Download Liesl van der Linden

Phenotypic and genetic evidence for tolerance to
bacterial wilt in Arabidopsis plants
Dave Berger
Plant Science Department
Forestry and Agricultural Biotechnology Institute (FABI)
University of Pretoria
Bacterial wilt
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Ralstonia solanacearum
Soil-borne vascular pathogen
Wide host range
Symptoms – wilting and necrosis
Species complex (Fegan & Prior 2005)
APSNET
Bacterial wilt on Eucalyptus trees
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: 369-373
APSNET
Identify mechanisms of plant resistance to
bacterial wilt
STRATEGY
Screen Natural Diversity of Arabidopsis thaliana for
resistance to bacterial wilt using Eucalyptus isolate of
Ralstonia solanacearum BCCF 402*
APSNET
* 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: 369-373
Dogma in
molecular plant pathology
Resistance / :
Immunity
gene-for-gene interactions
Tolerance
polygenic, QTLs of small effect
:
The pathosystem
Ralstonia solanacearum BCCF 402 (from Eucalyptus) vs Arabidopsis thaliana
mock inoculated
BCCF402
Be-0
Kil-0
Nd-1
Disease index
1.0
Be-0 + BCCF402
Kil0 + BCCF402
0.8
Nd1 + BCCF402
0.6
0.4
0.2
0.0
2
4
6
8
10
days post inoculation
12
14
A curious result
Disease index
1.0
Be-0 + BCCF402
Kil0 + BCCF402
0.8
Nd1 + BCCF402
0.6
0.4
0.2
0.0
log (cfu/g fresh weight)
2
4
6
8
10
days post inoculation
12
14
12
11
Kil-0
10
9
8
7
6
4
8
12
days post inoculation
16
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 99:11270-11274
Kil-0 does not show significant reduction in
yield/fecundity in response to R. solanacearum,
in contrast to Be-0
Support for Tolerance hypothesis
What is the genetic basis of tolerance?
Cross-fertilization of Kil-0 and Be-0
CAPS markers confirm cross-fertilization
ie F1 progeny are hybrids
LweI digestion of PCR products
(CAPS = cleaved amplified polymorphic sequences)
F1 progeny were susceptible to R. solanacearum BCCF402
Tolerance is recessive
F2 progeny segregate for tolerance:susceptibility in a 1: 3 ratio
Be-0
F2 progeny
Kil-0
Table 2.2. Segregation analysis of R. solanacearum isolate BCCF 402 resistance in the F2 progeny from crosses
between ecotypes Kil-0 (resistant) and Be-0 (susceptible).
Number of plants
Trial
1
2
Cross
Kil-0 × Be-0
Kil-0 × Be-0
a χ2 values were calculated for
Resistant
74
92
Susceptible
215
295
Expected Observed
Total ratio (R:S) ratio (R:S) χ2a
289
1:3
1:2.9
0.06
387
1:3
1:3.2
0.31
P
0.9>P>0.7
0.7>P>0.5
a segregation ratio of 1 resistant : 3 susceptible plants.
Kil-0 tolerance to R. solanacearum conferred by a single recessive gene
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
Kil-0 Be-0
Kil-0 Be-0
Tolerant F2 progeny
Susceptible F2 progeny
Kil-0 tolerance to R. solanacearum linked to RRS1
F3
Tolerance in Kil-0 is allelic to resistance in Nd-1
mock inoculated
inoculated
Bacterial numbers
Be-0
High
Kil-0
High
Nd-1
Low
F1
(Kil-0 X Nd-1)
Kil-0 tolerance conferred by RRS1 or tightly linked gene
High*
Susceptibility
Resistance
R. solanacearum
popP2
Be-0
Col-5
Tolerance
R. solanacearum
R. solanacearum
popP2
RRS1-R
popP2
RRS1-R
Nd-1
Kil-0
Nd-1
Effector triggered susceptibility
(ETS)
Effector triggered
immunity
(ETI)
( adapted from da Cunha et al. 2006)
Effector triggered
tolerance
(ETT)
Susceptibity
Predict: popP2 mutant
R. solanacearum
Tolerance
R. solanacearum
popP2
RRS1-R
Be-0
R. solanacearum
popP2
RRS1-R
Kil-0
Col-5
Effector triggered
susceptibility
(ETS)
ETT breaks down
( adapted from da Cunha et al. 2006)
Effector triggered
tolerance
(ETT)
Kil-0 tolerance requires R. solanacearum popP2 effector
mock
inoculated
BCCF402
BCCF402
ΔpopP2
BCCF402
ΔpopP2
pLAFR6::popP2
Kil-0
Be-0
Supports hypothesis that Kil-0 tolerance
conferred by RRS1 and not another
linked gene
Do AA sequences of RRS1 or popP2 explain difference between ETI and ETT?
Resistance
Tolerance
R. solanacearum
R. solanacearum
popP2
RRS1-R
popP2
RRS1-R
Nd-1
Kil-0
Effector triggered
immunity
(ETI)
Effector triggered
tolerance
(ETT)
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 GMI1000
Catalytic triad conserved
Autoacetylated lysine conserved
Do AA sequence differences in RRS1 explain difference between ETI and ETT?
1378 AA
Nd-1 (R )
% identity
Kil-0
(R )
98.9
Be-0
(S)
97.3
Col-0
(S)
91.8
RRS1 truncated in susceptible ecotypes
Only 8 AA difference between Nd-1 and Kil-0
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 interaction
Model of Effector triggered tolerance (ETT)
R. solanacearum
popP2
RRS1-R
R. solanacearum
popP2
RRS1-R
Bergelson lab
Rpm1 – Nd-1
fitness benefit at high inoculum levels
Kil-0
i.e. single gene tolerance (Genetics 2010)
Nd-1
Rps5 – no fitness benefit (New Phytol 2009)
Effector triggered
immunity
(ETI)
from da Cunha et al. 2006)
Effector triggered
tolerance
(ETT)
Acknowledgements
Students
Liesl van der Linden
Jane Bredenkamp
Collaborators
Yves Marco & Stephane Genin, CNRS/INRA, Toulouse, France
Katherine Denby, University of Warwick, UK
Sanushka Naidoo, Dept of Genetics, UP
Funding
NRF, South Africa
CNRS & Agropolis -South Africa exchange programme
Multiplication of R. solanacearum BCCF402 bacteria in A. thaliana
accessions Be-0 and Kil-0 is hrp-dependent.
(Hrp cluster encodes type III secretion system)
12
 Be-0
Kil-0 + BCCF402
11
Log (CFU/g fresh weight)
+ BCCF402
10
Be-0 + BCCF402
hrp
Kil-0 + BCCF402
hrp
9
8
7
6
5
4
3
0
2
4
6
8
Days After Inoculation
10
12