1. Group Meeting Andrew Diener 12-17-02
natural resistance to Fusarium oxysporum
cloning eds3
genetic screens for Fusarium mutants

2. (1) natural resistance to Fusarium oxysporum
RFO1 confers resistance to all crucifer F. oxysporum pathogens and is located at the end of chromosome 1.
RFO2 confers resistance to F. oxysporum f. sp. matthioli and is located in the middle of chromosome 5.
Does a RFO3 exist? If so, RFO3 confers resistance to F. oxysporum f. sp. conglutinans and is located on chromosome 1 cetromeric to RFO1.

3. Fusarium oxysporum Crucifer Pathogens
HOST forma specialis
cabbage conglutinans, race 1
conglutinans, race 2
radish raphani
stock matthioli, race 1
matthioli, race 2

4. Distribution of PKS sequences
+ = presence of homologous sequence
x = presence of heterologous sequence
–– = absent

5. Fusarium resistance CABBAGE
Resistance to F. oxysporum f. sp. conglutinans race 1 is a single dominant Mendelian trait.
J Agric Res (1930) 41: 1
TOMATO
Each of three races of F. oxysporum f. sp. lycospersici is recognized by a dominant resistance gene I-1, I-2 or I-3.
I-2 encodes a LZ-NBS-LRR protein.
Plant Cell (1997) 9: 521
Plant Cell (1998) 10: 1055

6. pellets are watered with
Peat Pellet Dip Inoculation
pellets are watered with
bud cell (conidia)
suspension
2 1/2
weeks
2 1/2
weeks
sow
inoculate
score

7. Disease Index 1 2 3 4 5 5 = unaffected 4 = stunted petioles1 2 3 4 5
5 = unaffected
4 = stunted petioles
3 = vascular chlorosis in older leaves
2 = yellows and death in older leaves
1 = restricted growth of young leaves
0 = death

8. } Arabidopsis genetics: mapping population “F1-selfed population”
Parents: Col-0 X Ty-0
Only the genotype of plants with a recessive phenotype are unambiguous. F1
“F1-selfed population”
1/4 of F2 plants are homozygous recessive.
(self)
All F2 chromosomes are recombinant. F2
1/4 of F2 chromosomes give informative recombination. }
1 C/C : 2 C/T : 1 T/T

9. Arabidopsis genetics: mapping population
Parents: Col-0 X Ty-0
The genotype of plants is unambiguous from both dominant and recessive phenotypes. F1 X
Ty-0
“F1-backcrossed population”
1/2 of F2 chromosomes are recombinant. F2
1/2 of F2 chromosomes give informative recombination.
1 C/T : 1 T/T

10. Parental Contribution to F2 genotype
50% Col-0
25% Col-0
50% Ty-0
75% Ty-0
recessive contribution from both parents
recessive contribution from Ty-0 parent only

11. Polygenic Traits mapping population 1 gene 2 genes 3 genes
F1 selfed / / /64
F1 backcrossed / / /8

12. Resistance to Fusarium oxysporum f. sp. matthioli
Unlike f. sp. conglutinans and f. sp. raphani, most ecotypes are completely resistant to f. sp. matthioli.
Ty-0 is a susceptible ecotype
Col-0 is completely resistant
Col-0 X Ty-0 mapping population
8 in 128 F2 plants from F1 selfed population were susceptible = 1/16 = 1/4 X 1/4 = two genes.
14 in 60 F2 plants from F1 backcrossed population were susceptible = 1/4 = 1/2 X 1/2 = two genes.

13. F2 : (F1:Col-0 X Ty-0) X Ty-0 X X X
Col-0
Ty-0
Flat #1

14. F2 : (F1:Col-0 X Ty-0) X Ty-0 X X X
Col-0
Ty-0
Flat #1

15. F2 : (F1:Col-0 X Ty-0) X Ty-0
Col-0
Ty-0
Flat #2

16. F2 : (F1:Col-0 X Ty-0) X Ty-0
Col-0
Ty-0
Flat #2

17. “F1 backcross” Col-0 Ty-0 F2 (F1 Col-0 X Ty-0) X Ty-0 number of plants
disease index

18. 5 1 Genetic Linkage to loci on chromosome 1 and 5 C : T C : T
PHYC
ciw9
ciw8
CTR1
nga280
nga111 1
F21M12
C : T
C : T
sensitive
resistent
sensitive
resistent
4 : : 7
7 : : 3
3 : : 6
3 : : 2
5 : : 9
7 : : 5
2 : : 3

19. Col-0 X Sg-1 Genotyping susceptible F2 plants from F1-backcross
chromosome 1
plant nga111 nga280
A3 S S
A9 S C
C5 C S
C7 C C
D2 S C
D3 S S
D6 S S
D7 S S
D8 S C
D9 C C
E2 S C
E3 S S
S 9 6
C 3 6
Col-0 X Sg-1
Fusarium oxysporum
f. sp. conglutinans

20. Ty-0

21. Number of
Plants
Disease Index

22. Future Plans
Just inoculated 240 Col-0 X Ty-0 F2 plants from F1-backcross.
Should obtain about 60 susceptible F2 plants and 60 strongly resistant F2 plants.

