Regulatory networks underlying barley interaction with rust fungi
Arnis Druka
Scottish Crop Research Institute
Inaugural Meeting, Edinburgh, October 1-3, 2008
Why barley?
• about 10,000 years long co-existence with humans
• economically important crop for Scotland, Northern Europe and Worldwide
• prime crop for malting and distilling
• alternative uses, approved by FDA as a Health Food
• spectacular natural diversity within the species complemented by extensive induced mutant
populations
• good genetics – inbreeder, relatively stable diploid genome, only 5 giga base pairs
• easy to grow, fast reproductive cycle
• can be genetically modified by several different transformation methods
• relatively far advanced genomics:
- over 500,000 ESTs
- 22K GeneChip by Affymetrix, two Agilent arrays, 15K and 44K,
- about 10,000 gene-based SNPs have been identified
- 1536-plex SNP genotyping platform has been developed
- genome sequencing is well under way
Outcomes of the barley – stem rust fungus interaction
compatible
incompatible
Major stem rust resistance gene Rpg1
Rpg1
Chr 7H
Puccinia graminis MCC
Compatible
Incompatible
cv Steptoe
cv Morex
Positional cloning
Mendelian trait
mapping
R or S
Brueggeman et al PNAS 2002
Identification of the Rpr1 locus
Resistant
Rpg1 / Rpg1
Pgt f.sp. tritici
MCC
in vitro
mutagenesis
selection
Fast neutrons
Susceptible
Rpg1 / Rpg1
Rpr1
Chr 4H
Mendelian trait
mapping
R or S
mRNA profiling
Zhang at al TAG 2006
Phenotyping barley and stem rust fungus interaction
?
HU03D17u_s_at
sensory transduction
histidine protein kinase
Benjamini-Hochberg FDR (0.05%)
HVSMEm0005P05r2_at
1.5
-log10(P-Value)
Contig4901_s_at
1
Contig6699_s_at
Contig7061_s_at
0.5
Contig13680_s_at
Rpr1
0
-5
-4
-3
-2
-1
0
1
2
3
4
5
log2(Fold Change)
Susceptible
Resistant
Rpg1
Rpg1
Contig13681_at
Contig14769_at
Druka et al TAG 2008
Puccinia graminis MCC
Compatible
Incompatible
cv Steptoe
cv Morex
Type ‘0’
Type ‘1’
Type ‘2’
Type ‘3’
Mapping infection types
Type ‘0’
IM
Morex - resistant
Type ‘1’
IM
Morex - resistant
Type ‘2’
IM
Morex - resistant
Type ‘3’
IM
Steptoe - susceptible
sensory transduction
histidine protein kinase
Morex effect
Type '2' phenotype
Morex effect
Co-localization
1H
2H
3H
4H
5H
6H
7H
locus A
5H
Co-regulation
1H
3H
2H
3H
locus B
4H
5H
6H
7H
sensory
transduction
histidine
kinase
Ppd-H1
35.5
Tef4
36.3
ABG358
37.8
CDO064
40.6
ABG459
42
MWG520A 43.4
snp_0847
44
DAK213A 44.8
Pox
48
snp_1073 48.6
ABC454
49.3
cMWG663A 50
JS188A
50.7
Adh8
51.4
snp_1061 58.4
snp_1054 60.4
MWG557 61.1
snp_1272 61.8
CDO537
63.3
ABG316C 65.5
snp_0474 66.3
olad_259 66.9
P40FA
66.9
Hvex1
67.6
snp_1121 68.3
snp_0545 70.3
snp_0265 73.8
snp_0407 74.4
bBE54D
75.1
snp_0650 76.5
snp_1449 78.5
snp_1435 79.2
His3C
81.6
2H
18
21
20
65
33
80
25
58
75
80
30
21
29
57
27
85
32
116
16
10
16
17
cyclic nucleotide and calmodulin-regulated
ion channel protein
RAR1
MLO1
heat shock protein
lipoxygenase
jasmonate-induced protein
defender against death 1 (dad1)
calcineurin-like phosphoesterase
26S proteasome non-ATPase
regulatory subunit 6
elicitor-responsive gene 3
DegP2 protease (DEGP2)
heat shock protein 70
endo-1,3;1,4-beta-D-glucanase
hypersensitive reaction-induced protein 3
heat shock factor binding protein 2
sensory transductionhistidinekinase
acid phosphatase
serine/threonine receptor kinase
Hsp90
NMD3
jasmonate-induced protein
MLA1
6H bin8
2H bin10
4H
nk
2HS*
6H*
7H*
2HS*
nk
2HS*
nk
nk
nk
5H
7H*
2H bin8
7H
nk
nk
nk
nk
5H
nec1
Barley – mildew
interaction
PCD
Conclusions
• A novel rust resistance enhancing locus, Rpr2 was identified by mapping barley-stem rust
interaction as a quantitative trait;
• Based on eQTL cluster analysis, Rpr2 locus possibly controls expression of genes associated
with PCD;
• Sensory transduction histidine kinase was identified as one of the candidate genes for the
Rpr2 locus based on eQTL mapping and mRNA profiling of the Rpr1 or rpr1 alleles carrying
barleys.
