SNARE proteins from the start of the release from the infection thread throughout the symbiosome development. Young SBs do not acquire vacuolar SNAREs, which shows that they do not have vacuolar identity. However, as nodules start to senesce, the SM starts to acquire vacuolar SNAREs, after which the symbiosomes fuse and form lytic compartments. Therefore, we suggest that the absence of vacuolar SNAREs on the SM prevents the formation of lytic compartments by which they are maintained as individual units. During senescence, the SBs acquire vacuolar SNAREs and are degraded as a result of vacuole-like formation in the senescent infected cells.

170


PS8-8 Genes involved in nodule meristem formation Irina V. Leppyanen1, Maria Osipova1,2, Ludmila Lutova2, and Elena Dolgikh1 1All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia; 2Department of Genetics, St.-Petersburg State University, 199034 St. Petersburg, Russia The formation of symbiotic nodules on legume roots starts with the reactivation of cell divisions in definite regions of the pericycle and root cortex, where later the nodule meristem is formed. The molecular mechanisms of nodule meristem formation are poorly investigated. The purpose of our work is to study the involvement of two genes Enod40 and WOX5 in nodule meristem development. The Enod40 is known to be expressed in pericycle and cortex cells, being the molecular marker of early steps of nodule development. The definite function of this gene in nodule development is poorly characterised. The WOX5 gene is active in the quiescence center of the root meristem. Until now, little is known about the role of this gene in nodule meristem development. Pea mutants with defects in nodule primordium/meristem development could be considered as a convenient model to study nodule meristem formation. Analysis of the Enod40 and WOX5 gene expression in such pea mutants allows us to elucidate the role of these genes in nodule meristem development. There are several Enod40 genes in different legume species, whereas attempts to find additional variants of the Enod40 have not been successful in pea. We have designed primers to estimate the Enod40 expression in pea. Sequence analysis of this gene in different pea cultivars and lines has been performed. At the same time, cloning of the WOX5 gene, not identified previously in the pea genome, has been carried out. The sequence analysis of the putative PsWOX5 has proved its similarity with MtWOX5 and AtWOX5 gene sequences. We have estimated the expression of the Enod40 and WOX5 genes at the different stages of symbiosis in wild-type and mutant pea roots inoculated with Rhizobium leguminosarum. The possible role of the PsWOX5 and Enod40 genes upon nodule development is discussed. This work was supported by RFBR 07-08-00700, CRDF RUXO-ST-012-00, and RFBR-NWO (06-04-89000) grants.

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PS8-9 Microscopic analysis of the effect of cellulase CelC2 overproduction by Rhizobium on infection thread initiation and bacterial release into host cells

Marta Robledo1, José I. Jiménez-Zurdo2, Encarna Velázquez1, Frank Dazzo3, Eustoquio Martínez1, and Pedro F. Mateos1 1Departamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, Spain; 2Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain; 3Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA In order to develop an effective Rhizobium-legume nodule symbiosis, bacteria must invade the host root. Two key processes of infectivity are the primary erosion of the plant root hair tip cell wall where the bacterial symbiont first penetrates to enter the host, leading to infection thread formation (Inf), and secondary, terminal disruptions of the infection thread wall within the invaded nodule host cells, allowing bacterial release (Bar) to establish their intracellular endosymbiotic state. These two processes of host wall degradation are highly localised and controlled to allow bacterial passage and release without host cell lysis. Here, we report on a cell-bound bacterial cellulase from Rhizobium leguminosarum bv. trifolii ANU843 that fulfils a significant role in both of these symbiotic infectivity events. CelC2 cellulase is a 1,4-β-D-endoglucanase with high substrate specificity for noncrystalline cellulose. The purified enzyme can completely erode the highly localised noncrystalline tip of the host root hair, making a complete hole, whose geometry and location match the entry point of primary host infection into white clover. ANU843CelC2- null mutants were unable to breach the host wall at the root hair tip and form infection threads [1]. Microscopy analysis of the overexpressed ANU843CelC2+ strain in the root hair tips and the nodule invasion zone revealed an exaggerated tip degradation of host root hairs and a high hydrolysis of the noncrystalline zone of the infection thread cell wall where the bacteria are released. Finally, some white clover plants inoculated with the CelC2-overproducing strain displayed a nodule histology of disorganised internal structures, showing that this strain invades these cells by an uncontrolled way, leading to infective, but ineffective, nodules. Our results document the crucial role of this cell-bound enzyme in the root-nodule symbiosis with legumes. [1] Robledo et al. (2008). Proc. Natl. Acad. Sci. USA 105:7064-7069.

172


PS8-10 Construction and analysis of a Phaseolus vulgaris sRNAs root library, after Rhizobium inoculation

Georgina Estrada-Navarrete, Pablo Peláez, Luis Servín, Yamile Márquez, Catalina Arenas, José Luis Reyes, Alejandra Covarrubias, Carmen Quinto, and Federico Sánchez Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, Mexico In plants, microRNAs (miRNAs) are 20-22 nucleotides in length, non-coding RNAs that generally repress genetic information at a posttranscriptional level. miRNAs play important roles in development and abiotic and biotic stress responses. Primary miRNA transcripts (pri-miRNAs) adopt stem-loop RNA secondary structures from which a specific 21-nucleotide duplex is excised by a Dicer-like endonuclease. The mature miRNA stably incorporated into an ARGONAUTE (AGO) protein complex that exerts an endonucleolytic cleavage on mRNAs based upon complementarity. Recently, a strong component of translational silencing by siRNA and miRNAs has also been discovered [1]. Auxin signalling pathway is importantly regulated by miRNAs, In particular, miR393 participates in resistance to bacterial infection by repressing the plant auxin response [2]. We have constructed a sRNA library from Phaseolus vulgaris roots, recently inoculated by Rhizobium tropici. Several miRNAs were cloned and characterized by bioinformatics and molecular approaches, including those that seem to be specific of the symbiotic interaction in P. vulgaris. In Arabidopsis, different members of the miR160 and miR167 families have as targets the transcription factors ARF10, ARF16, and ARF17, and ARF6 and ARF8, respectively. In P. vulgaris roots, Rhizobium inoculation and auxin addition induced both miR160 and miR167 family members. We will discuss the phenotypes that overexpression of the pre-miR67 family shows in transgenic roots in P. vulgaris. This work was partially supported by grants ININ208407 and CONACYT 42562-Q. [1] Brodersen et al. (2008). Science 320:1185-1190. [2] Navarro et al. (2006). Science 312:436-439.

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PS8-11 Nap and Pir genes are involved in control of actin rearrangements essential for infection thread formation and colonisation of Lotus japonicus root nodules

Keisuke Yokota1,5, Eigo Fukai1, Lene H. Madsen1, Anna Jurkiewicz1, Paloma Rueda1, Simona Radutoiu1, Mark Held2, Md Shakhawat Hossain2, Krzysztof Szczyglowski2, Mette W. Nielsen1, Giulia Morieri6, Allan Downie6, Giles Oldroyd6, Anna Maria Rusak1, Shusei Sato3, Satoshi Tabata3, Euan K. James4, Hiroshi Oyaizu5, Niels Sandal1, and Jens Stougaard1 1Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark; 2Agriculture and Agri-food Canada, SCPFRC, London, Ontario NV5 4T3, Canada; 3Kazusa DNA Research Institute, Chiba, 292-0818, Japan; 4University of Dundee, Dundee DD1 5EH, UK; 5Biotechnology Research Center, University of Tokyo, Tokyo 113-8657, Japan; 6Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK By surveying Lotus mutant lines with a defective infection process, we have selected a subclass, which, in addition to aberrant infections, have abnormal trichomes, a phenotype often associated with defects in endomembrane, microtubule, or actin-dependent morphogenesis. Since Nod factor-dependent re-organization of microtubules and actin filaments was suggested to be a prerequisite for successful symbiotic interaction, we further characterized two such mutants, pir (sym40) and nap (sym67), which both develop an excess of uncolonised nodule primordia when inoculated with Medicago loti. Formation and subsequent progression of infection threads into the root cortex is significantly impaired in these mutants; in addition to the symbiotic phenotype, both mutants show a short and distorted trichome phenotype on both flowers, leaves, and stems. Map-based cloning identified the L. japonicus genes, Nap1 and Pir1, as responsible for these phenotypes. Both NAP and PIR proteins are part of the SCAR/WAVE complex that mediates actin dynamics. Subsequent analysis of the actin structure in Lotus wild type and nap and pir mutants using Alexa-phalloidin staining showed a disturbed actin cytoskeleton organisation in the mutants: nap and pir root hairs had mostly short transverse actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Measurement of Ca2+ flux and Ca2+ spiking showed that the Ca2+ response to Nod factor application in nap and pir mutants is indistinguishable from the wild type. This suggests that the actin rearrangements act downstream of the Ca2+ response, or alternatively act in a parallel pathway.

174


PS8-12 Distribution of hydrogen peroxide in infection threads in a series of symbiotically defective mutants of pea (Pisum sativum) Fix- mutants

Anna Tsyganova1, Viktor Tsyganov1, Igor Tikhonovich1, and Nicholas Brewin2 1Laboratory of Molecular and Cellular Biology, All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia; 2Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Colonisation of host cells by Rhizobium bacteria involves the progressive remodelling of the plant cell walls [1]. Extensibility of the infection thread is apparently controlled by peroxide-driven protein cross-linking of plant matrix glycoproteins [2, 3]. We performed the comparative analysis of the hydrogen peroxide distribution in a panel of Fix- pea mutants, using the technique of precipitation with cerium chloride followed by electron microscopy. During development of infection threads in wild-type nodules, peroxide was first detectable in association with the infection thread wall, and subsequently its distribution spread to the luminal matrix. Peroxide was abundant in the lumen of the infection droplets. In the mutant lines SGEFix-1 (sym40) and RisFixV (sym42), where the premature senescence occurs, there was often an increased abundance of peroxide in infection threads and symbiosomes. Intriguingly, however, in mutant SGEFix-2 (sym33), which does not release bacteroids from infection droplets, there was the absence of hydrogen peroxide-induced cerium precipitates that were not detected in infection droplets nor around infection thread walls (although exceptionally there were presented by a few large precipitates on the outer surface of infection thread walls). A similar situation was observed in double mutants carrying gene sym33 RBT3 (sym33, sym40) and RBT4 (sym33, sym42), which also fail to release bacteria from infection droplets. These observations point towards a novel function of hydrogen peroxide during nodule development, i.e. during endocytosis of bacteria from infection droplets inside the host cytoplasm. This work was supported by the INTAS (YSF 04-83-3196), the Federal Science Agency of Russia (state contract N 02.442.11.7130), the Russian Foundation for Basic Research (RFBR) (05-04-49105), the UK Biotechnology and Biological Research Council, joint grants RFBR - Netherlands' Organization for Scientific Research (06-04-89000) and U.S. Civilian Research & Development Foundation (CRDF) - the Russian Ministry of Education and Science (RUXO-012-ST-06, DP2M12). [1] Brewin, N.J. (2004). Crit. Rev. Plant Sci. 23: 293-316. [2] Hérouart et al. (2002). Plant Physiol. Biochem. 40: 619-624. [3] Wisniewski et al. (2000). Mol. Plant-Microbe Interact. 13:413-420.

175


PS8-13 Stimulation of nodulation in Medicago truncatula Gaertn. by low concentrations of ammonium: quantitative reverse-transcriptase (qRT) PCR analysis of selected genes

Kevin Vessey, and Houman Fei Biology Department, Saint Mary's University, Halifax, NS B3H 3C3, Canada Previous studies have shown that low concentrations of ammonium stimulate nodulation in pea and white clover and that this enhancement may be due to an elevation in cytokinin to auxin levels in roots. Here, the effects of ammonium and nitrate at increasing concentrations (0.0, 0.1, 0.5, and 2.5 mM) on nodulation and expression of two N-uptake and 12 putative nodulation-related genes of the model symbiosis of Medicago truncatula– Sinorhizobium meliloti are investigated. After 3 weeks of hydroponic growth, whole plant nodulation was enhanced in all of the ammonium treatments and up to 3-fold in the 0.5 mM treatment compared to the zero-N control. Most informative was that specific nodulation (nodule/g root DW) was greatly stimulated in the 0.1 and 0.5 mM NH4

+ treatments, to a lower extent in the 0.1 mM NO3

- treatment, and inhibited in all other treatments. Gene expression at 6, 12, and 24 h after exposure to the mineral N treatments was investigated by quantitative, reverse-transcriptase (qRT)-PCR of 14 genes selected from the MtGI database. Expression of nitrogen transporter genes increased significantly with exposure to mineral N. Of three genes putatively associated with symbiosis signalling/nodule initiation, responses to treatments were mixed. There were no consistent significant responses of genes coding for an ABA-activated protein kinase or a gibberellin-regulated protein, but an ethylene-responsive element binding factor showed increased expression in various treatment/time points. Three auxin-responsive genes and three cytokinin-responsive genes showed various responses to ammonium and nitrate over the three sampling times. These results demonstrate that similar to pea and white clover, low concentrations of ammonium stimulates nodulation in M. truncatula and are consistent with the hypothesis that a relatively high ratio of cytokinin to auxin may be the mechanism by which low concentrations of ammonium lead to stimulation of nodulation in some legumes.

176


Session 9

Ecology and sustainable agriculture

Chaired by Manuel Megías

Sevilla, Spain


177

Session 9: Ecology and sustainable agriculture

S9-1 Bacterial group-II introns: new mobile elements in the rhizosphere Francisco Martínez-Abarca, Rafael Nisa-Martínez, María Dolores Molina-Sánchez, José I. Jiménez-Zurdo, Manuel Fernández-López, Fernando M. García-Rodríguez, Antonio Barrientos-Durán, E. Muñoz-Adelantado, Isabel Chillón-Gázquez, Alicia Ortigosa-Alcon, and Nicolás Toro Genetic Ecology Group, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain Group-II introns are large catalytic RNAs (Ribozymes) and a class of retroelements that splice via a lariat intermediate mechanism resembling that of nuclear spliceosomal introns. Intron mobility may occur either to intronless alleles (retrohoming) or to ectopic sites (retrotransposition) and is mediated by ribonucleoprotein complexes consisting of the Intron Encoded Protein and the excised lariat RNA [1]. They were initially found in mitochondrial and chloroplast genomes of lower eukatyotes and plants where their presence have been shown relatively abundant, and later identified in Bacteria and Archaebacteria (25% of eubacterial genomes). RmInt1 was the first group II intron described in the order Rhizobiales. It was identified in Sinorhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa) [2]. The natural insertion site for RmInt1 is ISRm2011-2, which is usually present in high copy number in the genome of virtually all S. meliloti isolates. RmInt1 is present in 90% of these S. meliloti isolates and also in other Rhizobium species in which RmInt1 is able to move efficiently. Both, its behaviour as modifiable specific retroelement and its successful dispersion in rhizobia render this genetic element as a biotechnological tool for targeting gene disruption and as a model for the study of group-II intron survival, evolution, and dissemination in nature. [1] Toro et al. (2007). FEMS Microbiol. Rev. 31:342-345. [2] Martinez-Abarca et al. (1998). Mol. Microbiol. 28:1295-1306

178


S9-2 The role of biological nitrogen fixation in cropping systems and its environmental impact

Mark B. Peoples Commenwealth Scientific and Industrial Research Organisation, CSIRO Plant Industry, Canberra ACT 2601, Australia Recent estimates suggest that 55 Million t of fixed nitrogen (N) might be contributed to agricultural systems by legumes each year (of which 21-22 Million t N may be fixed by grain legumes). However, the relative importance of legume inputs of fixed N varies considerably in different regions of the world, and it is clear that a sizable proportion of the human population now depends on the 85-90 Million t of fertilizer N applied each year to satisfy the N needs of major food crops. Because N is so susceptibility to loss processes when supplied in excess of crops' requirements, N fertilizers have been linked to many environmental hazards including groundwater contamination, and stratospheric ozone destruction. Some researchers argue that legumes offer a more environmentally sound and sustainable source of N to cropping systems via biological N2 fixation [1]. Clearly there are many advantages in legumes fixing N to produce high-quality protein for livestock and humans. However, the limited data available comparing the subsequent fate of N from legume residues with applications of fertilizer suggest that while the extent of N losses tend to be greater from fertilizer under irrigated agriculture, losses from both N sources may not differ as much as previously suspected in rainfed cropping systems [2]. The main environmental differences between legume and fertilizer sources of N appear to be derived from contributions to global warming associated with the energy use and CO2 emissions from fertilizer production and application [3]. It also needs to be recognized that N-fixing legumes play an important role in food production by providing rotational benefits for following crops that are unrelated to direct effects on N availability. These non-N benefits will be examined in relation to the impact of hydrogen emitted from nodules as a by-product of N2 fixation on soil biology. [1] Jensen & Hauggaard-Nielsen (2003). Plant Soil 252:177-186. [2] Crews & Peoples (2005). Nutrient Cycling in Agroecosys. 72:101-120. [3] Crews & Peoples (2004). Agric. Ecosys. Environ. 102:279-297.

179


PS9-1 Genetic engineering of the Rhizobium-legume interaction for bioremediation

Miguel Angel Caviedes Formento, Ignacio David Rodríguez Llorente, Alejandro Lafuente, Julián Delgadillo, Mohamed Dary, Bouchra Doukkali, Antonio J. Palomares(†), and Eloísa Pajuelo (†) In memoriam Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain The design of genetic traits through plant biotechnology allows the use of plants far beyond the production of food and fibers. Molecular farming expands the use of plants to the production of compounds and biofuels, or in phytoremediation. Legumes have been traditionally used in soil regeneration, given their capacity to increase the soil nitrogen content due to biological nitrogen fixation in nodules. More recently, legumes are getting an increasing interest in bioremediation. Our group has been working for the last years on the Rhizobium-legume symbiosis as a new tool in bioremediation of heavy metals polluted soils. Legumes accumulate As and heavy metals mainly in roots, being the main application of these plants in metal phytostabilisation. Furthermore, inoculation of legume plants with Rhizobium and a consortium of rhizosphere bacteria resistant to As and heavy metals produced a protective effect on the plant, together with a diminution of metal accumulated. We have developed transgenic plants in order to increase the Cu-phytoextraction potential. Modified bacterial copper resistance genes have been introduced into plants under the control of tissue-specific promoters. On the other hand, copper resistance genes have been transferred to Rhizobium in order to increase copper resistance and/or copper accumulation. Preliminary data on the bioremediation capacity of the "genetically engineered symbiosis" are presented.

180


PS9-2 Analysis of sustainable agriculture alternatives in Chihuahua, Mexico

L. Hernández1, M. Miranda1, José-Antonio Munive2, and María del Carmen Villegas1 1Centro de Investigación en Biotecnología Aplicada, Instituto de Politécnico Nacional, Tlaxcala, Tlax. 90700, Mexico; 2Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico Modern agriculture focuses on increasing crop yields to satisfy the needs of a growing population. Sustainable approaches look for the substitution of chemical inputs by an optimal use of natural resources. Biological Nitrogen Fixation (BNF) fits well in this model as it is a more environmentally clean way of satisfying plant nitrogen needs, although several restraints to the BNF rates have been reported [1, 2] They include poor nodulation capacity, competitive but inefficient indigenous rhizobia, and lack of response to inoculation under field conditions. However, an approach not often considered is the isolation and assessment of native efficient strains. The aim of this work was to evaluate the presence of microbial populations in agricultural lands in the Chihuahua State (Mexico), following the estimation of their biotechnological potential to improve the nutritional quality of soils under a native population enforcement strategy [3], as a sustainable approach to increase crop yields. However, due to frequent use of chemical fertilisation in this area, microbial communities have dramatically decreased. Soil samples were obtained from three different localities in the Chihuahua State from peanut, cotton, potato, chili, peach, apple tree, and alfalfa cultures. Also, root nodules were sampled from alfalfa, peanut, and bean plants. Mycorrhizal fungi population was found to be extremely small. Putative rhizobia were evaluated as potential biofertilizers considering their nitrogen-fixing rate (using the ARA technique), and biochemical tests (API galleries, temperature, pH, and salt tolerance). Genetic characterisation was performed using 16S rDNA and nifH sequencing analysis. Since native rhizobia seem to be affected on the nitrogen-fixing rate, and the size of the microbial communities is also weak, biofertilising strategies in this dry area have to change from the enforcement of native populations (already adapted to extreme climatic conditions) to the introduction of foreign bacteria, without damaging the ecological equilibrium. [1] Aguirre-Medina (2006). Biofertilizantes Microbianos: Experiencias Agronómicas del Programa Nacional del INIFAP en

México. Libro Técnico N° 2. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Campo Experimental Rosario Izapa, Tuxtla Chico, Chiapas, México.

[2] Villegas & Munive (2005). Biótica 2:55-106. [3] Hungria & Stacy (1997). Soil Biol. Biochem. 29:819-830.

181


PS9-3 Hydrogen fertilization: is this the benefit of crop rotation? Zhongmin Dong Department of Biology, St. Mary's University, Halifax, Nova Scotia, B3H 3C3, Canada Hydrogen gas (H2) is a natural and unavoidable byproduct of biological nitrogen fixation that occurs in most agricultural legumes. It is released into the soil around the nitrogen-fixing root nodules. In a typical legume crop, at peak growth, up to 7000 litres or more of H2 are produced per ha per day, representing an energy loss from the crop equivalent to 5% of its net photosynthetic carbon gain. This hydrogen triggers major changes in the biology of the soil surrounding the nodules, changes that have been shown to have significant positive impacts on the growth and yield of both legume and non-legume crops. This plant growth enhancement coupled with microbial changes in soils following "H2 fertilization" may -for the first time- provide an explanation of the beneficial effects of legumes in rotation with other crops besides the nitrogen leftover.

182


Session 9

Ecology and sustainable agriculture

Chaired by Manuel Megías

Sevilla, Spain


183


PS9-4 Effect of planting dates and genistein on nitrogen content and nodulation of three annual medics

Majid Amini Dehaghi1, Fatemeh Abedin2, and Jaber Karimi3 1Department of Agronomy, Faculty of Agriculture, Shahed University, Tehran, Iran; 2Department of Agronomy, Faculty of Agriculture, Tarbiat Modarres University, Tehran, Iran; 3Functional and Evolutionary Entomology, Gembloux Agricultural University, 5030 Gembloux, Belgium In order to study the effect of planting dates and genistein on the nitrogen content and nodulation of three annual Medicago species (Medicago polymorpha cv. Santiago, M. rigidula cv. Ragidula, and M. radiata cv. Radiata), an experiment was conducted at the Research Farm of Faculty of Agriculture (Shahed University, Tehran) (35°43'N and 51°8'E) during 2004-2005. The factors were arranged as split-split plots in a randomized complete block design with four replications. Planting dates (February 20, March 1, and March 11) were randomized to main plots and three annual medics were located in sub-plots and genistein (0 and 20 µM) were randomized to sub-sub-plot units. Plant nitrogen content, nodulation, and other traits were significantly species different and M. polymorpha was better than other species in respect of dry nodule weight, nodule number, nodule number in each cluster, nodule cluster number, and nodule diameter. Medicago rigidula had more resistance to cold than other varieties, and its forage yield and nitrogen percent was higher than those of M. polymorpha. Therefore, M. rigidula may be better suited for cold zones. Twenty µmol genistein had a remarkable effect on nodulation and nitrogen percent of annual medics when compared with controls. The result showed that genistein modified the negative effect of the low temperature environment on nodulation and nitrogen percent of annual medics. Nodulation and nitrogen percent increased in all varieties at the first planting date. This matter emphasizes that genistein has a considerable effect on the cold resistance establishment in varieties for improving nodulation and increasing plant nitrogen percent under farming conditions.

184


PS9-5 Effect of planting dates and genistein on quantitative and qualitative traits of three Medicago species

Majid Amini Dehaghi1, Fatemeh Abedin2, and Jaber Karimi3 1Department of Agronomy, Faculty of Agriculture, Shahed University, Tehran, Iran; 2Department of Agronomy, Faculty of Agriculture, Tarbiat Modarres University, Tehran, Iran; 3Functional and Evolutionary Entomology, Gembloux Agricultural University, 5030 Gembloux, Belgium In order to study the effect of planting dates and genestein on quantative and qualitative traits of three Medicago species, an experiment was conducted in 2004 and 2005. Three annual medic cultivars (Medicago polymorpha cv. Santiago, M. rigidula cv. Rigidula, and M. radiata cv. Radiata) that adapted to cold and temperature zones were cultivated at three planting dates (February 20, March 1, and March 11) with two levels of genistein concentrations (0 and 20 µM). The experiments were conducted in randomized, complete block design arrangement in factorial with four replications. M. rigidula had the highest forage and ash percent at the first planting date in two years. This result showed that M. rigidula has good resistance to cold conditions. Annual medics did not have good vegetative growth and suitable forage yield at the first planting date due to cold, but they produced better forage yield and more protein at the second planting date than the third planting date due to suitable environmental conditions and longer growth period. M. polymorpha and M. rigidula were better than M. radiata in aspect of dry forage production. Genistein increased forage yield in M. polymorpha at the second planting date and protein percent in M. radiata at the third planting date.

185


PS9-6 Sinorhizobium strains nodulating Medicago sativa in different Iranian regions

Majid Talebi Bedaf1, Masoud Bahar1, Ghodratollah Saeidi2, Alessio Mengoni3, and Marco Bazzicalupo3 Departments of 1Agricultural Biotechnology, and 2Agronomy and Plant Breeding, College of Agriculture, Isfahan University of Technology, Isfahan, Iran; 3Dipartimento di Biologia Evoluzionistica, Università degli Studi di Firenze, 50125 Firenze, Italy Alfalfa (Medicago sativa L.) is believed to have originated in north-western Iran and has a long history of coexistence with its bacterial symbiont Sinorhizobium in soils of Iran, but little is known about diversity of Sinorhizobium strains nodulating Iranian alfalfa genotypes. In this study, Sinorhizobium populations from eight different Iranian sites were sampled using three cultivar of M. sativa as trap-host plants. A total of 982 rhizobial strains were isolated and species were identified showing large prevalence of S. meliloti over S. medicae. Analysis of salt tolerance demonstrated a great phenotypic diversity, while it did not show significant differences between groups of strains isolated from different sites and cultivars. Genetic diversity of the Sinorhizobium isolates was analysed by BOX-PCR and ERIC sequences. Patterns of BOX-PCR fingerprinting were statistically analysed with AMOVA to evaluate the roles of plant variety and site of origin on the genetic variance observed. Results indicated that most of the total molecular variance was attributable to divergence among strains isolated from the same sites and cultivars (intra-population genetic variance). Moreover, results showed the presence of two groups of populations (western and north-western Iran). A significant genetic difference (1.97%) was found among sites; in contrast, only 0.77% of the genetic diversity was attributable to differences among cultivars, showing that the effect of the different sites of origin could be more relevant in shaping the population genetic diversity than the effect of different cultivars and individual plants.

186


PS9-7 Innovation technologies for commercial bioinoculants production and application in agriculture

Vladimir Chebotar1, Alexander Kazakov2, and Igor Tikhonovich1 1All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia; 2Bisolbi-Inter Ltd., 196608 St. Petersburg, Russia Development of bioinoculants for agricultural use usually represents a final step in research of plant-microbe interactions. However, selected strains should possess several properties necessary for mass production. They should grow in a wide range of temperatures and pH, effectively use available and cheap sources of carbon and nitrogen, such as molasses, corn liquor, nutrients derived from meat and fish, and wheat bran with production of high cells number. The commercial strains should be able to grow quickly and effectively produce compounds beneficial for plants. However, even the excellent bioinoculants do not guarantee effectiveness under field conditions. It is very important to develop the proper technology for the application of bioinoculant (seed treatment, foliar treatment, etc.). Usually more reasonable is to apply microbes in combinations with chemicals, such as herbicides, insecticides, fungicides or fertilizers, so bioinoculants should be compatible with these preparations. The field for application of bioinoculants depends on their properties and can cover agriculture, horticulture, gardening, landscape restoring, forestry, pastures, and fruit and vegetable storage. The Innovation Company Bisolbi-Inter organised in 2000 is producing microbial inoculants for agriculture: Extrasol™ biofertilizer, BisolbiFit™ for stimulation of plant growth, and BisolbiSan™ for plant protection from phytopathogens. The main advantage of company products is their ability to substitute chemical means that are usually dangerous for the environment for use on a whole cycle for foodstuff production: from seed treatment to after harvest treatment. Another advantage of the company is its close cooperation with the All-Russia Research Institute for Agricultural Microbiology. This facilitates constant improvement of products and selection of new products and technologies for marketing. Besides the Russian Federation, some products have been registered in Moldova, Kazakhstan, South Africa, Romania, Serbia, and Guinea.

187


PS9-8 Effects of plant age and soil toposequence on the δ15N and δ13C of Cyclopia and Aspalathus species in the fynbos of the Western Cape

Sipho T. Maseko, and Felix D. Dakora Faculty of Science, Tshwane University of Technology, Pretoria 0001, South Africa The dry matter of young shoots collected from field plants of Cyclopia genistoides, Aspalathus caledonensis, and an Aspalathus sp. at Koksrivier near Gansbaai was analysed for 15N/14N isotope ratio using mass spectrometry. The δ15N values obtained were used to assess the symbiotic performance of the three species occurring in the same habitat on a farmer's field. The data showed that the three species differed significantly in their levels of dependency on N2 fixation for their N nutrition. A comparison of 1997 C. genistoides plants with those planted in 2005 revealed marked differences in δ15N values of young shoots, with the older plants showing a greater dependency on N2 fixation for N nutrition compared to their younger counterparts. The effect of annual tea harvesting of C. genistoides by the farmer also showed significant differences in the δ15N of young shoots and pods when compared to unharvested plants. Similar studies done on cultivated Cyclopia subternata plants on Kanetberg mountains near Riversdale also showed marked differences between the δ15N values of plants with differing ages. Toposequence and seedlings versus cuttings were similarly found to affect the δ15N of C. subternata at Kanetberg. Our data suggest that the δ15N of symbiotic legumes can be altered by various environmental factors, including plant age, soil topography, and farmer practices.

