Wei Liu, Nor

1. Stripe rust resistance gene Yr10
encodes an evolutionary- conserved
and unique CC-NBS-LRR sequence
Wei Liu
1.State Key Laboratory of Crop Stress Biology in Arid Areas, NAFU
2.Lethbridge Research Centre, AAFC

2. Wheat stripe rust
 Caused by Puccinia striiformis
 Cool and humid climatic conditions
 Control methods

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

4. Example of wheat Gene Example of wheat Chromo. location Resistance typea
Gene Chromo. location Resistance typea
genotype genotype
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

5. Yr36 (Kinase-START gene)
Fu et al. 2009 Science 323:1357-1360

6. Lr34/ Yr18/ Pm38 (ABC transporter gene)
Krattinger et al. 2009 Science 323:1360-1363

7. Yr10
In China
Resistance to CYR32, CYR33 in seedling and adult stages
(Zeng et al.,2011, unpublished)
In Pacific Northwestern United States
Few races can overcome Yr10 -- (Chen et al., 2005)
In western Canada
Still provide resistance in soft white spring and winter
wheat --(Su et al.,2003)

8. Yr10
Moro as source of resistance
-Yr10 ( located on chromosome 1B)
(Metzger and Silbaugh,1970)
-YrMor (located on chromosome 4B)
(Chen and Line,1992)

9. Fielder
Moro
susceptible
resistant
parent
parent
(local strain)
Backcross 4
F2 plants

10. RAPD amplification of a 1100 bp polymorphic
fragment using primer OPE5
M c s s s s R S r r r r
 1100bp
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;

11. Segregation of E51100 tested on 874 lines derived
from Moro and Fielder
genotype phenotype
422 R 422 R
452 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

12. 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

13. Conclusion 1
 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
A polymorphic DNA fragment ≈1100bp was
detected to Yr10

14. Construction of an EcoR1 Subgenomic library
Screening of 350,000 recombinant clones were
hybridized by E51100
Seven independent clones identified
Two clones, 4B and 4E, were selected for
sequence analysis and characterization

15. 0 nt 834 1963 3663 nt
(1129 nt)
AF149113 ATG TGA
0 aa 843 aa
0 nt 834 1989 3630 nt
4B (1155 nt)
AF149112 ATG TGA
0 aa 824 aa
cDNA SRPL 1 406 nt
Fragments
SRLR 1 961 nt
ATG
SRLZ 1 1935 nt
TGA
Full
length
ATG 2475 bp TGA
cDNA
AF149114

16. Comparison of LRR domain
clone 4B clone 4E
xxLxxLxxLxxLxL/I xxLxxLxxLxxLxL/I
QTRSLPSKIRRLSL QSSNEEDVQPM CKIRRLSHVRSL
PTMSSLSHVRSLTV FSKDLSLL TV FSKDFSLL
SALSGFLVLRALDL SGCEEVGNHHM SELSSFLVLRALDL SGCTEVRDHNL
KDICNLFHLRYLSL EGTSITEIP KDICNLFHLRYMSL QGGCITEIP
KEISNLRLLQLLVI RSTKMKKFPSTF KEISNLQLLQVLDI RSTEIEKFPSTV
VQLGQLVFIDMGNR EVSRLLL VELRQLVFIDMGNG MVSASLL
KSMSTLPSLSSLAI GIGELREEDL KLMSTLPSLSSLAI GLTGLREEDL
QILGSMPSLHDLSI DVGYWERGRD QILGSMSSLRDLSL IVVHWGKVTG
KRLVIDSGSPFRSL TRFSIKGCGFIDF KRLVIGNGCPFQSL TRFNIKSIDGTGFMFAE
GTLQKLQILE-LSI FGKAIKDRFGDFQ GTLQKLQILE-LDL YVEKAKDQLGDFQ
FGLENLSSLEHVYV DARGRGIIPSQE FGLENLSSLEHVNV VARGGGSEGELINALE
KELDINPNKPTLTV KKVTPCSFTFVH

17. Expression of genomic sequences
4E specific 4E specific
forward reverse
4E 4E specific primers
clone Fragment size = 524 bp
LRR TGA
LRR start
LRR start
TGA
Size Amplified
4B = 359 bp
forward reverse
primer primer
LRR start
TGA
4E deletion Size Amplified =
4E 302 bp
forward reverse
primer primer

18. RT-PCR
4E primers  4E primers
4B 4E R R R R 4B 4E R R R R
524 bp
359 bp
302 bp

19. Promoter characterization
TSS
+1
4B
-1300
? ? +1
4E
-1300
TATA box
Caat box
Light & stress responsive elements Plant defense responsive elements
ABRE ElRE ER
MyB + LR WUN MeJA
LR LAMP TCA

20. 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.

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

22. 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

23. 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).

24. Relative expression levels in resistant Moro and
susceptible Fielder after inoculation with SRC-84
8 PR1.1
8
PAD4 6
M-84 F-84
M-84 F-84 4
6
2
4
0
2
-2
Relative gene expression (Log2)
0
-4
-2
-4 8 TLP1
Relative gene expression
M-84 F-84
EDS1 6
8 4
M-84 F-84
6 2
4 0
(Log2)
2 -2
0 -4
-2 8 Glu1
M-84 F-84
-4 6
8
AOS 4
M-84 F-84 2
6 0
4 -2
2 -4
0 8 Chi3
M-84 F-84
-2 6
4
-4
2
2 4 6 8 10 12 14 0
Days after inoculation -2
-4
2 4 6 8 10 12 14
Wang et al.,2011, unpublished Days after inoculation

25. Functional analysis of the wheat stripe
rust resistance gene Yr10
 Transgenesis
 BSMV-VIGS

26. Phenotypic reaction of Fielder, Moro (Yr10) and
transformed Fielder (Yr10) (T1)
Fielder
T1 SRC-84 strain
avirulent on Yr10
Moro

27. Silencing PDS in Moro

28. 4B clone (Yr10)
NBS
LRR
CC
γK2a γSLR γCLR

29. Phenotypic changes in Moro leaves infected
with P. striiformis f. sp. tritici.
A B C D E F G
Mock γ00 Mock γ00 γK2a γSLR γCLR
CDL-29 SRC-84 SRC-84

30. Relative gene expressions of Yr10 in silenced and
control Moro
Moro84 Moro29
Fold difference (log2) 2.5
1.5
0.5
-0.5
-1.5
-2.5
0 2 4 6 8 10 12 14
Post-innoculation (days)

31. 10 dpi
A B C
hr
h
hr
D E F
h h
h

32. 14 dpi
G H I
J K L

33. 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.

34. Conclusions 3
clone 4B exhibits the same
Transgenesis functional properties as Yr10
silencing 4B in Moro resulted
BSMV-VIGS in a susceptible interaction

35. 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.

36. 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.

37. 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.

38. Acknowledgement
Collaborators
André Laroche, Zhensheng Kang, Denis A. Gaudet
Michele Frick, Réné Huelb, Cory L. Nykiforuk,
Xiaomin Wang, François Eudes, Robert L. Conner
Alan Kuzyk, Qin Chen
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.