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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
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N
C
0,5mm
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Werner et al Nat Com 2014
Rickettsia Caulobacter
RhodopseudomonasBradyrhizobium
Methylobacterium
Azorhizobium
Agrobacterium
Rhizobium
Shinella
Ochrobactrum
Bartonella
Phyllobacterium
DevosiaMesorhizobium
Xanthomonas
Cupriavidus
Ralstonia
Burkholderia
Bordetella
Neisseria
Pseudomonas
a
b
g
Brucella
Sinorhizobium
Symbiotic N2-fixation have spread to many a- and
b-proteobacterial genera
Masson-Boivin et al TIM 2009
Symbiotic N2-fixation arose ca. 100 MYA in Angiosperms
Symbiosis genes (nod-nif)
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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
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Strategy
nod-nif kit+
rhizobium
legume selection pressurelateral transfer
soil bacteriumGenomeremodelling
Rejouer et analyser l’évolution d’un rhizobium en
laboratoire
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Strategy
Nodulation Extracell Infection Intracell Infection Nice
InfectionInfection site
R. solanacearumpSym
cycles of 21 days or 42 days (inoculation to Mimosa)selection
cycle
R. solanacearum
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Objectifs spécifiques
Evolutionary pathsof symbioticadaptation
Lab-selection of nitrogen fixation
Experimentalversus naturalevolution
Mutagenesiscassettes as a tool to manipulate
bacterialevolvability
Lab-evolutionof symbioticRalstonia
Partners 1, 2, 3 Partners 1, 2, 3
Partners 1, 2, 3Partners 1, 3
Partner 2
Partner 3
Institute Pasteur PARIS (E Rocha)
CEA-Genoscope EVRY (S Cruveiller)
Partner 1LIPM TOULOUSE)
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Task1 Evolutionary paths of symbiotic adaptation
R. solanacearumpSym
R. solanacearum
Phenotypic shifts
Sequencing (Illumina)
Genetic reconstruction
Capela et al MBE 2017
Nodulation
Infection +
Infection ++
Phenotype-genotype
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Task1 Evolutionary paths of symbiotic adaptation
R. solanacearumpSym
R. solanacearum
Plant signal
hrpB
hrpG
prhI
prhJ
prhAprhR
phcA
T3SSvsrD
vsrA
phcS
phcB
phcR
3OH-PAME
Flagellar motility
Signal?EPS
Metabolicsubstrates
Plant cell walldegradingenzymes
efpR
phcQ
hrcV
Capela et al MBE 2017
Nodulation
Infection +
Infection ++
Unpublished data
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Task3:Experimental versus natural evolution
RickettsiaCaulobacterRhodopseudomonas
BradyrhizobiumMethylobacterium
Azorhizobium
Agrobacterium
Rhizobium
Shinella
BrucellaOchrobactrum
Sinorhizobium
Bartonella
PhyllobacteriumDevosiaMesorhizobium
Xanthomonas
Cupriavidus taiwanensis (Mimosa)
Ralstonia solanacearum
Burkholderia
Bordetella
Neisseria
Pseudomonas
a
b
g
Synthetic Biology& experimental evolution
Lab evolutionpSym
pSym
Natural evolutionGenome sequencingPopulation geneticsof 58
Cupriavidus
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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 12-16 MY
mutualism no yes
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Sequencing +Assembly +Annotation
DeterminationCore +
PangenomePhylogeny
16S
Inference of events
ANIb
timingrootingSpecies delimitation
Pop genetics RecombinationGene gain/loss Positive select
diversity
Mutation analysis
(number, type, location)
selection type
Experimental data
Illumina or PacBio of 43 C. taiwanensis15 other Cupriavidus, 31
Ralstonia
https://www.google.fr/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&ved=0ahUKEwj13KTJl-TXAhVLOBQKHQI4C1YQjRwIBw&url=https://partychameleon.com.au/magic-parties-nsw-newcastle/&psig=AOvVaw0twnDHbRe_MVDK5NMuqMgw&ust=1512058646804772
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Task3:Experimental versus natural evolution
Clerissi et al, in revision
pRaltaanc
C. taiwanensis
Environmental conditions (soil, plants, microbes) 12-16MYA
LCA Ct
pRaltaLMG19424
Evolved Ralstonia
Lab-conditions (M. pudica, Jensen) (1-2 years 400
generations)
R. solanacearum
Nod- Inf- Fix-
Parallel changes
Other changes
Cupriavidus
Dominant trend of purifying selection
Excess of changes (substitutions and deletions) in plasmid
Mutations in the phc regulatory pathway
Many substitutions of unknown adaptive valuesConservation of the
recently acquired T3SSPositive selection in lipid and amino acid
transport and metabolism
ca. 1200 point mutations, 436 non synomymousRegulatory ewiring
(hrpG, vsrA, prhI, efpR) T3SS inactivation
Nod+ Inf+ Fix+Nod- Inf- Fix-
Nod+ Inf+ Fix-
Adaptation by mutations in the genetic background
No evidence of adaptation in plasmid
Maintenance of the imuA2B2C2 cassette
Other changes
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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
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Stéphane Cruveiller, Genoscope Evry
Eduardo Rocha, Institut Pasteur Paris
Cécile Pouzet, Imaging platform, Toulouse
Olivier Bouchez, GeT Plage, Toulouse
Delphine Capela
Marta Marchetti
Benoit Daubech
Chaitanya Gokhale, Max Planck Ploen
Philippe Remigi, Massey University
Jacques Batut
LIPM Toulouse
Tang Mingxing
Caroline Raboin
Collaborations
Camille Clerissi
Lukas Brichet