Next
• mapping Rpr2 in a different or F2-based population;
• precision mapping of the rust phenotype;
• identification of other phenotypes associated with Rpr2 locus;
• testing the histidine kinase gene as a candidate gene for the Rpr2 locus;
• functional assays of other genes associated with Rpr2: tilling,VIGS, in situ hybridization,
immunocytochemistry, ChIP, Y2H screens.
Enquiries about studentships on this project are welcome!
Arnis.Druka@scri.ac.uk
Variability (%)
1H
100
90
80
70
60
50
40
30
20
10
0
2H
3H
4H
69.4%
IT0
IT1
IT3
PC1
5H
6H
Steptoe
27.7%
IT2
PC2
2.6%
PC3
Factor
Mapping factor
scores
0.3%
Morex
PC4
Steptoe
Morex
Steptoe
7H
Wheat stem rust pathosystem in barley
Pgt-TTKS
Most of the
Pgt f.sp. tritici
pathotypes
MCC
Rpg1
rpg1
Chr 7H
Pgt f.sp.
secale
QCC
Rpg1
Rpg1
Rpg1
Rpg1
Rpg4
rpg4
rpg5
Rpg5
Chr 5H
Chr 5H
Brueggeman et al PNAS 2008
‘Wheat killer detected in Iran - Dangerous fungus on the move
from East Africa to the Middle East’
2008
fao.org
‘Deadly Wheat Fungus Threatens World's Breadbaskets’
2007
Science
‘Wheat fungus spreads out of Africa - Stem rust threatens key crops in Asia’
2007
Nature
‘Wheat Warning—New Rust Could Spread Like Wildfire’
2005
sciencenews.org
Project PIs
Robbie Waugh
Mike Kearsey
SCRI
University of Birmingham
Phenotyping
Brian Steffenson
University of Minnesota
Sequencing
Nicky Bonar
SCRI
Probe level analysis
Ilze Druka
University of Abertay, SCRI
GeneNetwork
Rob Williams
University of Tennesee
Animation
Anna Druka
University of Dundee
AFFX-CreX-3_at
16384
100pM
8192
AFFX-r2-P1-cre-5_at
AFFX-r2-P1-cre-3_at
4096
log2 MAS5.0
AFFX-CreX-5_at
2048
AFFX-BioDn-3_at
25pM
1024
AFFX-BioDn-5_at
AFFX-r2-Ec-bioD-5_at
512
AFFX-r2-Ec-bioD-3_at
256
AFFX-BioC-3_at
128
5pM
AFFX-BioC-5_at
AFFX-r2-Ec-bioC-5_at
64
AFFX-r2-Ec-bioC-3_at
32
AFFX-BioB-3_at
16
AFFX-BioB-5_at
1.5pM
8
AFFX-r2-Ec-bioB-5_at
AFFX-BioB-M_at
4
AFFX-r2-Ec-bioB-3_at
2
1
AFFX-ThrX-3_at
AFFX-r2-Ec-bioB-M_at
1
2
GEM
3
1
2
COL
3
1
2
RAD
3
AFFX-ThrX-5_at
0pM
AFFX-ThrX-M_at
AFFX-TrpnX-3_at
AFFX-TrpnX-5_at
AFFX-TrpnX-M_at
median expression
400
300
100
MAS5.0
background
50
30
BRC
ANT
INF
DEM22
END22
CAR16
CAR10
CAR5
PST
ROO
CRO
LEA
RAD
COL
GEM
ROO
CRO
LEA
RAD
COL
GEM
Optic
GP
Barke
HN
OW-R
OW-D
Morex
Steptoe
GPGP
GPMx
MxGP
MxMx
Morex
Golden
Promise
leaf
embryo
GP-Rpg1
GP(rpg1)
SM136
SM039
Morex
Steptoe
absolute expression
(log scale)
200
condition1
condition2
condition1
expression
condition1
condition2
gene1gene2
T1 T2 T3
gene1
T1 T2 T3
gene2
Chs
1H
3H
2H
T1 T2 T3
gene1
T1 T2 T3
gene2
transcription
factor
3H
locus B
gene1gene2 gene1gene2
Cfi
4H
5H
6H
7H
mRNA profiling:
seedling leaf
expression
in situ hybridization
Hybridization probe: Gene 2
Hybridization probe: Gene1
gene 1 gene 2
mRNA profiling:
isolated cells
cell type 2
expression
expression
cell type 1
