The symbiotic transcription factor MtEFD and cytokinins are positively acting in the Medicago truncatula and Ralstonia solanacearum pathogenic interaction.

• A plant-microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont Sinorhizobium meliloti. • Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts. • Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor. • The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.

[1]  A. Loraine,et al.  Identification of Cytokinin-Responsive Genes Using Microarray Meta-Analysis and RNA-Seq in Arabidopsis1[C][W][OA] , 2013, Plant Physiology.

[2]  M. Kojima,et al.  Cytokinins act synergistically with salicylic acid to activate defense gene expression in rice. , 2013, Molecular plant-microbe interactions : MPMI.

[3]  J. Weissenbach,et al.  Genome sequence of the plant pathogen Ralstonia solanacearum , 2002, Nature.

[4]  M. Van Montagu,et al.  Identification of Rhodococcus fascians cytokinins and their modus operandi to reshape the plant , 2009, Proceedings of the National Academy of Sciences.

[5]  M. Kojima,et al.  The cytokinin-activated transcription factor ARR2 promotes plant immunity via TGA3/NPR1-dependent salicylic acid signaling in Arabidopsis. , 2010, Developmental cell.

[6]  C. Town,et al.  Transcript Analysis of Early Nodulation Events in , 2006 .

[7]  Masayuki Higuchi,et al.  Cytokinin Signaling and Its Inhibitor AHP6 Regulate Cell Fate During Vascular Development , 2006, Science.

[8]  I. Maksimov,et al.  Changes in the Levels of IAA, ABA, and Cytokinins in Wheat Seedlings Infected with Tilletia caries , 2002, Russian Journal of Plant Physiology.

[9]  E. Martino,et al.  Biotic and Abiotic Stimulation of Root Epidermal Cells Reveals Common and Specific Responses to Arbuscular Mycorrhizal Fungi1[W] , 2009, Plant Physiology.

[10]  G. Weiller,et al.  A gene expression atlas of the model legume Medicago truncatula. , 2008, The Plant journal : for cell and molecular biology.

[11]  B. Usadel,et al.  The Lipopolysaccharide of Sinorhizobium meliloti Suppresses Defense-Associated Gene Expression in Cell Cultures of the Host Plant Medicago truncatula1[W][OA] , 2006, Plant Physiology.

[12]  H. Fukuda,et al.  Functional Analyses of LONELY GUY Cytokinin-Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis[W][OA] , 2009, The Plant Cell Online.

[13]  T. Boller,et al.  The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity. , 2014, The New phytologist.

[14]  Brian S. Yandell,et al.  Practical Data Analysis for Designed Experiments , 1998 .

[15]  M. Crespi,et al.  Cytokinin: secret agent of symbiosis. , 2008, Trends in plant science.

[16]  D. Shibata,et al.  Large-scale analysis of gene expression profiles during early stages of root nodule formation in a model legume, Lotus japonicus. , 2004, DNA research : an international journal for rapid publication of reports on genes and genomes.

[17]  C. Boucher,et al.  Characterization of the cis-Acting Regulatory Element Controlling HrpB-Mediated Activation of the Type III Secretion System and Effector Genes in Ralstonia solanacearum , 2004, Journal of bacteriology.

[18]  Elodie Sartorel,et al.  Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula. , 2007, Molecular plant-microbe interactions : MPMI.

[19]  J. Vasse,et al.  Xylem Colonization by an HrcV - Mutant of Ralstonia solanacearum Is a Key Factor for the Efficient Biological Control of Tomato Bacterial Wilt , 1998 .

[20]  M. Djordjevic,et al.  Crosstalk between the nodulation signaling pathway and the autoregulation of nodulation in Medicago truncatula. , 2011, The New phytologist.

[21]  Thomas D. Wu Large-scale analysis of gene expression profiles , 2002, Briefings Bioinform..

[22]  Interactions of beneficial and detrimental root-colonizing filamentous microbes with plant hosts , 2013, Genome Biology.

[23]  Jonathan D. G. Jones,et al.  The plant immune system , 2006, Nature.

