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Experimental evolution of the pathogen Ralstonia solanacearum:
molecular basis of adaptation to plants
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
Stéphane Genin
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P
R. solanacearum, a major plant pathogen that infects more than 200 plant species
- A wide range of solanaceous crops (tomato, pepper, potato, eggplant, tobacco )
- Model plants (Arabidopsis thaliana...)
- Other economically important crops (banana, groundnut…)
Wicker et al., 2007
Strains with novel pathogenic capabilities on Cucurbits
Emergence of new pathogenic variants in the field
The R. solanacearum host range is still expanding
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
T3SS and effectors
PrhR
PrhI PrhJ
HrpG
HrpB
Metabolic signals
>180 genes
Genetic determinants controlling pathogenicity and adaptation to the plant environment
Phytohormones
Motility
Plant cell wall degrading enzymes
EPS
Attachment
Protection responses
Others?? Metabolism
Experimental evolution as a novel approach to explore the molecular basis of adaptation to
plants
Experimental evolution, seeing evolution in action
Propagation of populations over hundreds/thousands of generations in a controlled environment.
a real time window on evolutionary processes
When the environment is changing, more adapted phenotypes are selected Mutations are random, beneficial mutations are selected
Next generation sequencing: to monitor the acquisition and fixation of adaptive mutations in clones of laboratory evolution experiments to gain a sequence level view of these evolutionary processes
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
PROJECT AIMS
1 Experimental evolution of a R. solanacearum GMI1000
clone on different host plants
2 Measuring adaptation of the ‘derived’ clones compared
to the ancestral GMI1000 clone.
3 Re-sequencing & genotypic characterization of the
‘derived’ clones
4 Characterization of the mutations conferring adaptive
traits on plants.
Does the bacterium rearrange its genome to adapt to different plants?
Can an adaptive mutation on a given host be detrimental on an other host (Trade-offs) ?
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
Serial Passage Experiments (SPE) on a different host plants
Ancestral clone
Derived clones
SPE1 SPE2 SPEn
≈ 300 Bacterial
generations GMI1000
strain -completely sequenced
Intermediate derived clones
Injection directly into the stem :
≈ 104 bacterial cells of the SPE(n-1)
Fossil record at -80 C
Plant
1. Tomato Marmande VR
2. Tomato Hawaï 7996
3. Eggplant Zebrina MM61
4. Eggplant MM134
5. Pelargonium Maverick Ecarlate
6. Bean Blanc précoce
7. Cabbage Bartolo
8. Melon Vedrantais
Susceptibility / resistance to GMI1000
Susceptible
Resistant
Susceptible
Resistant
Susceptible
Tolerant
Tolerant
Resistant
Botanical family
Solanaceae
Solanaceae
Solanaceae
Solanaceae
Geraniaceae
Fabaceae
Brassicaceae
Cucurbitaceae
Plant material
8 plant species 5 repeats (5 parallel populations of derived clones/plant)
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
Measuring fitness of derived clones by in planta competition with the ancestral clone
Tomato Cabbage Pelargonium
Adaptation depends on the plant species
Ancestral clone
Ancestral clone
Ancestral clone
lam
bda
derived clones from each
parallel experiment
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
a1 b1 d1 a2 c1 c3 c4 d2 e2
RSc1097 A,C A,C A,C T,C A,G Transcription regulator
RSc1622 Del 21 Transmembrane
RSc1976 T,C T,C T,C Amidophosphoribosyl transferase purF
RSc2508 A,- Hypothetical protein
RSc2736 T,C Transcription regulator transmembrane sensor histidine kinase phcS
RSc2750 C,G Transketolase tktA
RSc3062 T,C Transcription regulator
Gene NameGene ID
Derived clones on Tomato Marmande
after 26 SPE
Derived clones on Bean Blanc Precoce after 26 SPE Gene Description
Preliminary list of SNPs detected and confirmed by PCR-SANGER validation
very few SNPs per derived clone! But most are non-syn mutations
xxxx
xxxx
The RSxxxx gene is mutated in 4/5 parallel experiments in derived clones on bean
xxxx
xxxx
xxxx
xxxx
xxxx
Functional analysis of the RSxxxx gene
***:p
Conclusions
After 300 generations on a given plant, most of the derived clones show a fitness gain on this plant compared to the ancestral clone
The level of adaptation depends on the plant species
Very few SNPs were detected in the derived clones showing an enhanced fitness on plant
The RSxxxx gene (transcription regulator) mutation is associated to adaptation to bean
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
Alice Guidot Wei Jiang Patrick Barberis Christian Boucher Sébastien Carrère Jérôme Gouzy
Jean-Baptiste Ferdy Christophe Thébault Christophe Andalo
S e m i n a r P l a n t G e n o m i c s 2 0 1 2 – 3 r d - 4 t h - 5 t h A p r i l 2 0 1 2 – P o n t R o y a l e n P r o v e n c e
Experimental evolution of the pathogen Ralstonia solanacearum: molecular basis of adaptation to plantsDiapositive numéro 2Diapositive numéro 3Diapositive numéro 4Diapositive numéro 5Diapositive numéro 6Diapositive numéro 7Diapositive numéro 8Diapositive numéro 9Diapositive numéro 10Diapositive numéro 11Diapositive numéro 12Diapositive numéro 13