Dave Berger Plant Science Department Forestry and Agricultural Biotechnology Institute FABI University of Pretoria Ralstonia solanacearum Soilborne vascular pathogen Wide host range ID: 332283
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Slide1
Phenotypic and genetic evidence for tolerance to bacterial wilt in Arabidopsis plants
Dave BergerPlant Science DepartmentForestry and Agricultural Biotechnology Institute (FABI)University of PretoriaSlide2
Ralstonia solanacearum
Soil-borne vascular pathogen Wide host rangeSymptoms – wilting and necrosisSpecies complex (Fegan & Prior 2005)
Bacterial wilt
APSNETSlide3
Bacterial wilt on Eucalyptus trees
APSNET
AFRICA
Coutinho
TA, Roux J, Riedel KH,
Terblanche
J,
Wingfield
MJ (2000) First report of bacterial wilt caused by
Ralstonia
solanacearum
on eucalypts in South Africa. For
Pathol
30: 205-210
Roux
J,
Coutinho
TA,
Wingfield
MJ,
Bouillet
J-P (2000) Diseases of plantation Eucalyptus in the Republic of Congo. S
Afr
J
Sci
96: 454-456
Roux J,
Coutinho
TA,
Byabashaija
DM,
Wingfield
MJ (2001) Diseases of plantation Eucalyptus in Uganda. S
Afr
J
Sci
97: 16-18
BRAZIL
(2005) Susceptibility
to wilt associated with
Pseudomonas
solanacearum
among six species of Eucalyptus growing in equatorial Brazil. Austral Plant
Pathol
19: 71-76
CHINA
(
2009) Genetic diversity of
Ralstonia
solanacearum
strains from China. European Journal of Plant Pathology 125:
641-653
Fouche-Weich
J, Berger D,
Poussier
S,
Trigalet-Demery
D,
Coutinho
T (
2006) Molecular
identification of some African strains of
Ralstonia
solanacearum
from eucalypt and potato. Journal of General Plant Pathology 72: 369-373Slide4
Identify mechanisms of plant resistance to bacterial wilt
APSNET
STRATEGY
Screen Natural Diversity of Arabidopsis thaliana for resistance to bacterial wilt using Eucalyptus isolate of
Ralstonia
solanacearum
BCCF 402*
*
Fouche-Weich
J, Berger D,
Poussier
S,
Trigalet-Demery
D,
Coutinho
T (
2006) Molecular
identification of some African strains of
Ralstonia
solanacearum from eucalypt and potato. Journal of General Plant Pathology 72: 369-373Slide5
Dogma in
molecular plant pathologyResistance / : gene-for-gene interactionsImmunity
Tolerance : polygenic, QTLs of small effectSlide6
mock inoculated
Be-0
Kil-0
Nd-1
BCCF402
+ BCCF402
+ BCCF402
+ BCCF402
The
pathosystem
Ralstonia
solanacearum
BCCF 402 (from Eucalyptus)
vs
Arabidopsis thalianaSlide7
+ BCCF402
+ BCCF402
+ BCCF402
A curious result
Kil-0Slide8
Tolerance:
plant does not show a significant reduction in fitness despite high pathogen numbers in
planta
Resistance:
plant does not show a significant reduction in fitness
but severely restricts pathogen
numbers in
planta
Susceptibility:
plant shows a significant reduction in fitness and
has
high
pathogen numbers in
planta
Kover
and
Schaal
(2002) PNAS 99:11270-11274Slide9
Kil-0 does not show significant reduction in yield/fecundity in response to
R. solanacearum,in contrast to Be-0
Support for Tolerance hypothesisSlide10
What is the genetic basis of tolerance?
Cross-fertilization of Kil-0 and Be-0Slide11
CAPS markers confirm cross-fertilization ie
F1 progeny are hybrids
LweI
digestion of PCR products
(CAPS = cleaved amplified polymorphic sequences)
F
1
progeny were susceptible to
R.
solanacearum
BCCF402
Tolerance
is recessiveSlide12
Be-0
F
2 progeny
Kil-0
Kil-0 tolerance to
R.
solanacearum
conferred by
a single recessive gene
F
2
progeny segregate for
tolerance:
susceptibility
in a
1: 3 ratioSlide13
Kil-0 tolerance to
R. solanacearum linked to RRS1
Where in the Arabidopsis genome is the tolerance gene?