[24]  J. Gouzy,et al.  Expression Profiling in Medicago truncatula Identifies More Than 750 Genes Differentially Expressed during Nodulation, Including Many Potential Regulators of the Symbiotic Program1[w] , 2004, Plant Physiology.

[25]  S. Balzergue,et al.  NFP, a LysM protein controlling Nod factor perception, also intervenes in Medicago truncatula resistance to pathogens. , 2013, The New phytologist.

[26]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[27]  C. Boucher,et al.  The hrpB and hrpG regulatory genes of Ralstonia solanacearum are required for different stages of the tomato root infection process. , 2000, Molecular plant-microbe interactions : MPMI.

[28]  C. Ryu,et al.  Cytokinins and plant immunity: old foes or new friends? , 2011, Trends in plant science.

[29]  C. Town,et al.  Transcript Analysis of Early Nodulation Events in Medicago truncatula12[W] , 2005, Plant Physiology.

[30]  H. Kouchi,et al.  From defense to symbiosis: limited alterations in the kinase domain of LysM receptor-like kinases are crucial for evolution of legume-Rhizobium symbiosis. , 2011, The Plant journal : for cell and molecular biology.

[31]  D. Cook,et al.  A Legume Ethylene-Insensitive Mutant Hyperinfected by Its Rhizobial Symbiont , 1997, Science.

[32]  D. Regier,et al.  Cytokinin production by Agrobacterium and Pseudomonas spp , 1987, Journal of bacteriology.

[33]  G. Stacey,et al.  Effects of Endogenous Salicylic Acid on Nodulation in the Model Legumes Lotus japonicus and Medicago truncatula1[W] , 2006, Plant Physiology.

[34]  T. Bisseling,et al.  Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction. , 1997, Development.

[35]  Johanna Sjöberg,et al.  Arbuscular mycorrhizal fungi , 2005 .

[36]  S. Genin,et al.  Secreted proteins from Ralstonia solanacearum: a hundred tricks to kill a plant. , 2009, Current opinion in microbiology.

[37]  T. Pustovoĭtova,et al.  Changes in the Levels of IAA and ABA in Cucumber Leaves under Progressive Soil Drought , 2004, Russian Journal of Plant Physiology.

[38]  N. Melamed-Book,et al.  ROS production during symbiotic infection suppresses pathogenesis-related gene expression , 2012, Plant signaling & behavior.

[39]  A. Mithöfer Suppression of plant defence in rhizobia-legume symbiosis. , 2002, Trends in plant science.

[40]  W G Hines,et al.  Increased power with modified forms of the Levene (Med) test for heterogeneity of variance. , 2000, Biometrics.

[41]  A. Jauneau,et al.  Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development[C][W][OA] , 2009, Plant Physiology.

[42]  H. Delacroix,et al.  Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches , 2010, PloS one.

[43]  J. Vasse,et al.  Abortion of infection during the Rhizobium meliloti—alfalfa symbiotic interaction is accompanied by a hypersensitive reaction , 1993 .

[44]  A. Rashotte,et al.  The CRF domain defines Cytokinin Response Factor proteins in plants , 2010, BMC Plant Biology.

[45]  P. Poole,et al.  The rules of engagement in the legume-rhizobial symbiosis. , 2011, Annual review of genetics.

[46]  D. Burritt,et al.  Influence of plant hormones on virus replication and pathogenesis-related proteins inPhaseolus vulgarisL. infected with white clover mosaic potexvirus , 1998 .

[47]  Kathryn M. Jones,et al.  How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model , 2007, Nature Reviews Microbiology.

[48]  M. Delledonne,et al.  Expression Dynamics of the Medicago truncatula Transcriptome during the Symbiotic Interaction with Sinorhizobium meliloti: Which Role for Nitric Oxide?1[C][W][OA] , 2012, Plant Physiology.

[49]  J. Gouzy,et al.  Transcription Reprogramming during Root Nodule Development in Medicago truncatula , 2011, PloS one.

[50]  G. Bécard,et al.  Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. , 2001, Molecular plant-microbe interactions : MPMI.

[51]  C. Boucher,et al.  Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered , 1987, Journal of bacteriology.