188


PS9-9 Transposomics and stress-induced gene expression in Sinorhizobium meliloti: the case of the enigmatic tspO gene

Frans J. de Bruijn Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France In nature, bacterial growth is restricted by a wide variety of environmental factors. Factors of particular importance are the lack of essential nutrients, oxygen limitation, abbarent pH, oxidative stress, dessication, and osmolarity stress. Understanding how bacteria are able to sense and respond to their environment is fundamental to our understanding of microbial persistence in bulk soil, the rhizosphere of plants, and, in the case of symbiotic bacteria (for instance, rhizobia) in planta. A nutrient-deprivation-induced locus in Sinorhizobium meliloti strain 1021 was identified by use of a Tn5luxAB reporter gene transposon. The tagged locus is comprised of two open reading frames (ORFs), designated ndiA and ndiB for nutrient deprivation-induced genes A and B. The expression of the ndi locus was found to be induced by carbon and nitrogen deprivation, osmotic stress, oxygen limitation, and during entry into stationary phase. To identify regulatory components involved in the control of ndi gene expression, a second round of mutagenesis was performed on the primary ndiB::Tn5luxAB-tagged strain (C22) with transposon Tn1721. A double mutant strain was obtained that lacked transcriptional activity of the ndi locus under all inducing conditions. The Tn1721-tagged gene product showed a high degree of similarity to the tryptophan-rich sensory protein, TspO, from Rhodobacter sphaeroides, as well as to the mitochondrial benzodiazepine receptor, pK18, from mammals. In the former, TspO appears to be an outer membrane protein involved in porphyrin transport, and in the latter, TspO plays an important role in disease and diagnostics and is also involved in cholesterol and porphyrin uptake. Quantitative RT-PCR and microarray analyses did not support a regulatory role for TspO [M. Hrisztoskova, L. Sauviac and C. Bruand, unpublished data]. The tspO gene is also found in plants, where it also is involved in cholesterol uptake, porphyrin transport, and plant resistance to abiotic stress. A model for the action of TspO in S. meliloti is presented.

189


PS9-10 Sesbania virgata and Azorhizobium doebereinerae symbiosis: a model of high specificity and symbiotic efficiency for all strains

Fatima Maria de Souza Moreira, Ligiane Aparecida Florentino, Cleide Aparecida Bomfeti, Ana Paula Guimarães, Patrícia Gomes Cardoso, and Mário César Guerreiro Department of Soil Science, Federal University of Lavras, Lavras, MG, 37200-000, Brazil The genus Azorhizobium was first described by Dreyfus & Dommergues [1], represented by only one species: Azorhizobium caulinodans. This species was described on the basis of nodule isolates from both stems and roots of Sesbania rostrata in Africa. After 18 years, the second species of this genus was described: A. doebereinerae [2] based on isolates from Sesbania virgata root nodules occurring in Brazilian southeast soils. Several studies carried out in the Laboratory of Soil Microbiology of the Federal University of Lavras have showed that S. virgata, when inoculated with a variety of rhizobia species and soil samples, collected from different regions and ecosystems, only developed a symbiosis with A. doebereinerae strains and with soil samples collected near the root system of S. virgata. In all these cases, the symbiosis S. virgata and A. doebereinerae was always efficient and highly specific. For a better understanding about the role played by surface polysaccharides (SPSs) in the symbiosis between S. virgata and A. doebereinerae, one SPS mutant strain is being constructed based on the partial sequence of the oac locus (genes oac1, oac2, and oac3) described for A. caulinodans [3]. The primer construction for the amplification of these genes was done according to the sequence available in the NCBI Genbank database with accession number Z22611. The composition of the exopolysaccharides (EPSs) present in A. doebereinerae showed the elementary analyses of carbon and nitrogen as being 11.8% and 6%, respectively. The ash analysis was carried out and 36.78% was found, indicating the presence of a high level of metal ions. [1] Dreyfus & Dommergues (1980). C. R. Acad. Sc. Paris, Ser. D 291:767-770. [2] Moreira et al. (2006). Syst. Appl. Microbiol. 29:197–206. [3] Goethals et al. (1994). J. Bacteriol. 176:92-99.

190


PS9-11 Using plant genotypes to improve N2 fixation: compatibility of Trifolium subterraneum L. cultivars with soil rhizobia can influence symbiotic performance

Elizabeth Drew, Ross A. Ballard, and Nigel Charman South Australian Research and Development Institute, Adelaide, SA 5001, Australia Trifolium subterraneum (sub-clover) is the most important and most widely grown pasture legume in southern Australia, occurring on approximately 50% of the 23 M ha of improved pastures. We have shown that some widely sown cultivars of sub-clover are on average achieving only 50% of their symbiotic (N2 fixation) potential, due to their "incompatibility" with the large populations of naturalised clover rhizobia that have developed in many soils. This incompatibility has led us to select for high symbiotic performance within sub-clover germplasm. Variation in symbiotic performance (SP) was assessed for 49 genotypes of T. subterraneum in combination with four strains of rhizobia isolated from field soils. Plants were grown in N-free media in the greenhouse and their shoot dry weight measured and expressed as a percentage of the control treatment (Australian clover inoculant strain WSM1325). SP ranged from 7 to 90% between genotypes. Neither breeding history or geographic origin of genotypes was correlated with SP. Two cultivars with high (cv. Campeda) and low (cv. Clare) SP values overall, but similar growth with the control, were investigated in more detail. Campeda typically out performed Clare when inoculated with 30 soil populations of rhizobia (SP of 73% vs. 48%) and with 14 isolates from those soils (SP of 68% vs. 39%). The poor performance of Clare could be attributed to interruptions at multiple stages of the symbiotic association, from nodule initiation (less nodules), nodule development (pseudo-nodules) through to nodule function (from 0-60% of max N2 fixed/g nodule dry weight) depending on treatment. We continue to investigate the mechanisms controlling the compatibility of clover genotypes with populations of soil rhizobia, including the ability of the host to influence nodule occupancy by specific strains. Nonetheless, we believe that the findings already demonstrate that through the careful selection and use of clover genotypes by plant breeders, it should be possible to make significant gains in the SP of future sub-clover cultivars.

191


PS9-12 Enhancement of bioremediation of PAH and diesel fuel contaminated soils after inoculation of maize (Zea mays) of Azospirillum spp. and Pseudomonas stutzeri

Anna Gałązka, and Maria Król Department of Agricultural Microbiology, Institute of Soil Science and Plant Cultivation–State Research Institute, 24-100 Puławy, Poland PAHs are often found in contaminated soils and there is the need of developing techniques that can be applied in the remediation of these sites, where PAH, specially those with high molecular weight pose health and environmental risks [1]. Successful bioaugmentation requires not only a catabolically active inoculum but also a microbial strains or consortium that can survive well in the target environment. The survival of introduced microorganisms in soil depends on a broad range of biotic and abiotic factors [2]. Microbial degradation is the mean to remove PAHs from contaminated soils, especially the use of strains of bacteria which are able to degrade PAHs and use them as a source of carbon and energy and fix free nitrogen such as the strains of Azospirillum spp. and Pseudomonas stutzeri. The objective of this study was to investigate the effect of inoculation of plant with PAHs contaminated soil (chernozem, redzina, and gray-brown podsolic soil) treated with phytoremediation. The strains of Azospirillum spp. and Pseudomonas stutzeri were able to degrade PAHs as the only source of carbon and energy. The soil used in the experiment was collected from uncontaminated area of agricultural use. The following combinations of the artificially-contaminated soils were used: soil + plant, soil + plant + PAH (anthracene, phenanthrene and pyrene at the doses 100; 500; 1000 g·kg-1 of the soil) and soil + plant + PAH + inoculation. Diesel fuel was used at the doses 0.1%, 0.5%, and 1%. Experiment was carried out as a greenhouse pot study. Plants had been cultivated for 30 days and then harvested. The total number of various bacteria strains, fungi and enzymatic activity in soil before and after vegetation period, degradation of selected PAHs were the basis for the determination of the effectiveness of the phytoremediation. Obtained results suggest that inoculation of plants (esp. maize) with Azospirillum spp. and Pseudomonas stutzeri looks promising as a low-cost treatment method for PAHs contaminated soil. The differences in concentration between the inoculated or non-inoculated soils indicate that the presence of plant roots, in addition to the period of time, contributes to reduction in the bioavailability of selected PAHs. The research has confirmed higher total number of micoorganisms after inoculation of plants with all doses of PAHs. Also the better growth of under and over soil parts of the plants and higher PAHs degradation was observed. [1] Alexander M. (1999) Biodegradation and bioremediation. AP, San Diego, USA, pp. 252-258. [2] Boonchan et al. (2000) Appl. Environ. Microbiol. 66:1007-1019.

192


PS9-13 Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.)

Katarina Huić Babić1,2, Kristina Schauss1, Brigitte Hai1, Sanja Sikora2, and Michael Schloter1 1Helmholtz Zentrum München, German Research Center for Environmental Health, Department of Terrestrial Ecogenetics, Institute of Soil Ecology, 85764 Neuherberg, Germany; 2Department of Microbiology, Faculty of Agriculture, University of Zagreb, 10000 Zagreb, Croatia The inoculation of seeds with Rhizobium is known to increase nodulation, nitrogen uptake, growth, and yield response of crop plants. However, the effective symbiosis does not depend only on the capacity to fix elemental nitrogen from the air, but also on the internal nitrogen turnover in the root-rhizosphere complex. In the present study, a greenhouse experiment was set up to test the influence of inoculation with two indigenous Sinorhizobium meliloti strains, with different efficiency in terms of plant growth promotion, on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, and nifH and the archaeal amoA) was performed by real-time PCR in rhizosphere samples that had been taken before nodule formation, at the bud development stage, as well as at the late flowering stage. The results clearly demonstrate that the efficiency of rhizobial inocula is highly related to the abundance of the nifH genes in the late flowering phase of alfalfa. Low copy numbers of genes involved in ammonia oxidation as well as in denitrification at the same plant developmental stage indicate that the formed ammonia by the rhizobia at this time point is mostly used by the plant and is not transferred to the rhizosphere.

193


PS9-14 Cultivar specific differences in N cycling functional groups in the cotton rhizosphere

Oliver Knox1,2, and Gupta Vadakattu2 1Scottish Agricultural College, Crop and Soil Systems, Edinburgh EH9 3JG, UK; 2Commonwealth Scientific and Industrial Research Organisation, CSIRO Entomology, Urrbrae, SA 5064, Australia Within most farming systems there is currently a great deal of information being provided to maintain productivity and reduce environmental damage through better use and management of N and increased biological N inputs. In Australian cotton soils, we accessed the levels of non-symbiotic N fixation, potential nitrification rates, and populations of ammonium oxidiser (AO) bacteria, while undertaking studies of cotton cultivar-soil microbe interactions in the rhizosphere. Results indicate that there is a cultivar-based influence upon the rhizosphere microbial population, which is involved in different aspects of nitrogen cycling, including free-living N2-fixing microorganisms. However, it currently remains unclear as to whether the differences are the result of changes in activity or structure of the rhizosphere microbial community. If cultivars are producing different quantities of root exudates, then this might be expected to influence the overall activity of the microbiota. However, if the quality or chemical nature of the exudates is different then the rhizosphere microbial population is potentially being altered. It is likely that both factors will be involved in a more complex relationship that also encompasses subtle differences in root architecture and rhizosphere environment. Whatever the reason for the difference, it is clear that cotton cultivars are influencing processes involved in N cycling, which includes the free-living N2-fixing microorganisms within their rhizospheres. The observations made in the cotton system are an indicator that cultivar selection could be an important tool for integrating improved nitrogen fixation to assist in meeting plant nutritional demand under current agricultural practices.

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PS9-15 Role of AM fungi in the alleviation of salt-induced ionic, osmotic, and oxidative stresses in Cajanus cajan nodules

Neera Garg, and Geetanjali Department of Botany, Panjab University, Chandigarh-160014, India Salt stress affects various physiological and biochemical processes in legume nodules. The objective of the study was to evaluate the role of arbuscular mycorrhiza (AM) in moderating the toxic effects of salt stress on plant growth and nodular metabolism in Cajanus cajan (L.) Millsp. (pigeonpea). Exposure of plants to salinity stress reduced shoot and root dry weights. Nodular growth suffered remarkably and a marked decline in nodule biomass was observed under salt stress. Leghemoglobin content and acetylene reduction activity (ARA) also declined under saline conditions. Salt stress reduced nodular membrane stability and caused ionic imbalance, which resulted in reduced K+/Na+ and Ca2+/Na+ ratios in the nodules. Salinity induced increased synthesis and accumulation of osmolytes, such as proline and glycine betaine. Salt stress significantly increased the antioxidant enzyme (superoxide dismutase, catalase, and peroxidase) activities in the nodules of all plants. AM could significantly improve plant biomass, nodule dry mass, leghemoglobin content, and nitrogenase activity under salt stress. Activities of antioxidant enzymes increased markedly in nodules of mycorrhised plants. This study suggested a correlation between improved plant growth, increased functional efficiency of nodules, higher osmolyte accumulation, and enhanced antioxidant enzyme activities in the nodules of AM plants under stressed conditions relative those of uninoculated ones.

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PS9-16 Effect of bean inoculation with Rhizobium leguminosarum bv. phaseoli on plant nitrogen content

Jelena Marinković1, Mirjana Vasić1, and Mirjana Jarak2 1Institute of Field and Vegetable Crops, and 2Faculty of Agriculture, University of Novi Sad, 21000 Novi Sad, Serbia The use of microbial fertilizers provides cost-free plant nutrition with atmospheric nitrogen, increases yields, and grain protein quality due to the increased presence of essential amino acids, and enriches the soil with nitrogen to be utilised by the subsequent crops. Increase in mineral nitrogen fertilizers reduces the number of nodules formed by the strains used for inoculation, while increasng the number of nodules formed by native strains [1]. Another important benefit of inoculation in legume production is an increase in plant nitrogen content. Bean is a crop grown for its highly nutritious grain, which is one of the richest sources of vegetable protein in the human diet and one of the most widely used food items overall. For this reason, it is essential to work on improving bean grain quality, most importantly, grain protein content. The objectives of this study were to examine four strains of Rhizobium leguminosarum bv. phaseoli on four bean cultivars on the basis of nitrogen content in aerial plant part and in seeds. Field trials were established at the Institute of Field and Vegetable Crops in 2002 and 2003. Four bean cultivars were tested. Immediately before planting, bean seeds were inoculated with four R. leguminosarum bv. phaseoli strains. The study included five variants (four variants -seeds inoculated with R. leguminosarum bv. phaseoli strains and non-inoculated bean seeds). Plant samples were taken at the flowering stage and at the end of the bean growing season. The obtained results were statistically processed by the analysis of variance and LSD test. The average results show that the bean plants accumulated more nitrogen in 2003. The plant nitrogen content at flowering ranged from 96.26 to 207.62 mg/plant in 2002 and from 183.52 to 367.69 mg/plant in 2003. The results show that R. leguminosarum bv. phaseoli strains P4 and 3b/2 brought about the largest increases in nitrogen content in the above plant part. In both study years, inoculation increased nitrogen content in all the cultivars on average. R. leguminosarum bv. phaseoli strains P4 and 5b caused significant increases in nitrogen content in seeds in 2002, while strains 5b, 3b/2, and B2 in 2003. [1] Vargas et al. (2000). Biol. Fertil. Soil 32:228-233.

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PS9-17 Response of two sugarcane varieties to inoculation with Gluconacetobacter spp. in Mauritius

Jean-François Yvan Moutia1,2, Gunshiam Umrit1, Salem A. Saumtally1, and Jos Vanderleyden2 1Mauritius Sugar Industry Research Institute, Réduit, Mauritius; 2Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium Sugarcane production in Mauritius relies on the intensive use of inorganic fertilizers, which account for approximately 10% of the total production cost. Of these, nitrogen (N) fertilizers are of key importance both from the economic and environmental viewpoint. The use of N fertilizer in sugarcane cultivation in Mauritius has risen from 2500 t in the late 1940s to 11 000 t (representing EUR 5 million) in 1999. Today, the price of N fertilizer has increased to EUR 1120/t representing an increase of 158% over the past nine years. Due to the low efficiency of N utilization and the potential threat to the environment through contamination of surface and ground water, alternatives to N fertilization has been sought. In that context, two sugarcane varieties R570 and M1176/77 have been inoculated with Gluconacetobacter spp. to gauge their potential to obtain N from biological nitrogen fixation (BNF). Four different combinations of nitrogen fertilizer and bacterial inoculum were compared. Results indicated that relatively low bacterial populations have established themselves. The two varieties responded differently to the treatments. Growth and development of sugarcane in inoculated plots were comparable to or better than the control treatment. The sucrose content represented by the industrially recoverable sucrose content (IRSC) was not significantly different between treatments, indicating a beneficial effect following inoculation. The ∆15N signature as determined by Isotope Mass Ratio Spectrometry (IRMS) was also followed at regular intervals and a definite trend indicating fixation was observed. It was estimated that at the end of the first year, BNF had contributed 9.5% of the N requirement of R570 when the latter was inoculated with Gluconacetobacter spp. and received 35 kg N/ha. Growth and development in the ratoon crop of 2007/2008 has confirmed the promising results obtained in the virgin cane.

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PS9-18 Evaluation of the interaction bean genotypes, rhizobacteria, and environmental factors in Cuba

Lara Ramaekers1, Roseline Remans1, Jorge Luis Reyes2, German Hernandez3, Aurelio Garcia3, Vidalina Toscano3, Nancy Mendez3, Miguel Mulling3, Lazaro Galvez3, and Jos Vanderleyden1 1Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium; 2Estación Experimental Forestal, Piñar del Río, Cuba; 3Instituto de Suelos, Ministry of Agriculture, Ciudad de la Habana, Cuba In many parts of Latin America and Africa, common bean (Phaseolus vulgaris L.) forms a healthy and protein-rich centre piece of the daily diet. Demand is high, especially in poor regions, but production often does not meet demand due to suboptimal yields. Symbiotic Nitrogen Fixation (SNF) can contribute to improve bean production in a sustainable way. However, research is needed to improve the efficiency and stability of SNF in bean cultivation, especially under phosphorus (P)-deficient conditions. This research aims at enhancing N2 fixation in bean cultivation under low P conditions by studying the interaction between bean genotypes, Rhizobium, and plant growth-promoting rhizobacteria (PGPR). It also contributes to the evaluation of the agronomic relevance of selected genotype-PGPR combinations under field conditions in Cuba, a crucial step to bring PGPR inoculation into practice. In an on-station field experiment, four selected bean genotypes in combination with four nitrogen sources (N fertilizer, Rhizobium inoculation, Rhizobium and Azospirillum coinoculation, and no additional N source) were evaluated under low and high P conditions. The results show differential responsiveness to N mineral fertilizer and microbial treatments among the bean genotypes. Further, six "researcher plans, farmer manages" trials contributed to characterise stability of inoculation and genotype effects. Finally, the farmers' preferences and acceptance of microbial inoculation in bean cultivation in Cuba were assessed by a survey among local bean producers. Taken together, these results highlight the potential to improve bean production by microbial inoculation and the importance of the interaction between bean genotypes, agricultural practices, and environmental factors therein. Therefore, if recommendations are made to farmers with respect to Rhizobium-Azospirillum inoculation, it is crucial that local conditions are taken into account, including the farmer's management and resources, soil characteristics, and local bean variety preferences. [1] Remans et al. (2008). Plant Soil, in press (doi 10.1007/s11104-008-9606-4)

198


PS9-19 Toxic effects of arsenic on Sinorhizobium-Medicago symbiotic interaction: microscopic and molecular analysis

Ignacio D. Rodríguez-Llorente, Mohamed Dary, Alejandro Lafuente, Antonio J. Palomares (†), and Eloísa Pajuelo (†) In memoriam Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain Environmental contamination with arsenic is mainly associated with mining and smelting activities, but arsenic is also used in a wide variety of industrial applications (from computers to fireworks). The Rhizobium-legume symbiosis is a well-studied agronomically important process that has been recently proposed as an interesting tool in bioremediation. However, little is known about the effect of most common contaminants on this process. The phytotoxic effects of arsenic on nodulation of Medicago sativa have been examined in vitro using the highly arsenic-resistant and symbiotically effective Sinorhizobium sp. strain MA11. The bacteria were able to grow on plates containing As concentrations as high as 10 mM. Nevertheless, as little as 25 to 35 µM arsenite produced a 75% decrease in the total number of nodules, due to a 90% reduction in the number of rhizobial infections, as could be determined using the strain MA11 carrying a lacZ reporter gene. This effect was associated to root hair damage and a shorter infective root zone. However, once nodulation was established, nodule development seemed to continue normally, although earlier senescence could be observed in nodules of arsenic-grown plants [1]. We have also studied by TR-PCR the expression of several nodulation marker genes, such as NORK, MsEnod2, MsENOD40, MsCcs52, MtN6, and MsLeg in the presence or absence of arsenic. [1] Pajuelo et al. (2007). Environ Pollut. 154:230-211.

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PS9-20 Rhizobia from forest legumes in Mexico: genetic diversity and biotechnological potential

Yuriria Santoyo-Paez1,2, Luz Pérez-Melchor2, Moisés Carcaño-Montiel2, Lucía López-Reyes2, José Luis Contreras-Jiménez3, María del Carmen Villegas1, and José-Antonio Munive2 1Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional, Tlaxcala, Tlax. 90700, Mexico; 2Instituto de Ciencias and 3Herbario-Jardín Botánico, Benemérita Universidad Autónoma de Puebla, Puebla, Pue. 72570, Mexico In Mexico, forests used to cover a very important proportion of the country, but increasing deforestation is destroying them. The tropical forest covers the south and south-east of the country, and in the central region, in the high places, like the State of Puebla, temperate forests are dominant. These forests are characterised by year-round high temperatures, abundant moisture, mineral-poor soils, and high vegetal diversity, with a high proportion of legume trees. A main advantage of members of this family is their ability to establish a symbiotic interaction with nitrogen-fixing soil bacteria, named rhizobia. Nitrogen-fixing legumes are very useful to restore fertility in forest-disturbed soils, if these plants are inoculated with rhizobia selected strains, but these studies have never been conducted under Mexico's forests conditions. The objectives are to analyse the genetic diversity of root nodule bacteria from temperate forest legumes in Mexico and to select the most performant strains to be inoculated in nurseries for forest restoration. We have established a collection of 136 strains, from which most of them were capable of nodulating siratro plants, showing a great potential for increasing the development of forest legumes. The most efficient strains for nodulation and nitrogen fixation have been chosen for a nursery nodulation assay to make a collection of the best strains useful as inoculum for the restoration of disturbed forests. Inside these symbiotic bacteria, we found a great diversity, shown by 16S rDNA partial sequencing, ERIC-PCR patterns, and ARDRA. 16S rDNA sequencing analysis showed a most important presence of bacteria belonging to the genus Bradyrhizobium inside these forest species. ERIC-PCR and ARDRA confirmed a great diversitry inside these bacteria nodulating forest legume in the temperate forest of Mexico. This supports that temperate forest legumes investigated here are mainly associated with new Bradyrhizobium genospecies.

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PS9-21 Metabolic diversity of Rhizobium leguminosarum bv. trifolii population isolated from nodules of individual plant

Jerzy Wielbo1, Monika Marek-Kozaczuk1, Andrzej Mazur1, Agnieszka Kubik-Komar2, and Anna Skorupska1 1Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, University of Maria Curie Skłodowska, Lublin, Poland; 2Department of Applied Mathematics, University of Life Sciences, Lublin, Poland The adaptive potential of a bacterium is correlated with its genome complexity. Rhizobia tend to occupy highly complex soil habitats, so their large and multipartite genomes, which encode many "potentially useful" metabolic pathways, might be advantageous, thus enhancing the adaptive potential. Moreover, transition between saprophytic and symbiotic lifestyles further forces rhizobia to retain a large metabolic potential stored in their genomes. The objective of this study was to screen a variety of physiological abilities of R. leguminosarum bv. trifolii populations isolated from root nodules of several individual clover plants. On the basis of the observed diversity of 16-23S rDNA PCR-RFLP profiles, the 129 isolates were grouped into eight clusters. Four groups may be considered as prevalent, because they constituted from 13% to 29% of the strains in the tested population and were found in nodules of more than half of the selected plants, thus indicating a considerable genetic variation. Analyses of the metabolic profiles of strains belonging to different genetic groups using Biolog tests showed a bacterial metabolic diversity, observed even within one genetic group, especially in the acid and amino acid utilisation. Principal component analysis of metabolic traits revealed the existence of obvious differences in utilization of two large groups of substrates: (i) mono-, oligo-, and polysaccharides and (ii) organic acids, amino acids, and modified sugars. Despite the general weak similarity between genetic and metabolic classifications of isolates, a common profile of utilisation of several groups of substrates for some PCR-RFLP classes can be found. For strains belonging to dominant groups, the utilisation of "sugars" and "non-sugars" were balanced; numerous of these strains metabolised sugar substrates well. Moreover, they were able to grow relatively well on different media, suggesting that the most widespread rhizobia in native populations may be metabolically non-specialised, further indicating their genome versatility.

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PS9-22 Cadmium influence on legume-Rhizobium symbioses Viktor Tsyganov1, Alexander Zhernakov1, Olga Kulaeva1, Anna Tsyganova1, Andrei Belimov1, Alexander Zabolotny2, Tamara Budkevich2, Dmitrii Bazhanov3, František Baluška4, Maggie Knox5, Noel Ellis5, and Igor Tikhonovich1 1Laboratory of Molecular and Cellular Biology, All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia; 2Laboratory of Plant Ecology, V.F. Kuprevich Institute of Experimental Botany, and 3Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk, 220072 Belarus; 4Institute of Cellular and Molecular Botany, University of Bonn, 53115 Bonn, Germany; 5Department of Crop Genetics, John Innes Centre, Norwich NR4 7UH, UK Associations of legume plants with rhizobia are very sensitive to stresses. Cadmium is one of the most toxic heavy metals for plants. Surprisingly, studies of Cd influence on legume-Rhizobium symbiosis are very limited. We studied the influence of Cd in combination with Ni, Pb, and Sr in soil cultures. The treatment with heavy metals led to a decrease in fresh shoot biomass, pod number, and seed weight in Lupinus angustifolius, fresh shoot biomass and pod number in Lotus corniculatus, and seed weight in Trifolium pratense. At the same time, the specific nitrogen-fixing activity was decreased by 47% in T. pratense, was not changed in L. angustifolium, and was unexpectedly increased by 78% in L. corniculatus. These results indicate different effects of Cd on legumes with different types of nodules. To discover the molecular-genetic and cellular mechanisms underlying the tolerance of legume-Rhizobium symbioses to Cd, we performed EMS mutagenesis of the Pisum sativum line SGE and screened the mutant SGECdt with increased tolerance to Cd and decreased sensitivity to Cd stress [1]. Confocal microscopy revealed that SGECdt is able to maintain the cellular organization of root apices under Cd concentrations causing serious abnormalities in SGE. Using SSAP analysis, the gene cdt was localised on the pea VI linkage group. We performed a comparative analysis of the Cd influence on nodulation in SGE and SGECdt in hydroponic cultures. It was shown that, in contrast to SGE, the mutant is able to form symbiotic nodules under higher Cd concentrations. Light and electron microscopy revealed the main abnormalities caused by Cd stress in pea nodules. Recently, we have also initiated the analysis of the Cd influence on nodulation in the model legumes Lotus japonicus and Medicago truncatula. This work was supported with RFBR (08-04-01565; 08-04-90051), State Program of Oriented Basic Research of NAS of Belarus (51-30.06.2005), and grant of the President of Russia (5399.2008.4), INTAS (YSF 04-83-3143). [1] Tsyganov et al. (2007). Ann. Bot. 99: 227-237.

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Session 10

Comparative and functional genomics of nitrogen-fixing bacteria

Chaired by Jacques Batut Castanet Tolosan, France


203

Session 10: Comparative and functional genomics of nitrogen-fixing bacteria

S10-1 Minimal set of symbiotic rhizobial genes Christel Schmeisser1, Xavier Perret2, William J. Broughton2, Dagmar Krysziak1, Ruth A. Schmitz3, Sidney Brenner4, Axel W. Strittmatter5, Heiko Liesegang5, and Wolfgang R. Streit1 1Biozentrum Klein-Flottbek, Abteilung für Mikrobiologie und Biotechnologie, Universität Hamburg, 22609 Hamburg, Germany; 2Université de Genève, 1211 Genève, Switzerland; 3Biologiezentrum, Christian-Albrechts-Universität zu Kiel, 24118 Kiel, Germany; 4Institute of Molecular and Cell Biology, Singapore 138673; 5Laboratorium für Genomanalyse der Universität Göttingen, 37077 Göttingen, Germany Complete genome sequences of Azorhizobium caulinodans ORS 571, Bradyrhizobium sp. BTAi1, Bradyrhizobium sp. ORS278, Bradyrhizobium japonicum USDA110, Mesorhizobium loti MAFF303099, Mesorhizobium sp. BNC1, Rhizobium sp. pNGR234, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium medicae WSM419, Sinorhizobium meliloti 1021 as well as the symbiotic island of M. loti R7A symbiosis island are available. These were analysed to find classes of symbiotic genes common to all strains. Here the results will be discussed in relation to evolution of nodulation in legumes.

