Phenotypic and genetic evidence for tolerance to bacterial wilt in Arabidopsis plants

Dave BergerPlant Science Department

Forestry and Agricultural Biotechnology Institute (FABI)University of Pretoria

• Ralstonia solanacearum• Soil-borne vascular pathogen • Wide host range• Symptoms – wilting and necrosis• Species complex (Fegan & Prior 2005)

Bacterial wilt

APSNET

Bacterial wilt on Eucalyptus trees

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AFRICACoutinho 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: 205-210Roux J, Coutinho TA, Wingfield MJ, Bouillet J-P (2000) Diseases of plantation Eucalyptus in the Republic of Congo. S Afr J Sci 96: 454-456Roux 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

Identify mechanisms of plant resistance to bacterial wilt

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STRATEGYScreen 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: 369-373

Dogma in molecular plant pathology

Resistance / : gene-for-gene interactionsImmunity

Tolerance : polygenic, QTLs of small effect

mock inoculated

Be-0

Kil-0

Nd-1

BCCF402

2 4 6 8 10 12 140.0

0.2

0.4

0.6

0.8

1.0Be-0

Kil0

Nd1

days post inoculation

Dis

ease

inde

x

+ BCCF402

+ BCCF402

+ BCCF402

The pathosystemRalstonia solanacearum BCCF 402 (from Eucalyptus) vs Arabidopsis thaliana

2 4 6 8 10 12 140.0

0.2

0.4

0.6

0.8

1.0Be-0

Kil0

Nd1

days post inoculation

Dis

ease

inde

x

+ BCCF402

+ BCCF402

+ BCCF402

A curious result

4 8 12 166

7

8

9

10

11

12

days post inoculation

log

(cfu

/g fr

esh

wei

ght)

Kil-0

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

Be-0 F2 progeny Kil-0

Kil-0 tolerance to R. solanacearum conferred by a single recessive gene

F2 progeny segregate for tolerance:susceptibility in a 1: 3 ratio

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 Cross Resistant Susceptible TotalExpected ratio (R:S)

Observed ratio (R:S) χ2a P

1 Kil-0 × Be-0 74 215 289 1:3 1:2.9 0.06 0.9>P>0.72 Kil-0 × Be-0 92 295 387 1:3 1:3.2 0.31 0.7>P>0.5

a χ2 values were calculated for a segregation ratio of 1 resistant : 3 susceptible plants.

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-0

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 gene

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)

popP2RRS1-R

R. solanacearum

Tolerance

Kil-0

Effector triggered tolerance

(ETT)

popP2RRS1-R

R. solanacearum

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)

popP2RRS1-R

R. solanacearum

Predict: popP2 mutant

ETT breaks down

R. solanacearum

RRS1-R

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 gene

Tolerance

Kil-0

Effector triggered tolerance

(ETT)

popP2RRS1-R

R. solanacearum

Resistance

Nd-1

Effector triggered immunity

(ETI)

popP2RRS1-R

R. solanacearum

Do AA sequences of RRS1 or popP2 explain difference between ETI and 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 GMI1000Catalytic triad conservedAutoacetylated lysine conserved

RRS1 truncated in susceptible ecotypesOnly 8 AA difference between Nd-1 and Kil-0

Do AA sequence differences in RRS1 explain difference between ETI and ETT?

  Nd-1 (R )  % identityKil-0 (R ) 98.9

Be-0 (S) 97.3

Col-0 (S) 91.8

1378 AA

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 requiresRRS1 – popP2 interaction

Nd-1

( adapted from da Cunha et al. 2006)

Kil-0

Effector triggered tolerance

(ETT)

popP2RRS1-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)

popP2RRS1-R

R. solanacearum

CollaboratorsYves Marco & Stephane Genin, CNRS/INRA, Toulouse, FranceKatherine Denby, University of Warwick, UKSanushka Naidoo, Dept of Genetics, UP

StudentsLiesl van der LindenJane Bredenkamp

Acknowledgements

FundingNRF, South AfricaCNRS & Agropolis -South Africa exchange programme

0 2 6 8 12 143

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Days After Inoculation

Log

(CFU

/g fr

esh

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ght)

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)