Catherine MASSON-BOIVIN

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2 Catherine MASSON-BOIVIN INRA-CNRS REPLAY Replaying the evolution of rhizobia: towards a conceptual and practical framework for the design of new nitrogen-fixing plant symbionts

3 N C 0,5mm

4 Symbiosis genes (nod-nif) Cupriavidus Burkholderia Bordetella Xanthomonas Pseudomonas Ralstonia Neisseria b g a Phyllobacterium Devosia Mesorhizobium Agrobacterium Shinella Sinorhizobium Rhizobium Ochrobactrum Brucella Bartonella Azorhizobium Rickettsia Caulobacter Rhodopseudomonas Bradyrhizobium Methylobacterium Symbiotic N2-fixation arose ca. 100 MYA in Angiosperms Symbiotic N2-fixation have spread to many a- and b-proteobacterial genera Werner et al Nat Com 2014 Masson-Boivin et al TIM 2009

5 Objectifs Comprendre les mécanismes évolutifs ayant conduit à l émergence de symbiotes de légumineuses Développer un cadre conceptuel et pratique pour l évolution de nouveaux symbiotes fixateurs d azote

6 Strategy Rejouer et analyser l évolution d un rhizobium en laboratoire nod-nif kit + lateral transfer legume selection pressure soil bacterium Genome remodelling rhizobium

7 Strategy selection cycle cycles of 21 days or 42 days (inoculation to Mimosa) R. solanacearum R. solanacearum psym Infection site Nodulation Extracell Infection Intracell Infection Nice Infection

8 Objectifs spécifiques Evolutionary paths of symbiotic adaptation Experimental versus natural evolution Partner 1 LIPM TOULOUSE) Partners 1, 2, 3 Partners 1, 2, 3 Lab-evolution of symbiotic Ralstonia Lab-selection of nitrogen fixation Partners 1, 3 Mutagenesis cassettes as a tool to manipulate bacterial evolvability Partners 1, 2, 3 Partner 2 Institute Pasteur PARIS (E Rocha) Partner 3 CEA-Genoscope EVRY (S Cruveiller)

9 Task1 Evolutionary paths of symbiotic adaptation R. solanacearum R. solanacearum psym Phenotypic shifts Sequencing (Illumina) Genetic reconstruction Nodulation Infection + Infection ++ Phenotype-genotype Capela et al MBE 2017

10 Task1 Evolutionary paths of symbiotic adaptation R. solanacearum R. solanacearum psym Nodulation Infection + Infection ++ Plant signal Metabolic substrates Plant cell wall degrading enzymes prha prhr prhi prhj 3OH-PAME phcb phcs phcq phc R efpr phca hrpg hrpb hrcv Capela et al MBE 2017 Unpublished data Flagellar motility vsrd vsra Signal? EPS T3SS

11 Task3:Experimental versus natural evolution Pseudomonas Phyllobacterium Mesorhizobium Devosia Agrobacterium Shinella Sinorhizobium Rhizobium Ochrobactrum Brucella Bartonella Azorhizobium Methylobacterium Bradyrhizobium Caulobacter Rhodopseudomonas g a Xanthomonas b Rickettsia Ralstonia solanacearum Cupriavidus taiwanensis (Mimosa) Neisseria Burkholderia Bordetella psym psym Lab evolution Synthetic Biology & experimental evolution Natural evolution Genome sequencing Population genetics of 58 Cupriavidus

12 The two processes differed in fundamental points Experiment Natural evolution genus Ralstonia Cupriavidus lifestyle pathogen saprophyte conditions simplified and controlled complex and changing time span 1-2 years MY mutualism no yes

13 Illumina or PacBio of 43 C. taiwanensis 15 other Cupriavidus, 31 Ralstonia ANIb Species delimitation rooting 16 S timing Sequencing + Assembly + Annotation Determination Core + Pangenome Phylogeny Inference of events Experimental data Mutation analysis (number, type, location) selection type Pop genetics Recombination Gene gain/loss Positive select diversity

14 Task3:Experimental versus natural evolution pralta anc Positive selection in lipid and amino acid transport and metabolism Conservation of the recently acquired T3SS Many substitutions of unknown adaptive values Other changes Environmental conditions (soil, plants, microbes) 12-16MYA Cupriavidus Nod - Inf - Fix - pralta LMG19424 LCA Ct Mutations in the phc regulatory pathway Adaptation by mutations in the genetic background Excess of changes (substitutions and deletions) in plasmid No evidence of adaptation in plasmid Dominant trend of purifying selection Parallel changes Maintenance of the imua2b2c2 cassette Lab-conditions (M. pudica, Jensen) (1-2 years 400 generations) C. taiwanensis Nod + Inf + Fix + R. solanacearum Nod - Inf - Fix - T3SS inactivation Regulatory ewiring (hrpg, vsra, prhi, efpr) ca point mutations, 436 non synomymous Other changes Evolved Ralstonia Nod + Inf + Fix - Clerissi et al, in revision

15 Impacts A fundamental research project. A better understanding of the evolution of rhizobia Exploring pathogenesis-symbiosis relationships and transition Exploring strategies for plant-mediated selection experiments

16 LIPM Toulouse Collaborations Delphine Capela Marta Marchetti Benoit Daubech Jacques Batut Tang Mingxing Caroline Raboin Lukas Brichet Camille Clerissi Eduardo Rocha, Institut Pasteur Paris Stéphane Cruveiller, Genoscope Evry Olivier Bouchez, GeT Plage, Toulouse Cécile Pouzet, Imaging platform, Toulouse Philippe Remigi, Massey University Chaitanya Gokhale, Max Planck Ploen