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S10-2 Genome sequence of the β-Rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia

Claire Amadou1, Géraldine Pascal1, Sophie Mangenot2, Michelle Glew1, Cyril Bontemps1, Delphine Capela1, Sébastien Carrère1, Stéphane Cruveiller5, Carole Dossat2, Aurélie Lajus5, Marta Marchetti1, Véréna Poinsot3, Zoé Rouy5, Bertrand Servin4, Maged Saad1, Chantal Schenowitz2, Valérie Barbe2, Jacques Batut1, Claudine Médigue5, and Catherine Masson-Boivin1 1Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France; 2Commissariat à l'Energie Atomique/Institut de Génomique/Génoscope, Centre National de la Recherche Scientifique, UMR8030, 91057 Evry Cedex, France; 3Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique, Université Paul Sabatier, 31062 Toulouse Cedex. France; 4Laboratoire de Génétique Cellulaire, Institut National de la Recherche Agronomique/Ecole Nationale Vétérinaire, 31326 Castanet-Tolosan Cedex, France; 5Atelier de Génomique Comparative, Centre National de la Recherche Scientifique, UMR8030, 91057 Evry Cedex, France The first complete genome sequence of a β-proteobacterial nitrogen-fixing symbiont of legumes, Cupriavidus taiwanensis LMG19424 has been determined. The genome consists of two chromosomes of size 3.42 Mb and 2.50 Mb, and a large symbiotic plasmid of 0.56 Mb. The C. taiwanensis genome displays an unexpected high similarity with the genome of the saprophytic bacterium C. eutrophus H16, despite being 0.94 Mb smaller. Both organisms harbour two chromosomes with large regions of synteny interspersed by specific regions. By contrast, the two species host highly divergent plasmids with the consequence that C. taiwanensis is symbiotically proficient and less metabolically versatile. Altogether specific regions in C. taiwanensis compared to C. eutrophus cover 1.02 Mb and are enriched in genes associated with symbiosis or virulence in other bacteria. C. taiwanensis reveals characteristics of a minimal Rhizobium, including the most compact (35 kb) symbiotic island (nod and nif) identified so far in any Rhizobium. The atypical phylogenetic position of C. taiwanensis allowed insightful comparative genomics of all available rhizobia genomes. We did not find any gene that was both common and specific to all rhizobia, thus suggesting that a unique shared genetic strategy does not support symbiosis of rhizobia with legumes. Instead, phylodistribution analysis of more than 200 Sinorhizobium meliloti known symbiotic genes indicated large and complex variations of their occurrence in rhizobia and non-rhizobia. This led us to devise an in silico method to extract genes preferentially associated with rhizobia. We discuss how the novel genes we have identified may contribute to symbiotic adaptation.

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S10-3 Genome structure and symbiosis-related genes of the versatile nitrogen-fixing Azorhizobium caulinodans

Hiroshi Oyaizu Biotechnology Research Center, University of Tokyo, Tokyo 113-8657, Japan The whole genome sequence was determined for Azorhizobium caulinodans ORS571. This bacterium has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The 5.37-Mb genome consists of a single circular chromosome with average GC of 67% and numerous islands with varying GC contents. The genome potentially encodes 4717 proteins of which 96.3% have homologues and 3.7% are unique for A. caulinodans. The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants. This study was carried out under collaboration, among which Biotechnology Research Center (University of Tokyo), Center for Information Biology and DNA Data Bank of Japan, Department of Plant Systems Biology (Flanders Institute for Biotechnology, Gent), Department of Molecular Genetics (Ghent University), Kazusa DNA Research Institute (Japan), Department of Chemistry and Biochemistry (University of Oklahoma), Center for Biological Sequence Analysis (Technical University of Denmark), and Department of Civil and Environmental Engineering (Massachusetts Institute of Technology).

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S10-4 Transcriptomics and functional genomics to study the Bradyrhizobium japonicum-soybean symbiosis

Hans-Martin Fischer, Marion Koch, Andrea Lindemann, Socorro Mesa, Gabriella Pessi, Luzia Reutimann, and Hauke Hennecke Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zürich, 8092 Zürich, Switzerland Symbiotic nitrogen fixation by Bradyrhizobium japonicum endosymbiotic bacteroids living within root nodules of soybean host plants is controlled at multiple levels via a complex genetic network. Two linked regulatory cascades consisting of transcriptional regulators (FixLJ-FixK2 and RegSR-NifA) form the core of the network. Both cascades are activated by distinct low levels of environmental oxygen. Together they control transcription of large sets of genes that are directly involved in nitrogen fixation or encode other cellular functions associated with the microoxic life style in nodules. While in the past individual target genes of both cascades were identified predominantly with classical genetic tools, we have exploited more recently the availability of the B. japonicum genome sequence and a whole-genome microarray to study specific regulons at the genome-wide level. This strategy combined with biochemical and bioinformatics tools led to the comprehensive and expanded definition of the FixJ, FixK2, RegR and NifA regulons [1, 2; unpublished data]. Notably, not only many new (direct and indirect) target genes of individual regulators were found, but also a previously unrecognized regulatory link between the two cascades and a novel role of FixK2 in the response of B. japonicum to oxidative stress. In addition, comparative microarray experiments with bacteroids and free-living cells revealed that many (but not all) of the genes induced in bacteroids are those that are up-regulated in oxygen-limited cultures [3]. Both classes have the potential to comprise genes encoding novel symbiotic functions. Accordingly, a number of candidate genes are currently under further functional analysis. Examples of newly identified genes that are required for efficient symbiosis include a novel NifA-regulated ferredoxin gene (fdxN) [1] and RegR-controlled genes encoding an RND-type multidrug efflux system. Thus, the combination of transcriptomics with functional genomics is a promising approach to obtain new insights into the biology of symbiotic nitrogen fixation. [1] Hauser et al. (2007). Mol. Genet. Genomics 278:255-271. [2] Lindemann et al. (2007). J. Bacteriol. 189:8928-8943. [3] Pessi et al. (2007) Mol. Plant-Microbe Interact. 20:1353-1363.

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PS10-1 A novel mechanism of quorum sensing gene regulation in Rhizobium leguminosarum

Marij Frederix, Anne Edwards, and J. Allan Downie Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Rhizobia regulate their gene expression in a population density-dependent manner by a mechanism called quorum sensing (QS). Small signalling molecules (acyl homoserine lactones [AHLs]) are synthesised at a low level by a LuxI-type AHL synthase, until a certain threshold ("quorum") is reached, causing the activation of a LuxR-type transcriptional regulator. In Rhizobium leguminosarum bv. viciae 3841, there are two QS systems that influence the symbiotic interaction with legumes. The cin QS system acts as a master regulator, controlling the rhi QS system through the LuxR-type transcriptional regulator rhiR. The rhi QS system controls the expression of the rhi genes, which have been shown to influence legume infection. In addition to this, the cin QS system also regulates the expression of genes involved in exopolysaccharide biosynthesis and biofilm formation. One of these genes is plyB, which encodes an extracellular glycanase. Although mutation of cinI decreases rhiR and plyB expression, addition of CinI-made AHLs exogenously is not essential for activating these genes. In trying to understand the molecular mechanism behind the AHL-independent gene regulation by the cin QS system, we have identified a novel gene, cinS.

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Session 10

Comparative and functional genomics of nitrogen-fixing bacteria

Chaired by Jacques Batut Castanet Tolosan, France


209


PS10-2 Functional analysis of a second type-III secretion system in Rhizobium sp. NGR234

Nadia Bakkou, and Xavier Perret Microbiology Unit, Plant Biology Department, University of Geneva, 1211 Genève 4, Switzerland Type-III secretion systems (T3SS) are used by Gram-negative bacteria to deliver into the cytoplasm of eukaryotic cells various effector proteins, which subvert host defences. Known to be important determinants of pathogenicity, T3SS were also found to play a role in symbiotic interactions between rhizobia and legumes. In contrast to the secreted effectors, most of the membrane-associated proteins that constitute the secretion apparatus are highly conserved and are called Rhc in rhizobia. The genome of Rhizobium sp. NGR234 codes for two complete T3SS. Genes for T3SS-I, which are carried by the symbiotic plasmid pNGR234a, are under the transcriptional control of the NodD1-SyrM2-NodD2-TtsI and flavonoid-dependent regulatory cascade that also activates most of the nodulation genes in NGR234. Depending on the legume host, the nodulation outer proteins (NOPs) secreted by the T3SS-I modulate the ability of NGR234 to form functional symbiotic associations. Recently, the analysis of the 2.43- Mb plasmid (pNGR234b) revealed a second gene cluster (T3SS-II) predicted to encode all of the nine conserved Rhc proteins. Homology searches showed that Rhc proteins of the T3SS-II matched components of a second and cryptic T3SS encoded by the chromosome of Pseudomonas syringae pv. phaseolicola strain 1448A. Analysis by RT-PCR showed that, unlike the genes of the symbiotically active T3SS-I locus, the transcriptional activity of the promoter that controls the major operon of the T3SS-II cluster is weak and independent of flavonoids and/or the presence of a functional TtsI. In order to assess the activity of the T3SS-II locus and its possible role in symbiosis, strains which lack either one (NGR∆T3SS-II or NGRΩrhcN) or both of the secretion systems (NGR∆rhcNΩT3SS-II) were compared for protein secretion, membrane proteins, and efficiency of nodulation and nitrogen fixation on a number of legume hosts. So far, none of the assays confirmed that the T3SS-II is functional and/or plays a significant role in symbiotic associations with legume hosts. Although these results indicate that this locus is probably not directly involved in symbiosis, Southern and PCR analyses confirmed that T3SS-II genes are conserved in a number of Sinorhizobium fredii strains related to NGR234.

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PS10-3 HasA-HasR, a putative second heme-acquisition system mediated by hemophores in Sinorhizobium meliloti 1021

Federico Battistoni, V. Amarelle, and Elena R. Fabiano Laboratorio de Ecología Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Universitad de la República, Montevideo 11600, Uruguay When iron is scarce, some bacteria possess highly efficient heme uptake mechanisms that provide bacteria with nutritional iron. Moreover, heme has been considered as a major iron source for bacteria. One of the strategies used by Gram-negative bacteria for heme acquisition relies on the secretion of heme-carrier proteins, known as hemophores. These proteins bind free heme or heme bound to hemoproteins and deliver it to specific outer membrane TonB-dependent receptors, which, in turn, release it to the bacterial periplasm [1]. The first hemophore to be discovered was HasA from Serratia marcescens. In this bacterium, heme bound to HasA is recognized and internalised by the HasR outer membrane receptor [1]. Rhizobia can use heme, hemoglobin, and leghemoglobin as sole iron sources [2]. In Sinorhizobium meliloti 242, an iron-regulated heme-binding outer membrane protein (ShmR) was identified and characterised [3]. In silico analysis of the S. meliloti 1021 genome reveals the presence of two putative TonB-dependent heme receptors: ShmR and Smc04205. Smc04205 is homologous to HasR from S. marcescens (HasRmar) and, accordingly, we renamed it as HasRmel. Bioinformatics studies demonstrated that HasRmel belongs to an operon. Moreover, a putative regulatory operon that comprises an anti-σ factor (HasSmel) and an extracytoplasmic function σ factor (HasImel) is present uspsteam of HasRmel. The aim of the present work is to determine the function of HasRmel. We could not detect HasRmel expression in the conditions assayed. Taking into consideration that one possibility is that the conditions required for HasRmel expression are different from the ones we tested andthat the conditions are actually unknown, we decided to construct a knock-out mutant in the putative repressor of the HasRmel expression: the HasImel anti-σ factor. Bioinformatics and biochemical studies will be presented. [1] Wandersman & Delepelaire (2004). Annu. Rev. Microbiol. 58:611-647. [2] Noya et al. (1997). J. Bacteriol. 179:3076-3078. [3] Battistoni et al. (2002). Appl. Environ. Microbiol. 68:5877-5881.

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PS10-4 Effect of salt stress in the growth of chickpea mesorhizobia Clarisse Cordeiro Brígido, and Solange Oliveira Departamento de Biologia, Instituto de Ciências Agrárias Mediterrânicas, Universidade de Évora, 7002-554 Évora, Portugal Production of grain legumes is severely reduced in salt-affected soils. Chickpea, a major food legume and important source of protein in many countries, is highly sensitive to salinity. The isolation and characterisation of rhizobial strains tolerant to saline stress conditions may contribute to better understand their behaviour as a community in soils and lead to a more efficient symbiosis with the plant in unfavourable soils [1]. Depending on the species, mesorhizobia can tolerate 1-2% NaCl. The objective of the present study was to evaluate the tolerance to salt stress of Portuguese chickpea rhizobia, isolated in a national survey, as well as to investigate the changes in their protein expression induced by salinity. Ninety-seven chickpea rhizobia isolates, close to several Mesorhizobium species, as indicated by the 16S rRNA gene-based phylogeny, were evaluated. Tolerance of isolates to salt stress was evaluated by quantification of bacterial growth in YMB medium with 1.5% and 3% NaCl. Comparing to the growth at control conditions, 12 isolates grew above 25% at 1.5% NaCl, whereas 54 isolates could grow between 10 to 25% at the same salt concentration. However, under a higher salt concentration (3% NaCl), only five isolates grew above 10%. Interestingly, some isolates showed an identical growth at both NaCl concentrations. Plasmid profile analysis of the 97 isolates revealed plasmid numbers between 0 and 6. Total protein analysis by SDS-PAGE revealed the overproduction of a 15-kDa protein under salt stress (1.5% NaCl) in some tolerant isolates. Additional studies are in course to identify genes involved in tolerance to salt stress. These results show a diversity of responses to salt stress by rhizobia, and suggest that selection of adapted strains to stress conditions can contribute to the development of inoculants for successful chickpea growth. [1] Laranjo & Oliveira (2006). In Microbial Biotechnology in Agriculture and Aquaculture, Vol II, R.C. Ray (Ed.). Enfield,

Science Publisher, pp. 225-260.

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PS10-5 Characterisation of a novel acyl carrier protein SMc01553 from Sinorhizobium meliloti 1021

Yadira Dávila-Martínez, Ana Laura Ramos-Vega, Sandra Contreras, Sergio Encarnación, Otto Geiger, and Isabel M. López-Lara Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, Mexico Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. ACPs are small acidic proteins that contain a 4'-phosphopantetheine prosthetic group. This group is transferred from coenzyme A (CoA) to the ACP by holo-ACP-synthase (AcpS). Beside the four different ACPs known in rhizobia [1], the genome of Sinorhizobium meliloti 1021 reveals two additional open reading frames (ORFs) that possibly codify for new ACPs [2, 3]. One of these ORFs is codified from the gene smc01553. Interestingly, smc01553 is in an 6.5-kb segment of DNA that is the result of a very recent event of gene transfer and appears to have been duplicated between the chromosomal DNA and the pSymB megaplasmid [2, 3]. SMc01553 overexpressed in Escherichia coli can be labelled in vivo with [3H]-β-alanine, a biosynthetic building block of 4'-phosphopantetheine. SMc01553 and His-SMc01553 have been purified from E. coli cultures and incubated with CoA and S. meliloti His-AcpS. The mass spectrometric analysis of the reaction products confirmed the in vitro 4'-phosphopantetheine incorporation. Since malonyl-ACP is the loading unit in fatty acid and some polyketide syntheses, the capacity of malonylation of SMc01553 is being compared with that of other rhizobial ACPs. Although experiments of RT-PCR show expression of smc01553, polycloclonal antibodies created against His-SMc01553 could not detect the protein, indicating a low abundance in the conditions tested (less than 0.02% of the total protein). We have created a deletion mutant that lacks both of the 6.5-kb duplicated DNA regions and no phenotypes in free-living or symbiotic conditions were associated with the mutant. The gene smc01553 is predicted to form an operon with smc01554. Currently, we are testing if SMc01554 is involved in loading the novel ACP SMc01553. [1] López-Lara & Geiger (2002). FEMS Microbiol. Lett. 208:153-162. [2] Finan et al. (2001). Proc. Natl. Acad. Sci. USA 98:9889-9894. [3] Galibert et al. (2001). Science 293:668-672.

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PS10-6 Transcriptional responses of Bradyrhizobium japonicum to environmental changes

Kathrin Lang1, Susanne Zehner1, Tobias Günther1, Felix Hauser2, Andrea Lindemann2, and Michael Göttfert1 1Institute of Genetics, Technische Universität Dresden, 01069 Dresden, Germany; 2Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zürich, 8092 Zürich, Switzerland In the rhizosphere, rhizobia compete with other bacteria for resources. They also have to withstand changing environmental conditions. Rhizobial strains differ in their ability to cope with extreme pH values, high salt concentrations, or an elevated temperature. We are studying the effect of these parameters on the transcriptome of Bradyrhizobium japonicum, the symbiont of soybean. After heat shock for 15 minutes at 43°C, more than 150 genes were up-regulated. This included the already known heat shock genes. Approximately 250 genes were down-regulated. The pH of the growth medium strongly influenced the expression pattern. After incubation for 4 hours at pH 8.0, a large number of genes were differentially expressed (fold change ≥2) if compared to data acquired at pH 6.9. In addition to the mentioned parameters, the transcriptome is also influenced by flavonoids released by the plants. Genistein, a soybean signal, activates the LysR-type regulator NodD1 and the two-component regulatory system NodVW. Both systems are required for efficient expression of nodulation genes. Within the wild type, approximately 100 genes are inducible by genistein. The majority of these genes is not preceded by known promoter elements. In contrast to nodD1, nodW is essential for induction of most of the genes. Seven of the nodW-independent genes seem to be involved in transport processes; the product of the eighth gene contains a patatin-like domain. Our data indicate that genistein has a much broader function than mere induction of nod genes and so far uncharacterised regulators are involved in genistein-dependent responses.

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PS10-7 A new regulator involved in symbiosis in Bradyrhizobium japonicum USDA110

Benjamin Gourion, P. Stiefel, Sylvia Balsiger, Julia Vorholt, Hauke Hennecke, and Hans-Martin Fischer Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zürich, 8092 Zürich, Switzerland The Bradyrhizobium japonicum signalization/regulation cascades leading to the infection of its host plant, to the development of bacteroids, and to nitrogen fixation are subject to extensive work. As a result, numerous genes required for the symbiotic lifestyle are now characterised. Recently, by a targeted approach, a new bacterial regulator has been identified in the strain B. japonicum USDA110spc4 as being involved in symbiosis. A mutant strain carrying an insertion in the corresponding gene is less efficient in infection. Compared to the wild type, it induces the formation of fewer nodules, and the nitrogen fixation activity is diminished based on the acetylene reduction assay. These effects are more or less severe, depending on the host plant species. It is now our aim to determine the reason of the observed phenotype. To this end, we plan to investigate the regulon of this regulator in vitro and in planta. The identification of its targets could lead to the discovery of new bacterial traits involved in symbiosis.

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PS10-8 Sinorhizobium meliloti 1021 mutations that link nitrogen and osmotic stress responses

Uriel Koziol1, Svetlana N. Yurgel2, Michael L. Kahn2, and Francisco Noya1 1Laboratorio de Ecología Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay; 2Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA Sinorhizobium meliloti 1021 shows a marked halotolerance when compared to other rhizobia. After exposure to osmotic stress, S. meliloti responds by accumulating glutamate, which is also a key intermediate of ammonium assimilation. The mechanism underlying this accumulation has not yet been elucidated. During a screen for Tn5-induced mutants sensitive to sodium citrate, we identified one mutant, 4D6, which showed increased sensitivity to sodium citrate and also to sodium chloride. The Tn5 insertion in 4D6 was in ntrY, which encodes a sensor protein that, together with the downstream response regulatory protein NtrX, previously have been described as being involved in the response to nitrogen and osmotic stresses in other species. The mutant has a normal nodulation and fixation phenotype when tested in alfalfa plants. As expected, when the expression of the whole operon ntrYX was reconstituted, halotolerance was restored. However, deleting ntrX, or the adjacent ntrB or ntrC genes, which are involved in the response to nitrogen stress, did not fully establish the halosensitivity found in 4D6. Furthermore, expression of ntrY alone was not enough to restore halotolerance in 4D6. Taken together, these results suggest that gene(s) downstream of ntrYX might be affected in 4D6. We are currently investigating the role of other genes belonging to the ntrYX operon in halotolerance. The results contained in this work show, for the first time, a link between the response to salts and the response to nitrogen stress in S. meliloti. In natural conditions, these two stresses may overlap in time and location in the rhizosphere.

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PS10-9 Sinorhizobium meliloti genome re-annotation, gene regulation networks, and gene co-transcription data integrated into the RhizoGate portal

Elizaveta Krol1, Jan Baumbach3, Jochen Blom2, Burkhard Linke2, Kai Runte2, Danijel Wetter2, Delphine Capela4, Melanie Barnett5, Alexander Goesmann2, and Anke Becker1 1Institute for Biology III, Universität Freiburg, 79104 Freiburg, Germany; 2Bioinformatics Resource Facility, Centrum for Biotechnology, and 3Genome Informatics, Faculty of Technology, Bielefeld University, 33501 Bielefeld, Germany; 4Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France; 5Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA Sinorhizobium meliloti is extensively studied as a model microorganism for molecular genetics of plant-microbe interactions. The genome was sequenced and annotated in 2001. Over the last years, a large amount of S. meliloti transcriptome and proteome data became available, new tools for gene prediction were established, and genomes of related bacteria were sequenced. This constitutes an archive of data, which paved the way for re-annotation of the genome, new postgenomic strategies, and integration of the biological data obtained in high-throughput experiments into one single portal. We have coordinated the re-annotation of the genome conducted by the S. meliloti consortium improving the annotation and EC number assignment. The RhizoGate portal was established that integrates the recent genome annotation database GenDB and the transcriptome database EMMA (http://www.cebitec.uni-bielefeld.de/brf/software/brfsoftware.html). In order to visualise, classify, and reconstruct the regulatory interaction networks in S. meliloti, the interactive platform SmRegNet is currently being established that will be connected to the GenDB and EMMA databases. Presently, the SmRegNet database contains 133 experimentally confirmed single regulatory interactions that involve 330 target genes and 61 regulatory proteins, and 17 regulons that comprise 1109 target genes. We also conducted an experimental study using a high-throughput approach to analyse potential co-transcribed genes in the S. meliloti genome identified by bioinformatics methods and confirmed co-transcription in 75 gene groups consisting of 225 genes. These data, combined with already published data on co-transcription in S. meliloti, will be applied for generation and training of a new operon prediction tool, which will be also available on the RhizoGate portal. A data warehouse based on IGetDB (http://www.cebitec.uni-bielefeld.de/groups/brf/software/wiki/IGetDBWiki) for queries of data from different resources is an integrative part of the portal.

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PS10-10 The complete genome sequence of Rhizobium sp. NGR234: more secretion systems equates with more legumes nodulated?

Dagmar Krysciak1, Axel Strittmatter2, Heiko Liesegang2, Christel Schmeisser1, Ruth A. Schmitz3, Gerhard Gottschalk2, William J. Broughton2, Xavier Perret4, and Wolfgang R. Streit1 1Biozentrum Klein-Flottbek, Abteilung für Mikrobiologie und Biotechnologie, Universität Hamburg, 22609 Hamburg, Germany; 2Laboratorium für Genomanalyse der Universität Göttingen, 37077 Göttingen, Germany; 3Biologiezentrum, Christian Albrechts Universität zu Kiel, 24118 Kiel, Germany; 4Laboratoire de Biologie Moléculaire des Plantes Supérieures, Université de Genève, 1211 Genève, Switzerland Rhizobium sp. NGR234 occupies a special niche in the Rhizobiales -it forms symbiotic, nitrogen-fixing associations with more legumes than any other rhizobia. In contrast, Sinorhizobium meliloti, one of NGR234's closest relatives, has a very restricted host range. Both bacteria possess a "symbiotic" plasmid that carries most genes necessary for the interaction with plants, extremely large second plasmids (2,430 Mb in NGR234 (pNGR234b) vs. 1,688 Mb in S. meliloti (pSymB), respectively) as well as chromosomes of similar sizes. We have now established the complete nucleotide sequence of pNGR234b and the NGR234 chromosome (NGR234c). Comparisons of the two mega-plasmids pNGR234b vs. pSymB reveal that pNGR234b carries many loci involved in protein secretion as well as a vast family of sRNAs. A second type-III secretion system, a type-IV secretion system as well as two type-IV pili are also present. Since these are main differences between the two genomes, it seems likely their regulation in a novel manner largely explains variations in host range.

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PS10-11 Tolerance of Mesorhizobium type strains to thermal and osmotic stress

Marta Sofia Laranjo, and Solange Oliveira Instituto de Ciências Agrárias Mediterrânicas, Departamento de Biologia, Universidade de Évora, 7002-554 Évora, Portugal One of the most widespread problems facing agriculture is the degradation of the soil quality due to desiccation and salinity. Environmental factors, such as temperature and soil salinity, reduce survival and growth of rhizobia in the soil and inhibit rhizobia-legume symbiosis [1], resulting in lower legume productivity [2]. In order to obtain symbiotically efficient inoculant strains, tolerant to environmental stress conditions, it is important to study the genes involved in stress response. The genus Mesorhizobium comprises 12 species (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. plurifarium, M. septentrionale, M. temperatum, M. thiogangeticum, and M. tianshanense) that nodulate a wide variety of host legumes, such as chickpea, a legume with high agronomic importance. The optimum growth temperature described for mesorhizobia is 25-30°C [3]. Regarding osmotic stress, previous studies indicate that mesorhizobia can tolerate 1-2% NaCl, depending on the species [3]. The aim of the present study was to evaluate the tolerance to thermal and osmotic stresses of Mesorhizobium-type strains. Tolerance to continuous cold (15°C) and heat (37°C) stresses, as well as tolerance to heat shock (48°C), was evaluated. Tolerance to osmotic stress (1.5 and 3% NaCl) was also evaluated. Most mesorhizobia strains revealed a higher tolerance to cold than to heat stress. M albiziae and M. plurifarium showed tolerance to heat shock. M. plurifarium revealed tolerance to 1.5% NaCl. M. thiogangeticum showed higher growth with 1.5% NaCl. SDS-PAGE analysis of total proteins revealed at least five overexpressed proteins between 40-85 kDa under osmotic stress. The overexpression of a 60-kDa protein, probably GroEL, was detected under heat stress. Differences in groEL mRNA levels were investigated by RNA gel blot analysis using sensitive and tolerant strains. Further studies are in course to elucidate the function of overproduced proteins in thermal and osmotic stress tolerance. Funding: FCT-fellowship to M.L. (SFRH/BPD/27008/2006). [1] Zahran, H.H. (1999). Microbiol. Mol. Biol. Rev. 63:968-989. [2] Miller & Wood (1996), Annu. Rev. Microbiol. 50:101-136. [3] Chen et al. (2005) In Bergeys Manual of Systematic Bacteriology, 2nd ed., D.J. Brenner, N.R. Krig, and J.T. Staley (Eds.).

New York, Springer, pp. 403-408.

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PS10-12 Characterisation of the RpoH1 σ factor of Sinorhizobium meliloti under pH stress

Daniella K. Cavalcanti de Lucena, Alfred Pühler, and Stefan Weidner Department of Genetics, Faculty of Biology, University of Bielefeld, 33501 Bielefeld, Germany Transcription initiation is the major step in the regulation of gene expression in prokaryotes. Although transcription is effected by the core RNA polymerase, an interchangeable σ factor is responsible for promoter recognition, thus directing the holoenzyme to specific genes. Σ factors are therefore indispensable for transcription initiation, for they enable the specific binding of RNA polymerase to gene promoters. Different σ factors are activated in response to different environmental conditions and the number of σ factors varies considerably between bacterial species. Alternative σ factors are implicated in the transcription of specific regulons associated with environmental or physiological changes. There are 14 genes that code for potentially functional σ factors in the genome of Sinorhizobium meliloti strain 1021. In this work, pH stress conditions were assessed for the differential expression of a number of S. meliloti σ factors. The role of the heat shock σ factor RpoH1 was primarily analysed through the confection of a knockout mutant, followed by transcription profiling experiments. Moreover, time-course microarray analyses were performed with the intent of identifying the genetic circuits under RpoH1 regulation in pH stress conditions. Research on gene expression regulation by S. meliloti σ factors will help in achieving a better understanding on the role of alternative σ factors and the identification of distinct σ-regulated genes involved in stress response.

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PS10-13 The extracytoplasmic σ factor RpoE4 is involved on oxidative and osmotic stress response in Rhizobium etli

Jaime M. Martínez-Salazar1, Miguel A. Ramírez-Romero2, Sergio Encarnación3, Emmanuel Salazar3, and Javier Rivera1 1Programa de Ingeniería Genómica, 2Programa de Genómica Evolutiva, and 3Programa Genómica Funcional, Centro de Ciencias Genómicas, Cuernavaca, Morelos 62271, Mexico The free-living soil rhizobia in the rhizosphere or as a nitrogen-fixing bacteroid, are continuously challenged by changes on nutrient availability and by different conditions that cause starvation, heat, salt, and osmotic and oxidative stresses. Several strategies to adapt to different conditions have been found. One of them is the gene expression controlled at the transcriptional level by σ factors. The σ factor binds to the core RNA polymerase and recognise specific promoters. Genome analysis of Rhizobium etli reveals 23 σ factors: one σ70 (housekeeping), two σ54 (symbiosis), two σ32 (heat and oxidative stress), and 18 extracytoplasmic (ECF) σ factors, four of them are σE-like (oxidative stress). In order to gain insights about the role of ECF σ factors in R. etli, transcriptional fusions (ECF-σ-uidA) as well as mutants were constructed. Phenotypic analysis of mutants shows that rpoE1, rpoE2, and rpoE3 were slightly sensitive to Paraquat (oxidative stress), while rpoE4 was sensitive to oxidative (H2O2 and Paraquat) and osmotic (NaCl and sucrose) stresses. The others mutants did not show a clear phenotype. The σ-uidA activity shows that 16 ECF σ factors were expressed at different levels in root nodules, in aerobic and microaerobic conditions. To identify the rpoE4-regulated genes, we compare the global expression of the rpoE4::Sp mutant and rpoE4 overexpression profiles with those of the parental strain in microarray experiments. We identified approximately 47 genes under the control of RpoE4, including xth1, tcrX, and rpoH2, which encode proteins with a possible role in stress response (exonuclease III, transcriptional regulator, and heat shock σ factor, respectively), also a possible Mn catalase and several membrane proteins. Partial financial support was provided by grant IN224002 and IN201006 from PAPIIT-DGAPA, UNAM.

