Regulatory networks underlying barley interaction with rust fungi
Arnis DrukaScottish 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
compatible incompatible
Outcomes of the barley – stem rust fungus interaction
R or S
Brueggeman et al PNAS 2002
Major stem rust resistance gene Rpg1
Mendelian trait mapping
Positional cloning
Rpg1 Chr 7H
Compatiblecv Steptoe
Incompatiblecv Morex
Puccinia graminis MCC
Identification of the Rpr1 locus
SusceptibleRpg1 / Rpg1
ResistantRpg1 / Rpg1
Zhang at al TAG 2006
Fast neutrons
in vitromutagenesis
Pgt f.sp. tritici
MCC
selection
mRNA profiling
R or S
Mendelian trait mapping
Rpr1 Chr 4H
-5 -4 -3 -2 -1 0 1 2 3 4 50
0.5
1
1.5
log2(Fold Change)
-log1
0(P-
Valu
e)HU03D17u_s_at
HVSMEm0005P05r2_at
Contig4901_s_at
Contig6699_s_at
Contig7061_s_at
Contig13680_s_at
Contig13681_at
Contig14769_at
ResistantRpg1
SusceptibleRpg1
?
Rpr1
Phenotyping barley and stem rust fungus interaction
sensory transduction histidine protein kinase
Benjamini-Hochberg FDR (0.05%)
Druka et al TAG 2008
Type ‘0’
Type ‘1’
Type ‘2’
Type ‘3’
Compatiblecv Steptoe
Incompatiblecv Morex
Puccinia graminis MCC
Mapping infection types
Type ‘0’
Type ‘1’
Type ‘2’
Type ‘3’
IM
IM
IM
IM
Morex - resistant
Morex - resistant
Morex - resistant
Steptoe - susceptible
Type '2' phenotype
sensory transduction histidine protein kinase
Morex effect
Morex effect
1H 2H 3H 4H 5H 6H 7H
3H locus B
Co-regulation
5H
1H 2H 3H 4H 5H 6H 7H
locus A
Co-localization
2H ion protein RAR1 2H bin10MLO1 4Hheat shock protein nk lipoxygenase 2HS* jasmonate-induced protein 6H* defender against death 1 (dad1) 7H* calcineurin-like phosphoesterase 2HS*
regulatory elicitor-responsive gene 3 2HS* DegP2 protease (DEGP2) nk heat shock protein 70 nk endo-1,3;1,4-beta-D-glucanase nk
hypersensitive reaction-induced protein 3 5H heat shock factor binding protein 2 7H* sensory transduction histidine kinase 2H bin8 acid phosphatase 7H serine/threonine receptor kinase nk Hsp90 nk NMD3 nk jasmonate-induced protein nk MLA1 5H
cyclic nucleotide and calmodulin-regulatedchannel 6H bin8 18
21
6520
33802558
26S proteasome non-ATPase subunit 6 nk 75
8030212957278532
11616101617
Ppd-H1 35.5Tef4 36.3ABG358 37.8CDO064 40.6ABG459 42MWG520A 43.4snp_0847 44DAK213A 44.8Pox 48snp_1073 48.6ABC454 49.3cMWG663A 50JS188A 50.7Adh8 51.4snp_1061 58.4snp_1054 60.4MWG557 61.1snp_1272 61.8CDO537 63.3ABG316C 65.5snp_0474 66.3olad_259 66.9P40FA 66.9Hvex1 67.6snp_1121 68.3snp_0545 70.3snp_0265 73.8snp_0407 74.4bBE54D 75.1snp_0650 76.5snp_1449 78.5snp_1435 79.2His3C 81.6
sensory transduction
histidine kinase
nec1
PCD
Barley – mildew interaction
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 [email protected]
1H 2H 3H 4H 5H 6H 7H
Steptoe
Morex
Morex
Steptoe
Steptoe
0102030405060708090
100
PC1 PC2 PC3 PC4Factor
Varia
bilit
y (%
) 69.4%
2.6%
27.7%
0.3%
IT0IT1IT3
IT2
Mapping factor scores
Wheat stem rust pathosystem in barley
rpg1
MCCMost of the Pgt f.sp. tritici pathotypes
Rpg1
QCC
Rpg4 rpg4
Rpg1 Rpg1
Pgt f.sp. secale
Rpg1 Rpg1
rpg5 Rpg5
Pgt-TTKS
Brueggeman et al PNAS 2008
Chr 5H Chr 5HChr 7H
‘Wheat killer detected in Iran - Dangerous fungus on the move from East Africa to the Middle East’
‘Deadly Wheat Fungus Threatens World's Breadbaskets’
‘Wheat fungus spreads out of Africa - Stem rust threatens key crops in Asia’
‘Wheat Warning—New Rust Could Spread Like Wildfire’2005
sciencenews.org
2008fao.org
2007Nature
2007Science
Project PIsRobbie Waugh SCRIMike Kearsey University of Birmingham
PhenotypingBrian Steffenson University of Minnesota
SequencingNicky Bonar SCRI
Probe level analysisIlze Druka University of Abertay, SCRI
GeneNetworkRob Williams University of Tennesee
AnimationAnna Druka University of Dundee
1248
163264
128256512
1024204840968192
16384AFFX-CreX-3_atAFFX-CreX-5_atAFFX-r2-P1-cre-5_atAFFX-r2-P1-cre-3_at
AFFX-BioDn-3_atAFFX-BioDn-5_atAFFX-r2-Ec-bioD-5_atAFFX-r2-Ec-bioD-3_at
AFFX-BioC-3_atAFFX-BioC-5_atAFFX-r2-Ec-bioC-5_atAFFX-r2-Ec-bioC-3_at
AFFX-BioB-3_atAFFX-BioB-5_atAFFX-r2-Ec-bioB-5_atAFFX-BioB-M_atAFFX-r2-Ec-bioB-3_atAFFX-r2-Ec-bioB-M_at
AFFX-ThrX-3_atAFFX-ThrX-5_atAFFX-ThrX-M_atAFFX-TrpnX-3_atAFFX-TrpnX-5_atAFFX-TrpnX-M_at
100pM
25pM
5pM
1.5pM
0pM
log2
MA
S5.
