Stripe rust resistance gene Yr10 encodes an evolutionary- conserved

and unique CC-NBS-LRR sequence

1.State Key Laboratory of Crop Stress Biology in Arid Areas, NAFU

2.Lethbridge Research Centre, AAFC

Wei Liu

Wheat stripe rust

Caused by Puccinia striiformis

Cool and humid climatic conditions

Control methods

Around 50 stripe rust resistance genes have been officially named but only a handful of these resistance genes have been deployed.

Yr-gene

Gene Example of wheat genotyp

eChromo. location Resistance typea Gene Example of wheat genot

ypeChromo. location Resistance typea

Yr1 Chinese 166 2AL RS, AS Yr24 K773 1BS RS, AS

Yr2 Heines VII 7B RS, AS Yr25 Strubes Dickkopf 1D RS, AS

Yr3a Cappelle Desprez 5BL RS, AS Yr26 R55 1BS RS, AS

Yr3b Hybrid 46 5BL RS, AS Yr27 Ciano 79 2BS RS, AS

Yr3c Minister 5BL RS, AS Yr28 W7984 4DS RS, AS

Yr4a Cappelle Desprez 3B RS, AS Yr29 Pavon 76 1BL NRS, AP

Yr4b Hybrid 46 3B RS, AS Yr30 Opata 85 3BS NRS, AP

Yr5 T.spelta Album 2BL RS, AS Yr31 Pastor 2BS RS, AS

Yr6 Heines Kolben 7BS RS, AS Yr32 Carstens V 2AS RS, AS

Yr7 Lee 2BL RS, AS Yr33 Batavia 7DL RS, AS

Yr8 Compair 2DL RS, AS Yr34 WAWHT2046 5AL NRS, AP

Yr9 Clement 1RS/1BL RS, AS Yr35 98M71 6BS RS, AS

Yr10 Moro 1BS RS, AS Yr36 RSL65 6BS NRS, HTAP

Yr11 Joss Cambier / RS, AP Yr37 S14 2DL RS, AS

Yr12 Mega / RS, AP Yr38 Line 03524 6AS RS, AS

Yr13 Maris Huntsman / RS, AP Yr39 Alpowa 7BL NRS, HTAP

Yr14 Hobit / RS, AP Yr40 T5DL.5DS-5MgS 5DS RS, AS

Yr15 G-25 1BS RS, AS Yr41 Chuannong19 2BS RS, AS

Yr16 Capelle Desprez 2DL NRS, AP Yr42 03M119-71A 6AL/6AS RS,AS

Yr17 VPM1 2AS RS, AS Yr43 IDO377s 2BL RS, AS

Yr18 Jupateco 73R 7DS NRS, HTAP Yr44 Zak 2BL RS, AS

Yr19 Compair 5B RS, AS Yr45 PI181434 3DL RS, AS

Yr20 Fielder 6D RS, AS Yr46 RL6007 4DL NRS, AP

Yr21 Lemhi 1BL RS, AS Yr47 AUS28183 5BS RS, AS

Yr22 Lee 4D RS, AS Yr48 PI610750 5AL RS,AS

Yr23 Lee 6D RS, AS

Yr36 (Kinase-START gene)

Fu et al. 2009 Science 323:1357-1360

Lr34/ Yr18/ Pm38 (ABC transporter gene)

Krattinger et al. 2009 Science 323:1360-1363

Yr10

In China

Resistance to CYR32, CYR33 in seedling and adult stages

(Zeng et al.,2011, unpublished)

In Pacific Northwestern United StatesFew races can overcome Yr10 -- (Chen et al., 2005)

In western CanadaStill provide resistance in soft white spring and winter wheat --(Su et al.,2003)

Yr10

Moro as source of resistance

-Yr10 ( located on chromosome 1B)

(Metzger and Silbaugh,1970)

-YrMor (located on chromosome 4B)

(Chen and Line,1992)

Moro resistant

parent

Fielder susceptible

parent

(local strain)

F2 plantsBackcross 4

M c s s s Rs S r r r r

1100bp

RAPD amplification of a 1100 bp polymorphic fragment using primer OPE5

R, bulk of Cot fractionated DNA from 14 resistant line BC4F5; S, bulk of Cot fractionated DNA from 4 susceptible line BC4F5; r, Cot fractionated DNA from individual resistant line BC4F5; s, Cot fractionated DNA from individual susceptible line BC4F5;