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PS10-14 ISSfr2, a new insertion sequence isolated from the region upstream of the nodA in Ensifer fredii HH103

Carmen García Trigueros, Manuel Megías, and Hamid Manyani Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain Sequence analysis of the DNA region upstream of the nodA nod box of Ensifer fredii HH103 identified a 4376-bp fragment that presents a clearly different genetic arrangement from homologous regions in the related strains E. fredii USDA257 and Rhizobium sp. NGR234. BLAST analysis of this region showed the presence of three open reading frames and a 1543-bp DNA fragment that has never been described before in E. fredii and other related strains. This new DNA region contains all the typical features of an insertion element belonging to the IS1111 family of insertion sequences [1]. This IS contains a single 1103-bp open reading frame encoding a transposase homologous to the IS110/IS116 and IS1111 family transposases. Based on these data and according to the ISfinder database directions for new name attributions, we have named this new element, ISSfr2. The insertion sequence ISSfr2 was not found in the region upstream of the nodA gene in E. fredii USDA257 even though E. fredii HH103 is phylogenetically closely related to the former, suggesting that the ISSfr2 insertion upstream of the nodA gene in E. fredii HH103 has occurred recently. Therefore, we attempted to evaluate the prevalence of the ISSfr2 insertion upstream of the nodA gene among soybean-nodulating Chinese strains, by using different approaches. PCR-based strategies and hybridisation assays were performed with genomic DNA from 24 fast-growing soybean symbionts isolated from four different regions of China [2]. From these analyses, we have found that the presence or absence of ISSfr2 in fast-growing soybean-nodulating rhizobia isolated from Chinese soils is not related to any geographical origin or specific phenotypic characteristic. No experimental evidence of ISSfr2 transposition is available at present, although in vitro transposition assays of ISSfr2 are being performed. [1] Mahillon & Chandler (1998). Microbiol. Mol. Biol. Rev. 62:725-774. [2] Thomas-Oates et al. (2003). Syst. Appl. Microbiol. 26:453-465.

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PS10-15 New insights into the regulation mediated by the FixLJ-FixK2-FixK1 cascade in Bradyrhizobium japonicum

Socorro Mesa1, Felix Hauser1, Markus Friberg2, Emmanuelle Malaguti1, Hans-Martin Fischer1, and Hauke Hennecke1 Institutes of 1Microbiology and 2Computational Science, Swiss Federal Institute of Technology, ETH-Zürich, 8092 Zürich, Switzerland Symbiotic N2 fixation in Bradyrhizobium japonicum is controlled by a complex transcription factor network which is composed of two linked regulatory cascades (RegSR-NifA and FixLJ-FixK2) [1]. In the FixLJ-FixK2 cascade, the FixK2 protein plays a key role as distributor of the "low-oxygen" signal that is perceived and transduced by the superimposed two-component regulatory system FixLJ [2]. FixK2 activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQP, fixGHIS), but also further regulatory genes (rpoN1, nnrR, and fixK1), thus expanding the downstream end of the cascade. Transcriptome analyses performed have led to a comprehensive and expanded definition of the FixJ, FixK2, and FixK1 regulons, which consist of 26, 204, and 29 genes, respectively, specifically regulated in microaerobically grown cells. Particular attention was addressed to the FixK2-dependent genes, which included a bioinformatics search for putative FixK2-binding sites on DNA (FixK2 boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified FixK2 as the activator [3], we validated as direct targets eight new genes. Interestingly, the adjacent, but divergently oriented fixK1 and cycS genes shared the same FixK2 box for activated transcription into both directions. This recognition site may also be a direct target for the FixK1 protein, because activation of the cycS promoter required an intact fixK1 gene and either microoxic or anoxic, denitrifying conditions. Two other, unexpected results emerged from this study: (i) specifically FixK1 seemed to exert a negative control on genes that are normally activated by the N2 fixation-specific transcription factor NifA; (ii) endosymbiotic bacteroids expressed 166 FixK2-activated genes that are not at the same time dependent on FixJ, suggesting a FixK2-mediated regulation, uncoupled from FixJ, which operates in symbiosis. [1] Sciotti et al. (2003). J. Bacteriol. 185:5639-5642. [2] Nellen-Anthamatten et al. (1998). J. Bacteriol. 180:5251-5255. [3] Mesa et al. (2005). J. Bacteriol. 187:3329-3338.

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PS10-16 Structural characterisation of the manganese uptake regulator of Sinorhizobium meliloti 1021 and its interaction with DNA

Raúl R. Platero1, B. Garat2, Beatriz Gomes Guimarães3, and Elena R. Fabiano1 1Laboratorio de Ecología Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Unidad Asociada a Facultad de Ciencias, and 2Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Universitad de la República, Montevideo 11600, Uruguay; 3Laboratório Nacional de Luz Síncrotron, Campinas, SP 13083-970, Brazil Bacterial homeostasis of transition metals is essential for successful interactions with hosts. The Fur (ferric uptake regulator) is the prototype of a family of proteins that sense divalent metals and, subsequently, regulate the expression of genes implicated in its acquisition, transport, and storage. There is a great diversity in metal selectivity within the Fur family, including sensors for iron, zinc, manganese, and nickel. The molecular basis of the biological metal selectivity of these regulators is not understood [1]. A Fur homologue was identified in Sinorhizobium meliloti and shown to mediate the Mn-dependent regulation of the manganese transport operon mntABDC(sitABCD). The protein binds to the promoter region of the mntABCD operon and blocks its transcription. Therefore, this Fur homologue was renamed Mur (Manganese uptake regulator [2]. The MntABCD transport system is expressed by the free-living and symbiotic forms of rhizobia [3]. The aim of the present work was to investigate structural changes in the Mur protein upon DNA binding. Near and far UV Circular Dichroism (CD), Dynamic Light Scattering (DLS), and Size Exclusion Chromatography (SEC) analysis were carried out. As observed by DLS and SEC, the presence of divalent metals and reducing agents had a great influence in the oligomerisation state of the protein. In the presence of the target DNA, we observed the formation of various Mur-DNA complexes. Stochiometry of Mur-DNA complexes were strongly influenced by environmental conditions. However, after DNA addition, only slight changes were observed in the Mur CD spectrum, suggesting that DNA binding had no dramatic structural effect on Mur. Crystallographic studies of Mur alone and in complex with its target DNA are being conducted in order to determine the molecular basis of the Mur-DNA interaction. Financial assistance: ANII, DINACYT-FCE, AMSUD-Pasteur (Uruguay), and SBBq (Brasil). RP is a PhD student of the PEDECIBA program, Uruguay. [1] Lee & Helmann (2007). Biometals 20:485-499. [2] Platero et al. (2007). Appl. Environ. Microbiol. 73:4832-4838. [3] Djordjevic et al. (2003). Mol. Plant-Microbe Interact. 6:508-524.

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PS10-17 The Rhizobium etli RpoH1 and RpoH2 σ factors are regulated differentially and are involved in different but overlapping stress conditions

Jaime M. Martínez-Salazar1, Mario Sandoval-Calderón2, Xianwu Guo2, Santiago Castillo-Ramírez2, Alma Reyes2, Maria G. Loza2, Javier Rivera1, Xochitl Alvarado-Affantranger3, Federico Sánchez3, Víctor González2, Guillermo Dávila2, and Miguel A. Ramírez-Romero2 1Programa de Ingeniería Genómica, 2Programa de Genómica Evolutiva, Centro de Ciencias Genómicas and 3Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, Mexico The physiological role and the transcriptional regulation of Rhizobium etli rpoH1 and rpoH2 σ factor genes were studied. Both genes were able to complement the temperature- sensitive phenotype of an Escherichia coli rpoH mutant. The R. etli rpoH1 mutant was sensitive to heat shock, sodium hypochlorite, and hydrogen peroxide, whereas the phenotype of the rpoH2 mutant was indistinguishable from the wild type. The double mutant rpoH2-rpoH1 showed an increased sensitivity to heat shock and oxidative stress compared to the single rpoH1 mutant. This suggests that RpoH1 is the main σ factor for stress, but a complete protective response is achieved with the participation of RpoH2. In symbiosis with bean plants, the R. etli rpoH1 and rpoH2-rpoH1 mutants still elicit nodule formation, but exhibited reduced nitrogenase activity and cell viability. Nodules formed with R. etli rpoH1 and rpoH2-rpoH1 mutants showed premature senescence, compared with the nodules infected with the rpoH2 and wild-type strains. We showed that RpoH1 regulates the expression of nifNd, nifKf, and nifNf genes. Both rpoH genes are induced under microaerobic growth conditions and in stationary phase of growth, but not in response to heat shock. The rpoH genes have different regulatory regions. In rpoH1, there are a σ70 and a probable σ32 or σE promoters. In rpoH2, we detect three promoters, two probably σE dependent and the other without similarity to known binding sites for σ. In conclusion, both RpoH1 and RpoH2 have overlapping functions, but the differences in their transcriptional regulation could lead them to act in specific conditions. This work was supported by CONACyT grants N-028, N-U46333-Q for emergent areas, IN220307 and IN201006 from PAPIIT-DGAPA Universidad Nacional Autónoma de México.

225


PS10-18 Genetic characterisation of potassium transport systems in Sinorhizobium meliloti

Ana Domínguez-Ferreras, Socorro Muñoz, José Olivares, María J. Soto, and Juan Sanjuán Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain The ability of rhizobia to tolerate osmotic stress is related to its survival, persistence, and, therefore, performance in symbiosis with their legume hosts in environmentally constrained soils. Potassium accumulation is one of the most frequent and earliest responses of rhizobia to an osmotic upshift [1], but very little is known about the actual transport systems involved in this process. The mutation of a potassium transport system in Rhizobium tropici leads to decreased osmotolerance and reduced nitrogen fixation efficiency in symbiosis with Phaseolus vulgaris [2]. In silico analysis of the Sinorhizobium meliloti 1021 genome revealed the presence of four putative potassium uptake systems: Trk, Kdp, Kup1, and Kup2. We have addressed the functional characterisation of these systems by constructing the four possible simple mutants and, in some cases, double and triple mutants. The growth abilities of the resulting mutants were determined in media containing different NaCl or sucrose concentrations. The influence of pH and potassium levels in the growth media on the osmotolerance of the mutants was also determined. Our data indicate that both Trk and Kup1 are required for bacterial growth, even in osmotically balanced media. Furthermore, potassium accumulation during adaptation to high NaCl concentrations is mainly triggered by the Trk system. Nevertheless, Kdp is also involved in the osmoadaptation process and its relative importance increases in media containing low potassium concentrations. The relevance of the different potassium transport systems was also studied during the establishment of symbiosis with alfalfa plants. Trk and Kup1 are important for this process and the lack of both systems results in delayed nodulation in non-stressed conditions. Supported by grants BIO2005-08089-C02-01 and BOS2002-04182-C02-01 (DGI-MEC, Spain). ADF was supported by postgraduate fellowships from MEC and CSIC. [1] Zahran, H.H. (1999). Microbiol. Mol. Rev. 63:968-989. [2] Nogales et al. (2002). Mol. Plant-Microbe Interact. 15:225-232.

226


Session 11

New tools to study biological nitrogen

fixation

Chaired by Helge Küster Bielefeld, Germany


227

Session 11: New tools to study biological nitrogen fixation

S11-1 Postgenomic strategies and databases to explore the symbiotic lifestyle of Sinorhizobium meliloti

Anke Becker Institute of Biology III, University of Freiburg, 79104 Freiburg, Germany Complete genome sequences of a number of rhizobia have recently become available and constitute an archive of data that paved the way for postgenomic strategies. Many gene products deduced from the genome sequences have no experimentally proven function or even lack a functional assignment based on similarity measures. Exploitation of genome data requires new strategies that will broaden our knowledge of the genetic basis of the rhizobial lifestyle. Rhizobial functional genomics has entered the "omics" era just after the first genome sequences became available in the year 2000. These approaches comprise high-throughput methods that allow to monitor gene expression at RNA and protein levels in a highly parallel fashion. Such transcriptome and proteome studies have been complemented by metabolome analyses. Apart from profiling methods, several other high-throughput strategies for functional analysis of rhizobial genes were established, including mutagenesis, cloning, and gene fusion strategies. After the first years of development and expedition, these techniques became standard tools in rhizobial research resulting in an enormous quantity of data. A portal for data integration and meta-analysis will be introduced and examples of the application of this platform will be given.

228


S11-2 Monitoring dynamics of transcriptional regulation of Nod factor signalling genes using an improved DsRED-E5 variant

René Geurts, Natalya Saveljeva, Gerben Bijl, Silvester de Nooyer, Stephane De Mita, and Ton Bisseling Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands Key regulatory genes of Nod factor signalling have been identified in different legume species by means of genetics. Under nitrogen-limiting conditions, these genes are expressed in the susceptible zone of the root and the encoded proteins are essential for Nod factor perception and signalling. Perception of Nod factors subsequently leads to transcriptional activation of other symbiotic genes. Based on homology and microsynteny however, it can concluded that putative orthologues of Nod factor signalling (NFS) genes are present in many non-legume plant species. For several of these, it was demonstrated that they -at least partially- can functionally complement corresponding legume mutants. This suggests that, with the exception of the Nod factor receptors, sequence differences in other NFS proteins are the result of genetic drift rather than specific evolution within the legume lineage. In contrast to coding sequences, cis-regulatory elements (CREs) have a higher degree of freedom to change. The spatial expression of virtually every developmental regulatory gene is controlled by sets of modular independent CREs, and changes in one CRE does not affect the function of other CREs. Therefore, changes of a single CRE results generally in minimal pleiotropic effects. We investigated whether in case of legumes changes in NFS gene expression is a prerequisite for symbiosis with Rhizobium. To study this, we used Medicago truncatula and Poplar trichocarpa (poplar). NFS mutants of M. truncatula affected in symbiotic signalling were trans-complemented using promoter constructs of the non-legume P. trichocarpa. Subsequently, the temporal and spatial regulation was studied of these promoter regions that contain legume specific CRE(s), essential for nodulation. To do so, we made use of an improved fluorescent timer reporter construct that consists of a tandem repeated DsRED-E5 tagged with a nuclear localization signal. DsRED-E5 has a prolonged maturation time, by which it has a bright green fluorescence for a period of 24 h, which subsequently shifts to red. By quantifying the ratio between green and red fluorescence dynamics in transcriptional regulation of NFS genes in response to Nod factors is studied.

Session 11

New tools to study biological nitrogen

fixation

Chaired by Helge Küster Bielefeld, Germany


229


PS11-1 Genomic comparison of Bradyrhizobium japonicum strains with different symbiotic N2-fixing capabilities: a QTL approach for N2-fixing efficiency in rhizobia

Manabu Itakura1, Kazuhiko Saeki2, Hirofumi Omori3, Tadashi Yokoyama4, Takakazu Kaneko5, Satoshi Tabata5, Takuji Ohwada6, Shigeyuki Tajima7, Toshiki Uchiumi8, Keina Honnma9, Konosuke Fujita9, Hiroyoshi Iwata10, Yuichi Saeki11, Yoshino Hara1, Seishi Ikeda1, Shima Eda1, Hisayuki Mitsui1, and Kiwamu Minamisawa1 1Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan; 2Department of Biological Sciences, Faculty of Science, Nara Women's University, Nara 850-6503, Japan; 3Department of Biology, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan; 4Tokyo University of Agriculture and Technology, Tokyo 184-8588,Japan; 5Kazusa DNA Research Institute, Chiba 292-0818, Japan; 6Department of Agricultural and Life Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan; 7Department of Life Science, Kagawa University, Takamatsu 760-8521, Japan; 8Department of Chemistry and BioScience, Faculty of Science, Kagoshima University, Kagoshima 890-0065, Japan; 9Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; 10National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan; 11Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan Comparative genomic hybridisation (CGH) was carried out for nine strains of Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium associated with soybeans and other eight members in Bradyrhizobiaceae by DNA macroarray of B. japonicum USDA110. The CGH clearly discriminated genomic variations of B. japonicum strains, but similar CGH patterns were observed in other members in Bradyrhizobiaceae. Most variable regions were 14 genomic islands (GIs) (4 kb-97 kb) and low G+C regions on the USDA110 genome, which were missing in several strains of B. japonicum and other members in Bradyrhizobiaceae. The CGH profiles of B. japonicum were classified into three genome types, namely 110, 122, and 6. DNA sequences around the boundary regions showed that at least seven GIs were missing in genome type 122 as compared with type 110. Strains belonging to the genome types 110, 122, and 6 formed separate clades by phylogenetic analysis for internal transcribed sequences (ITSs). These results demonstrate that GIs were horizontally inserted into the ancestor genome of the type 110 after the divergence of the types 110 and 122 strains. To examine the possible functional relationships of GIs, a multiple regression analysis was conducted using linear models of correlation coefficients between the existence of genomic regions and the parameters regarding symbiotic nitrogen fixation with soybeans. Consequently, variable genomic regions, including GIs were assigned to enhance symbiotic N2 fixation in B. japonicum USDA110. Although the overall gene expression of the variable regions showed low levels, a part of the regions were up-regulated in symbiosis. This strategy is regarded as a rhizobial QTL approach for N2-fixing efficiency. [1] Uchiumi et al. (2004). J. Bacteriol. 186:2439-2448. [2] Wei et al. (2008) DNA Res., in press.

230


PS11-2 Molecular characterization of Tnt1 insertion mutants of Medicago truncatula impaired in symbiotic nitrogen fixation

Catalina Pislariu1, Jeremy Murray1, Jiangqi Wen1, Mingyi Wang1, Xiaofei Cheng1, Shulan Zhang1, Bruce Roe2, Pascal Ratet3, and Michael Udvardi1 1The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA; 2Advanced Center for Genome Technology, University of Oklahoma, Norman, OK 73019-3051, USA; 3Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France Complex interactions between leguminous plants and rhizobia result in the development of nitrogen-fixing root nodules. With a relatively small, diploid genome (haploid size approximately 550 Mbp), Medicago truncatula has emerged as one of the preferred model legumes for genomic and functional genomics studies. Significant progress towards completing the Medicago genome sequence, as well as the development of new genetic tools and resources are contributing to a better understanding of the molecular mechanisms underlying nodule development and symbiotic nitrogen fixation (SNF) in legumes. Tobacco retrotransposon (Tnt1)-tagging is an efficient approach to introduce multiple insertions per plant, for saturation mutagenesis in Medicago [1]. So far, approximately 7800 Tnt1 insertion lines have been generated in the R108 genotype and more than 200 of these appear to be compromised in SNF. To identify Tnt1 insertion sites in mutants of interest, Thermal Asymmetric InterLaced-PCR (TAIL-PCR) has been used to generate approximately 6000 flanking sequence tags (FSTs), of which 3373 are from 207 putative symbiotic mutants. FST analysis has identified insertion alleles of the following known symbiotic genes: NIN, ERN1, SUNN, MtHMGR1, MtSST1, and LYK3. To develop a comprehensive FST database for all Tnt1 insertion lines, we are testing a three-dimensional pooling strategy for deep sequencing using adaptor-ligation PCR for high-throughput 454 sequencing. To complement on-going FST sequencing, we have established a PCR-based approach to identify Tnt1 insertions in genes of interest. Using this approach we identified mutants with Tnt1 insertions in the following symbiotic genes: NIN, NSP1, NSP2, DMI1, DMI3, NFP, and MtSST1. Currently, we are developing a database that contains information about the Tnt1 mutant lines, including photos of different phenotypes and FSTs associated with each line. When released, the database will be a valuable resource for the scientific community, not only for research on SNF, but also for more general functional genomics studies in Medicago. [1] Tadege et al. (2005). Trends Plant Sci. 10:229-235.

231


PS11-3 Direct screening of mutants impaired in arbuscular mycorrhiza development is a new tool for pea (Pisum sativum L.) symbiotic genes identification

Oksana Shtark1, Vladimir Zhukov1, Eugenia Ovchinnikova1, Alexandra Krasheninnikova1, Timofey Nemankin1, Alexey Borisov1, Vivienne Gianinazzi-Pearson2, Noel Ellis3, and Igor Tikhonovich1 1All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia; 2Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique, UMR1088-5184, Plante-Microbe Environnement, Université de Bourgogne, 21065 Dijon Cedex, France; 3John Innes Centre, Norwich NH4 7UH, UK Arbuscular mycorrhiza (AM) is an ancient symbiotic system formed by 80-90% of terrestrial plants and fungi belonging to the phylum Glomeromycota. The symbiosis is beneficial for plants due to improved minerals and water acquisition and can be used in sustainable agriculture. Until recently, the only approach used for identification of legume genes controlling AM development was based on the fact that legumes have a common genetic system as well responsible for nitrogen-fixing symbiosis formation. Particularly, in pea (Pisum sativum L.), eight "common" symbiotic genes were identified [1]. But AM-specific pea genes have not been revealed to date. Only recently, the first three AM-specific legume mutants were identified in Medicago truncatula using a direct screening of mutagenized plant population [2]. In this study, the direct screening of M2 pea population after ethyl methanesulfonate mutagenesis has been initiated to identify the required genes in P. sativum. By using a "nurse plant" inoculation system [3], specially modified for pea plants and the local conditions, the first putative mutants presumably defective in AM development were isolated. Their progeny was involved in tests for stability of the revealed phenotypes and analysis of root nodule formation. The fact that the majority of the analyzed plants impaired in AM formation had normal nodulation phenotypes can indicate that mutation has occurred in the specific gene controlling AM development. One of the putative mutants is probably impaired in a new "common" symbiotic gene for both AM and nodule formation. The genetic analysis of the mutants isolated and justified, which is in progress now, will let to identify new pea genes controlling the development of AM, but not nitrogen-fixing nodules. This work was supported by the grants of RFBR (07-04-01171, 07-04-01558, 07-04-13566, 06-04-89000-NWOC_a), Government of Russian Federation (HIII-5399.2008.4, 02.512.11.2182), Burgundy Administration (07 9201-40 S 3623), NWO (047.018.001).

[1] Borisov et al. (2007). Appl. Biochem. Microbiol. 43:237-243. [2] March et al. (2006). In Abstract book of 3rd International Conference on Legume Genomics and Genetics, Brisbane,

Australia, p. 52. [3] Rosewarne et al. (1997). Mycol. Res. 101:966-970.

232


PS11-4 RIVET analysis reveals symbiotic regulation of quorum sensing genes in Sinorhizobium meliloti

Mengsheng Gao, and Max Teplitski Soil and Water Science Department, Cancer and Genetics Research Complex, University of Florida, Institute of Food and Agricultural Sciences, Gainesville, FL 32610, USA The need for functional analysis of rhizobial genes during symbiosis has led to advances in the development of tools for monitoring expression of these genes in plant tissues. A recombinase-based IVET (RIVET) reporter system was adapted in Sinorhizobium meliloti to track expression of genes during different stages of symbiotic interactions [1]. In addition to visualising changes in gene regulation using GUS staining, RIVET also allows quantitative measurement of gene expression during different stages of interactions with the legume hosts. In proof-of- principle in vitro experiments, the resolution of the nodC-tnpR reporter was detected within 4 hours of exposure to micromolar levels of the nod operon inducer luteolin. In contrast, responses of the nodC::gus reporter fusion required at least 8 hours to reach detectable levels. The nodC RIVET reporter was also resolved after the overnight incubation in the rhizosphere. RIVET assays demonstrated that a cell division gene ftsZ2 was not strongly expressed in the rhizosphere, but was activated inside the nodules and on agar surfaces. Rhizosphere expression of sinI, a gene encoding autoinducer synthase was monitored by the sinI-tnpR-GUS reporter. The resolution of the sinI RIVET reporter was low in the pre-quorate microcolonies, and then increased with time, presumably as colonies reached quorate levels. The sinI and an autoinducer-regulated gene expG were activated inside the nodules. These observations suggest that the tnpR-based RIVET could be used a sensitive tool for documenting rhizobial gene expression in planta. [1] Gao & Teplitski (2008). Mol. Plant-Microbe Interact. 21:162-170.

Session 12

Nodule functioning

Chaired by Eva Kondorosi Gif sur Yvette, France


233

Session 12: Nodule functioning

S12-1 Terminal differentiation of nitrogen-fixing bacteroids induced by antimicrobial plant peptides in Medicago truncatula nodules

Peter Mergaert1, Willem Van de Velde1, Grigor Zehirov2, Hironobu Ishihara2, Agnes Szatmari1, Andrea Nagy3, Benoît Alunni1, Eszter Schuler1, Gergely Maroti3, Attila Kereszt3, Toshiki Uchiumi2, and Eva Kondorosi1,3 1Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France; 2Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, Kagoshima, 890 0065, Japan, 3Institute for Plant Genomics, Human Biotechnology and Bioenergy, Bay Zoltan Foundation For Applied Research, 6726 Szeged, Hungary The nodules on legume roots are colonised with intracellular and nitrogen- fixing rhizobia, called bacteroids, which are differentiated bacteria adapted to symbiotic life. In legumes of the IRLC clade (e.g. Medicago truncatula), bacteroids undergo several morphological and cellular modifications such as membrane permeabilisation and genome endoreduplication, supporting an important cell enlargement. They also lost capacity for growth and are thus terminally differentiated bacteria. Intriguingly, none of these microsymbiont features are observed for rhizobia nodulating legumes outside the IRLC clade (such as Lotus japonicus). Bacteroids of recombinant or natural rhizobia able to nodulate both legume types (IRLC and non-IRLC) are differentiated in a way depending to the host plant, demonstrating that not the bacterial genetic background but plant factors present in IRLC legumes and absent in non-IRLC legumes induce the terminal bacteroid differentiation. Transcriptome analysis in M. truncatula and L. japonicus nodules allowed identification of candidate genes coding for the factors inducing the terminal bacteroid differentiation. Expression of these genes is absolutely restricted to nodules and is associated with bacteroid differentiation. They do not exist in L. japonicus. The genes encode several hundreds of different peptides that have potential antimicrobial activity and that fall in three classes: cysteine-rich peptides are the most abundant with more than 350 different members; glycine-rich peptides form a family of at least 20 members; and acidic peptides are represented with at least eight members. We postulate that intracellular rhizobia in the nodules of IRLC legumes are challenged with a battery of these antimicrobial peptides that induce the terminal bacteroid differentiation. Testing this hypothesis relies on localising the peptides in nodules and analysing their in vitro activity on Sinorhizobium meliloti, the microsymbiont of M. truncatula, and their in planta activity in L. japonicus nodules.

234


S12-2 Role of amino acids in bacteroid development and nitrogen fixation Philip Poole, Jurgen Prell, and James White Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK We have shown previously that the two broad solute range amino acid transporters, Aap and Bra, are essential for productive nitrogen fixation in pea nodules. It was proposed that amino acids must cycle between the plant and the bacteroid to be able to assimilate nitrogen. One model considered was that an amino acid, such as glutamate or GABA must enter the bacteroid and this could be secreted back to the plant as alanine or aspartate. A complication is that the broad solute range of these transporters means the model is unconstrained, making it difficult to distinguish between the roles of individual amino acids. However, the Bra transporter has been shown to use two different solute binding proteins, one BraC is very broad, while BraC3 is specific for aliphatic amino acids. Therefore, in an aap braC background, Bra only transports aliphatic amino acids via BraC3 and plants inoculated with this strain are Fix+. Furthermore, expression of the Pseudomonas aeruginosa Bra and the Escherichia coli LIV, which is specific for leucine, isoleucine, and valine, in an aap bra background results in Fix+ plants. Thus, bacteroids become dependent on the plant for the provision of leucine, valine, and isoleucine, so that a symbiotic auxotrophy occurs in bacteroids. This is remarkable because aap bra mutants are not auxotrophic in free-living cultures. The regulation of aliphatic amino acid synthesis in bacteroids and the importance of symbiotic auxotrophy in development of nitrogen-fixing symbioses are discussed.

235


PS12-1 Oligopeptide transporters of Rhizobium etli fulfil an essential role during symbiotic nitrogen fixation and free-living growth under stress conditions

Maarten Vercruysse, Maarten Fauvart, Debkumari Bachaspatimayum, Kristien Braeken, Maru Belete, Karen Vos, and Jan Michiels Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium Rhizobium etli is a Gram-negative root-colonising soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. An efficient symbiosis requires an extensive molecular communication between plant and bacteria. Although a lot of research has been devoted to the initial steps of the interaction, little is known about the signals that are important during the late steps of the symbiosis. In search of novel symbiotic genes, a genome-wide screening for R. etli symbiotic mutants was performed, revealing two oligopeptide ABC transporters (opp and opt) involved in the uptake of oligopeptides. Plants nodulated by R. etli strains containing mutations in the opp and/or opt genes showed a significant reduction in symbiotic nitrogen fixation activity. Further phenotypic analysis of the opp and opt mutants revealed a difference in antibiotic resistance compared to the wild type and a decreased osmotolerance, demonstrating the importance of both oligopeptide transporters in stress resistance during free-living growth.

236


PS12-2 Investigating the role and biosynthesis of lipopolysaccharide very-long-chain-fatty-acids in the Sinorhizobium meliloti-alfalfa symbiosis

Andreas F. Haag1, Silvia Wehmeier1, Victoria L. Marlow1, Euan K. James2, and Gail P. Ferguson1 1School of Medicine, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK; 2College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK Sinorhizobium meliloti forms a beneficial interaction with legumes, such as alfalfa, and the BacA protein is essential for S. meliloti to persist within the plant cell. We determined previously that a S. meliloti bacA null mutant has a 50% reduction in its LPS very-long-chain fatty acid (VLCFA) content and this led to the hypothesis that the altered LPS could account for the symbiotic defect of this mutant. To investigate this further, we constructed and characterised S. meliloti mutants lacking either AcpXL (VLCFA acyl carrier protein) or LpxXL (VLCFA acyl transferase). We found that, despite lacking the LPS VLCFA in the free-living state, these mutants can form a successful symbiosis. However, we found that in the absence of either AcpXL or LpxXL, S. meliloti produces aberrantly shaped bacteroids that appear to be prematurely senescing; bacteroid alterations were observed throughout the entire 4-weeks of plant growth. These findings provide evidence that the LPS VLCFA modification plays an important role in bacteroid development. Since the acpXL and lpxXL genes form part of a larger cluster of genes predicted to be involved in LPS VLCFA biosynthesis, we also constructed and are currently characterising mutants in these other genes. Our initial analyses suggest that at least three other genes in the cluster are involved in LPS biosynthesis. Since VLCFAs are produced by a number of different bacterial species, which form persistent bacterial host interactions, these findings could provide valuable insights into the biosynthesis and role of VLCFAs.

