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

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