Transcript Document

Group Meeting
Andrew Diener
12-17-02
(1) natural resistance to Fusarium oxysporum
(2) cloning eds3
(3) genetic screens for Fusarium mutants
(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.
Fusarium oxysporum
Crucifer Pathogens
HOST
cabbage
radish
stock
forma specialis
conglutinans, race 1
conglutinans, race 2
raphani
matthioli, race 1
matthioli, race 2
Distribution of PKS sequences
Probe
PKS1
PKS3
PKS4
PKS6
PKS7
PKS8
PKS9
PKS10
PKS11
PKS12
PKS13
PKS14
Fusari um oxysporum forma specialis
conglutinans conglutinans raphani matthioli
lini
race1
race2
+
+
–––
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radicis lycopersici
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+
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+
–––
+
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+
+
+ = presence of homologous sequence
x = presence of heterologous sequence
–– = absent
Fusari um
grami nearum
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x
x
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x
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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
Peat Pellet Dip Inoculation
pellets are watered with
bud cell (conidia)
suspension
2 1/ 2
weeks
sow
2 1/ 2
weeks
inoculate
score
Disease Index
0
1
5
4
3
2
1
0
=
=
=
=
=
=
2
3
4
unaffected
stunted petioles
vascular chlorosis in older leaves
yellows and death in older leaves
restricted growth of young leaves
death
5
Arabidopsis genetics: mapping population
Parents:
Col-0
“F1-selfed
population”
X
Ty-0
Only the genotype of plants
with a recessive phenotype
are unambiguous.
F1
1/4 of F2 plants are
homozygous recessive.
(self)
F2
}
1 C/C : 2 C/T : 1 T/T
All F2 chromosomes are
recombinant.
1/4 of F2 chromosomes
give informative
recombination.
Arabidopsis genetics: mapping population
Parents:
Col-0
F1
X
X
“F1-backcrossed
population”
Ty-0
Ty-0
The genotype of plants is
unambiguous from both
dominant and recessive
phenotypes.
1/2 of F2 chromosomes are
recombinant.
F2
1 C/T : 1 T/T
1/2 of F2 chromosomes
give informative
recombination.
Parental Contribution to F2 genotype
50% Col-0
50% Ty-0
25% Col-0
75% Ty-0
recessive contribution
from both parents
recessive contribution
from Ty-0 parent only
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
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.
F2 : (F1:Col-0 X Ty-0) X Ty-0
X
X
X
Col-0
Flat #1
Ty-0
F2 : (F1:Col-0 X Ty-0) X Ty-0
X
X
X
Col-0
Flat #1
Ty-0
F2 : (F1:Col-0 X Ty-0) X Ty-0
Col-0
Flat #2
Ty-0
F2 : (F1:Col-0 X Ty-0) X Ty-0
Col-0
Flat #2
Ty-0
25
“F1 backcross”
Column 2
20
15
Col-0
Column 2
10
5
Data #1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0
20
Colum n 1
Column 4
10
5
F2
(F1 Col-0 X Ty-0)
Column 4
X
Ty-0
Data #1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0
8
Colum n 1
6
Ty-0
4
Column 3
2
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Column 3
number
of
plants
15
disease index
Colum n 1
Genetic Linkage to loci on chromosome 1 and 5
C:T
1
sensitive
F21M12
resistent
7 : 7 10 : 3
5
C:T
sensitive
resistent
4:7
7:7
3 : 11
8:6
PHYC
3:9
10 : 2
ciw9
7:7
9:5
CTR1
ciw8
nga280
nga111
5:8 4:9
2 : 12 11 : 3
Genotyping susceptible F2 plants from F1-backcross
chromosome 1
plant nga111 nga280
A3
A9
C5
C7
D2
D3
D6
D7
D8
D9
E2
E3
S
S
C
C
S
S
S
S
S
C
S
S
S
C
S
C
C
S
S
S
C
C
C
S
S
C
9
3
6
6
Col-0 X Sg-1
Fusarium oxysporum
f. sp. conglutinans
Ty-0
10
8
Ty-0
6
4
2
0
0
1
2
3
4
5
4
5
4
5
25
Number
of
Plants
20
Col-0
15
10
5
0
0
1
2
3
100
80
60
Ty-0 x Col-0 (F2)
40
20
0
0
1
2
3
Disease Index
Future Plans
Just inoculated 240 Col-0 X Ty-0 F2 plants from F1backcross.