237


PS12-3 Soybean nitrogen fixation is dependent on the activity of the novel peribacteroid membrane-bound transcription factor, GmSAT1

Patrick C. Loughlin1, David A. Day2, Stephen D. Tyerman1, Elena Fedorova3, and Brent N. Kaiser1 1School of Agriculture Food and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia; 2Faculty of Science, University of Sydney, Sydney, NSW 2006, Australia; 3Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, 6703 HA, Wageningen, The Netherlands GmSAT1 (Glycine max symbiotic ammonium transporter1) is expressed in mature soybean nodules and was initially isolated in a yeast complementation screen by its ability to rescue an ammonium transport mutant 26972c [1]. GmSAT1 is not a typical membrane transport protein per se as it contains a basic Helix-Loop-Helix DNA-binding domain and a predicted single C-terminal transmembrane spanning domain. In yeast and soybean, GmSAT1 has previously been shown to localise to the plasma membrane and PBM, respectively, although the exact mechanism of GmSAT1 in ammonium transport remains controversial [2]. To help address this question, we have generated GmSAT1-silenced soybean plants and using GFP fusions alongside immunogold labelling and Western blot analysis, we examined the subcellular localisation of GmSAT1 in both yeast and soybean. Lack of GmSAT1 disrupted nodule activity, where plants were nitrogen deficient and their nodules, although abundant, poorly developed with abnormal ultrastructure. Nodules lacked leghemoglobin and infected cells were small, highly vacuolated with limited numbers of symbiosomes. N-terminal GmSAT1-GFP fusions in yeast demonstrate that the protein is localised to the periphery of the cell in small intracellular vesicles as well as co-localised in the nucleus. In the nodule, immunolocalisation confirmed GmSAT1 was localised to both the PBM and nucleus of infected cells. Western blot analysis of yeast and nodule protein extracts demonstrated the presence of both a full-length GmSAT1 protein in microsomal fractions, but also a smaller truncated protein in the soluble fraction. Protein banding patterns, N-terminal GFP tagging, and split-ubiquitin analysis suggest GmSAT1 is cleaved next to the membrane-spanning domain and the bHLH domain released and delivered to the nucleus. Our results in both yeast and soybean nodules suggest GmSAT1 behaves as a membrane-bound transcription factor that is essential for the formation and/or maintenance of soybean nodule symbiosomes and for effective nitrogen fixation in general. [1] Kaiser et al. (1998). Science 281:1202-1206. [2] Marini et al. (2000). Mol. Microbiol. 35:378-385.

238


Session 12

Nodule functioning

Chaired by Eva Kondorosi Gif sur Yvette, France


239


PS12-4 Physiological aspects of nodulation and nodule functioning under osmotic constraints

Chedly Abdelly Laboratoire d'Adaptation des Plantes aux Stress Abiotiques, Centre de Biotechnologie, 2050 Hammam-Lif, Tunisie The limitation of symbiotic nitrogen fixation (SNF) by osmotic constraints (salinity and water deficit stress) restricts the development of a sustainable agriculture. The enhancement of legume productivity requires the development of salinity-tolerant symbiosis. Exploration of the variability in osmotic stress responses would permit not only to identify some tolerant genotypes, but also to determine useful criteria for genetic improvement of osmotic stress tolerance. The analysis of several works suggests that the two components implied in the sensitivity of the symbiosis legume/Rhizobium to osmotic constraints are nodule development, growth and function, as well as the aptitude of plants to supply nodules with photosynthates. However, the mechanisms of action of osmotic constraints on these components remain incompletely elucidated. In the present study, some data are analysed to emphasise physiological and biochemical features associated with tolerance relating of some symbiosis. Nodule number and size, carbohydrate utilisation in the nodules, and the antioxidant capacity of these symbiotic organs are implied in the osmotic constraint tolerance of symbiotic nitrogen fixation.

240


PS12-5 MtRAP2.4: about ethylene, nodule development, and nodule senescence

Katrien D'haeseleer1,2, Willem Van de Velde3, Juan Carlos Pérez Guerra4, Annick De Keyser1,2, Christa Verplancke1,2, Sofie Goormachtig1,2, and Marcelle Holsters1,2 1Department of Plant Systems Biology, Flanders Institute for Biotechnology, and 2Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium; 3Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France; 4Bayer BioScience N.V., 9052 Gent, Belgium Age-dependent senescence of nodules represents the final stage of the symbiotic interaction between legumes and rhizobia. Via cDNA-AFLP analysis of different developmental stages of fixing and senescing nodules, several tags involved in ethylene production and perception were identified, supporting the hypothesis of ethylene having a positive role in natural nodule senescence [1]. MtRAP2.4, an AP2 transcription factor, whose expression is upregulated during nodule development and nodule senescence, is thought to be involved in ethylene production throughout nodule development as preliminary results show overproduction of ethylene in 35S::MtRAP2.4 transgenic roots. This hypothesis correlates with the observation of diminished nodulation and reduced shoot growth in overexpression plants and the increased nodule numbers in knock-down plants obtained via Agrobacterium rhizogenes-mediated transformation using the RNAi and CREST approach. The role of MtRAP2.4 and ethylene in senescence is being investigated via MtRAP2.4 expression driven by the Lb1 promoter. Preliminary observations of an increased number of full senescent nodules, suggest that MtRAP2.4 and ethylene are involved in nodule senescence. [1] Van de Velde et al. (2006). Plant Physiol. 141:711-720.

241


PS12-6 Nitric oxide formed by bacteroidal nitrate reduction affects nifH expression in soybean root nodules

Cristina Sánchez1, Toshiki Uchiumi2, Lourdes Girard3, David J. Richardson4, Eulogio J. Bedmar1, and María J. Delgado1 1Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain; 2Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, Kagoshima 890-0065, Japan; 3Programa de Genómica Funcional de Procariotes, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62271, Mexico; 4School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK Nitric oxide (NO) has recently gained interest as a major signalling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defence response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported [1]. By using electron paramagnetic resonance, it has been previously found that hypoxia and nitrate induces NO production in soybean root nodules [2]. However, the physiological relevance of NO formation in nodules is unknown. In this work, we have analysed nitric oxide production in soybean nodules by using the NO-specific fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). We found that NO production was induced in nodules of soybean plants by flooding and nitrate. Moreover, RT-PCR analysis showed that nifH expression was strongly reduced in nodules subjected to nitrate and flooding conditions. When nodules were incubated in the presence of the NO scavenger CPTIO, induction of NO accumulation as well as inhibition of nifH expression, in response to flooding and nitrate, was not observed. Infection of soybean roots with a Bradyrhizobium japonicum strain having a mutation in the napA gene, which encodes the periplasmic nitrate reductase, led to the formation of nodules where not induction of NO formation was observed in response to nitrate and flooding conditions. These conditions did not affect nifH expression and nitrogenase activity in nodules formed by the napA mutant strain. These results suggest that NO formed through bacteroidal nitrate reduction by the periplasmic Nap enzyme in soybean nodules in response to nitrate and flooding conditions has a negative effect on both activity and expression of nitrogenase. This work was supported by grants AGL2006-13848-CO2-02/AGR from Ministerio de Educación y Ciencia, 107PICO312 from CYTED; the Junta de Andalucía (BIO-275), the CSIC (2007GB0035) and from CSIC/CONACYT (2005MX0032) for collaborations with UEA (UK) and Centro de Ciencias Genómicas (México). C.S. was supported by a fellowship from the CSIC (I3P). [1] Baudouin et al. (2006). Mol. Plant-Microbe Interact. 19:970-975. [2] Meakin et al. (2007). Microbiology 153:411-419.

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PS12-7 Which phosphatases are involved in the regulation of legume symbiotic nitrogen fixation under environmental osmotic constraint?

Jean-Jacques Drevon1, Laurie Amenc1, Jihene Ghidaoui1, Carlos Molina2, Mainassera Zaman1, Peter Winter2, and Guenter Kahl2 1Institut National de la Recherche Agronomique-Montpellier-SupAgro, UMR1222, Rhizosphère et Symbiose, 34060 Montpellier Cedex, France; 2Plant Molecular Biology, Johann-Wolfgang Goethe University, 60439 Frankfurt, Germany; 3Laboratoire des Grandes Cultures, Institut National de la Recherche Agronomique de Tunisie, 2080 Ariana, Tunisia The search for genes involved in the adaptation of nodulated-legume to osmotic constraint was performed with SuperSAGE trancription profiling of RNA extracted from roots and nodules of chickpea grown in hydroaeroponic culture, either with or without exposure to 25 mM NaCl. Among approximately 1500 genes in roots and 237 nodule genes in nodules that changed their expression after exposure to salinity, 35 phosphatase genes were identified, with eight occurring only in roots and nine only in nodules. The latter corresponded to one trehalose-P phosphatase, one acid phosphatase, one protein phosphatase of type 1 (PP1) and two PP1 catalytic subunits (β and ε), two protein phosphatases of type 2A (PP2A), three PP2A-regulatory subunits, two protein phosphatases of type 2C (PP2C), including one with homology to the gene coding for the nodule-specific LjNPP2C1 [1]. With in situ RT-PCR, this latter PP2C and the above PP1 were found to express in the nodule cortex. The intensity of the fluorescent signals of PP1 increased in the nodule inner cortex under salinity. Since this increase in PP1 expression correlates with increases in nodule permeability [2], and the related cell-expansion in the nodule inner cortex [3], it is suggested that PP1 is involved in the osmoregulation of the nodule permeability to O2 diffusion that is associated with changes in nodule nitrogenase activity under environment constraint. It is concluded that diverse nodule phosphatases are involved in the nodule functioning under osmotic constraints, and that in situ RT-PCR can help to understand their functions. [1] Kapranov et al. (1999). Proc. Natl. Acad. Sci. USA 96:1738-1743. [2] L'Taief et al. (2007). J. Plant Physiol. 164:1028-1036. [3] Serraj et al. (1995). Plant Cell Environ. 18:455-462.

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PS12-8 Coordination between carbon and nitrogen metabolism in nitrogen-fixing bean nodules

Miguel Lara Flores1, Lourdes Blanco1, Sonia Silvente1, Yadira Gaona1, Carroll P. Vance2, and Pallavolu Maheswara Reddy1 1Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, Mexico; 2United States Department of Agriculture-Agricultural Research Service, Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA A cDNA clone of the asparagine synthetase (AS), called PvNAS2, and two different cDNA clones of PvNADH-GOGAT were isolated from bean nodules. Southern blot analysis indicated that a small gene family encodes AS. Northern blot analysis demonstrated that PvNAS2 expression is induced in nodules, during the early days of nitrogen fixation. Investigations with the PvNAS2 promoter:gusA fusion revealed that the expression of PvNAS2 is solely localised to vascular traces and outer cortical cells, but never detected in the central nitrogen-fixing zone of the nodule. Each clone of PvNADH-GOGAT-I (7.4 kb) and PvNADH-GOGAT-II (7.0 kb), represents a unique gene in the bean genome. PvNADH-GOGAT-II expression is higher than PvNADH-GOGAT-I during nodule development. In situ hybridisation and promoter expression analyses demonstrated that the NADH-GOGAT-I and NADH-GOGAT-II genes are differentially expressed in bean nodules. Regulation studies of these three enzymes revealed that PvNAS2 expression is down-regulated when carbon availability is reduced and the addition of glucose resulted in its induction, leading to the increased asparagine production and in the reduction of ureide content in nodules. Expression of PvNADH-GOGAT-I, but not PvNADH-GOGAT-II is strongly inhibited by ureides. Since in bean the symbiotically assimilated nitrogen is transported from nodules to the shoot in the form of ureides, this result indicated that ammonia assimilation in bean nodules take places through glutamine synthetase and PvNADH-GOGAT-II, but not PvNADH-GOGAT-I. In light of the above results, a model is proposed in which a favourable environment is created for the efficient transfer of the amido group of glutamine for the synthesis of ureide in bean nodule.

244


PS12-9 Isolation and analysis of ineffective nodulation mutants in Medicago truncatula

Beatrix Horváth1, Gábor Halász1, Ágota Domonkos1, Krisztina Miró1, John Marsh2, Giles E.D. Oldroyd2, and Péter Kaló1 1Agricultural Biotechnology Center, 2100 Gödöllő, Hungary; 2Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, UK In order to dissect the later stages of the nitrogen-fixing interaction between Sinorhizobium meliloti and Medicago truncatula, we have screened fast-neutron and ethyl methanesulfonate-mutagenised M. truncatula plants and identified mutants with ineffective (Fix-) symbiotic phenotypes: development of white non-fixing nodules, symptoms of nitrogen starvation, and retarded growth. The ineffective mutants were classified into three main categories based on their nodule morphology; they developed small or large white, and senescence nodules. In order to determine whether bacteria are released or retained in the infection threads and nodule cells enable to host bacteria and provide the proper environment for rhizobia, we analysed the mutant plants with toluidine-blue staining or following inoculation with S. meliloti 1021, which constitutively expresses the LacZ gene. We tested the mutants for their ability to support rhizobial nitrogenase activity and expression profiles of several nodulin genes. We have started experiments to test the allelic relationship between eight mutants which showed complete reduction in nitrogenase activity in their nodules. Based on the phenotype and transcriptional profile of the mutants, the mutated genes are positioned in the symbiotic process. In order to define the molecular identity of these Fix genes, we have determined the map position of the mutant loci and started the cloning of three genes.

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PS12-10 Phenotypic characterisation of Fix- symbiotic mutants in Medicago truncatula

Hossein Khademian1, Willem Van de Velde1, Viviane Cosson1, Patricia Durand1, Million Tadege2, Kirankumar S. Mysore2, Eva Kondorosi1, Pascal Ratet1, and Peter Mergaert1 1Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France; 2Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA The symbiosis between the soil bacterium Sinorhizobium meliloti and the legume Medicago truncatula leads to the formation of nitrogen-fixing nodules on the roots of the host, which the bacteria colonise as intracellular symbionts. Symbiotic host cells are highly differentiated cells, adapted to accomodate the endosymbiont. They originate from meristem cells and their differentiation is driven by endoreduplication. Rhizobia infect the developing nodules via infection threads and, thereafter, enter host cells via endocytosis. These intracellular rhizobia differentiate to nitrogen-fixing bacteroids. In M. truncatula, bacteroid differentiation also involves genome endoreduplication, supporting cell enlargement, membrane modifications, and a lost capacity for growth: bacteroids are terminally differentiated. A large population of M. truncatula lines mutagenised with the retrotransposon Tnt1 or by T-DNA mutagenesis was screened for symbiotic mutants and approximately 40 lines were identified that formed non-functional (Fix-) nodules. We are currently characterising the Fix- phenotypes of confirmed mutants by cell biology and molecular methods. Bacterial infection of nodules was followed by histological staining for the β-galactosidase marker introduced on a plasmid in S. meliloti. The formation of infection threads and of symbiotic host cells and the differentiation of bacteroids was observed by microscopy and flow cytometry analysis. The expression of molecular markers of nodule formation was measured by RT-qPCR and Western blotting. Among the 13 mutants analysed until now, we found three different phenotypic classes. In one type, the nodules were filled with infection threads without formation of symbiotic cells and bacteroid uptake. In a second class, symbiotic cells were formed and infected with bacteroids, but those did not properly differentiate. And finally, in the last type of mutants, symbiotic cells and bacteroids differentiated normally.

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PS12-11 Regulation of nitrogen acquisition by the N demand of the whole plant in Medicago truncatula

Marc Lepetit1, Sandrine Ruffel1, Christian Jeudy2, Sandra Freixes2, Pascal Tillard1, Alain Gojon1, and Christophe Salon2 1Biochimie et Physiologie Moléculaire des Plantes, UMR 5004 INRA-CNRS-SupAgro-UM2, 34060 Montpellier Cedex, France; 2Unité de Génétique et Ecophysiologie des Légumineuses, Institut National de la Recherche Agronomique, UMR-LEG, 21065 Dijon, France Legumes may acquire nitrogen from NO3-, NH4

+, and N2 (through symbiosis with Rhizobium). Mechanisms involved in the regulation of the acquisition of these sources by the N demand of the plant have been compared in the model legume Medicago truncatula [1]. Using split-root systems, we demonstrated that NO3- and NH4

+ uptake as well as N2 fixation are under the control of systemic signalling of the plant N status. Indeed, irrespective of the nature of the N source, N acquisition by one side of the root system is repressed by high N supply to the other side. Transcriptome analysis facilitated the identification of in excess of 3000 genes that were regulated by systemic signalling of the plant N status. However, detailed scrutiny of the data revealed that the differential gene expression was highly dependent on the N source. Further investigation using short-term (4 days)-localized N starvation in split-root system has shown that the compensation by a rapid up-regulation of the N intake of root remained supplied is only observed in NO3-fed plant, but not in N2-fed plant. In non-limited conditions, nodule N2 fixation is likely to be at its maximum capacity and, therefore, cannot be increased by the release of systemic repression. Under such a limitation, N2-fixing plants display long-term (14 days) morphological responses associated to important changes of C allocation within the plant. Nodule biomass is increased progressively, resulting in a compensatory response to N limitation, indicating that nodule development is a major target of regulatory pathways related to the N status. However, this adaptative response is less efficient than the one observed in NO3-fed plant (rapid increase of both uptake capacity and root proliferation), probably because the development of new and efficient symbiotic structures takes several days and requires substantial C supply by the plant. [1] Ruffel et al. (2008). Plant Physiol. 146:2020-2035.

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PS12-12 Regulation of ABC transport systems in Rhizobium leguminosarum bv. viciae 3841

Geraldine Mulley1, James P. White1, Alexandre Bourdès2, A. Jurgen Prell2, Kim Findlay2, J. Allan Downie2, and Philip S. Poole2 1School of Biological Sciences, University of Reading, Reading RG6 6AJ, UK; 2Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Perturbation of intracellular amino acid pools by mutation of the large subunit of GOGAT (gltB) in Rhizobium leguminosarum prevents growth using a broad range of amino acids, including glutamate, by severely limiting uptake via the Aap and Bra ABC transport systems. In the absence of C4-dicarboxylates, aspartate uptake via the dicarboxylate transport system (Dct) [1] alleviates the inhibition of amino acid transport and enables growth using this amino acid as a sole nitrogen source. The gltB mutant (RU2307) is able to nodulate pea roots and colonise infection threads, but bacteroid development is disrupted by an inability to transport amino acids, thereby preventing nitrogen fixation. Overexpression of aapJQMP on a complementing plasmid enables growth using glutamate and is sufficient to rescue normal bacteroid development and nitrogen fixation by restoring amino acid transport. A suppressor mutant of RU2307 (RU2386) able to grow using glutamate as a nitrogen source formed effective nodules on pea plants and showed extremely high levels of amino acid transport in vitro. Complementation of RU2386 with gltBD restored ammonium assimilation while amino acid transport remained elevated, indicating that the suppressor mutation is independent of the mutation in gltB. Microarray analysis of RU2386 revealed specific regulation of a subset of ABC transport operons, with the most strongly up-regulated genes encoding structural components of the Aap and Bra. 454 FLX sequencing of the RU2386 genome identified a missense mutation in hfq (P66R). Sequencing of the hfq gene from a further 12 suppressor strains revealed five with missense mutations (V37F (x2), C44F, K58R, and H59Y) and one with a nonsense mutation (Q6stop). Transport and growth phenotypes of the suppressor strains suggest at least two additional loci participate in a regulatory pathway that involves the RNA chaperone Hfq and affects expression and/or mRNA stability of a subset of ABC transport genes in R. leguminosarum. [1] Reid et al. (1996). Microbiology 142:2603-2612.

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PS12-13 Expression of defense genes of Lotus japonicus in symbiosis with Mesorhizobium loti

Ei-ichi Murakami1, Maki Nagata1, Ken-ichi Kucho2, Mikiko Abe2, Akihiro Suzuki3, Shiro Higashi2, and Toshiki Uchiumi2 1Graduate School of Science and Engineering, and 2Faculty of Science, Kagoshima University, Kagoshima 890-0065, Japan; 3Faculty of Agriculture, Saga University, Saga 840-8502, Japan The inoculation of Rhizobium induces production of nitric oxide (NO), which is an important molecule in the initiation of the plant defence, in the root of its host plant. Lipopolysaccharide (LPS) is required to establish both the symbiotic and pathogenic relations [1, 2]. LPSs of many bacterial species are known to induce NO production and plant defence [3]. Many mutants in LPS fail to develop normal symbiosis or induce successful disease symptom with host plants. These analogies suggest that there exist a common mechanism between symbiotic and pathogenic responses of host plants. We analysed the expression of defence genes during symbiosis. Although defence genes are not identified yet in Lotus japonicus, we focused on four genes homologous to the pathogenesis-related 1 (PR1) and three genes homologous to the flavin-dependent monooxygenase 1 (FMO1). L. japonicus plants were grown by symbiotic nitrogen fixation or by ammonium chloride as nitrogen nutrient for 28 days. The expression levels of these seven genes were estimated by quantitative RT-PCR. The enhanced expression of one of the PR1 was detected in stems and roots of the nodulated plants, but not in leaves and nodules. This result suggests that the symbiosis with Rhizobium induces a systemic resistance of L. japonicus, except for leaves and nodules. [1] Campbell et al. (2002). Proc. Natl. Acad. Sci. USA 99:3938-3943. [2] Hendrickt & Sequeira (1984). Appl. Environ. Microbiol. 48:94-101. [3] Zeidler et al. (2004). Proc. Natl. Acad. Sci. USA 101:15811-15816.

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PS12-14 Analysis of the molecular basis of nickel homeostasis in Rhizobium leguminosarum bv. viciae

Belén Brito1, Rosabel Prieto1, Ezequiel Cabrera1, Anabel Hidalgo1, Juan Imperial1,2, José Palacios1, and Tomás Ruiz-Argüeso1 1Laboratorio de Microbiología, Departamento de Biotecnología, and Centro de Biotecnología y Genómica de Plantas, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain; 2Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain Nickel is an essential element for different biological processes. Enzymes with nickel atom(s) in their active site participate in urea hydrolysis, oxidation of molecular hydrogen, and detoxification of superoxide anion radicals. Nickel transport is a challenge for microorganisms since, although essential, high intracellular levels of this metal are toxic. For this reason, bacteria have developed high affinity nickel transporters as well as nickel-specific detoxification systems. In Rhizobium leguminosarum bv. viciae, a nickel-dependent hydrogenase is induced in pea (Pisum sativum) bacteroids. Symbiotic hydrogen uptake is limited by nickel availability to the bacteroids [1], and this nickel-dependent limitation is partly dependent on the plant host [2]. Proteins involved in nickel transport for hydrogenase synthesis have not been described in R. leguminosarum. One of the putative candidates for this role is HupE, a predicted membrane protein that shows a histidine-rich domain similar to those involved in nickel transporters, like HoxN from Ralstonia eutropha or NixA from Helicobacter pylori. In R. leguminosarum bv. viciae UPM791, the gene hupE is part of the hydrogenase gene cluster located in the symbiotic plasmid, whereas a second hupE-like gene, hupE2, lies in the pRLUPM791b plasmid. In addition, this bacterium contains an rcnRA system in the chromosome. In this work, we report the characterisation of HupE/HupE2 as a novel class of nickel transporter involved in hydrogen oxidation. In these proteins, histidine residues essential for the HupE functionality have been identified. On the other hand, analysis of expression of the rcnRA operon has revealed that these genes are overexpressed in response to high levels of nickel or cobalt in the culture medium. This mode of regulation is consistent with the purported role of these genes as part of a Ni/Co efflux system. [1] Brito et al. (1994). J. Bacteriol. 176:5297-5303. [2] Brito et al. (2008). Mol. Plant-Microbe Interact. 21:597-604.

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PS12-15 Jasmonate in legume and actinorhizal root nodule development Anna Zdyb1,2, Kirill Demchenko3, Cornelia Göbel2, Peter Grzeganek2, Ivo Feussner2, Bettina Hause4, and Katharina Pawlowski1,2 1Department of Botany, Stockholm University, Stockholm, 106 91 Sweden; 2Plant Biochemistry, Göttingen University, 37077 Göttingen, Germany; 3Komarov Botanical Institute, Russian Academy of Sciences, 197376 St.-Petersburg, Russia, 4Leibniz Institute for Plant Biochemistry, 06120 Halle, Germany Two groups of plants can enter root nodule symbioses with nitrogen-fixing soil bacteria. Rhizobia induce nodules on legume roots, while Gram-positive mycelial actinomycetes of the genus Frankia induce nodules on the roots of plants from eight angiosperm families, mostly woody shrubs, collectively called actinorhizal plants. Both types of root nodule symbioses go back to a common ancestor. Jasmonates are oxylipin signalling compounds that play a role in vegetative and propagative plant development and in plant defence and have recently shown also to be involved in plant root symbioses. We compared jasmonate levels in roots and nodules of two legumes, Medicago truncatula and Lotus japonicus, and two actinorhizal species, Datisca glomerata and Casuarina glauca. We also compared the localisation of enzymes involved in jasmonate biosynthesis in actinorhizal and legume nodules. In all three plant species examined, lipoxygenases (LOXs), which catalyses the first step, as well as allene oxide cyclase (AOC), which catalyses the third step in jasmonate biosynthesis, were restricted to uninfected cells. These results are discussed with regard to the role of jasmonate in mature nodules, and in nodule induction. This work was supported by the EU (Marie Curie RTN INTEGRAL).

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PS12-16 Analysis of the endosymbiotic forms of the promiscuous Rhizobium sp. NGR234

Maged Saad, Nadia Bakkou, and Xavier Perret Microbiology Unit, Plant Biology Department, University of Geneva, 1211 Genève 4, Switzerland Symbiotic nitrogen fixation by bacteroids, the endocellular form of rhizobia, requires the coordinated activation of a number of functions that are otherwise silent in the free-living state. Bacteroids also need to adapt to the peculiar environment of nodule cells, which may differ significantly between plants. Amongst rhizobia, Rhizobium sp. strain NGR234 has the distinct ability to initiate nodule formation on roots of more than 120 genera of legumes, as well as to fix nitrogen in association with plants that form either determinate or indeterminate types of nodules. Complementary molecular approaches were used to characterise the endosymbiotic forms of NGR234. Bacteria grown in free-living conditions or isolated from nitrogen-fixing as well as senescent nodules were analysed by electron microscopy. Reference proteomes for each of these distinct cell populations were compared, and are currently matched against the recently completed genome sequence of NGR234. To correlate protein profiles with gene expression, the transcription levels of reference genes that include loci involved in nodulation, nitrogen fixation, and various housekeeping functions were quantified by real time-PCR. Transcription of genes within nodules is primarily under the control of two parallel regulatory networks, which differ significantly from those found in the established models of Bradyrhizobium japonicum or Sinorhizobium meliloti.

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PS12-17 Legume nodule development results in symbiosis-specific auxotrophy for aliphatic amino acids in the bacterial partner

Jurgen Prell, James P. White, Ramakrishnan Karunakaran, and Philip S. Poole Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK A double mutant in the general amino acid ABC transporters, AapJQMP and BraDEFGC, of Rhizobium leguminosarum 3841 was described with a severely impaired symbiotic phenotype on pea plants [1]. Infected plants are strongly reduced in dry weights because overall acetylene reduction and bacteroid protein amounts per plant are drastically reduced, but single bacteroids fix nitrogen at wild-type level. This showed that amino acids must move between the plant and bacteroid for wild-type nitrogen fixation to occur, however, which amino acids are involved was unclear because of the wide range of solutes transported by the two ABC transporters. Recent advances demonstrate that when only the orphan SBP BraC3 of the Bra ABC transporter is expressed, then wild-type nitrogen fixation occurs. BraC3 has a very specific transport activity for the aliphatic amino acids alanine, leucine, isoleucine, and valine. One explanation is that pea bacteroids become auxotrophs for the branched chain amino acids specifically during symbiosis and depend on their supply from the plant. Microarray data showed that key enzymes of the biosynthetic pathways of the branched chain amino acids (e.g., ilvC, ilvE2, leuC, and leuD) are severely down-regulated in bacteroids. This suggests that bacteroids are unable to synthesise the carbon skeletons of these amino acids in sufficient amounts. We hypothesised that mutants in the biosynthetic pathways should be unaffected during symbiosis. An IlvD and a LeuD mutant were both unable to nodulate pea plants. However, nodulation of the LeuD mutant could be rescued by the addition of leucine to the nodulation medium and then infected plants fixed nitrogen and had dry weights identical to wild-type infected plants. Additionally, the phenotype of the Aap/Bra mutant bacteroids could not be rescued by the expression of glutamate dehydrogenase, an ammonium- assimilating enzyme missing in R. leguminosarum, which emphasises that the symbiotic auxotrophy is not general, but very specific for the synthesis of the branched chain amino acids. [1] Lodwig et al. (2003). Nature 422:722-726.

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PS12-18 Characterisation of enf (enhanced nitrogen fixation) mutants of Lotus japonicus

Akihiro Suzuki1, Akiyoshi Tominaga1, Ayaka Yamauchi1, Kouichi Futsuki1, Toshiki Uchiumi2, Mikiko Abe2, Ken-ichi Kucho2, Shiro Higashi2, Masatsugu Hashiguchi3, Ryo Akashi3, Shusei Sato4, Takakazu Kaneko4, Satoshi Tabata4, Toyoaki Anai1, and Susumu Arima1 1Department of Agricultural Sciences, Saga University, Saga 840-8502, Japan; 2Department of Chemistry and Bioscience, Kagoshima University, Kagoshima 890-0065, Japan; 3Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan; 4Kazusa DNA Research Institute, Chiba 292-0818, Japan Endogenous concentration of the phytohormone abscisic acid (ABA) affects the number of root nodule of leguminous plants. Indeed, the nodule number on the root of the model legume Lotus japonicus treated with abamine, a specific inhibitor of ABA biosynthesis, was drastically increased [1, 2]. Based on the working hypothesis that the root nodule number of leguminous plants, which show lower concentration of endogenous ABA, increase, we carried out a screening to isolate the ABA-insensitive mutants of L. japonicus. Using all the isolated candidates of L. japonicus, root nodule formation was analyzed 30 days after inoculation with Mesorhizobium loti MAFF303099. In some mutant lines, the root nodule number was clearly increased compared with that of wild type ('Miyakojima MG-20'). Moreover, surprisingly, the nitrogen fixation activity (with an acetylene reduction assay (ARA)) of these mutants was strongly increased. ABA-related mutants #12 and #18 were designated as enf1 (enhanced nitrogen fixation 1) and enf2, respectively. This screening strategy was applied to soybean for isolation of mutants that show higher nitrogen fixation activity. Approximately 3,000 M2 soybean seeds mutagenized by X-ray were germinated on the filter paper containing 100 µM ABA. Eventually, 80 mutant candidates of ABA low-sensitive (insensitive?) were obtained. Using 20 candidates of soybean, preliminary experiments to analyse root nodule formation and acetylene-reducing activity were conducted. The nitrogen-reducing activity 28 days after inoculation in some mutant candidates was strongly increased compared with that of wild-type soybean (Bay). Field experiments to analyse growth, symbiotic phenotypes, and yield of seeds are currently going on. [1] Suzuki et al. (2004). Plant Cell Physiol. 45:914-922. [2] Nakatsukasa-Akune et al. (2005). Mol. Plant-Microbe Interact. 18:1069-1080.

