Recruitment of a lineage-specific virulence regulatory pathway promotes intracellular infection by a plant pathogen experimentally evolved into a legume symbiont

Ajuts: We are grateful to Lidwine Trouilh for helping in NimbleGen microarray hybridizations and Loic Escoriza for mutant construction. J.P.C. and C.C. were supported by the Initiative d'Excellence IDEX UNITI Actions Thématiques Stratégiques program (RHIZOWHEAT 2014) and by the French National Research Agency (ANR-12-ADAP-0014-01). This work was supported by funds from the French National Institute for Agricultural Research (Plant Health and the Environment Division), the French National Research Agency (ANR-12-ADAP-0014-01) and the French Laboratory of Excellence project TULIP (ANR-10-LABX-41). The complete collections of events generated for all the clones from this study are available on the Microscope platform (https://www.genoscope.cns.fr/agc/microscope/expdata/NGSProjectEvo.php, SYMPA tag).Ecological transitions between different lifestyles, such as pathogenicity, mutualism and saprophytism, have been very frequent in the course of microbial evolution, and often driven by horizontal gene transfer. Yet, how genomes achieve the ecological transition initiated by the transfer of complex biological traits remains poorly known. Here we used experimental evolution, genomics, transcriptomics and high-resolution phenotyping to analyze the evolution of the plant pathogen Ralstonia solanacearum into legume symbionts, following the transfer of a natural plasmid encoding the essential mutualistic genes. We show that a regulatory pathway of the recipient R. solanacearum genome involved in extracellular infection of natural hosts was reused to improve intracellular symbiosis with the Mimosa pudica legume. Optimization of intracellular infection capacity was gained through mutations affecting two components of a new regulatory pathway, the transcriptional regulator efpR and a region upstream from the RSc0965-0967 genes of unknown functions. Adaptive mutations caused the downregulation of efpR and the over-expression of a downstream regulatory module, the three unknown genes RSc3146-3148, two of which encoding proteins likely associated to the membrane. This over-expression led to important metabolic and transcriptomic changes and a drastic qualitative and quantitative improvement of nodule intracellular infection. In addition, these adaptive mutations decreased the virulence of the original pathogen. The complete efpR/RSc3146-3148 pathway could only be identified in the genomes of the pathogenic R. solanacearum species complex. Our findings illustrate how the rewiring of a genetic network regulating virulence allows a radically different type of symbiotic interaction and contributes to ecological transitions and trade-offs

Endosymbiotic Sinorhizobium meliloti modulate Medicago root susceptibility to secondary infection via ethylene

A complex network of pathways coordinates nodulation and epidermal root hair infection in the symbiotic interaction between rhizobia and legume plants. Whereas nodule formation was known to be autoregulated, it was so far unclear whether a similar control is exerted on the infection process. We assessed the capacity of Medicago plants nodulated by Sinorhizobium meliloti to modulate root susceptibility to secondary bacterial infection or to purified Nod factors in split-root and volatile assays using bacterial and plant mutant combinations. Ethylene implication in this process emerged from gas production measurements, use of a chemical inhibitor of ethylene biosynthesis and of a Medicago mutant affected in ethylene signal transduction. We identified a feedback mechanism that we named AOI (for Autoregulation Of Infection) by which endosymbiotic bacteria control secondary infection thread formation by their rhizospheric peers. AOI involves activation of a cyclic adenosine 30,50-monophosphate (cAMP) cascade in endosymbiotic bacteria, which decreases both root infectiveness and root susceptibility to bacterial Nod factors. These latter two effects are mediated by ethylene. AOI is a novel component of the complex regulatory network controlling the interaction between Sinorhizobium meliloti and its host plants that emphasizes the implication of endosymbiotic bacteria in fine-tuning the interaction

Experimental Evolution of a Plant Pathogen into a Legume Symbiont

Following acquisition of a rhizobial symbiotic plasmid, adaptive mutations in the virulence pathway allowed pathogenic Ralstonia solanacearum to evolve into a legume symbiont under plant selection