gene 1
gene 2
gene 1 gene 2
Correlating the phenotype
Pearson product-moment correlation: Type ’0’ to 22,840 mRNA abundance measures
Ranked by r
kelch repeat-containing F-box protein
IQ calmodulin-binding protein
Acyl-coenzyme A oxidase 1.2
peroxisomal
expressed protein
DNA methyltransferase DMT106
hypothetical protein
NB-ARC protein
None
Sulfotransferase
beta-14-mannan synthase
reverse transcriptase
NB-ARC protein
F-box protein
None
Acid phosphatase
Rpg1
RNA recognition motif
Centrin
alpha-dioxygenase
Late embryogenesis abundant group
1 protein
signal peptide peptidase
expressed protein
proline-rich protein
UDP-glucoronosyl and UDP-glucosyl
transferase
UPF0016 protein
polygalacturonase
Ranked by IrI
0.86
0.86
-0.85
-0.84
-0.82
-0.82
0.81
0.79
-0.78
-0.76
0.76
-0.74
0.73
0.72
0.71
0.71
-0.69
0.69
0.67
0.67
0.66
0.65
0.65
-0.64
-0.63
-0.62
kelch repeat-containing F-box protein
IQ calmodulin-binding protein
NB-ARC protein
None
reverse transcriptase
F-box protein
None
Acid phosphatase
Rpg1
Centrin
alpha-dioxygenase
Late embryogenesis abundant group
1 protein
signal peptide peptidase
expressed protein
proline-rich protein
polygalacturonase
UPF0016 protein
UDP-glucoronosyl and UDP-glucosyl
transferase
RNA recognition motif
NB-ARC protein
beta-14-mannan synthase
Sulfotransferase
hypothetical protein
DNA methyltransferase DMT106
expressed protein
Acyl-coenzyme A oxidase 1.2
peroxisomal
0.86
0.86
0.81
0.79
0.76
0.73
0.72
0.71
0.71
0.69
0.67
0.67
0.66
0.65
0.65
-0.62
-0.63
-0.64
-0.69
-0.74
-0.76
-0.78
-0.82
-0.82
-0.84
-0.85
Rpg1 expression
Is this expression mapping or genotyping?
Morex - resistant
Rpg1 genotype
Signal values of individual probes across 150 segregants
Significant linkage
Meta-analysis of the Rpg1 expression
Golden Promise (rpg1)
100
MAS5.0
background
50
30
BRC
ANT
INF
DEM22
END22
CAR16
CAR10
CAR5
PST
ROO
CRO
LEA
RAD
COL
GEM
ROO
CRO
LEA
RAD
COL
GEM
Optic
GP
Barke
HN
OW-R
OW-D
Morex
Steptoe
GPGP
GPMx
MxGP
MxMx
GP-Rp g1
GP(rpg1)
SM136
SM039
Morex
Steptoe
absolute expression
(log scale)
Morex
Golden
Promise
leaf
embryo
lower 10% of the
expression
~1200 genes
200
LRS value distributions of the cis- and trans- regulated genes
100
80
60
40
20
cis
trans
100000
Number of GeneChip probe sets
Linkage probability frequency distribution
11939
10000
7135
4969
3982
3339
2866
2518
2195
1949
1719
992
1000
616
LRS range
173
100
>10 >20 >30 >40 >50 >60 >70 >80 >90 >100 >150 >200 >300
Rice chromosome 5 (LRS>30)
Rice chromosome 5 (LRS>30)
1H
2H
3H
35000000
4H 5H 6H 7H
30000000
30000000
25000000
25000000
Mb (rice)
Mb (rice)
35000000
20000000
15000000
1H
2H
3H
4H
5H
6H
7H
20000000
15000000
10000000
10000000
5000000
5000000
0
0
0
50
100
150
200
250
chromosome/markers (barley)
300
350
0
100
200
300
400
500
chromosome/markers (barley)
600
700