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PS12-19 Localization of peptides belonging to two large families with homology to antimicrobial peptides in Medicago truncatula nodules

Willem Van de Velde, Eszter Schuler, Eva Kondorosi, and Peter Mergaert Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France The symbiosis between legume plants and bacteria, known as rhizobia, leads to the formation of root nodules that are colonised by rhizobia fixing atmospheric nitrogen. The rhizobia in nodules, called bacteroids, are differentiated bacteria adapted to symbiotic life. In legumes of the IRL clade (e.g. Medicago truncatula), bacteroids display several morphological and cellular modifications, such as genome endoreduplication, supporting an important cell enlargement and membrane permeabilisation. In addition, they lose all capacity for growth upon release from the nodule and are, as a consequence, terminally differentiated. Intriguingly, none of these microsymbiont features are observed for rhizobia nodulating legumes outside the IRL clade (like Lotus japonicus). Transcriptome analysis of M. truncatula and L. japonicus nodules allowed identification of candidate genes coding for the plant factors inducing the terminal bacteroid differentiation. Expression of these genes is associated with bacteroid differentiation and, moreover, they do not exist in L. japonicus. Encoded proteins have functional signal peptides and could, therefore, be targeted to the bacteroids. The mature peptides have potential antimicrobial activity and fall in three classes: cysteine-rich peptides, glycine-rich peptides, and acidic peptides. Using a dual cell biology approach consisting of peptide-specific antibodies, on the one hand, and endogenous promoter driven CHERRY translational peptide fusions, on the other hand, we have studied the localisation of members of the cysteine and glycine-rich families in M. truncatula nodules.

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PS12-20 Regulation of Sinorhizobium meliloti nitrogen stress response in free-living cells and in symbiosis with alfalfa

Svetlana N. Yurgel1, Jennifer Topham2, and Michael L. Kahn1,2 1Institute of Biological Chemistry, and 2School of Molecular Biosciences, Pullman, WA 99164, USA The nitrogen-fixing symbiosis between rhizobia and legumes is a model of coevolved nutritional complementation. The plants reduce atmospheric CO2 by photosynthesis and provide carbon compounds to associated bacteria; the rhizobia use these compounds to reduce (fix) atmospheric N2 to ammonia, a form of nitrogen the plants can use. A key feature of symbiotic N2 fixation is that N2 fixation is uncoupled from bacterial nitrogen stress metabolism, so that the rhizobia generate "excess" ammonia and release this ammonia to the plant. In the symbiosis between Sinorhizobium meliloti and alfalfa, we have found mutations in GlnD, the major bacterial nitrogen stress response sensor, that lead to a symbiotic relationship in which nitrogen is fixed (Fix+) but the symbiosis is not effective (Eff-) in substantially increasing plant growth. This phenotype implicates the nitrogen stress response in some aspect of symbiotic communication. Analysis of free-living S. meliloti strains carrying mutations in several genes related to nitrogen stress response regulation (glnD, glnB, ntrC, ntrA, and glnK) showed that catabolism of nitrogen-containing compounds depended on the NtrC and GlnD components of the nitrogen stress response cascade. Our data indicate that mutations deleting either or both of the glnK or glnB genes, which remove the capacity to produce the respective PII proteins, GlnK or GlnB, do not have the same effect on the symbiotic phenotype. The glnB and glnK mutants and the double glnB/glnK mutant have some effect on the nitrogen stress response, although the data indicate that the PII proteins can partially substitute for each other to generate an active nitrogen stress response. However, only mutants of GlnD had the unusual Fix+Eff- symbiotic phenotype. These results indicate that bacterial nitrogen stress regulation is important to symbiotic productivity, and suggest that GlnD may act in a novel way that bypasses GlnK/PII and GlnB/PII to influence symbiotic behaviour.

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Closing remarks

Summary talk Nitrogen fixation, nodulation, and plant developmental genes and genomics help paint an integrative picture Peter M. Gresshoff Australian Research Council Centre of Excellence for Integrative Legume Research, University of Queensland, Brisbane, QLD 4072, Australia Biotechnology and functional genomics work hand-in-hand with plant physiology, biology, and breeding to advance the ability of legume plants to produce more and better seed, and all with less input and improved sustainability. We now have available a modern toolbox of genomic tools, ranging from quantitative trait loci (QTLs as they are called) controlling properties such as disease resistance, root structure, oil, isoflavone and protein content to whole-genome sequences, metabolite profiling, and systems biology. This summary presentation will encapsulate the advances presented at the 8th European Nitrogen Fixation meeting, paralleling the advances with the prokaryotic partners

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Genomics of Nitrogen-Fixing Organisms

Organised by Peter Young

York, UK

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Programme of Workshop 1 9.00 – 10.30 Session 1 9.00 – 9.10 Peter Young (York, UK): Welcome and introduction 9.10 – 9.30 Danny Vereecke (Gent, Belgium): Arabinosylation in Azorhizobium: Nod

factors and beyond? 9.30 – 9.50 Wolfgang Streit (Hamburg, Germany): The complete genome sequence

of Rhizobium sp. NGR234: more secretion systems equates with more legumes nodulated?

9.50 – 10.10 Lucie Miché (Montpellier, France): Diversity and population genomics of Aeschynomene photosynthetic symbionts

10.10 – 10.30 Xavier Bailly (York, UK): Population genomics of Sinorhizobium medicae 10.30 – 11.00 Coffee break 11.00 – 12.20 Session 2 11.00 – 11.20 Marina Roumiantseva (St Petersburg, Russia): Comparative genomics of

alfalfa nodulating bacteria from intra- to interspecies level. 11.20 – 11.40 Pablo Vinuesa (Cuernavaca, Mexico): Genome-wide selection of primer

pairs amplifying highly informative amplicons for multilocus sequence analyses

11.40 – 12.00 Alessio Mengoni (Firenze, Italy): Integrating Biolog phenomic analysis with genomic approaches to explore the diversity of natural Sinorhizobium meliloti strains

12.00 – 12.20 Barbara Reinhold-Hurek (Bremen, Germany): Functional genomic analyses of the diazotrophic grass endophyte Azoarcus sp. strain BH72

12.30 – 13.30 Sandwich lunch 13.30 – 15.10 Session 3 13.30 – 13.50 Martin Krehenbrink (Paris, France): Protein secretion in Rhizobium: what

is out there? 13.50 – 14.10 Michael Göttfert (Dresden, Germany): Transcriptional responses of

Bradyrhizobium japonicum to environmental changes 14.10 – 14.30 José Jiménez Zurdo (Granada, Spain): The Sinorhizobium meliloti

RNome: identification and characterization of new small non-coding RNAs

14.30 – 14.50 Ruth Schmitz-Streit (Kiel, Germany): Genome-wide Screening for non-coding RNAs in Methanosarcina mazei strain Gö1

14.50 – 15.10 Anke Becker (Freiburg, Germany): SmRegNet: an interactive platform for the analysis of gene regulatory networks in Sinorhizobium meliloti

15.10 – 15.30 Socorro Mesa (Zürich, Switzerland): Appraisal of the regulons controlled by the FixLJ-FixK2-FixK1 cascade in Bradyrhizobium japonicum

15.30 End of workshop + Coffee break together with welcome coffee

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S1-1 Arabinosylation in Azorhizobium: Nod factors and beyond? Danny Vereecke1,2, Philippe De Backer1,2, Wim D’Haeze2, Christa Verplancke1,2, Annick De Keyser1,2, and Marcelle Holsters1,2

1Department of Plant Systems Biology, Flanders Institute for Biotechnology, and 2Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium Azorhizobium caulinodans ORS571, a microsymbiont of the tropical legume Sesbania rostrata, produces highly decorated Nod factors that carry a D-arabinofuranosyl group at the reducing N-acetylglucosamine residue of the lipochitooligosaccharide backbone. Within the large number of Nod factor structures that have been elucidated, only the Nod factors from the microbial partners of S. rostrata have this modification. D-arabinose occurs rarely in nature and its biosynthesis in prokaryotes is best described for Mycobacterium species, where it is incorporated into the arabinogalactan and (lipo)arabinomannan polymers of the cell wall. Functional analysis of A. caulinodans has shown that the genes noeCHOP that are part of the nodA operon, are essential for Nod factor arabinosylation. Interestingly, noeCHO are shared between Azorhizobium and Mycobacterium and biochemical studies in Mycobacterium have shown that these genes are required for the production of the arabinose donor, decrapenylphosphoryl- D-arabinose. The alleged arabinosyl transferase gene noeP of A. caulinodans however is not conserved in Mycobacterium, consistent with the presumably very different nature of the arabinosylation substrates in both organisms. Unexpectedly, in the absence of enhanced Nod factor production, overexpression of noeP in A. caulinodans causes growth retardation, raising the possibility of the occurrence of other substrates. Using comparative genomics, we analysed the origin of the noeCHO genes as a first step to address the question if besides Nod factors other molecules are arabinosylated in Azorhizobium.

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S1-2 The complete genome sequence of Rhizobium sp. NGR234: more secretion systems equates with more legumes nodulated?

Dagmar Krysciak1, Axel Strittmatter2, Heiko Liesegang2, Christel Schmeisser1, Ruth A. Schmitz3, William J. Broughton2, Xavier Perret4, and Wolfgang R. Streit1 1Biozentrum Klein-Flottbek, Abteilung für Mikrobiologie und Biotechnologie, Universität Hamburg, 22609 Hamburg, Germany; 2Laboratorium für Genomanalyse der Universität Göttingen, 37077 Göttingen, Germany; 3Biologiezentrum, Christian Albrechts Universität zu Kiel, 24118 Kiel, Germany; 4Laboratoire de Biologie Moléculaire des Plantes Supérieures, Université de Genève, 1211 Genève, Switzerland Rhizobium sp. NGR234 forms symbiotic, nitrogen-fixing associations with more legumes than any other rhizobia. Its genome consists of three replicons. It harbours a symbiotic plasmid (pNGR234a) with a size of 0.56 Mb that carries most genes necessary for the interaction with plants. Furthermore, the microbe harbours an extremely large second plasmid (pNGR234b) plus a chromosome. While the sequence of pNGR234a was already established 11 years ago [1], we have now established the complete nucleotide sequence of pNGR234b and the NGR234 chromosome (NGR234c). The megaplasmid has a size of 2.43 Mb and the chromosome a size of 3.92 Mb. The overall genome size and structure is comparable to the genome of Sinorhizobium meliloti strain 1021. However, comparisons of the megaplasmid of pNGR234b vs. the pSymB of S. meliloti reveal that pNGR234b carries many loci involved in protein secretion as well as a vast family of sRNAs. A second type-III secretion system, a type-IV secretion system as well as two type-IV pili are also present. Since these are main differences between the two genomes, it seems likely their regulation in a novel manner largely explains variations in host-range. [1] Freiberg et al. (1997). Nature 387:394-401.

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S1-3 Diversity and population genomics of Aeschynomene photosynthetic symbionts

Lucie Miché, Lionel Moulin, Clémence Chaintreuil, and Gilles Béna Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France Tropical aquatic legumes of the genus Aeschynomene are nodulated by Bradyrhizobium spp. that possess several peculiarities compared to more "classical" rhizobia interacting with legumes: (i) Aeschynomene symbionts are photosynthetic, a trait that is unique among rhizobia, and nodulation efficiency is highly dependent on photosynthetic activity [1]; (ii) besides root nodulation, photosynthetic bradyrhizobia can also induce nodulation on the stem of their hosts; (iii) whole-genome analysis of two reference strains (ORS278 and BTAi1) highlighted the absence of canonical nodABC genes in these bacteria [2], a surprising result that raises the question of the mechanisms involved in Aeschynomene/photosynthetic bradyrhizobia interactions. A large sampling campaign was done to study the diversity of photosynthetic bradyrhizobia nodulating several species of Aeschynomene in Africa and Central America. Phylogenetic analyses based on both recA marker and whole-genome AFLP studies confirmed that photosynthetic strains form a separate cluster among bradyrhizobia. Moreover, a new cluster of strains that are not photosynthetic, but are able to stem-nodulate A. indica was found. Six representative strains were fully sequenced by the new Solexa (Illumena) pyrosequencing technology that generates millions of 36-bp oligomers. Data were assembled and comparative genomics carried out, in order to study the core genome and accessory genes involved in photosynthetic Bradyrhizobium spp. adaptation to their peculiar ecological niches. [1] Giraud & Fleischman (2004). Photosynth. Res. 82:115-130. [2] Giraud et al. (2007). Science 316:1307-1312.

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S1-4 Population genomics of Sinorhizobium medicae strains based on the analysis of partial sequences of environmental genomes

Xavier Bailly, Elisa Giuntini, Ryan Lower, Peter W. Harrison, and J. Peter W. Young Department of Biology, University of York, York YO10 5YW, UK The increase in sequencing throughput is revealing the vast polymorphism of bacterial species at a genomic scale, based on projects focusing on reference strains. Identifying the environmental constraints and the evolutionary processes that shape such diversity patterns rescales the questions of microbial ecology. These issues raise two main methodological problems because they require representative samples of in-situ populations to be addressed: (i) reference genomes, when they are available, might include a subset of the supra-genome of their species, limiting the insights obtained from complete genome hybridisation approaches; (ii) obtaining the complete genome of a sufficient number of strains is still difficult for logistical reasons and due to cultivation problems (e.g., Frankia strains). The analysis of partial sequences of environmental genomes might provide an interesting solution to both problems. In order to assess the efficiency of this approach, we obtained low coverage (i.e., less than 1X) genome sequences from Sinorhizobium medicae strains isolated from nodules of Medicago lupulina plants located within a one square-meter area. The meta-genome we observed is analyzed in the light of both the genome of S. medicae strain WSM 419 and the polymorphism patterns previously observed from populations of Medicago symbionts.

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S2-1 Sinorhizobium comparative genomics: from intra- to inter-species level

Evgeny Andronov1, Viktoria Belova1, Larissa Sharypova1, Liza Krol2, Anke Becker2, Marina Roumiantseva1, and Boris Simarov1 1Laboratory of Genetics of Microorganisms, All-Russion Research Institute for Agricultural Microbiology, Pushkin 8, 196608, St.Petersburg, Russia; 2Institute for Genome Research, Centre for Biotechnology, Bielefeld University, 33501 Bielefeld, Germany The reference strain CXM1-105 of Sinorhizobium meliloti and five isolates native to the Aral sea region different in salt tolerance were genomically hybridised to the Sm6kPCR biochip of the fully sequenced Rm1021. In all these strains, the three chromosome divergent regions (from 15 kb to 80 kb) corresponded to the three chromosomal genome islands (GIs) in silico predicted for S. meliloti Rm1021 [1] were identified. All three GIs were evaluated for: (i) the structure organization; (ii) the putative function of genes corresponding to the COG classification; and (iii) the expression profiles under different conditions. GIs inserted near or into chromosomal tRNA genes contained phage-related integrases, characterized by significantly lower GC% content and abundance with IS elements. GIs genes involve in general function control, amino acid metabolism, inorganic ion transport, and defence mechanisms. No significant alterations in GIs gene expression profiles could be revealed under salinity, phosphate starvation and nitrogen-fixing symbiosis [2]. Cloning and sequencing of the flanking sequences (FS) of all three chromosome divergent regions (GIs) of the reference S. meliloti strain CXM1-105 provided the precise boundaries for these three regions. Constructed FS primers were used to screen S. meliloti isolates native to different gene centres of alfalfa for the presence/absence of these GIs. PCR screening revealed that the majority of the isolates were free from GIs. Finally, the S. meliloti Rm1021 and S. medicae WSM419 [unpublished data] genome sequences were aligned and compared with the microarray data. The genome rearrangements (escape "microarray scope") in diversification at intraspecies and interspecific level are discussed. [1] Mantri & Williams (2004). Nucleic Acids Res. 32:D55-D58. [2] Becker et al. (2004). Mol. Plant-Microbe Interact. 17:292-303.

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S2-2 Genome-wide selection of primer pairs amplifying highly informative amplicons for multilocus sequence analyses

Bruno Contreras-Moreira, Iraís Figueroa-Palacios, Agustín Avila Casanueva, Enrique Zozaya, Bernardo Sachman, and Pablo Vinuesa Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, 62210 Morelos, Mexico Multilocus Sequence Analysis (MLSA) is the novel standard in bacterial phylogenetic systematics. However, very few attempts have been made in order to identify the markers (PCR amplicons) with optimal phylogenetic properties for MLSA. We present the rationale and experimental results of a phylogenomic analysis pipeline designed to select such amplicons. The pipeline consists of two interrelated analysis blocks. The first one is designed to estimate a robust species phylogeny based on carefully selected members of the full set of orthologous proteins shared by the target microbial group of interest. The gene families are assembled based on BLASTP bidirectional best hits coupled with MCL clustering. The sizes of the core and pan-genomes are estimated from these data. Maximum likelihood trees are inferred for each of the resulting alignments of orthologous gene products. Several parameters and tree support statistics are calculated for each alignment and tree. Only the most informative trees/alignments are then used for phylogenetic congruence analyses. This filtering allows the identification of highly informative, phylogenetically "well-behaved" and congruent source alignments and trees to infer robust species trees using supermatrix and supertree approaches. The second part is designed to find potential PCR primer pairs binding to the genes selected in the first block producing theoretical (in silico) amplicons of a certain size range, which yield highly informative gene trees that are congruent with the previously estimated (genomic) species tree. Key thermodynamic parameters of each primer pair are also computed. The theoretical amplicons are ranked by their phylogenetic information content and the thermodynamic parameter values of the corresponding oligonucleotides. We have experimentally validated the pipeline by successfully developing the PCR protocols for sesven novel markers, used for biogeographic and evolutionary genetic analyses of a large and diverse collection of Bradyrhizobium isolates. The relative performance of four previously used and the seven novel markers is quantitatively assessed.

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S2-3 Integrating Biolog phenomic analysis with genomic approaches to explore the diversity of natural Sinorhizobium meliloti strains

Emanuele G. Biondi1, Marco Bazzicalupo1, Enrico Tatti2, Carlo Viti2, and Alessio Mengoni1 1Department of Evolutionary Biology, University of Firenze, 50125, Firenze, Italy; 2Department of Agricultural Biotechnology, Section of Microbiology, University of Florence, 50144, Firenze, Italy Sinorhizobium meliloti is a soil bacterium that fixes atmospheric nitrogen in plant roots. The study of its natural populations has revealed high genetic diversity that, by using comparative genomic hybridization (CGH), was estimated to account for 5-10% of the coding sequences. This massive genomic diversity therefore suggests also a high phenotypic differentiation among different strains of the species. Here, the laboratory strain Rm1021 and four natural isolates were analyzed for their colony and cell morphology, doubling time and ability to nodulate Medicago truncatula plants. Then, the phenotypic diversity of the five strains of S. meliloti was systematically assayed using the Phenotype Microarray technique (Biolog). A total number of 571 different growth conditions was tested, including 190 carbon sources, 95 nitrogen sources, 59 phosphorus sources, 35 sulfur sources, and tolerances to different osmolytes and pH conditions. Obtained results showed that most of the variable phenotypes were due to carbon source utilization and tolerance to osmolytes and pH, while few or no differences were scored for nitrogen source and phosphorus and sulfur sources utilization, respectively. Concerning the variability in tolerance to sodium nitrite and ammonium sulfate among the tested strains, an association with genomic polymorphism from CGH analysis was observed. In conclusion, the overall results suggest that the study of functional (phenotypic) variability of S. meliloti could be a new step in the investigation of genetic polymorphism of rhizobia whose results could help in elucidating their evolutionary dynamics and their adaptation to such diverse environments as plant root nodules and soil.

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S2-4 Functional genomic analyses of the diazotrophic grass endophyte Azoarcus sp. strain BH72

Abhijit Sarkar, Lena Hauberg, Lucie Miché, Andrea Krause, and Barbara Reinhold-Hurek Laboratory of General Microbiology, Faculty of Biology and Chemistry, University Bremen, 28334 Bremen Azoarcus sp. strain BH72 is a nitrogen-fixing endophyte that is capable of infecting rice roots and expressing nitrogenase genes in planta. Although endophytic bacterial cells may reach high numbers, plants do not show obvious symptoms of plant disease. This raises the question of mechanisms of compatibility between host and bacterium. The comparative genome sequence analysis of strain BH72 revealed several clues to this question [1]. Strain BH72 appears to be "disarmed" compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactonesûbased communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment. Several platforms for functional genomic analyses were established for strain BH72. In cooperation with Anke Becker (University of Bielefeld), an oligonucleotide-based transcriptome microarray covering 3992 protein-coding genes was developed. First results of the analysis of differential gene expression related to nitrogen fixation and the regulon of key regulatory proteins will be presented. With regard to proteome analysis, a reference map of abundant proteins in two-dimensional gel electrophoresis has been established, and bacterial proteins expressed in rice roots could be identified. Moreover, a protocol for rapid insertional mutagenensis and generation of transcriptional fusions was developed and applied to study in planta gene expression. [1] Krause et al. (2006). Nat. Biotechnol. 24:1385-1391.

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S3-1 Protein secretion in Rhizobium: what is out there? Martin Krehenbrink1,2, Anne Edwards1, and J. Allan Downie1 1Department of Molecular Microbiology, John Innes Centre, Norwich NH4 7UH, UK; 2Unité de Génétique Moléculaire, Institut Pasteur, 75724 Paris, France The genome sequence of Rhizobium leguminosarum bv. viciae 3841 was analyzed for the presence of protein secretion systems and their potential substrates. In addition to the general export pathway (GEP) and a twin-arginine translocase (TAT) system, there are four Type-I systems, one putative Type-IV system, three Type-V autotransporters, and one Type-VI system. Mutations affecting the PrsDE (Type I) and TAT systems affected the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatics analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type-I substrates, 12 of which are secreted via the PrsDE secretion system. Although none of the identified extracellular proteins are essential for symbiosis, many of them have predicted functions compatible with an accessory role in the infection of the legume host. The TAT mutant was defective in symbiosis and formed partially infected nodules incapable of nitrogen fixation. Analysis of the genome sequence using TAT signal prediction programs and genomic comparisons identified over 70 possible TAT substrate proteins, of which 27 were predicted to be periplasmic substrate-binding proteins of ABC uptake systems.

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S3-2 Transcriptional responses of Bradyrhizobium japonicum to environmental changes

Kathrin Lang1, Susanne Zehner1, Tobias Günther1, Felix Hauser2, Andrea Lindemann2, and Michael Göttfert1 1Institut für Genetik, Technische Universität Dresden, 01069 Dresden, Germany; 2Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zürich, 8092 Zürich, Switzerland In the rhizosphere, rhizobia compete with other bacteria for resources. They also have to withstand changing environmental conditions. Rhizobial strains differ in their ability to cope with extreme pH values, high salt concentrations or an elevated temperature. We are studying the effect of these parameters on the transcriptome of Bradyrhizobium japonicum, the symbiont of soybean. After heat shock for 15 minutes at 43°C, more than 150 genes were up-regulated. This included the already known heat shock genes. About 250 genes were down-regulated. The pH of the growth medium strongly influenced the expression pattern. After incubation for 4 hours at pH 8.0 a large number of genes were differentially expressed (fold change ≥2) if compared to data acquired at pH 6.9. In addition to the mentioned parameters, the transcriptome is also influenced by flavonoids released by the plants. Genistein, a soybean signal, activates the LysR-type regulator NodD1 and the two-component regulatory system NodVW. Both systems are required for efficient expression of nodulation genes. Within the wild type, about 100 genes are inducible by genistein. The majority of these genes is not preceded by known promoter elements. In contrast to nodD1, nodW is essential for induction of most of the genes. Seven of the nodW-independent genes seem to be involved in transport processes; the product of the eighth gene contains a patatin-like domain. Our data indicate that genistein has a much broader function than mere induction of nod genes and so far uncharacterised regulators are involved in genistein-dependent responses.

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S3-3 The Sinorhizobium meliloti RNome: identification and characterization of new small non-coding RNAs

José I. Jiménez-Zurdo1, Omar Torres-Quesada1, Coral del Val2, Elena Rivas3 and Nicolás Toro1 1Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain; 2Escuela Técnica Superior de Ingenierías, Informática, Universidad de Granada, 18071 Granada, Spain; 3Howard Hughes Medical Institute, Ashburn, VA 20147, USA Post-genomic research has revealed that the functional untranslated RNA species (collectively referred to as the RNome) are more abundant and diverse than expected in both, prokaryotic and eukaryotic, organisms. In bacteria, most of these newly identified noncoding RNAs are small transcripts (50-400 nucleotides; sRNAs), which generally act as posttranscriptional regulators of gene expression in response to environmental stimuli. Riboregulation involves either the interaction of the sRNA with specific proteins or, most often, basepairing with complementary sequence stretches within 5'-UTR regions of trans-encoded target mRNAs. These antisense interactions ultimately influence the translation and/or stability of the message and require the binding of the riboregulator to the RNA chaperone Hfq. Most of the sRNA genes currently annotated in databases have been identified in Escherichia coli. Nonetheless, in the past few years, the number of sRNAs predicted in other bacterial species (i.e. animal pathogens) has substantially increased [1]. However, the RNomes of the nitrogen-fixing legume endosymbiotic bacteria have remained largely unexplored. We have recently conducted a pioneering genome-wide search for sRNA genes in Sinorhizobium meliloti, the symbiont of alfalfa [2]. First, the intergenic regions (IGRs) were compiled and used as queries to interrogate related α-proteobacterial genomes. The resulting pairwise alignments were then individually scanned with the programs QRNA and RNAz as complementary predictive tools to identify conserved and thermodynamically stable secondary structures, likely corresponding to RNA molecules. Verification of the bioinformatics predictions by Northern hybridization experiments and 5'-end mapping led to the identification of eight previously unknown loci expressing small transcripts and organized in independent transcription units. Seven of these transcripts accumulate differentially under free-living and endosymbiotic conditions. These results predict a role for these sRNAs as trans-acting riboregulators. Details on the computational screen and characterization of the identified sRNAs will be presented. [1] Livny & Waldor (2007). Curr. Opin. Microbiol. 10:96-101. [2] del Val et al. (2007). Mol. Microbiol. 66:1080-1091.

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S3-4 Screening for non-coding RNAs in the archaeal diazotroph Methanosarcina mazei

Dominik Jäger1, Jens Thomsen1, Claudia Ehlers1, Cynthia Sharma2, Jörg Vogel2, Heiko Liesegang3, Wolfgang Hess4, and Ruth A. Schmitz-Streit1 1Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, 24118 Kiel, Germany; 2RNA Biologie, Max-Planck-Institut für Infektionsbiologie Berlin, 10117 Berlin, Germany; 3Laboratorium für Genomanalyse, Georg-August-Universität Göttingen, 37077 Göttingen, Germany; 4Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany Small non-coding RNAs (sRNAs) are an emerging field of research as their global impact in regulatory processes becomes more and more obvious. Noncoding RNAs have been identified in all three domains of life. In Eukarya and Bacteria, functions have been assigned for many sRNAs, however still little is known about sRNAs in Archaea. To get an insight into potential regulatory roles of sRNAs in Archaea, we chose the methanogenic archaeon Methanosarcina mazei strain Gö1 as model system, which is able to fix molecular nitrogen. It is a perfect candidate as, due to its high ecological importance in biogenic methane production, many aspects of the organism's adaptation to different stress situations are currently under investigation (e.g. carbon stress [1], nitrogen limitation [2], and osmotic stress [3]. The goal of this work is to identify noncoding RNAs in M. mazei using two different approaches: (i) computational screens, based on comparative analysis of the three Methanosarcina genomes (M. acetivorans, M. barkeri, and M. mazei) and (ii) direct sequencing of M. mazei cDNA populations by massive parallel sequencing technology. In silico screens predicted more than 600 potential sRNAs in the 4.1-Mb genome of M. mazei. Direct sequencing of M. mazei cDNA populations using the 454-sequencing system resulted in 44 000 reads in total, approximately 14 000 reads of which exclusively correspond to intergenic regions. Results from both approaches will be compared and discussed. [1] Hovey et al. (2005). Mol. Genet. Genomics 273:225-239. [2] Veit et al. (2006). Mol. Genet. Genomics 276:41-55. [3] Pflüger et al. (2007). FEMS Microbiol. Lett. 277:79-89.

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S3-5 SmRegNet: an interactive platform for the analysis of gene regulatory networks in Sinorhizobium meliloti

Elizaveta Krol1, Delphine Capela2, Melanie Barnett3, Jochen Blom4, Jan Baumbach4, Alexander Goesmann4, Sven Rahmann4, and Anke Becker1 1Institute for Biology III, Universität Freiburg, 79104 Freiburg, Germany; 2Laboratoire des Interactions Plantes Microorganismes, Centre National de la Recherche Scientifique-Institut National de la Recherche Agronomique, UMR2594-441, 31326 Castanet-Tolosan Cedex, France; 3Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA; 4Bioinformatics Resource Facility, Centrum for Biotechnology, Bielefeld University, 33501 Bielefeld, Germany The web-based software environment RegNet is an ontology-based data warehouse designed to facilitate the genome-wide reconstruction of transcriptional regulatory interactions in prokaryotes [1, 2]. The database can hold comprehensive information on transcriptional regulation of gene expression derived from experimental studies, computational predictions, and accumulating literature knowledge. During the data import and integration process, links to the corresponding genome annotations and DNA binding sites of transcriptional regulators are generated. The web interface offers several types of search options. The results of a search are displayed in a table-based style and include a visualization of the genetic organization of the respective gene region. Information on DNA binding sites of transcriptional regulators is depicted by sequence logos. The results can also be displayed by several layouters implemented in the graphical user interface, e.g. allowing the visualization of genome-wide network reconstructions and the homology-based inter-species comparison of reconstructed gene regulatory networks. Based on this platform, we have established SmRegNet that includes available experimental information on the regulation of gene expression and co-transcription in Sinorhizobium meliloti. So far, it includes 133 regulatory interactions that involve 330 regulated genes and 61 regulatory proteins of S. meliloti. A future objective is extension of the data content and integration of data from other rhizobia. [1] Baumbach (2007). BMC Bioinformatics 8:429. [2] Baumbach et al. (2007). J. Biotechnol. 129:279-89.

