Ancient host adaptation modulated

the actual resistance-breaking ability

of the Rice yellow mottle virus

Directeur : Denis FARGETTECo-directeur : Eugénie HEBRARD

UMR RPBDirecteur : Michel NICOLEÉquipe Durabilité des Résistances

Nils POULICARD

Emerging disease in Africa

Kenya 1966

Rice yellow mottle disease

Induced yield losses 20-100%

Rice yellow mottle virus

Fargette et al, 2004Fargette et al, 2008

S3

S2

S1

S4

S5

S2/S3

S1

S4

S5

Emergence in East Africa

Dissemination to the rest of Africa

5' 3'VPg ORF1

ORF2b

ORF2a ORF4

Genus Sobemovirus

(+) ssRNA (4,4kb)

Host range restricted to Oryzae species

Mechanical transmission (insects and human)

Non-transmitted by seeds

Molecular-dating : Emergence = ~ 150 years ago

High resistance of rice toward RYMV

Khush G.S. 1997

~500.000 years

safe inoculatedsafe infected

Resistant plantsSusceptible plants

Recessive resistance

High resistance : no symptoms, no multiplication

Resistance gene Rymv1 = translation initiation factor eIF(iso)4G Albar et al, 2006

E309K rymv1-2

N-terminaldomain

Δ322-324 rymv1-3

C-terminaldomain

eIF(iso)4G

rymv1-2 rymv1-3

Breakdown of the resistance alleles rymv1-2 (O. sativa indica)

rymv1-3 (O. glaberrima)

Resistance breakdown(RB)

Direct interaction between VPg and eIF(iso)4GVPg

eIF(iso)4GHébrard et al. 2010Traore et al, unpublished

Interaction strongly affected by resistance mutations

*

X*

Interaction restored by resistance-breaking mutations

*

Infectedsusceptible

plant

mechanicalinoculation

Resistantplant

SPFEIYGKFR EANSEEYDES LRHGVQYAEY DFSGDTIRAS SNTWVRERER YHAEERRKSG QPSWADRFGD DSGEDVDIE5241 48

5' 3'VPg ORF1

ORF2b

ORF2a ORF4

Hébrard et al. 2006Pinel-Galzi et al, 2007Traore et al, unpublished

S3

S2

S1

S4

S5

S2/S3

S1

S4

S5

rymv1-2 : + / -

rymv1-2 : + / -

rymv1-2 : ++

rymv1-2 : ++O. sativarymv1-2

Traore et al, unpublished

Pinel-Galzi et al, 2007

Breakdown of the resistance alleles rymv1-2 (O. sativa indica)

rymv1-3 : ++

rymv1-3 : + / -

rymv1-3 : - -

rymv1-3 : - -

O. glaberrimarymv1-3

rymv1-3 (O. glaberrima)

• dN/dS < 1 : codon under conservative selection• dN/dS = 1 : codon under neutral selection• dN/dS > 1 : codon under diversifying selection (positive selection)

Genetic signatures

Codon 49 is under strong diversifying selection

0

5

10

15

20

25

1

dN/d

S

1

+26

+

49+62codon Pinel-Galzi et al 2007

VPg

Pinel-Galzi et al 2007

Genetic signatures

VPg

Eas

t-A

fric

a

S5-

S6

100

90

84

100

99

Wes

t -A

fri c

aC

entr

al-A

fric

a

S2-

S3

Sa

S1

S4

S1-

ca

99

100

98

91

100

99

99

100

90

100

83

86

SPFEIYGKFREANSEEYDESLRHGVEYAEYDFSGDTIRASSNTWVRERERYHAEERRKSGQLSWADRFGDDSGEDVDIE1 10 20 30 40 50 60 70

...........................V....................TK...............E............. BF1

................................................TK...............E............. CI63

................................................TK............................. Ma203 (n = 1)

................................................TK............................. CIa

................................................TK............................. SL4

.........................Q..................................................... Ma77

.........................Q..................................................... Ma202 (n = 1)

.........................Q..................................................... Ma206 (n = 1)

.........................Q..................................................... Ma145 (n = 1)

.........................Q...................................P................. CIb

.........................Q...................................P................. CI4 (n = 12)

................................................TK..................S.......... BF5 (n = 1)

.........................Q..................................................... Ma10

.....................K..........................TK............................. Ni1

.....................K......................................................... Ni2

.....................K.......................................P................. Nia

............................................................................... Mg1

............................................................................... Mg2

.........................K.E................................................... Mg16 (n = 19)

.............................................................P................. Tz5

............................................................................... Tz8

............................................................................... Tz230 (n = 1)

.........................V..................................................... Tz225 (n = 1)

.........................Q.......................K...........P.........D....... Tz11

.........................Q.......................K...........P.........D....... Tz202

.........................Q...................................P................. Tz127

.................................................K............................. Tz209 (n = 3)

.........................Q.......................K............................. Tz18

............................................................................... Tz3

49

S2/S3

TS1

T/E

S4

E

S5

E

rymv1-2 : - -

rymv1-2 : + / -

rymv1-2 : ++

rymv1-2 : ++

rymv1-3 : ++

rymv1-3 : + / -

rymv1-3 : - -

rymv1-3 : - -

rymv1-3 (O. glaberrima)

Traore et al, unpublished

Pinel-Galzi et al, 2007

Breakdown of the resistance alleles rymv1-2 (O. sativa indica)

Portères R. , 1951Semon et al. 2005Traore et al. 2010

Geographic distribution of Oryzae glaberrima and polymorphism at codon 49

T49 = adaptation to O. glaberrima ?