[52]  P. Abad,et al.  Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes. , 2012, The New phytologist.

[53]  Bruno Müller,et al.  Cytokinin signaling networks. , 2012, Annual review of plant biology.

[54]  G. Oldroyd,et al.  Crosstalk between jasmonic acid, ethylene and Nod factor signaling allows integration of diverse inputs for regulation of nodulation. , 2006, The Plant journal : for cell and molecular biology.

[55]  F. Frugier,et al.  EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[W] , 2008, The Plant Cell Online.

[56]  R. O’Malley,et al.  Expansin Message Regulation in Parasitic Angiosperms: Marking Time in Development , 2000, Plant Cell.

[57]  S. Genin Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum. , 2010, The New phytologist.

[58]  Ron Edgar,et al.  Gene Expression Omnibus ( GEO ) : Microarray data storage , submission , retrieval , and analysis , 2008 .

[59]  F. Ariel,et al.  Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[W][OA] , 2012, Plant Cell.

[60]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[61]  A. Bellec,et al.  MtQRRS1, an R-locus required for Medicago truncatula quantitative resistance to Ralstonia solanacearum. , 2013, The New phytologist.

[62]  M. Crespi,et al.  The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[W] , 2006, The Plant Cell Online.

[63]  P. Royston A Remark on Algorithm as 181: The W‐Test for Normality , 1995 .

[64]  R. Wing,et al.  LysM-Type Mycorrhizal Receptor Recruited for Rhizobium Symbiosis in Nonlegume Parasponia , 2011, Science.

[65]  Murray Grant,et al.  Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. , 2011, Annual review of phytopathology.

[66]  S. Genin,et al.  Pathogenomics of the Ralstonia solanacearum species complex. , 2012, Annual review of phytopathology.

[67]  N. McRoberts,et al.  Are green islands red herrings? Significance of green islands in plant interactions with pathogens and pests , 2007, Biological reviews of the Cambridge Philosophical Society.

[68]  Kun Xu,et al.  The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations. , 2008, The Plant journal : for cell and molecular biology.

[69]  M. Valls,et al.  Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era. , 2013, Molecular plant pathology.

[70]  Karam B. Singh,et al.  The B-3 Ethylene Response Factor MtERF1-1 Mediates Resistance to a Subset of Root Pathogens in Medicago truncatula without Adversely Affecting Symbiosis with Rhizobia1[W][OA] , 2010, Plant Physiology.

[71]  P. Gamas,et al.  Symbiosis-specific expression of two Medicago truncatula nodulin genes, MtN1 and MtN13, encoding products homologous to plant defense proteins. , 1998, Molecular plant-microbe interactions : MPMI.

[72]  N. Melamed-Book,et al.  NPR1 Protein Regulates Pathogenic and Symbiotic Interactions between Rhizobium and Legumes and Non-Legumes , 2009, PloS one.

[73]  G. Oldroyd,et al.  Ethylene Inhibits the Nod Factor Signal Transduction Pathway of Medicago truncatula , 2001, The Plant Cell Online.

[74]  F. Ariel,et al.  MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula. , 2011, The Plant journal : for cell and molecular biology.

[75]  Frédérique Bitton,et al.  CATdb: a public access to Arabidopsis transcriptome data from the URGV-CATMA platform , 2007, Nucleic Acids Res..

[76]  J. Heslop-Harrison,et al.  Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. , 1970, Stain technology.

[77]  Frédéric Barras,et al.  Genome Sequence of the Plant-Pathogenic Bacterium Dickeya dadantii 3937 , 2011, Journal of bacteriology.

[78]  C. Baron,et al.  The plant response in pathogenesis, symbiosis, and wounding: variations on a common theme? , 1995, Annual review of genetics.

[79]  Clarke,et al.  Influence of white clover mosaic potexvirus infection on the endogenous cytokinin content of bean , 1999, Plant physiology.

[80]  M. Valls,et al.  Integrated Regulation of the Type III Secretion System and Other Virulence Determinants in Ralstonia solanacearum , 2006, PLoS pathogens.

[81]  K. Niehaus,et al.  Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant , 1993, Planta.

[82]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.