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S3-6 Appraisal of the regulons controlled by the the FixLJ-FixK2-FixK1 cascade in Bradyrhizobium japonicum

Socorro Mesa1, Felix Hauser1, Markus Friberg2, Emmanuelle Malaguti1, Hans-Martin Fischer1, and Hauke Hennecke1 Institutes of 1Microbiology and 2Computational Science, Swiss Federal Institute of Technology, ETH-Zürich, 8092 Zürich, Switzerland Symbiotic N2 fixation in Bradyrhizobium japonicum is controlled by a complex transcription factor network that is composed by two linked regulatory cascades (RegSR-NifA and FixLJ-FixK2) [1]. In the FixLJ-FixK2 cascade, the FixK2 protein plays a key role as distributor of the low-oxygen signal that is perceived and transduced by the superimposed two-component regulatory system FixLJ [2]. FixK2 activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQP and fixGHIS), but also further regulatory genes (rpoN1, nnrR, and fixK1), thus expanding the downstream end of the cascade. Transcriptome analyses have led to a comprehensive and expanded definition of the FixJ, FixK2, and and FixK1 regulons, which respectively consist of 26, 204, and 29 genes, specifically regulated in microaerobically grown cells. Particular attention was addressed to the FixK2-dependent genes, which included a bioinformatics search for putative FixK2 binding sites on DNA (FixK2 boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified FixK2 as the activator [3], we validated as direct targets eight new genes. Interestingly, the adjacent, but divergently, oriented fixK1 and cycS genes shared the same FixK2 box for activated transcription into both directions. This recognition site may also be a direct target for the FixK1 protein, because activation of the cycS promoter required an intact fixK1 gene and either microoxic or anoxic, denitrifying conditions. Two other, unexpected results emerged from this study: (i) specifically FixK1 seemed to exert a negative control on genes that are normally activated by the N2 fixation-specific transcription factor NifA; (ii) a larger number of genes are expressed in a FixK2-dependent manner in endosymbiotic bacteroids than in culture-grown cells, pointing to a possible symbiosis-specific control. [1] Sciotti et al. (2003). J. Bacteriol. 185:5639-5642. [2] Nellen-Anthamatten et al. (1998). J. Bacteriol. 180:5251-5255. [3] Mesa et al. (2005). J. Bacteriol. 187:3329-3338.

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Workshop 2

Towards Integration for Efficient Biological Nitrogen

Fixation Technology: Is a Straightforward Track

between Lab Bench and Dining Table Feasible?

Organised by Youssef Yanni

Kafr El-Sheikh, Egypt

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Programme of Workshop 2 14.00 – 14.25 Ivan Kennedy (Sydney, Australia): Constraints and spurs to application of

biological nitrogen fixation in the global ecosystem 14.25 – 14.50 Jean Jacques Drevon (Montpellier, France): Nodular diagnosis for

integrated improvement of legume symbiotic nitrogen fixation. 14.50 – 15.15 Marina Roumiantseva (St Petersburg, Russia): Alfalfa-Rhizobium salt

tolerant symbioses: achievements and opportunities. 15.15 – 15.40 Coffee break 15.40 – 16.05 Philippe de Lajudie (Montpellier, France): Investigating and promoting

new local legume symbioses for development in West African and Mediterranean countries

16.05 – 16.30 Stewart Smith (Milwaukee, USA): Innovation, technology transfer, and development for global crop production

16.30 – 16.50 Youssef Yanni (Kafr El-Sheikh, Egypt): Performance of common bean and fababean-rhizobial combinations under water stress east and west of the Nile delta

16.50 – 17.20 General discussion and Conclusions

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S1 Constraints and spurs to application of biological N2 fixation in the global ecosystem

Ivan R. Kennedy SUNFix Centre for Nitrogen Fixation, Faculty of Agriculture, Food and Natural Resources, University of Sydney, Sidney, NSW 2006, Australia Just over a decade ago [1], Robert Burris exhorted key international researchers everywhere to more effectively apply the documented wealth of what was already known about BNF –rich body of useful biological and agricultural information. This information has been substantially augmented in the ten years since, particularly in the molecular area, but with only a partial response to the call that was his main objective. Yet the onset of continuing high prices from "peak oil", higher fertilizer prices, and the need for adaptation of farming practices as a result of climate change amplify the consequences of not responding to Burris' call as a matter of urgency in the private and the public sphere. Why has the response been so weak? This presentation will examine and analyze the constraints to more widespread application of BNF, identifying some promising prospects that may now spur its more widespread application. These include the possibly ongoing commodity boom prompted in part by the biofuels versus food debate, new technology for more widespread application of rhizobia to crop and forage legumes and the prospects that may allow farmers to apply plant growth promoting rhizobacteria as a measure that may overcome some of the obstacles to the competition for land in the choice between cereals and legumes. However, there is a pressing need for more coordination between field and laboratory research, a topic on which this presentation will provide some practical prescriptions based on our results in this area. [1] Kennedy & Cocking (1997). Biological Nitrogen Fixation: The Global Challenge and Future Needs, Sidney, SUNFix Press.

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S2 Nodular diagnosis for integrated improvement of legume symbiotic nitrogen fixation

Jean-Jacques Drevon1, Fatma Tajini2, Catherine Pernot1, Hélène Vailhe1, and Mustapha Trabelsi2 1Institut National de la Recherche Agronomique-Montpellier-SupAgro, UMR1222, Biochimie du Sol et de la Rhizosphère, 34060 Montpellier Cedex, France; 2Ecole Supérieure d'Agriculture, 7030 Mateur, Tunisia The nodular diagnosis consists into measuring the nodulation of a legume in an area of production. Its objective is to assess the spatial and temporal variations in the expression of the legume potential for symbiotic nitrogen fixation, and to search for local factors that might limit this symbiosis. The sampling of the field sites where measurements are performed is based on the participation of farmers to the restitution meeting for each cultural campaign. The nodular diagnosis show a large partial variation in bean nodulation either in cereal- or in horticultural- farming systems of southern France. Also a large temporal variation of nodulation was found during 10 years in bean-wheat rotation with the Syndicat des Producteurs de Haricot à Cassoulet. In some sites of Lauragais (France) and Mateur (Tunisia), where low nodulation was presumably due to deficiency of native specific rhizobia, the inoculation with efficient rhizobia increased nodulation and stabilized yield above 2 t/ha under irrigation. The relation of the nodular diagnosis with legume improvement for the rhizobial symbiosis was tested by the participatory assessment of bean recombinant inbred lines contrasting for their efficiency in use of phosphorus for symbiotic nitrogen fixation in organic horticultural farming of Hérault valley. In some sites, soil-P bioavailability significantly increased in the rhizosphere of the most efficient lines. It is concluded that the nodular diagnosis contributed to the integrated approach of the previous Fysame and Aquarhiz projects of the INCOMED program of the European Union. As a prospect the PhytaN pre-project will be presented to adapt the legume-rhizobia symbiosis to low-P soils under osmotic constraints in mediterranean areas of Africa and Europe.

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S3 Alfalfa-Rhizobium salt-tolerant symbioses: achievements and opportunities

Marina Roumiantseva, and Boris Simarov Laboratory of Genetics of Microorganisms, All-Russian Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St.Petersburg, Russia Recently, there has been a scientific and practical demand for the development of salt-tolerant symbioses in preventing land degradation and desertification. Alfalfa is a moderate salinity-tolerant legume can establish symbioses with root nodule bacteria (Sinorhizobium meliloti), which contribute N to plant and through that soil fertility is finally increased. Rhizobia nodulating alfalfa are reputed to be eight times more salt tolerant than the host plant did [1]. A representative collection of S. meliloti isolates trapped from soil samples and recovered from nodules of wild alfalfa species native from four centres of origin in Russia, Kazakhstan, and Uzbekistan were examined by ISRm1021 fingerprinting, IGS-PCR-RFLP, and PCR-RFLP with the primers on several sym genes and by microarray approach. At about 187 chromosomal and plasmid genes are presumably implicated in salt stress protection in rhizobia according to genome hybridizations of isolates differing in salinity with Sm6kPCR biochip of fully sequenced Rm1021. Finally, a group of "salt-sensitive" isolates has accumulated much more divergent DNA regions than a group of isolates possessed higher level of salt tolerance. Nodule-trapped S. meliloti from salinized areas were significantly different in PCR-RFLP of betBC and betS genes from other alfalfa isolates collected at centres of origin that suffered from drought, but not from salinity. It was established that particular rhizobia symbionts could significantly improve alfalfa adaptability toward salinity through increasing yield of plant dry mass and seed production. The ability of alfalfa to accumulate NaCl in green mass or in root tissues was related to particular inoculants. The contribution of plant variety, rhizobia genotypes, salinity, and their community interaction to shoot the dry weight of M. sativa varieties was evaluated and the supreme role of plant-Rhizobium symbiotic interaction was highlighted. Generated data strongly suggest that symbiotic pairs of rhizobia inoculants and host plant varieties should be constructed for particular environments. [1] Ibragimova et al. (2006). Microbiology 75:77-81 (in Russian).

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S4 Investigating and promoting new local legume symbioses for development in West African and Mediterranean countries

Philippe de Lajudie1, Marc Neyra1, Antoine Galiana1, Angèle N'Zoué1, Abdoulaye Sy1, Flore Molouba1, Clémence Chaintreuil1, Lionel Moulin1, Christine Le Roux1, Odile Domergue1, Philippe Jourand1, Adeline Rénier1, C. Merabet2, A. Bekki2, M. Gueye3, Samba Sylla3, I. NDoye3, Diegane Diouf3, T. Wade3, H. Sow4, P. Houngnandan5, A.M. Zoubeirou6, I. Yattara7, O. Sacko7, T. Atallah8, F. Zakhia8, Mohamed Mars9, Mosbah Mahdhi9, Habib Jeder9, A. Filali-Maltouf10, S.H. Mohamed11, and Bernard Dreyfus1 1Laboratoire des Symbioses Tropicales et Méditerranéennes, CIRAD/INRA/IRD/SupAgro/UM2, UMR113, 34398 Montpellier Cedex, France; 2Université d'Oran, Es-Senia, Algeria; 3IRD/ISRA/UCAD, Dakar, Senegal; 4Association sénégalaise pour la promotion du développement à la base, Dakar, Senegal; 5Université d'Abomey Calavi, Cotonou, Benin; 6Faculté des Sciences, Université Abdou Moumouni, Niamey, Niger; 7Université du Mali, Bamako, Mali; 8Faculté d'Agronomie, Université Libanaise, Beirut, Lebanon; 9Faculty of Sciences, Gabès, Tunisia; 10Rabat University, Rabat, Morocco; 11Sebha University, Sebha, Libya In the context of climate change, increasing earth population, and burst of energy cost, legumes should contribute more to both food security and sustainable management of natural resources (water and soils) in the next years. A collaborative work with research groups in several developing countries during the past 20 years focused on investigation and sampling of local wild legumes (herbs, shrubs, and trees) with environmental/agronomic/forestry potential in West Africa and in the Mediterranean region. New symbiotic systems were discovered, resulting in new models for fundamental research, and new applications. This is, for one part, due to their associated microsymbionts, often belonging to unexpected bacterial groups with original physiological/metabolic properties, i.e. photosynthesis, free-living nitrogen fixation, methylothrophy, tolerance to extreme environmental conditions (salinity, aridity, heavy metals, a,d hydrocarbon breakdown), stem nodulation, and beneficial associations with non-legume plants (cereals). This may account for their wider adaptation to a variety of plant species and ecological habitats than previously thought, opening new insights for the domestication of these "multipurpose rhizobia". Indeed, new arable soils are required wordwide, often from degraded lands, affected by aridity, salinity, mining activities, and pollution. Rhizobia may thus participate as tools. Several examples picked up from our diversity investigations over recent years will be presented to illustrate either success stories of beneficial use of these new symbioses or reasonably good perspectives of application of research in different aspects, soil fertility regeneration/maintenance, food crop production optimization (i.e. green manure, nematode control, and associated cultures), and sustainable environmental management. We will present how federations farmers organizations at the local, regional, and national levels became active collaborative partners in these studies, and how results can be efficiently disseminated to their end-user members (small farmers, NGOs, foresters, agronomists, cattle breeders, industrials, etc).

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S5 Innovation, technology transfer, and development for global crop production

R. Stewart Smith EMD Crop BioScience, Inc., Milwaukee, WI 53209, USA The application of microbial and plant/microbial signal molecule technologies continues to provide significant opportunities for enhancing global legume production. The type of product formulation and application will likely vary between countries depending on commercial techniques, distribution infrastructure, and grower management capabilities and practices. However, the innovative process (from concept validation, thru development and successful commercial introduction) should basically remain the same. Legume inoculants have now been expanded to include both the rhizobia bacteria and the appropriate LCO (Nod factor) signal molecule with enhanced plant development and yield features (i.e. Optimize brand product). Successful technology transfer opportunities have expanded with such novel technologies designed to take the basic science through applied science and into crop-enhancing products. The development process must begin with statistically significant field responses to confirm efficacy, however successful product introduction must provide a convenient product delivering a ROI (Return On Investment) of three or greater for the grower. Obtaining benefits from the technology and the frequency for obtaining claimed responses, including yield, establish the product value in the market. With proper pricing structure included in the business plan, and providing the stated benefits for the crop while giving the grower at least a return of currency three times that of the product cost, should result in a successful product launch.

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S6 Performance of common bean and Faba bean-rhizobial combinations under water stress east and west of the Nile delta

Youssef Garas Yanni1, Mohamed Zidan2, Carmen Vargas3, and Frank Dazzo4

Departments of 1Microbiology and 2Plant Nutrition, Sakha Agricultural Research Station, Kafr El-Sheikh, Egypt; 3Department. of Microbiology and Parasitology, University of Seville, 41012 Sevilla, Spain; and 4Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA The symbiotic performance of some common and Faba bean rhizobial strains collected from slightly to highly saline and dry soils of east and west Nile delta were tested in 80 field experiments during the Faba bean seasons 2004/2005, 2005/2006, and 2006/2007 and the common bean seasons 2005, 2006, and 2007. We tested eight registered Faba bean cultivars and a lot of non-identified ones (produced by farmers and stored to be used in subsequent seasons) against nine strains of Rhizobium leguminosarum bv. vicea, and two registered common bean cultivars and non-identified ones against nine Rhizobium phaseoli strains. The used salt-tolerant strains were identified using the "Capillary T-RFLP analysis of the 16S rDNA", then re-tested for authenticity and efficiency as symbiotic micropartners in greenhouse before use in field experimentation. Field soil textures ranged from calcareous, sandy-loam in the west to sandy, sandy-loam, and clay-loamy in the east, either salt-affected and/or often suffering from moderate to severe shortage in irrigation water. Soil water contents were maintained at 100% or 50-60% of their field capacities all over the experimental course (main-plot treatments); three ascending doses of N2-fertilizer (split-plot treatments), and inoculation or not (split-split plot treatments). The symbiotic performances of the isolates varied according to strain/cultivar combinations, as increased plant growth, seed yield, straw, harvest index (% seed yield/seed + straw), and efficiency of used agronomic N fertiliser (kg seed yield/kg N fertilized). Superiority of the researcher package of recommendations over the conventional agro-economical management of the cooperator farmers was found attributable to the optimized crop management practices containing, in addition to inoculation, proper field stand densities, optimized NPK fertilization, integrated pest management, use of highly responsive varieties, and efficiency of researchers to disseminate knowledge to both the extension specialists and the cooperating farmers through a fluent feed/feedback information system.

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Workshop 3

Microbial fate inside and outside plants: what

makes symbiosis beneficial?

Organised by Igor Tikhonovich

St Petersburg, Russia

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Programme of Workshop 3 14.00 – 15.35 Session 1 14.00 – 14.25 Erik Limpens (Wageningen, The Netherlands): Symbiosome

development, a matter of membrane identity 14.25 – 15.50 Peter M. Gresshoff (Brisbane, Australia): Systemic and local regulation

of legume nodulation; receptor kinases, novel signals, targets and activated states

14.50 – 14.15 Kiwamu Minamisawa (Sendai, Japan): Ethylene-mediated interactions between plants and bacteria: rhizobitoxine production and ACC deaminase

15.15 – 15.40 Alex Borisov (St Petersburg, Russia): Legume genes controlling the fate of microbes inside beneficial plant-microbe systems

15.40 – 15.55 Coffee break 15.55 – 17.05 Session 2 15.55 – 16.20 Nikolai Provorov (St Petersburg, Russia): Host-directed evolution of

beneficial microbes 16.20 – 16.35 Svetlana Yurgel (Pullman, USA): Regulation of Sinorhizonium meliloti

nitrogen stress response in free-living cells and in symbiosis with alfalfa 16.35 – 16.50 Alessio Mengoni (Firenze, Italy): Sinorhizobium meliloti populations in

soil, nodules and plant tissues: what is their ecological meaning? 16.50 – 17.05 Natalya Savelieva (St Petersburg, Russia): The evolution of symbiotic

pathway

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S1-1 Symbiosome development, a matter of membrane identity Erik Limpens1, Elena Fedorova1,2, Sergey Ivanov1,2, and Ton Bisseling1

1Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands; 2K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Science, Moscow 127276, Russia Plants can accommodate a range of microbes inside their cells. A common theme in the accommodation of these microbes is the creation of a special host-derived membrane compartment in which the microbes are housed. A striking example of this is the accommodation of nitrogen-fixing Rhizobium bacteria as new (transient) organelles, called symbiosomes, inside the cells of legume root nodules. The bacteria are thought to enter the cells via an endocytosis-like process by which they become individually surrounded by a plant membrane, the symbiosome membrane. In the model legume Medicago truncatula, these symbiosomes remain separate units that divide and differentiate into their mature nitrogen-fixing forms and almost completely fill the infected cells. The formation of symbiosomes is crucial to allow an efficient nitrogen-fixing symbiosis: however, hardly anything is known about the molecular basis of symbiosome formation. Symbiosome formation shows a striking homology to the invasion of animal cells by pathogenic bacteria. Such bacteria manipulate the endocytic pathway of their host to create a pathogen-containing vacuole that prevents its fusion with lysosomes. Similarly, symbiosomes remain separate compartments that do not fuse to form lytic compartments. To understand the mechanism of symbiosome formation, we investigated the involvement of key components of the endocytic pathway during symbiosome formation in M. trucatula. Several key membrane identity markers, such as Rab GTPases and SNARE proteins, were studied to get insight into the identity of the symbiosome membrane in relation to the host endomembrane system. We show that symbiosomes do not interact with Rab5-labeled endosomes or acquire Rab5 at any stage during their development. Symbiosomes do acquire Rab7 from an early stage, which labels both (late) endosomes and the tonoplast. However, (young) symbiosomes do not have vacuolar identity as they lack vacuolar SNARE proteins, which control the fusion of vesicles with the vacuole. In contrast, symbiosomes retain plasma membrane SNARE proteins throughout their development. Therefore, the symbiosome membrane appears to have a plasma membrane-late endosome mosaic identity. As the nodule ages the symbiosis eventually breaks down in the process of senescence, which starts with the fusion of symbiosomes and formation of lytic compartments. At this stage, the symbiosomes do acquire vacuolar SNARE proteins. This indicates that the lack of vacuolar SNARE proteins on the symbiosome membrane is a mechanism to prevent the formation of lytic compartments.

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S1-2 Systemic and local regulation of legume nodulation; receptor kinases, novel signals, targets and activated states

Peter M. Gresshoff, Arief Indrasumunar, Yu-Hsiang Lin, Mark Kinkema, Sureeporn Nontachaiyapoom, Qunyi Jiag, Akira Miyahara, Mikiko Miyagi, DongXue Li, Bandana Biswas, Meng-Han Lin, Dugald Reid, Bernard Carroll, Pick Kuen Chan, Tripty Hirani, Attila Kereszt, and Brett Ferguson Australian Research Council Centre of Excellence for Integrative Legume Research, University of Queensland, St. Lucia, QLD 4072, Australia Nodule formation in legumes is minimally controlled at two levels: (i) the locally-acting perception of the mitogenic signal from the Rhizobium bacterium, and (ii) the systemically-acting Autoregulation of Nodulation (AON), which involves closely related CLAVATA1-like LRR receptor kinases (GmNARK/LjHAR1/MtSUNN) expressed in phloem of most legume tissues; yet its biological activity in nodulation is almost exclusively within the leaf. To dissect AON, the soybean NARK promoter was analyzed to reveal tissue expression domains and a putative phloem-specifying promoter domain. Purified kinase domain of GmNARK was able to autophosphorylate and transphosphorylate itself as well as the newly discovered GmKAPP (kinase-associated protein phosphatase). A bioassay for the AON shoot-derived inhibitor was developed to indicate that the inhibiting principle was extractable, Bradyrhizobium-induced, NARK-dependent, RNAase A- and Proteinase K-resistant, and of small size. Downstream molecular events from GmNARK revealed coordinate expression of genes leading to the synthesis of jasmonic acid. Mutations in LjHAR1 also lead to inhibited main root growth, which was reversed by ethylene insensitivity (ETR1 controlled) in shoots, suggesting a dual role in nodulation and root growth. Increased nodulation, nitrogen gain, and ability to nodulate effectively at suboptimal Bradyrhizobium titers were achieved after overexpression of the GmNFR1 gene. Cloning of GmNFR1 and GmNFR5, complementation of mutants, and nodulation efficiency analysis suggest that the AON circuit acts by perception of the Nod factor-signalling cascade and subsequently targeting its efficiency. [1] Kinkema et al. (2006). Funct. Plant Biol. 33:770-785.

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S1-3 Ethylene-mediated interactions between plants and bacteria: rhizobitoxine production and ACC deaminase

Kiwamu Minamisawa1, Masayuki Sugawara1, Shin Okazaki1, Noriyuki Nukui1, Hiroshi Ezura2, Shima Eda1, and Hisayuki Mitsui1

1Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan; 2Gene Research Center, Tsukuba University, Tsukuba, 305-8572, Japan Ethylene is a gaseous plant hormone that is produced and sensed by plants in response to a wide variety of environmental and developmental cues, such as germination, root formation, pathogen attack, and rhizobial nodulation. Plant-associated bacteria have at least two different strategies for lowering ethylene biosynthesis in plants: inhibitor production and intermediate degradation for ethylene biosynthesis. Bradyrhizobium elkanii produces rhizobitoxine, which strongly inhibits 1-aminocyclopropane-1-carboxylate (ACC) during ethylene biosynthesis in plants. Rhizobitoxine production reduces ethylene evolution from the associated legume roots and enhances nodule formation. B. elkanii rtxAC genes were essential as dihydrorhizobitoxine synthase and desaturase for rhizobitoxine biosynthesis, respectively. A legume, Lotus japonicus, with a mutated ethylene receptor gene produced markedly higher numbers of rhizobial infection threads and nodule primordia. Thus, endogenous ethylene in roots negatively regulates the formation of nodule primordia. The published genome sequences suggest that Burkholderia sp. and Xanthomonas sp. possesss a putative rhizobitoxine transposon on the genome. Plant growth-promoting rhizosphere bacteria, such as Enterobacter cloacae, have ACC deaminase, which degrades ACC, a precursor of a phytohormone ethylene. ACC deaminase gene (acdS) in Mesorhizobium loti was highly expressed in symbiosis. Indeed, the ACC deaminase was verified to enhance nodulation and competitiveness to the host legume by using a disruption mutant of the acdS gene. Rhizobacterial acdS genes were generally induced by ACC via the σ70 promoter, However, M. loti acdS gene was positively regulated by NifA2 and low oxygen with other symbiotic nitrogen fixation genes, such as nifH. The mode of gene expression suggests the participation of M. loti acdS in the establishment of mature nodules by interfering with the production of ethylene. From genome sequences of plant-associated bacteria, we found many genes homologous to rtxAC and acdS, and want to discuss their significance on ethylene-mediated interactions in rhizosphere and endosphere bacteria. [1] Sugawara et al. (2006). Biotechnol. Adv. 24:382-388. [2] Nukui et al. (2006). Appl. Environ. Microbiol. 72:4964-4969. [3] Uchiumi et al. (2004). J. Bacteriol. 186:2439-2448.

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S1-4 Genetic system of legumes controlling interactions with beneficial soil microflora: from fundamentals to applications

Alexey Y. Borisov1, T.N. Danilova1, O.A. Grishina1, Oksana Y. Shtark1, G.A. Akhtemova1, Alexandra A. Krasheninnikova1, A. Moloshionok2, A.E. Kazakov1, T.S. Naumkina2, A.G. Vasilchikov2, Vladimir K. Chebotar1, Vivienne Gianinazzi-Pearson3, and Igor A. Tikhonovich1

1All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia; 2Institute of Grain Legumes and Groat Crops, 303112 Orel, Russia; 3Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique, UMR1088-5184, Plante-Microbe Environnement, Université de Bourgogne, 21065 Dijon Cedex, France The existence of common legume plant genes implicated in interactions with both arbuscular mycorrhizal fungi and beneficial rhizosphere bacteria (including nodule bacteria) creates a theoretical basis for the exploitation of such a plant-microbe system in sustainable agriculture. Great genetic variability by effectiveness of such a system was demonstrated for pea, and therefore, the possibility and necessity of doing breeding to improve the symbiotic potential of legume crops was clearly shown. This, in turn, posed a question of development of new types of complex inoculants to select highly symbiotically effective plants during breeding processes. The field trials (performed during the years 2000-2006) demonstrated highly beneficial effects of such a kind of complex inoculation on plant biomass production and protein content in seeds. Exploitation of such systems in agriculture will allow to decrease application of mineral fertilizers and chemical means for plant protection and will improve the quality of agricultural produce. This work was supported by the grants of RFBR (07-04-01171, 07-04-01558), NWO grant 047.018.001, The EC grant FOOD-CT-2004-506223, Governmental contracts of Russian Ministry of Science and Education (02.512.11.2182, NSc-5399.2008).

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S2-1 Host-directed evolution of beneficial microbes Nikolai A. Provorov All-Russia Research Institute for Agricultural Microbiology, Pushkin 8, 196608 St. Petersburg, Russia Using the model of N2-fixing symbiosis formed by nodule bacteria (rhizobia) with legumes, we present evolution in symbiotic microbes as a programmed process directed by the host plants. Operation of the host-induced microevolutionary factors is based on partners' symbiotic feedbacks, which may be negative (at the early stages of symbiosis development) or positive (at the late stages). We simulate different types of natural selection induced in microbial populations by these feedbacks: Darwinian selection (during differential multiplication/extinction of microbial genotypes inside in planta and ex planta habitats), frequency-dependent selection (during competition for host nodulation), and group selection (during differential multiplication of microbes in in planta habitats). The model analysis suggests that under the conditions of clonal in planta propagation of strains contrasting in symbiotic activity (Fix+ and Fix-), evolution of mutualism may be presented using the models of reciprocal partners' altruism leading to group (inter-deme, kin) selection in favor of Fix+ strains in the host-associated rhizobia populations. Under conditions of mixed (Fix+/Fix-) bacteria propagation in planta, frequency-dependent selection in cooperation with positive partner's feedbacks ensures a stable maintenance or even domination of host-beneficial (Fix+) rhizobia genotypes.

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S2-2 Regulation of Sinorhizobium meliloti nitrogen stress response in free-living cells and in symbiosis with alfalfa

Svetlana N. Yurgel1, Jennifer Topham2, Michael L. Kahn1,2

1Institute of Biological Chemistry, and 2School of Molecular Biosciences, Pullman, WA 99164, USA The nitrogen-fixing symbiosis between rhizobia and legumes is a model of coevolved nutritional complementation. The plants reduce atmospheric CO2 by photosynthesis and provide carbon compounds to associated bacteria; the rhizobia use these compounds to reduce (fix) atmospheric N2 to ammonia, a form of nitrogen the plants can use. A key feature of symbiotic N2 fixation is that N2 fixation is uncoupled from bacterial nitrogen stress metabolism, so that the rhizobia generate "excess" ammonia and release this ammonia to the plant. In the symbiosis between Sinorhizobium meliloti and alfalfa, we have found mutations in GlnD, the major bacterial nitrogen stress response sensor, that lead to a symbiotic relationship in which nitrogen is fixed (Fix+) but the symbiosis is not effective (Eff-) in substantially increasing plant growth. This phenotype implicates the nitrogen stress response in some aspect of symbiotic communication. Analysis of free-living S. meliloti strains carrying mutations in several genes related to nitrogen stress response regulation (glnD, glnB, ntrC, ntrA, and glnK) showed that catabolism of nitrogen-containing compounds depended on the NtrC and GlnD components of the nitrogen stress response cascade. Our data indicate that mutations deleting either or both of the glnK or glnB genes, which remove the capacity to produce the respective PII proteins, GlnK or GlnB, do not have the same effect on the symbiotic phenotype. The glnB and glnK mutants and the double glnB/glnK mutant have some effect on the nitrogen stress response, although the data indicate that the PII proteins can partially substitute for each other to generate an active nitrogen stress response. However, only mutants of GlnD had the unusual Fix+Eff- symbiotic phenotype. These results indicate that bacterial nitrogen stress regulation is important to symbiotic productivity, and suggest that GlnD may act in a novel way that bypasses GlnK/PII and GlnB/PII to influence symbiotic behaviour.