T49 E49

Correlation between T49 and repartition area of O. glaberrima

Molecular signatures of adaptations

dN/dS > 1 : diversifying selection in all RYMV genome

T49 = unique signature of adaptation to O. glaberrima ?

VPgORF1

ORF2b

ORF2a ORF4

P1

IFEL

55

79

165191192

REL

556679

104

125162165191192214

Coat Protein

Sel

55

7981

116

165191

216218

IFEL

21

66

110

REL

26

45

5466

8599

110

Sel

12

2642454753546674798185

101110

IFEL REL Sel

45203226

Protease VPg

IFEL

49

REL

49

Sel

2126495062

Polymerase

IFEL

114

237

REL

114123134136

186187190233237

370389400437441

Sel

114

174179

324

signature of co-variations in RYMV genome?

no co-variation between codon 49 of the VPg and the others positions under diversifying selection

Competitions of isolates polymorphic at codon 49 in susceptible plants

- 3 cultivars O. sativa indica- 3 cultivars O. glaberrima

WestAfrica

CentralAfrica

EastAfrica

TTTTTTEETEEETEEEEEEEEEEEEEEE

T49 = signature of the adaptation to O. glaberrima

Competitions O. sativa indica O. glaberrima

Tz209 / CIa CIa (T) CIa (T)

Mg16 / CIa Mg16 (E) CIa (T)

Tz8 / BF1 BF1 (T) BF1 (T)

CI4 / CIa CI4 (E) CIa (T)

Ma10 / BF1 BF1 (T) BF1 (T)

Tz209 / BF5 BF5 (T) BF5 (T)

Mg16 / BF5 Mg16 (E) BF5 (T)

Tz8 / Ni1 Ni1 (T) Ni1 (T)

CI4 / BF5 BF5 (T) BF5 (T)

Validation by mutagenesis : CIa*T49E

Competition O. sativa indica O. glaberrima

CIa*T49E / CIa CIa*T49E (E) CIa (T)

E49T49

1,00E+09

1,00E+10

1,00E+11

1,00E+12

1

vira

l AR

N c

op

ies

/ mg

of

leav

es

a aa a

real time-PCR

CIa CIa*T49E

O. s

at.

O. g

lab.

O. s

at.

O. g

lab.

T49 = signature of the adaptation to O. glaberrima

T49 = adaptation signature to O. glaberrima

CIa (49T) and CIa*T49E inoculated at the same concentration to resistant O. sativa indica (rymv1-2) O. glaberrima (rymv1-3)

DAS-ELISA

+ 42 dpiIR64

(susceptible)

O. sativa indicarymv1-2

O. glaberrimarymv1-3

CIa (T49) 100% 5 % 95 %

CIa*T49E 100% 40 % 0 %

Polymorphism at codon 49 and resistance-breaking

Codon 49 influence the resistance-breaking of rymv1-2 and rymv1-3

intensification of O. sativa cultivation: since XIXth centuries

introduction of O. sativa: VII-Xth centuries in East Africa

XVth century in West Africa

Ancient host adaptation and actual resistance-breaking ability of RYMV

O. glaberrima

rymv1-3

O. sativa indica

rymv1-2

+++ + / -

- - - +++

domestication of O. glaberrima: ~ 3.500 years ago

T49

T49

O. glaberrima

O. sativa indica

East AfricaWest Africa

E49E49

Evolutionary history of RYMV could influence the adaptation to resistant plants

Ancient host adaptation could modulate the actual adaptation ability of pathogens

Conclusion

Perspectives

Competitions in susceptible plants O. sativa indica / O. glaberrima

- 3 phylogenetic branches of T49- competitions with enclosed-isolates

Role of T49 in the adaptation of O. glaberrima ?

- interaction VPg / eIF(iso)4G ?

Role of the host genetic background ?

- introgression of the rymv1-3 resistance allele in O. sativa indica genetic background)?