Etude genetique chez R. meliloti du catabolisme des calystegines et de la trigonelline, metabolites secondaires vegetaux

INIST T 73193 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Exploration et analyse de la diversité des rhizobia (nouveaux outils et concepts)

Suite à l'identification de b-protéobactéries nodulantes, nous avons confirmé le statut symbiotique de ces nouveaux rhizobia. R. taiwanensis LMG19424 a été choisi et caractérisé comme b-rhizobium modèle en vue du séquençage de son génome. Afin d'explorer davantage leur diversité, nous avons développé une méthode moléculaire d'identification des rhizobia qui s'affranchit des tests de nodulation en laboratoire. Grâce à une approche innovante par puce à ADN, utilisant comme sondes des motifs conservés de nodC et l'hybridation croisée d'une cible avec plusieurs sondes, nous avons pu détecter la présence de ce gène dans des cultures bactériennes ou des échantillons végétaux (nodules), évitant ainsi l'étape d'isolement du symbionte. L'analyse par clustering hiérarchisé des patterns d'hybridation permet également d'apporter une information phylogénétique sur le gène détecté. Cette approche originale et rapide est applicable à la détection d'autres gènes et fonctions bactériennes d'intérêtLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Etude moléculaire de la diversté symbiotique des rhizobia (de l'analyse du gène nodA à l'identification de rhizobia au sein des bèta-Protéobactéries)

Les rhizobia sont des bactéries du sol capables d'établir une symbiose fixatrice d'azote avec des légumineuses. Le séquençage et l'analyse phylogénétique du gène de nodulation nodA d'une collection de rhizobia taxonomiquement et symbiotiquement divers a permis d'établir l'existence d'une étroite corrélation entre la séquence protéique NodA et la présence de trois substitutions particulières des FNs. Nous avons développé une méthode de prédiction de la présence de ces substitutions basée sur l'analyse phylogénétique et statistique de la séquence NodA. La caractérisation du gène nodA parmi des souches de Burkholderia et Ralstonia isolées de nodules de légumineuses nous a conduit à démontrer l'existence de véritables rhizobium au sein des béta-protéobactéries, révélant ainsi l'étonnante diversité taxonomique des rhizobia. La construction de filtres ADN dédiés à leur identification et à leur caractérisation symbiotique a été entreprise à partir d'une collection de gènes nod spécifiques aux rhizobia.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Symbiotic nitrogen fixation by rhizobia-the roots of a success story.

By evolving the dual capacity of intracellular survival and symbiotic nitrogen fixation in legumes, rhizobia have achieved an ecological and evolutionary success that has reshaped our biosphere. Despite complex challenges, including a dual lifestyle of intracellular infection separated by a free-living phase in soil, rhizobial symbiosis has spread horizontally to hundreds of bacterial species and geographically throughout the globe. This symbiosis has also persisted and been reshaped through millions of years of history. Here, we summarize recent advances in our understanding of the molecular mechanisms, ecological settings, and evolutionary pathways that are collectively responsible for this symbiotic success story. We offer predictions of how this symbiosis can evolve under new influences and for the benefit of a burgeoning human population

Évolution expérimentale de l'infection intracellulaire chez les symbiotes de légumineuses

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Experimental Evolution as a Tool to Investigate Natural Processes and Molecular Functions

International audienceThe experimental evolution (EE) of microbes has allowed evolutionary biologists to examine adaptive processes in real time, generating novel insights into fundamental laws of evolution. Less appreciated is the potential of this approach to advance our understanding of microbial cells and molecular processes as a complement to traditional molecular genetics. The tracking of mutations underlying phenotypic changes offers the opportunity for detailed molecular analyses of novel phenotypes. This provides a breadth of information on diverse biological systems and may retrace key past events of natural history. Here, we highlight how the field has advanced our understanding of gene regulation, antibiotic resistance, and host–microbiome interactions to exemplify how EE can be used to provide new light on microbial systems