Segregation of E51100 tested on 874 lines derived from Moro and Fielder

genotype phenotype422 R 422 R452 S 452 S

Segregation of E51100 tested on 131 lines (BC2F3 and BC3F3) between Moro and Fielder

genotype phenotype

104 R 104 R

27 S 27 S

Assessment of Specific 1100 bp DNA Fragment Fragment present Fragment absent

Moro Corrigin(Yr10) Fielder Corrigin PI178383 QT3960(Yr10) Capp. D. QT3960 Jacmar 79W93(Yr10) Compair 79W93 T. spelta line 415 T. spelta Avocet (Yr10) Avocet

Conclusion 1

A polymorphic DNA fragment ≈1100bp was detected to Yr10

Perfect linkage of Yr10 gene with the phenotype in 874 segregating lines Fragment E-5 had a week homology to the P-loop of the L6 rust resistance gene in flax and score (94, P< 1.4x10-4) E-5 was present in the cultivars carrying Yr10

Two clones, 4B and 4E, were selected for sequence analysis and characterization

Construction of an EcoR1 Subgenomic library

Screening of 350,000 recombinant clones were hybridized by E51100

Seven independent clones identified

TGA

TGA4B

ATG

ATG

0 nt

0 nt 3630 nt

3663 nt

834

834 1963

1989

(1155 nt)

(1129 nt)

0 aa 843 aa

0 aa 824 aa

AF149113

AF149112

cDNAFragments

ATG

TGA

SRPL

SRLZ

SRLR

1935 nt

1

1

1

406 nt

961 nt

Full length cDNA

ATG

TGA

2475 bp

AF149114

Comparison of LRR domain

xxLxxLxxLxxLxL/I QTRSLPSKIRRLSL QSSNEEDVQPM PTMSSLSHVRSLTV FSKDLSLL SALSGFLVLRALDL SGCEEVGNHHM KDICNLFHLRYLSL EGTSITEIP KEISNLRLLQLLVI RSTKMKKFPSTF VQLGQLVFIDMGNR EVSRLLL KSMSTLPSLSSLAI GIGELREEDL QILGSMPSLHDLSI DVGYWERGRD KRLVIDSGSPFRSL TRFSIKGCGFIDF GTLQKLQILE-LSI FGKAIKDRFGDFQ FGLENLSSLEHVYV DARGRGIIPSQE

clone 4B xxLxxLxxLxxLxL/I

clone 4E xxLxxLxxLxxLxL/I CKIRRLSHVRSL

FSKDFSLL SELSSFLVLRALDL SGCTEVRDHNL KDICNLFHLRYMSL QGGCITEIPKEISNLQLLQVLDI RSTEIEKFPSTVVELRQLVFIDMGNG MVSASLLKLMSTLPSLSSLAI GLTGLREEDL QILGSMSSLRDLSL IVVHWGKVTG KRLVIGNGCPFQSL TRFNIKSIDGTGFMFAE GTLQKLQILE-LDL YVEKAKDQLGDFQ FGLENLSSLEHVNV VARGGGSEGELINALE KELDINPNKPTLTV KKVTPCSFTFVH

TV

Expression of genomic sequences

LRR

4Eclone

4E specificreverse

4E specific primersFragment size = 524 bp

4B

4E

LRR start

LRR start

TGA

TGA

Size Amplified= 359 bp

Size Amplified = 302 bp

forwardprimer

reverseprimer

forwardprimer

reverseprimer

4E deletion

LRR start

TGA

4E specificforward

RT-PCRRT-PCR

359 bp

524 bp

302 bp

4B R R RR4E

4E primers

4B R R RR4E

4E primers

TATA box

ABRE

LR

MyB + LR

Caat box

ERElRE

MeJAWUN

LAMP TCA

Light & stress responsive elements Plant defense responsive elements

4B

+1

-1300

TSS

4B

+1

-1300

TSS

4E

+1

-1300

???

Promoter characterizationPromoter characterization

Linkage of Clones 4B and 4E

Clone 4E was very closely linked to the resistance

phenotype in a random subsample of 55 lines of

the BC2F3 and BC3F3 segregating population as 42

lines (76%) were identified as resistant on the

genotype and 43 (78%) resistant on the

phenotype.