Denis FargetteEugénie HébrardAgnès Pinel-GalziJamel AribiNils PoulicardChristelle LiretteStelly Mississipi

UMR Génome et Développement des PlantesÉquipe Génomique appliquée au Riz

Alain GhesquièreLaurence AlbarFlorence VignolDeless Thiemele

UMR Résistance des Plantes aux Bioagresseurs Équipe Durabilité des Résistances

Gnissa KonateOumar TraoréDrissa Sérémé

Burkina-Faso Côte d’Ivoire Madagascar

Séré YacoubaFatogoma Sorho

Sissi Rakotomalala

Tanzanie

Zakaria KanyekaEmmanuel Sangu

Rémy FroissartThierry MichonBenoît MouryMichel Nicole

Histoire évolutive du RYMV

R = 0.77

0

2

4

6

8

10

12

2.00 2.50 3.00 3.50 4.00Distance Geographique (log)

Génome entier

Dis

tan

ce G

én

éti

qu

es

(%n

t)

S3

S2

S1

S4

S5

Fargette et al, 2004

Afriquede l’Est

Afriquede l’Ouest

0

2

4

6

8

10

AfriqueCentrale

Div

ersi

té n

ucle

otid

ique

(%

nt)

Nombre total de sites

S5S4S1ACS1AOS2S3

Fargette et al, 2008

Emergence : ~150 ans

Propagation d’Est en Ouest

Emergence en Afrique de l’Est

Why CIa is not able to overcome the rymv1-2 resistance allele ?

Contrasted rymv1-2 resistance-breaking ability

Pinel-Galzi et al, 2007

VPg48

E49

inoculation CIa (49T) no emergence of resistance-breaking (RB) mutations

T49T49 X

*48G is lethal in CIa

X

directed mutagenesis CIa validation of 48E RB mutations

Antagonistic epistasis between T49 and rymv1-2 RB mutations

*48G is lethal in CIa isolate. Influence of T49?

Directed mutagenesis: Substitution of T49 to E49:

0

1

2

CIa*48G *48G49E CIa*48G *48G49E

0

1.0

2.0

CIa*48G(49T)

CIa*48G*49E

CIa*48G(49T)

CIa*48G*49E

O. sativa indica(susceptible)

O. sativa indicarymv1-2

OD

(40

5nm

)

DAS-ELISA30 dpi

T49

X

T49 strongly limits the emergence of rymv1-2 RB mutations

1,0E+00

1,0E+02

1,0E+04

1,0E+06

1,0E+08

1,0E+10

1,0E+12

1,0E+14

1 2 3 4 5 6

vira

l RN

A c

opie

s / m

g of

leav

es

a

b b c

CI4*48

I*48

G*48

E

Capacités de contournement contrastées entre souches

CI4

E

CIa

T

1,0E+00

1,0E+02

1,0E+04

1,0E+06

1,0E+08

1,0E+10

1,0E+12

1,0E+14

1 2 3 4 5

vira

l RN

A c

opie

s / m

g of

leav

es

CIa*48

I*48

E

a

b

c

VPg*48I*49T

VPg*48G*49T

VPg*48E*49T

dilution

Interaction test(two hybrid assay)

VPg*48I*49E

VPg*48G*49E

VPg*48E*49E

eIF(iso)4G rymv1-2

Polymorphism at codon 49 and resistance-breaking

H52Y mutation involved in the resistance-breaking of rymv1-2 and rymv1-3

T49

*52Y

RB rymv1-3O. glaberrima

E49 RB rymv1-2O. sativa indica

RB rymv1-2O. sativa indica

X RB rymv1-3O. glaberrima

No antagonismbetween E49 and *52Y RB mutation

0

1

2

CIa*52Y CIa*49E*52Y CIa*52Y CIa*49E*52Y

CIa*52Y(49T)

CIa*52Y*49E

CIa*52Y(49T)

CIa*52Y*49E

O. sativa indicarymv1-2

O. glaberrimarymv1-3

0

1.0

2.0

OD

(40

5nm

)

Influence of the host genetic background ?E49 X O. glaberrima

Influence of the polymorphism at codon 49?

Validation by mutagenesis: - CIa(49T)*52Y - CIa*49E*52Y

E3605.5 A°

A303

E360

D303

Effet de la mutation A303D dans eIFiso4G d’O. glaberrima

O. glaberrima

O. sativa susceptible

susceptible

MIF4G

A303D

1,0E+00

1,0E+02

1,0E+04

1,0E+06

1,0E+08

1,0E+10

1,0E+12

1,0E+14

1 2 3 4 5 6

vira

l RN

A c

opie

s / m

g of

leav

es

CI4*48

I*48

G*48

E

ab

c

d

Évaluation de la multiplication virale sur plantes sensibles

le WT a la meilleure multiplication

*48E est le plus contre-sélectionné

réversion CI4*48E en CI4*48G (et non mutations compensatoires)

CI4S1

30dpi

VPg

VPg*48I

VPg*48G

VPg*48E

dilutiondilution

eIF(iso)4G HUMUM HUAM

dilution

Growth controlInteraction assay

Promotor Histidine

VPgDBD

Yeast strain leu- trp- his-

pGBKVPg

pGADiso4G

Transformation(-leu-trp)

Interaction test(-His)

No interactionNo transcription of the reporter gene

yeast death

VPg

Trp

4G

Leu

Yeast double hybrid assay

DBD AD

pGBKVPg

pGADiso4G

4G

Leu

ADVPg

Trp

DBD

iso4GAD

Interaction Transcription of the reporter gene

yeast growth

Promotor Histidine

VPgDBD

iso4GAD