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 welcome!Arnis.Druka@scri.ac.uk

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