23. enhanced disease susceptibility mutants
pad4
eds5/sid1
eds16/sid2
eds12
npr1
eds10
eds4
eds11
eds6
eds9
eds3
eds-N26
Mutants obtained from direct genetic screens for
increased susceptibility to either Pseudomonas syringae or Erysiphe orontii
Genetics (1996) 143: 973
Genetics (1998) 149: 537
Plant J (2000) 24: 205

24. Susceptibility to Fusarium oxysporum
Col-0 (wild type) R
pad4 S
eds5/sid1 S
eds16/sid2 S
eds12 R
npr1 R
eds10 S
eds4 S
eds11 R
eds6 R
eds9 R
eds3 S
eds-N26 S
R = resistance
S = sensitive

25. Mutants with defective accumulation of salicylic acid are required for Fusarium resistance.
Col-0 (wild type) R
pad4 S
eds5/sid1 S
eds16/sid2 S
eds12 R
npr1 R
eds10 S
eds4 S
eds11 R
eds6 R
eds9 R
eds3 S
eds-N26 S }
known to be
required for
inducible SA
accumulation

26. Mutants with defective Systemic Acquired Resistance are not required for Fusarium resistance.
Col-0 (wild type) R
pad4 S
eds5/sid1 S
eds16/sid2 S
eds12 R
npr1 R
eds10 S
eds4 S
eds11 R
eds6 R
eds9 R
eds3 S
eds-N26 S
SA-dependent
PR-1 induction
not required
for resistance }
Plant J (2002) 29: 11

27. Mutants with defective Induced Systemic Resistance are required for Fusarium resistance.
Col-0 (wild type) R
pad4 S
eds5/sid1 S
eds16/sid2 S
eds12 R
npr1 R
eds10 S
eds4 S
eds11 R
eds6 R
eds9 R
eds3 S
eds-N26 S
resistance
requires
ISR-dependent
genes }
Plant J (2002) 29: 11

28. Undefined eds mutants with enhanced susceptibility to Fusarium
Col-0 (wild type) R
pad4 S
eds5/sid1 S
eds16/sid2 S
eds12 R
npr1 R
eds10 S
eds4 S
eds11 R
eds6 R
eds9 R
eds3 S
eds-N26 S

29. Cloning eds3 Previous work with eds3: (Kristen’s Work)
isolated for Eds to Pseudomonas syringae
segregated in F2 150 Eds+ : 51 Eds– in outcross to Ler
Genetics (1996) 143: 973.
claimed to be 14 cM telomeric from LFY3
TAIR database, from Meinke?

30. Eds to Pseudomonas syringae
3 days post inoculation:
O.D.
0.0002
MOCK
Col-0
Ler
eds3 (Col-0)

31. Eds to Fusarium oxysporum
wild type
pad4
esd3
edsN26
1 X 106
1 X 105
1 X 105
conidia / mL

32. Genotyping of Eds– plants from a F1 selfed population
C = Col-0/Col-0 H = Col-0/Ler L = Ler/Ler

33. Candidate gene approach:
WRKY51 is located exactly where eds3 is claimed to be: about 14 cM telomeric from LFY3.
WRKYs are transcriptional regulators which are regulated by pathogen recognition and regulate other disease-related genes.
WRKY51 is one of three unusual WRKYs and lacks conservation at amino acids residues important for DNA binding.
We have PCR amplified the WRKY51 sequence from wildtype and eds3 DNA as two overlapping fragments.

34. Fusarium oxysporum haploid genome 40-50 Megabasepairs
8,000-13,000 genes
sequenced genome
(Fusarium graminearum)
simple transformation
targeted knockouts

35. Fusarium mutants have reduced virulence.
pks8
pacC
fmk2
fmk1 WT
3106
1106
Susceptible
Sg-1 ecotype
inoculated with dilutions of f. conglutinans, race 2 mutants
3105
conidia/mL
1105
3104
1104
mock

36. (2) genetic screens for Fusarium mutants
Loss of virulence
Loss of root adhesion
Plate assays

37. Loss of virulence
inoculation OR

38. Loss of root adhesion
15-30’
mutant:
{fmk1}

40. Acknowledgements Fred Ausubel Corby Kistler
Julia Dewdney Simone Ferrari
Kristin Hladun Sachiko Miyata
Joulia Plotnikova
Mary Wildermuth
Corby Kistler
USDA Cereal Disease Lab
University of Minnesota
Support is provided by a grant from the National Institutes of Health.
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