Hypothesis:
Tolerance conferred by allele of the RRS1 gene which confers R to a tomato isolate
F3
Tolerant F2 progeny
Susceptible F2 progeny
Kil-0
Kil-0
Be-0
Be-0Slide14
Tolerance in Kil-0 is allelic to resistance in Nd-1
mock
inoculated
inoculated
Be-0
Kil-0
Nd-1
F1
(Kil-0 X Nd-1)
Bacterial numbers
High
High
Low
High*
Kil-0 tolerance conferred by RRS1 or tightly linked geneSlide15
Susceptibility
Resistance
Col-5
Nd-1
( adapted from
da
Cunha et al. 2006)
R.
solanacearum
popP2
Be-0
Nd-1
Effector
triggered susceptibility
(ETS)
Effector triggered
immunity
(ETI)
popP2
RRS1-R
R.
solanacearum
Tolerance
Kil-0Effector triggered tolerance(ETT)popP2RRS1-RR. solanacearumSlide16
Susceptibity
Col-5
( adapted from
da
Cunha et al. 2006)
R.
solanacearum
popP2
Be-0
Effector triggered
susceptibility
(ETS)
Tolerance
Kil-0
Effector triggered
tolerance
(ETT)
popP2
RRS1-R
R.
solanacearum
Predict: popP2 mutant
ETT breaks down
R. solanacearumRRS1-RSlide17
mock inoculated
BCCF402
pLAFR6::
popP2
Kil-0
Be-0
BCCF402
ΔpopP2
BCCF402
ΔpopP2
Kil-0 tolerance requires
R.
solanacearum
popP2 effector
Supports hypothesis that Kil-0 tolerance conferred by RRS1 and not another linked geneSlide18
Tolerance
Kil-0
Effector triggered
tolerance
(ETT)
popP2
RRS1-R
R.
solanacearum
Resistance
Nd-1
Effector triggered
immunity
(ETI)
popP2
RRS1-R
R.
solanacearum
Do AA sequences of RRS1 or popP2 explain difference between ETI and ETT?Slide19
Do AA sequence differences in popP2 explain difference between ETI and ETT?
R.solanaceraum BCCF402 elicits ETI in Nd-1 and ETT in Kil-0.R.solanaceraum GMI1000 elicits ETI in Nd-1.Only 4 AA difference between PoP2 of BCCF402 and GMI1000Catalytic triad conservedAutoacetylated lysine conservedSlide20
RRS1
truncated in susceptible ecotypesOnly 8 AA difference between Nd-1 and Kil-0Do AA sequence differences in RRS1 explain difference between ETI and ETT?
Nd-1 (R )
% identity
Kil-0 (R )
98.9
Be-0 (S)
97.3
Col-0 (S)
91.8
1378 AASlide21
Conclusion: Gene-for-gene tolerance in Kil-0
R.solanacearum inoculation of Kil-0 plants:
Kil-0 did not wilt but had high bacterial numbers in
planta
Plant biomass yield, seed number, germination not reduced
Kil-0 response distinct from “resistant” ecotype Nd-1
Genetic evidence Kil-0 tolerance conferred by RRS1
Knockout/complementation evidence that Kil-0 requires
RRS1 – popP2 interactionSlide22
Nd-1
( adapted from da Cunha et al. 2006)
Kil-0
Effector triggered
tolerance
(ETT)
popP2
RRS1-R
R.
solanacearum
Model of Effector triggered tolerance (ETT)
Bergelson
lab
Rpm1 – fitness benefit at high inoculum levels
i.e. single gene tolerance (Genetics 2010)
Rps5 – no fitness benefit (New
Phytol
2009)
Nd-1
Effector triggered
immunity
(ETI)
popP2
RRS1-RR. solanacearumSlide23
Collaborators
Yves Marco &
Stephane
Genin
,
CNRS/INRA
, Toulouse, France
Katherine
Denby
, University of Warwick, UK
Sanushka
Naidoo
, Dept of Genetics, UP
Students
Liesl
van
der Linden
Jane
Bredenkamp
Acknowledgements
FundingNRF, South AfricaCNRS & Agropolis -South Africa exchange programmeSlide24Slide25Slide26
Multiplication of
R. solanacearum BCCF402 bacteria in A. thaliana
accessions Be‑0 and Kil-0 is hrp-dependent.
+ BCCF402
+ BCCF402
+ BCCF402
+ BCCF402
hrp
hrp
Be-0
Kil-0
Be-0
Kil-0
(
Hrp
cluster encodes type III secretion system)