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S2-3 Sinorhizobium meliloti populations in soil, nodules and plant tissues: What is their ecological meaning? Francesco Pini, Emanuele G. Biondi, Lorenzo Ferri, Marco Bazzicalupo, and Alessio Mengoni Dipartimento di Biologia Evoluzionistica, Università degli Studi di Firenze, 50125 Firenze, Italy Strains of the species Sinorhizobium meliloti are ubiquitous in soils and they specifically form symbiotic nitrogen-fixing nodules on the roots of leguminous plants such as alfalfa (Medicago spp.). S. meliloti is a model species to study symbiotic nitrogen-fixation and it has been investigated as a model system for also bacterial population genetics. So far, most of the studies on S. meliloti ecology have been performed on bacteria isolated from nodules of different Medicago species (plant trapping). Actually, the current model for lifestyle of S. meliloti is based on the alternation of free-life in soil and symbiosis with host plant species. In this classical model, due to the biased bacterial sampling (only strains from nodules are isolated) the role and evolutionary significance of free living and nodule-forming strains in a given S. meliloti population cannot be satisfactorily clarified. Actually, almost nothing is known about the presence and especially the genetic diversity of S. meliloti populations free living in soil and endophytically colonizing plants others than legumes [1]. To begin to answer these fundamental questions in the ecology of S. meliloti, we assayed the endophytic ability of wild-type and mutant strains on M. truncatula plants and we developed new PCR tools, based on qPCR and T-RFLP of S. meliloti-specific gene variants, for counting of S. meliloti cells, and analysis of their genetic polymorphism in environmental samples. Populations of S. meliloti present in the DNA extracted from soil, nodules, and leaves of leguminous (alfalfa) and grass species, were analyzed and the relationships between the populations detected in the different habitats were investigated. [1] Chi et al. (2005). Appl. Environ. Microbiol. 71:7271–7278.

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S2-4 The evolution of symbiotic pathway Natalya Saveljeva1, Gerben Bijl2, René Geurts2, Ludmila Lutova1, and Ton Bisseling2

1St. Petersburg State University, St. Petersburg 199034, Russia; 2Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703 HA Wageningen, The Netherlands The evolution of plant symbiotic interactions is one of the interesting scientific questions. An evolutionary queue frequently occurring is based on changes in gene expression, a process named regulatory evolution. Gene expression may evolve through changes in either the activity or the deployment of the proteins (primarily transcription factors) that govern gene expression, or in the cis-regulatory sequences that modulate the expression of individual genes. Also in case of legumes, changes in expression could be a prerequisite for symbiosis with Rhizobium. This work focuses on the identification of specific cis-regulatory elements (CREs) that govern symbiotic legume genes and are absent in orthologous counterparts of non-legumes plant species. Two model plants were used: Medicago truncatula (legume) and Poplar trichocarpa (non-legume). Mutants in the legume Medicago affected in symbiotic signalling where trans-complemented using promoter constructs of the non-legume P. trichocarpa. This revealed that at least for one gene, encoding a calcium/calmodulin kinase (CCaMK), specific cis-regulatory elements are lacking in the poplar construct. To compare both CCaMK promoter regions, reporter constructs were used. Experiments with GUS as reporter showed that both promoters are active in root tissues. To study the temporal and spatial regulation of both promoters, we used the DsRED-E5 reporter construct. DsRED-E5 has a prolonged maturation time, by which it has a bright green fluorescence for a period of 24 h, which subsequently shifts to red. By quantifying the ratio between green and red fluorescence, promoter regulation can be studied at the cellular level. Preliminary results using this reporter showed a strong up-regulation of the Medicago CCaMK promoter in roots of Medicago during symbiotic infection. Next, the regulation of the poplar promoter will be studied.

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List of attendees

293

Last name, first name Email address Affiliation Country Abdelly, Chedly [email protected] Centre de Biotechnologie de Borj

Cédria Tunisia

Abe, Mikiko [email protected] Kagoshima University Japan

Abedin, Fatemeh [email protected] Tarbiat Modarres University Iran

Alexandre, Ana [email protected] University of Évora Portugal

Amini Dehaghi, Majid [email protected] Shahed University Iran

Babic, Katarina [email protected] University of Zagreb Croatia

Baca, Beatriz Eugenia [email protected] Universidad Autónoma de Puebla Mexico

Bailly, Xavier [email protected] University of York UK

Bakkou, Nadia [email protected] University of Geneva Switzerland

Baldani, Ivo [email protected] Embrapa Agrobiologia Brazil

Baldani, Vera [email protected] Embrapa Agrobiologia Brazil

Barnett, Melanie [email protected] Stanford University USA

Batut, Jacques [email protected] Centre National de la Recherche Scientifique

France

Battistoni, Federico [email protected] Instituto de Investigaciones Biologicas

Uruguay

Bazzicalupo, Marco [email protected] University of Florence Italy

Becker, Anke [email protected] University of Freiburg Germany

Berry, Alison [email protected] University of California, Davis USA

Biabani, Abbas [email protected] Gorgan University of Agricultural Sciences and Natural Resources

Iran

Bijl, Gerardus J.M. [email protected] Wageningen University The Netherlands

Billiau, Kenny [email protected] VIB, Ghent University Belgium

Bisseling, Ton [email protected] Wageningen University The Netherlands

Bogusz, Didier [email protected] Institut de Recherche pour le Développement

France

Bonaldi, Katia [email protected] Institut de Recherche pour le Développement

France

Bonatto, Ana Claudia [email protected] Universidade Federal do Paraná Brazil

Bonilla, Ildefonso [email protected] Universidad Autónoma de Madrid Spain

Bonnet, Mariette [email protected] ETH Zürich Switzerland

Borisov, Alexey [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Brau, Lambert [email protected] Murdoch University Australia

Brewin, Nick [email protected] John Innes Centre UK

Brígido, Clarisse [email protected] University of Évora Portugal

Broughton, William [email protected] University of Geneva Switzerland

Burbano Roa, Claudia [email protected] University of Bremen Germany

Capela, Delphine [email protected] Centre National de la Recherche Scientifique

France

Capoen, Ward [email protected] John Innes Centre UK

Cavalcanti de Lucena, Daniella Karine

[email protected] University of Bielefeld Germany

Caviedes Formento, Miguel Angel

[email protected] University of Sevilla Spain

Chaintreuil, Clémence [email protected] Institut de Recherche pour le Développement

France

Chamber, Manuel [email protected] Junta de Andalucia Spain

Charpentier, Myriam [email protected] Ludwig-Maximilians-Universität München

Germany

Chebotar, Vladimir [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Chen, Wen-MIng [email protected] National Kaohsiung Marine University

Taiwan

Chiurazzi, Maurizio [email protected] CNR Italy

Clemente, Maria [email protected] ISV, Centre National de la Recherche Scientifique

France

294

Cosme, Ana [email protected] BSRG - IBB Portugal

Crespi, Martin [email protected] ISV, Centre National de la Recherche Scientifique

France

Dakora, Felix [email protected] Tshwane University of Technology South Africa

Dávila-Martínez, Yadira [email protected] CCG, Universidad Autonóma de México

Mexico

de Bruijn, Frans J. [email protected] INRA/Centre National de la Recherche Scientifique

France

De Cock, Martine [email protected] VIB, Ghent University Belgium

de Lajudie, Philippe [email protected] Institut de Recherche pour le Développement

France

De Meyer, Sofie [email protected] Ghent University Belgium

De Ron, Antonio M. [email protected] MBG- Consejo Superior de Investigaciones Científicas

Spain

De Vos, Paul [email protected] Ghent University Belgium

Deakin, William [email protected] Geneva University Switzerland

Dean, Dennis [email protected] Virginia Tech USA

Debkumari, Bachaspatimayum

[email protected] CMPG – Katholieke Universiteit Leuven

Belgium

del Castillo Madrigal, Inmaculada


Delgado Lopez, Elizabeth [email protected] Benemerita Universidad Autonoma de Puebla

Mexico

Delgado, María J. [email protected] Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas

Spain

Demchenko, Kirill [email protected] Komarov Botanical Institute, Russian Academy of Sciences

Russia

Den Herder, Griet [email protected] Ludwig-Maximilians-Universität München

Germany

D'haeseleer, Katrien [email protected] VIB, Ghent University Belgium

Ding, Yiliang [email protected] John Innes Centre UK

Dixon, Ray [email protected] John Innes centre UK

Dolgikh, Elena [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Dong, Zhongmin [email protected] St. Mary's University Canada

Downie, J Allan [email protected] John Innes Centre UK

Dragomir, Carmen [email protected] Universitatea de Ştiinte Agricole Romania

Dresler-Nurmi, Aneta [email protected] University of Helsinki Finland

Drew, Elizabeth [email protected] South Australian Research and Development Institute

Australia

Dreyfus, Bernard [email protected] Institut de Recherche pour le Développement

France

Eardly, Bertrand [email protected] Penn State University, USA USA

Elmerich, Claudine [email protected] Institut Pasteur France

Endre, Gabriella [email protected] Biological Research Center Hungary

Fauvart, Maarten [email protected] CMPG, Katholieke Universiteit Leuven

Belgium

Fedorova, Elena [email protected] Wageningen University The Netherlands

Ferguson, Gail [email protected] University of Aberdeen UK

Ferreira Santos, Patricia [email protected] Stockholm University Sweden

Ferreira, Paulo [email protected] Universidade Federal do Rio de Janeiro

Brazil

Ferri, Lorenzo [email protected] University of Florence Italy

Fischer, Hans-Martin [email protected] ETH Zürich Switzerland

Forchammer, Karl [email protected] University of Tübingen Germany

Frederix, Marij [email protected] John Innes Centre UK

Frugier, Florian [email protected] Centre National de la Recherche Scientifique

France

Fuentes, Luis [email protected] Universidad Autónoma de Puebla Mexico

Gałązka, Anna [email protected] Institute of Soil Science and Plant Cultivation

Poland

Gallina, Onofrio [email protected] Consejo Superior de Spain

295

Investigaciones Científicas

Gamas, Pascal [email protected] Centre National de la Recherche Scientifique-INRA

France

Gao, Mengsheng [email protected] University of Florida USA

Garg, Neera [email protected] Panjab University India

Gehlot, Hukam [email protected] J.N.Vyas University India

Geurts, René [email protected] Wageningen University The Netherlands

Gillis, Moniek [email protected] Ghent University Belgium

Giraud, Eric [email protected] Institut de Recherche pour le Développement

France

Giuntini, Elisa [email protected] University of York UK

Glöer, Jens [email protected] CAU Kiel Germany

Gobbato, Enrico [email protected] John Innes Centre UK

González, Victor [email protected] Centro de Ciencias Genómicas-UNAM

Mexico

González-Andrés, Fernando

[email protected] Universidad de León Spain

Goormachtig, Sofie [email protected] VIB, Ghent University Belgium

Gossmann, Jasmin Anna [email protected] University of Münich Germany

Göttfert, Michael [email protected] TU Dresden Germany

Gourion, Benjamin [email protected] ETH-Zürich Switzerland

Gresshoff, Peter [email protected] ARC Centre for Integrative Legume Research

Australia

Guinel, Frederique [email protected] Wilfrid Laurier University Canada

Gultyaev, Alexander [email protected] Leiden University The Netherlands

Gupta, Rajan Kumar [email protected] Government Post Graduate College India

Haag, Andreas [email protected] University of Aberdeen UK

Hartmann, Anton [email protected] Helmholtz Zentrum München, German Research Center for Environmental Health

Germany

Hashimoto, Masahito [email protected] Kagoshima University Japan

Hauberg, Lena [email protected] Universität Bremen Germany

Hayashi, Makoto [email protected] Ludwig-Maximilians-Universität München

Germany

Heckmann, Anne B. [email protected] University of Aarhus Denmark

Hemerly, Adriana [email protected] Federal University of Rio de Janeiro Brazil

Hervé, Christine [email protected] LIPM France

Hirsch, Sibylle [email protected] John Innes Centre UK

Holsters, Marcelle [email protected] VIB, Ghent University Belgium

Huergo, Luciano [email protected] UFPR Brazil

Hynes, Michael [email protected] University of Calgary Canada

Ivanov, Sergey [email protected] Wageningen University The Netherlands

James, Euan [email protected] University of Dundee UK

Janczarek, Monika [email protected] Marie-Curie Sklodowska University Poland

Jean-Jacques, Drevon [email protected] Institut National de la Recherche Agronomique-SupAgro Montpellier

France

Jiménez-Zurdo, José I. [email protected] Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas

Spain

Kaiser, Brent [email protected] The University of Adelaide Australia

Kalita, Michal [email protected] University of M. Curie-Sklodowska Poland

Kalo, Peter [email protected] Agricultural Biotechnology Center Hungary

Karojet, Silke [email protected] Max-Planck-Institute of Molecular Plant Physiology

Germany

Kennedy, Ivan [email protected] University of Sydney Australia

Khademian, Hossein [email protected] Centre National de la Recherche Scientifique

France

296

Kim, Jiyoung [email protected] John Innes Centre UK

Klaus, Dörte [email protected] Laboratoire des Interactions Plantes Micro-organismes

France

Knox, Oliver [email protected] Scottish Agricultural College UK

Kondorosi, Eva [email protected] ISV, Centre National de la Recherche Scientifique

France

Koziol, Uriel [email protected] Instituto de Investigaciones Biologicas Clemente Estable

Uruguay

Krause, Andrea [email protected] University of Bremen, FB2 Germany

Krehenbrink, Martin [email protected] Institut Pasteur France

Krishnen, Ganisan [email protected] University of Sydney Australia

Krol, Elizaveta [email protected] Freiburg University Germany

Król, Maria [email protected] Institute of Soil Science and Plant Cultivation

Poland

Krusell, Lene [email protected] University of Aarhus Denmark

Krysciak, Dagmar [email protected] University Hamburg Germany

Kucho, Ken-ichi [email protected] Kagoshima University Japan

Küster, Helge [email protected] Bielefeld University Germany

Kutkowska, Jolanta [email protected] Maria Curie-Skłodowska University Poland

Labeeuw, Hendrik [email protected] VIB, Ghent University Belgium

Lara-Flores, Miguel [email protected] CCG, Universidad Nacional Autónoma de México

Mexico

Laranjo, Marta Sofia [email protected] University of Évora Portugal

Lepetit, Marc [email protected] INRA France

Lerner, Anat [email protected] Hebrew University of Jerusalem Belgium

Lillo, Alessandra [email protected] Wageningen University The Netherlands

Limpens, Erik [email protected] Wageningen University The Netherlands

Lin, Min [email protected] Biotechnology Research Institute, Chinese Academy of Agricultural Sciences

China

Lindström, Kristina [email protected] University of Helsinki Finland

Lutova, Ludmila [email protected] St. Petersburg State University Russia

Madsen, Lene H. [email protected] University of Aarhus Denmark

Magori, Shimpei [email protected] University of Tokyo Japan

Malek, Wanda [email protected] University M. Curie-Sklodowska Poland

Maltempi De Souza, Emanuel

[email protected] UFPR Brazil

Manyani, Hamid [email protected] University of Sevilla Spain

Markmann, Katharina [email protected] Ludwig-Maximilians-Universität München

Germany

Marczak, Małgorzata [email protected] Maria Curie-Skłodowska University Poland

Marinković, Jelena [email protected] Institute of Field and Vegetable Crops

Serbia

Martens, Miet [email protected] Ghent University Belgium

Martinez-Salazar, Jaime [email protected] CCG, Universidad Autónoma de México

Mexico

Mateos, Pedro [email protected] University of Salamanca Spain

Mazur, Andrzej [email protected] University of Maria Curie Skłodowska

Poland

Megias, Esau [email protected] University of Sevilla Spain

Megias, Manuel [email protected] University of Sevilla Spain

Mengoni, Alessio [email protected] University of Florence Italy

Mergaert, Peter [email protected] Centre National de la Recherche Scientifique

France

Merritt, Kyle [email protected] Mapleton Agri Biotec Australia

Mesa, Socorro [email protected] ETH-Zürich Switzerland

Mhamdi, Ridha [email protected] CBBC Tunisia

Miché, Lucie [email protected] Institut de Recherche pour le Développement

France

297

Michiels, Jan [email protected] CMPG, Katholieke Universiteit Leuven

Belgium

Miclea, Sebastian Paul [email protected] Hungarian Academy of Science, Biological Research Center

Hungary

Mierzwa, Bożena [email protected] Maria Curie-Skłodowska University Poland

Minamisawa, Kiwamu [email protected] Tohoku University Japan

Miyahara, Akira [email protected] John Innes Centre UK

Moënne-Loccoz, Yvan [email protected] Université Lyon 1 France

Moling, Sjef [email protected] Wageningen UR The Netherlands

Moreira, Fatima [email protected] Federal University of Lavras Brazil

Morieri, Giullia [email protected] John Innes Centre UK

Mortier, Virginie [email protected] VIB, Ghent University Belgium

Mosbah, Mahdhi [email protected] Faculty of Science of Gabès Tunisia

Moutia, Jean-Francois [email protected] CMPG, Katholieke Universiteit Leuven

Belgium

Mrkovaski, Nastasija [email protected] Institute of Field and Vegetable Crops

Serbia

Mulas, Daniel [email protected] Universidad de León Spain

Mulley, Geraldine [email protected] University of Reading UK

Munive, José-Antonio [email protected] BUAP Mexico

Murakami, Ei-ichi [email protected] Kagoshima University Japan

Nag, Papri [email protected] Jadavpur University India

Nordlund, Stefan [email protected] Stockholm University Sweden

Normand, Philippe [email protected] Université Lyon France

Nouwen, Nico [email protected] Institut de Recherche pour le Développement

France

N'zoué, Angèle Affoué [email protected] Institut de Recherche pour le Développement

France

Oelkers, Karsten [email protected] Australian National University Australia

Oetjen, Janina [email protected] Universität Bremen Germany

Oliveira, Solange [email protected] University of Évora Portugal

Olroyd, Giles [email protected] John Innes Centre UK

Osipova, Maria [email protected] St-Petersburg State University Russia

Ott, Thomas [email protected] Ludwig-Maximilians-Universität München

Germany

Ovchinnikova, Evgenia [email protected] Wageningen University The Netherlands

Oyaizu, Hiroshi [email protected] University of Tokyo Japan

Palacios, Jose Manuel [email protected] Universidad Politécnica de Madrid Spain

Parniske, Martin [email protected] Ludwig-Maximilians-Universität München

Germany

Pawlowski, Katharina [email protected] Stockholm University Sweden

People, Mark [email protected] CSIRO Australia

Perret, Xavier [email protected] University of Geneva Switzerland

Peters, John [email protected] Montana State University USA

Pietraszewska, Anna [email protected] SILS, University of Amsterdam The Netherlands

Pini, Franceso [email protected] University of Florence Italy

Pislariu, Catalina [email protected] The Samuel Roberts Noble Foundation

USA

Platero, Raul [email protected] Institute of Biological Investigation Clemente Estable

Uruguay

Pobigaylo, Nataliya [email protected] Freiburg University Germany

Poole, Philip [email protected] John Innes Centre UK

Portz, Daniela [email protected] Bayer CropScience AG Germany

Prell, Jurgen [email protected] John Innes Centre UK

Provorov, Nikolay [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Pule-Meulenberg, Flora [email protected] Tshwane University of Technology South Africa

298

Queiroux, Clothilde [email protected] Université de Lyon 1 France

Quinto , Carmen [email protected] Universidad Nacional Autonoma de Mexico

Mexico

Radutoiu, Simona [email protected] University of Aarhus Denmark

Ramaekers, Lara [email protected] CMPG, Katholieke Universiteit Leuven

Belgium

Ramirez, Miguel [email protected] CCG, Universidad Autonoma de México

Mexico

Räsänen, Leena [email protected] Universtiy of Helsinki Finland

Ratet, Pascal [email protected] ISV, Centre National de la Recherche Scientifique

France

Reeve, Wayne [email protected] Murdoch University Australia

Reinhold-Hurek, Barbara [email protected] University Bremen Germany

Reis, Veronica [email protected] Embrapa Agrobiologia Brazil

Robledo, Marta [email protected] Universidad de Salamanca Spain

Rodino, Ana Paula [email protected] MBG, Consejo Superior de Investigaciones Científicas

Spain

Rodriguez, Dulce [email protected] IFAPA, Consejeria de Agricultura y Pesca

Spain

Rodriguez-Llorente, Ignacio


Rogers, Christian [email protected] John Innes Centre UK

Rombauts, Stephane [email protected] VIB, Ghent University Belgium

Roumiantseva, Marina [email protected] Research Institute for Agricultural microbiology

Russia

Rubio, Luis [email protected] University of California-Berkeley USA

Ruiz-Sainz, Jose E. [email protected] University of Sevilla Spain

Salinas Berna, Paloma [email protected] Universidad de Alicante Spain

Sanchez, Federico [email protected] Universidad Nacional Autonoma de Mexico

Mexico

Sandal, Niels [email protected] University of Aarhus Denmark

Santoyo-Paez, Yuriria [email protected] CIBA-IPN Mexico

Sarkar, Abhijit [email protected] University of Bremen Germany

Savelyeva, Natalya [email protected] Wageningen University The Netherlands

Schmitz-Streit, Ruth [email protected] University of Kiel, Institute for Microbiology

Germany

Selão, Tiago [email protected] Stockholm University Sweden

Shinano, Takuro [email protected] NARCH Japan

Shtark, Oksana [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Sikora, Sanja [email protected] Faculty of Agriculture University of Zagreb

Croatia

Silva, Krisle [email protected] Federal University of Lavras Brazil

Singh, Manoj [email protected] Banaras Hindu University India

Sinharoy, Senjuti [email protected] University of Calcutta India

Skorupska, Anna [email protected] Maria Curie-Skłodowska University Poland

Slavny, Peter [email protected] John Innes Centre UK

Smith, Stewart [email protected] EMD Crop BioScience USA

Soto, María José [email protected] Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas

Spain

Spaepen, Stijn [email protected] CMPG, Katholieke Universiteit Leuven

Belgium

Spruyt, Karel [email protected] VIB, Ghent University Belgium

Stajkovic, Olivera [email protected] Institute of Soil Science Serbia

Stougaard, Jens [email protected] University of Aarhus Denmark

Streit, Wolfgang [email protected] University of Hamburg Germany

Sun, Jongho [email protected] John Innes Centre UK

Suzuki, Akihiro [email protected] Saga University Japan

Svistoonoff, Sergio [email protected] Institut de Recherche pour le Développement

France

299

Taulé, Cecilia [email protected] Instituto de Investigaciones Biológicas Clemente Estable

Uruguay

Teaumroong, Neung [email protected] Suranaree University of Technology Thailand

Teixeira, Pedro [email protected] Stockholm University Sweden

Tian, Chang Fu [email protected] Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique

France

Tikhonovich, Igor [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Tittabutr, Panlada [email protected] Suranaree University of Technology Thailand

Tomitani, Akiko [email protected] Japan Agency for Marine-Earth Science and Technology

Japan

Torres-Quesada, Omar [email protected] Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas

Spain

Town, Chris [email protected] J Craig Venter Institute USA

Tsyganov, Viktor [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Tsyganova, Anna [email protected] All-Russia Research Institute for Agricultural Microbiology

Russia

Uchiumi, Toshiki [email protected] Kagoshima Univeristy Japan

Untergasser, Andreas [email protected] Wageningen University The Netherlands

Van de Elsacker, Suzanne

[email protected] Ghent University Belgium

Van de Velde, Willem [email protected] ISV, Centre National de la Recherche Scientifique

France

Vanderleyden, Jos [email protected] CMPG, Katholieke Universiteit Leuven

Belgium

Vanderlinde, Elizabeth [email protected] University of Regina Canada

Ventura Guedes, Helma [email protected] Embrapa Agrobiologia Brazil

Vercruysse, Maarten [email protected] CMPG, Katholieke Universiteit Leuven

Belgium

Vereecke, Danny [email protected] VIB, Ghent University Belgium

Vessey, Kevin [email protected] Saint Mary's University Canada

Vestergaard, Gitte [email protected] University of Aarhus Denmark

Villegas, Maria del Carmen

[email protected] Instituto Politecnico Nacional Mexico

Vintila, Simina [email protected] Stockholm university Sweden

Vinuesa, Pablo [email protected] CCG, Universidad Autónoma de México

Mexico

Wang, Dong [email protected] Stanford University USA

Wang, He [email protected] Stockholm University Sweden

Wdowiak-Wrobel, Sylwia [email protected] Maria Curie Skłodowska University Poland

Wehmeier, Silvia [email protected] University of Aberdeen UK

Weidner, Stefan [email protected] Universität Bielefeld Germany

Wibroe Nielsen, Mette [email protected] University of Aarhus Denmark

Willems, Anne [email protected] Ghent University Belgium

Williams, Paul [email protected] Becker Underwood Ltd UK

Wisniewski-Dyé, Florence [email protected] Université Lyon 1 France

Xavier, Ilungo [email protected] Novozymes BioAg Canada

Xie, Fang [email protected] John Innes Centre UK

Yamaura, Masatoshi [email protected] Kagoshima University Japan

Yanni, Youssef Garas [email protected] Sakha Agricultural Research Station Egypt

Yoneyama, Tadakatsu [email protected] University of Tokyo Japan

Yoshida, Ken-ichi [email protected] Kobe University Japan

Young, Peter [email protected] University of York UK

Yurgel, Svetlana [email protected] Washington State University USA

Zehner, Susanne [email protected] TU Dresden Germany

General Information

301

Conference venue The Conference takes place in the Aula Academica located in the Voldersstraat 9 in the heart of the city. The Aula consists of several spacious rooms allowing a comfortable organisation of the different parts of the Conference. Registration takes place in the Academic Council Foyer, which also houses a permanent secretariat. The sessions are held in the auditorium that can accommodate 300 attendees. In the Peristilium, the poster sessions, sandwich lunches, and coffee breaks are organised. Also there is room for sponsor exhibitions and a computer corner with access to the Internet. It is possible to have small meetings of up to 20 people in the VIP Salon. Contact the staff at the registration desk in case you would like to organise such a meeting.

Workshops Workshop 1 and Workshop 2 are organised in the Academic Council Room, whereas Workshop 3 is organised in the auditorium. Registration/Information desk/Message board Registration takes place in the Academic Council Foyer, which also houses the permanent secretariat. A message board is located at the registration desk. Organising secretariat The Professional Congress Organiser Evident takes care of registration, information on transportation to and from Ghent, assistance in hotel bookings, and the permanent secretariat on-site. Phone: +32 (0)479 608082 ask for Celine Monbailliu. Languages The official language in Gent is Flemish, but most citizens also speak French, English and/or German. The official Conference language is English. Speakers and Chairs The speakers are asked to upload their presentation file from a memory key or CD at least 2 hours before their talk or on the day before if their presentation is in the morning to allow checking compatibility. To the first speakers of Workshop 1 on Saturday August 30, we ask to upload your presentations at 8:00 am or preferentially to send your file in advance to Hendrik Labeeuw via a secured ftp location. The session chairs should be present in the auditorium at least 15 minutes before the beginning of their session. Speakers are asked to report to their chairs in due time before their lecture starts. Badges The name badge issued on registration is required for admission to the scientific sessions, the walk through Gent and the Conference dinner. If the badge is lost, please contact the registration desk.

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Coffee breaks, lunches and poster sessions The registration fee includes the abstract book, welcome coffee during registration, a welcome reception, lunches, coffee and refreshments during the breaks, food and drinks during the poster sessions, a guided walk through the historical centre of Gent and a Conference banquet and party. Not included are travel and accommodation expenses, and dinner on Sunday. The poster sessions, sandwich lunches and coffee breaks are organised in the Peristilium. Lunch on Monday and Tuesday is served in the student restaurant “ De Brug” located in the Sint-Pietersnieuwstraat 45 at a 15-minute walk from the Aula.

Posters Poster size: portrait format, maximum 90 cm wide and 130 cm high. All posters have been ordered alphabetically by presenting author and been assigned a number that you can find in the abstract book (PSx-y, x=session; y= poster number). This number is mentioned on the poster board. Posters can be mounted with clips provided by the conference organiser on Saturday, August 30 or at the latest at 17:00 on Sunday, August 31. The posters will be displayed throughout the meeting. Authors presenting posters are requested to be available for answering questions during their poster session. Poster presentations are on Sunday (even numbers) from 18:00 until 20:00 and on Monday (uneven numbers) from 18:20 until 21:00. All posters must be dismantled by Tuesday, September 2 at 19:00.

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Social events The guided walk through Gent on Sunday starts and ends at the Aula. The Conference dinner takes place in the cultural centre “ De Centrale” located in Ham 74 at a 20-minute walk from the Aula.

Bank and currency exchange The Belgian currency is the Euro. All prices in Belgium include taxes and service. In the town centre there are banks and exchange offices. In most places, credit cards are accepted. Cash points open 24/7 are located all over the city.

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Getting around The centre of Gent is quite small, so you can walk around. As the city council made the centre free of cars, it is now a very welcoming and open area. The public transportation system in Gent is excellent. It consists of trams, busses, and the unusual trolleybus. A single ticket costs € 1.50 if bought on the bus/tram/trolleybus or € 1.20 if bought from ticket machines or small kiosks called lijnwinkels. If the bus/tram/trolleybus stop has a ticket machine, you will have to buy the ticket there, because the driver will not sell you one in this case. A ticket is valid for a one-hour's travel on all trams and buses. The trams are the quickest and most comfortable way to travel, especially from the railway station to the city centre. The transportation company is De Lijn. In the Lijnwinkel kiosk (located near Sint-Pieters train station), you can get a free map of the city and its surroundings, with all bus, trolleybus and tram lines. Please contact the staff at the registration desk for assistance. Emergency phone numbers Police 100 Fire department 101 Ambulance 101 Meeting issues + 32 (0) 479 608082 Celine Monbailliu + 32 (0) 477 136820 Danny Vereecke Insurance and liability Neither the organisers, nor the Aula Academica take any responsibility for injury or damage involving persons or property during the Conference. Participants are advised to take their own health and travel insurance. Tourist Information Office Dienst Toerisme Gent Botermarkt 17a (Raadskelder) B-9000 GENT Tel: +32 (0)9 266 52 32 Fax: +32 (0)9 225 62 88 e-mail: [email protected] Conference website http://nfix2008.psb.ugent.be/

mailto:[email protected]

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NOTES

Dear friends and participants Welcome16.00 –18.00 Session 3: Nitrogenase and regulation of...

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