Should obtain about 60 susceptible F2 plants and 60
strongly resistant F2 plants.
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
Susceptibility to Fusarium oxysporum
Col-0 (wild type)
pad4
eds5/sid1
eds16/sid2
eds12
npr1
eds10
eds4
eds11
eds6
eds9
eds3
eds-N26
R
S
S
S
R
R
S
S
R
R
R
S
S
R = resistance
S = sensitive
Mutants with defective accumulation of salicylic
acid are required for Fusarium resistance.
Col-0 (wild type)
pad4
eds5/sid1
eds16/sid2
eds12
npr1
eds10
eds4
eds11
eds6
eds9
eds3
eds-N26
R
S
S
S
R
R
S
S
R
R
R
S
S
}
known to be
required for
inducible
SA
accumulation
Mutants with defective Systemic Acquired
Resistance are not required for Fusarium
resistance.
Col-0 (wild type)
pad4
eds5/sid1
eds16/sid2
eds12
npr1
eds10
eds4
eds11
eds6
eds9
eds3
eds-N26
R
S
S
S
R
R
S
S
R
R
R
S
S
}
SA-dependent
PR-1 induction
not required
for resistance
Plant J (2002) 29: 11
Mutants with defective Induced Systemic
Resistance are required for Fusarium resistance.
Col-0 (wild type)
pad4
eds5/sid1
eds16/sid2
eds12
npr1
eds10
eds4
eds11
eds6
eds9
eds3
eds-N26
R
S
S
S
R
R
S
S
R
R
R
S
S
}
resistance
requires
ISR-dependent
genes
Plant J (2002) 29: 11
Undefined eds mutants with
enhanced susceptibility to
Fusarium
Col-0 (wild type)
R
pad4
eds5/sid1
eds16/sid2
eds12
npr1
eds10
eds4
eds11
eds6
eds9
eds3
eds-N26
S
S
S
R
R
S
S
R
R
R
S
S
Cloning eds3
(Kristen’s 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?
Eds to Pseudomonas syringae
3 days post inoculation:
O.D.
0.0002
MOCK
Col-0
Ler
eds3 (Col-0)
Eds to Fusarium oxysporum
wild type
pad4
esd3
edsN26
1 X 106
1 X 105
conidia / mL
1 X 105
Genotyping of Eds– plants from a F1 selfed population
Chrom os om e
M ark e r
5A3
5A5
5A7
5C6
5D4
6B4
2A3
2B6
4B3
3A2
3A5
3C3
4B4
4B5
4B8
8A7
8D8
8A8
8B3
8B7
8C7
C:H:L
5
CIW10
C
H
H
C
C
C
C
H
C
H
L
C
H
C
7:5:1
5
M UB3
C
C
C
C
C
C
C
C
C
H
C
C
L
L
H
C
L
H
C
13:3:3
5
K1L20
C
C
C
C
C
C
C
C
C
C
H
C
C
L
L
H
C
L
H
H
C
14:4:3
5
5
CIW9
C
H
H
C
H
C
CTR1
C
H
L
H
C
H
C
C
H
L
H
L
L
H
C
5:4:2
C
4:5:2
C = Col-0/Col-0
5
5
4
CIW8
H
H
PHYC
H
H
nga1107
C
H
H
H
H
0:4:0
4
4
4
CIW6
H
L
CIW5
H
C
H
L
H
C
H
H
H
1:3:0
1:3:0
0:2:2
1:3:0
H = Col-0/Ler
CIW7
L = Ler/Ler
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.
Fusarium oxysporum
haploid genome
40-50 Megabasepairs
8,000-13,000 genes
sequenced genome
(Fusarium graminearum)
simple transformation
targeted knockouts
Fusarium mutants have reduced virulence.
pks8 pacC fmk2 fmk1 WT
3106
1106
3105
conidia/mL
1105
Susceptible
Sg-1
ecotype
3104
1104
mock
inoculated with
dilutions of f.
conglutinans, race 2
mutants
(2) genetic screens for Fusarium mutants
Loss of virulence
Loss of root adhesion
Plate assays
Loss of virulence
inoculation
OR
Loss of root adhesion
15-30’
mutant:
{fmk1}
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.
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