0
GEM COL RAD1 2 3 1 2 3 1 2 3
PS
TC
AR
5C
AR
1 0C
AR
16E
ND
2 2D
EM
2 2
GE
MC
OL
RA
DLE
AC
RO
RO
O
INF
AN
TB
RC
200
100
5030
absolute expression (log scale) MAS5.0
background
SM
03 9S
M13 6
GE
MC
OL
RA
DLE AC
RO
RO
O
Morex
Mx M
xM
x GP
GPM
xG
P GP
GP( rp g1 )
GP- R
p g1
Golden Promise
leafembryo
St ep toe
Mo re x
Ste pto e
Mor ex
OW
-DO
W- R
HN
Bar ke
GP
Op tic
300
400 median expression
gene1 T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3gene2gene1 gene2
gene1gene2gene1gene2
condition1
condition2
gene1 gene2
condition2condition1condition1
expr
essio
n
1H 2H 3H 4H 5H 6H 7H
3H locus B
transcriptionfactorChs Cfi
mRNA profiling: seedling leaf
gene 1 gene 2Hybridization probe: Gene1
in situ hybridization
Hybridization probe: Gene 2
expr
e ssi
o n
gene 1 gene 2gene 1 gene 2
cell type 1 cell type 2
mRNA profiling:isolated cells
expr
essi
on
exp r
essi
on
Correlating the phenotype
kelch repeat-containing F-box protein 0.86IQ calmodulin-binding protein 0.86Acyl-coenzyme A oxidase 1.2 peroxisomal -0.85expressed protein -0.84DNA methyltransferase DMT106 -0.82hypothetical protein -0.82NB-ARC protein 0.81None 0.79Sulfotransferase -0.78beta-14-mannan synthase -0.76reverse transcriptase 0.76NB-ARC protein -0.74F-box protein 0.73None 0.72Acid phosphatase 0.71Rpg1 0.71RNA recognition motif -0.69Centrin 0.69alpha-dioxygenase 0.67Late embryogenesis abundant group 1 protein 0.67signal peptide peptidase 0.66expressed protein 0.65proline-rich protein 0.65UDP-glucoronosyl and UDP-glucosyl transferase -0.64UPF0016 protein -0.63polygalacturonase -0.62
kelch repeat-containing F-box protein 0.86IQ calmodulin-binding protein 0.86NB-ARC protein 0.81None 0.79reverse transcriptase 0.76F-box protein 0.73None 0.72Acid phosphatase 0.71Rpg1 0.71Centrin 0.69alpha-dioxygenase 0.67Late embryogenesis abundant group 1 protein 0.67signal peptide peptidase 0.66expressed protein 0.65proline-rich protein 0.65polygalacturonase -0.62UPF0016 protein -0.63UDP-glucoronosyl and UDP-glucosyl transferase -0.64RNA recognition motif -0.69NB-ARC protein -0.74beta-14-mannan synthase -0.76Sulfotransferase -0.78hypothetical protein -0.82DNA methyltransferase DMT106 -0.82expressed protein -0.84Acyl-coenzyme A oxidase 1.2 peroxisomal -0.85
Ranked by r Ranked by IrI
Pearson product-moment correlation: Type ’0’ to 22,840 mRNA abundance measures
Is this expression mapping or genotyping?Rpg1 expression
Rpg1 genotype
Signal values of individual probes across 150 segregants
Morex - resistant
Significant linkage
PST
CAR
5CA
R10
CAR
16E N
D22
DEM
2 2
GEM
COL
RAD
LEA
C RO
ROO
INFAN
TBR
C200
100
50
30
absolute expression (log scale) MAS5.0
background
S M039
SM136
GEM
COL
RAD
LEA
CRO
ROO
Morex
MxMx
MxGP
GPM
xGP
GP
GP(rp g1
)GP
-Rpg1
Golden Promiseleafembryo
Ste ptoeM
orex
Stepto e
Mor ex
OW
-DO
W- R
H N BarkeG
PO
ptic
Golden Promise (rpg1)
Meta-analysis of the Rpg1 expression
lower 10% of the expression~1200 genes
LRS value distributions of the cis- and trans- regulated genes
cis trans
100
80
60
40
20
Linkage probability frequency distribution
173
616992
11939
71354969 3982 3339 2866 2518 2195 1949 1719
100
1000
10000
100000
>10 >20 >30 >40 >50 >60 >70 >80 >90 >100 >150 >200 >300
LRS range
Num
ber o
f Gen
eChi
p pr
obe
sets
Rice chromosome 5 (LRS>30)
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
0 100 200 300 400 500 600 700
chromosome/markers (barley)
Mb
(ric
e)
Rice chromosome 5 (LRS>30)
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
0 50 100 150 200 250 300 350
chromosome/markers (barley)
Mb
(ric
e)
1H 2H 3H 4H 5H 6H 7H 1H 2H 3H 4H 5H 6H 7H