Group Meeting

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Group Meeting Andrew Diener 12-17-02

1. natural resistance to Fusarium oxysporum
2. cloning eds3
3. genetic screens for Fusarium mutants

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(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.
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Fusarium oxysporum Crucifer Pathogens
HOST forma specialis cabbage conglutinans,
race 1 conglutinans, race 2 radish raphani s
tock matthioli, race 1 matthioli, race 2
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Distribution of PKS sequences
presence of homologous sequence x
presence of heterologous sequence absent
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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
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Peat Pellet Dip Inoculation
pellets are watered with bud cell
(conidia) suspension
2 1/2 weeks
2 1/2 weeks
sow
score
inoculate
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Disease Index
5
0
1
2
3
4

• 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

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Arabidopsis genetics mapping 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.
??1/4 of F2 chromosomes give informative
recombination.
F2

1 C/C 2 C/T 1 T/T
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Arabidopsis genetics mapping population
Parents Col-0 X Ty-0
The genotype of plants is unambiguous from both
dominant and recessive phenotypes.
F1
Ty-0
X
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
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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
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Polygenic Traits
mapping population 1 gene 2 genes 3
genes F1 selfed 1/4 1/16
1/64 F1 backcrossed 1/2
1/4 1/8
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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.
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F2 (F1Col-0 X Ty-0) X Ty-0
X
X
X
Col-0
Ty-0
Flat 1
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F2 (F1Col-0 X Ty-0) X Ty-0
X
X
X
Col-0
Ty-0
Flat 1
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F2 (F1Col-0 X Ty-0) X Ty-0
Col-0
Ty-0
Flat 2
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F2 (F1Col-0 X Ty-0) X Ty-0
Col-0
Ty-0
Flat 2
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F1 backcross
Col-0
F2 (F1 Col-0 X Ty-0) X Ty-0
number of plants
Ty-0
disease index
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Genetic Linkage to loci on chromosome 1 and 5
C T
C T
resistent
resistent
sensitive
sensitive
4 7 7 7
7 7 10 3
3 11 8 6
3 9 10 2
5 8 4 9
7 7 9 5
2 12 11 3
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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 D
7 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
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Ty-0
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Number of Plants
Disease Index
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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.
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enhanced disease susceptibility mutants
pad4 eds5/sid1 eds16/sid2 eds12 npr1 eds10 eds4 ed
s11 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
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Susceptibility to Fusarium oxysporum
Col-0 (wild type) R pad4 S eds5/sid1 S eds16/s
id2 S eds12 R npr1 R eds10 S eds4 S eds11
R eds6 R eds9 R eds3 S eds-N26 S
R resistance S sensitive
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Mutants with defective accumulation of salicylic
acid are required for Fusarium resistance.
Col-0 (wild type) R pad4 S eds5/sid1 S eds16/s
id2 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
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Mutants with defective Systemic Acquired
Resistance are not required for Fusarium
resistance.
Col-0 (wild type) R pad4 S eds5/sid1 S eds16/si
d2 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
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Mutants with defective Induced Systemic
Resistance are required for Fusarium resistance.
Col-0 (wild type) R pad4 S eds5/sid1 S eds16/s
id2 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
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Undefined eds mutants with enhanced
susceptibility to Fusarium
Col-0 (wild type) R pad4 S eds5/sid1 S eds16/s
id2 S eds12 R npr1 R eds10 S eds4 S eds11
R eds6 R eds9 R eds3 S eds-N26 S
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Cloning eds3
(Kristens Work)
Previous work with eds3
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?
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Eds to Pseudomonas syringae
3 days post inoculation
O.D. 0.0002
MOCK
Col-0
Ler
eds3 (Col-0)
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Eds to Fusarium oxysporum
wild type
pad4
esd3
edsN26
1 X 106
1 X 105
1 X 105
conidia / mL
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Genotyping of Eds plants from a F1 selfed
population
C Col-0/Col-0 H Col-0/Ler L Ler/Ler
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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.
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Fusarium oxysporum haploid genome 40-50
Megabasepairs 8,000-13,000 genes sequenced
genome (Fusarium graminearum) simple
transformation targeted knockouts
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Fusarium mutants have reduced virulence.
WT
fmk1
fmk2
pacC
pks8
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
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(2) genetic screens for Fusarium mutants
Loss of virulence
Loss of root adhesion
Plate assays
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Loss of virulence
inoculation
OR
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Loss of root adhesion
15-30
mutant fmk1
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(No Transcript)
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Acknowledgements
Fred Ausubel 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.
This