Fluorescent in situ hybridization of Moro metaphase chromosomes to localize probe 4B

Conclusion 2 Two distinct genomic clones 4B and 4E have

been isolated and characterized

4B and 4E are 84% identical and similar level of homology, they seem to be the same gene cluster. 4E represents a non-expressed pseudogene

Clone 4B located on the distal end of the short arm of chromosome pair 1B, the same reported genetic location of Yr10.

Clone 4B corresponds to Yr10

The deduced aa sequence of Yr10 exhibits highly conserved homologs from Dasypyrum breviaristatum (ACC64518), Aegilops tauschii (AAM47268),

Brachypodium distachyon (XP_003577469), Sorghum bicolor (XP_002450764) and Oryza sativa (EAY81279).

-4

-2

0

2

4

6

8M-84 F-84

PAD4

-4

-2

0

2

4

6

8M-84 F-84

EDS1

-4

-2

0

2

4

6

8M-84 F-84

AOS

Days after inoculation2 4 86 10 12 14

Relative gene expression (Log2)

-4

-2

0

2

4

6

8M-84 F-84

TLP1

-4

-2

0

2

4

6

8M-84 F-84

Glu1

Relative gene expression (Log2)

-4

-2

0

2

4

6

8M-84 F-84

PR1.1

-4

-2

0

2

4

6

8M-84 F-84

Chi3

2 4 86 10 12 14Days after inoculationWang et al.,2011, unpublished

Relative expression levels in resistant Moro and susceptible Fielder after inoculation with SRC-84

Functional analysis of the wheat stripe rust resistance gene Yr10

Transgenesis

BSMV-VIGS

Fielder

T1

Moro

Fielder

T1

Moro

SRC-84 strain

avirulent on Yr10

Phenotypic reaction of Fielder, Moro (Yr10) and transformed Fielder (Yr10) (T1)

Silencing PDS in Moro

4B clone (Yr10)

CCNBS LRR

γK2a γSLR γCLR

A B C D E F G

Mock Mock γK2a γ00 γSLRγ00 γCLR

CDL-29 SRC-84SRC-84

Phenotypic changes in Moro leaves infected with P. striiformis f. sp. tritici.

Relative gene expressions of Yr10 in silenced and control Moro

-2.5

-1.5

-0.5

0.5

1.5

2.5

0 2 4 6 8 10 12 14

Post-innoculation (days)

Fol

d di

ffer

ence

(lo

g2)

Moro84 Moro29

A B C

FE

hr

h

hhr

h h

10 dpi

D

G H I

J K L

14 dpi

Silencing of Yr10 transcripts and stripe rust infection levels in Moro wheat leaves transfected with BSMV-VIGS constructs and subsequently inoculated with strains of Pst.

Conclusions 3

Transgenesis clone 4B exhibits the same functional properties as Yr10

BSMV-VIGSsilencing 4B in Moro resulted in a susceptible interaction

Summary

• a polymorphic DNA fragment (E51100) was perfectly linked to Yr10

• two highly related genomic sequences 4B and 4E from Moro wheat expresses the seedling Yr10 gene for resistance to P. striiformis and 4B is the only expressed sequence and corresponds to Yr10 gene.

Summary

• Yr10 is the first and unique CC-NBS-LRR class of genes reported to provide seedling stripe rust resistance gene identified while Yr18/Lr34 and Yr36 represent APR genes.

• Yr10 shows its functionality using transgensis and gene silencing.

Discussions

• Whether Yr10 homologs in rice, maize and sorghum would confer reistance to wheat stripe rust or other pest is currently unknown

• Clone 4E, a Yr-type of pseudogene, may represent

an adaptive mechanism in wheat to counter the rapid virulence shifts in the pathogen by accelerating the evolution of specific resistance genes capable of recognizing new pathogen forms.

Acknowledgement

Collaborators

Michele Frick, Réné Huelb, Cory L. Nykiforuk, Xiaomin Wang, François Eudes, Robert L. ConnerAlan Kuzyk, Qin Chen

Denis A. GaudetZhensheng Kang,André Laroche,

Funding

This study is financially supported by grants from Alberta Agricultural Research Institute and AAFC A-base projects to AL and 111 Project from the Ministry of Education of China (Grant No.B07049) to ZK.