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Stripe rust resistance gene Yr10 encodes an evolutionary- conserved and unique CC-NBS-LRR sequence
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Stripe rust resistance gene Yr10 encodes an evolutionary- conserved and unique CC-NBS-LRR sequence

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Wei Liu, Nor

Wei Liu, Nor

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  • 1. Stripe rust resistance gene Yr10encodes 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-geneAround 50 stripe rust resistance geneshave been officially named but only ahandful of these resistance genes havebeen deployed.
  • 4. Example of wheat Gene Example of wheat Chromo. location Resistance typeaGene Chromo. location Resistance typea genotype genotypeYr1 Chinese 166 2AL RS, AS Yr24 K773 1BS RS, ASYr2 Heines VII 7B RS, AS Yr25 Strubes Dickkopf 1D RS, ASYr3a Cappelle Desprez 5BL RS, AS Yr26 R55 1BS RS, ASYr3b Hybrid 46 5BL RS, AS Yr27 Ciano 79 2BS RS, ASYr3c Minister 5BL RS, AS Yr28 W7984 4DS RS, ASYr4a Cappelle Desprez 3B RS, AS Yr29 Pavon 76 1BL NRS, APYr4b Hybrid 46 3B RS, AS Yr30 Opata 85 3BS NRS, APYr5 T.spelta Album 2BL RS, AS Yr31 Pastor 2BS RS, ASYr6 Heines Kolben 7BS RS, AS Yr32 Carstens V 2AS RS, ASYr7 Lee 2BL RS, AS Yr33 Batavia 7DL RS, ASYr8 Compair 2DL RS, AS Yr34 WAWHT2046 5AL NRS, APYr9 Clement 1RS/1BL RS, AS Yr35 98M71 6BS RS, ASYr10 Moro 1BS RS, AS Yr36 RSL65 6BS NRS, HTAPYr11 Joss Cambier / RS, AP Yr37 S14 2DL RS, ASYr12 Mega / RS, AP Yr38 Line 03524 6AS RS, ASYr13 Maris Huntsman / RS, AP Yr39 Alpowa 7BL NRS, HTAPYr14 Hobit / RS, AP Yr40 T5DL.5DS-5MgS 5DS RS, ASYr15 G-25 1BS RS, AS Yr41 Chuannong19 2BS RS, ASYr16 Capelle Desprez 2DL NRS, AP Yr42 03M119-71A 6AL/6AS RS,ASYr17 VPM1 2AS RS, AS Yr43 IDO377s 2BL RS, ASYr18 Jupateco 73R 7DS NRS, HTAP Yr44 Zak 2BL RS, ASYr19 Compair 5B RS, AS Yr45 PI181434 3DL RS, ASYr20 Fielder 6D RS, AS Yr46 RL6007 4DL NRS, APYr21 Lemhi 1BL RS, AS Yr47 AUS28183 5BS RS, ASYr22 Lee 4D RS, AS Yr48 PI610750 5AL RS,ASYr23 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. Yr10In ChinaResistance 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 winterwheat --(Su et al.,2003)
  • 8. Yr10Moro 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 susceptibleresistant 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 SSegregation 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 absentMoro Corrigin(Yr10) Fielder CorriginPI178383 QT3960(Yr10) Capp. D. QT3960Jacmar 79W93(Yr10) Compair 79W93T. spelta line 415 T. speltaAvocet (Yr10) Avocet
  • 13. Conclusion 1 Perfect linkage of Yr10 gene with thephenotype in 874 segregating lines Fragment E-5 had a week homology to the P-loop of the L6 rust resistance gene in flax andscore (94, P< 1.4x10-4) E-5 was present in the cultivars carrying Yr10A 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 identifiedTwo clones, 4B and 4E, were selected forsequence 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 aacDNA SRPL 1 406 ntFragments SRLR 1 961 nt ATG SRLZ 1 1935 nt TGAFulllength ATG 2475 bp TGAcDNAAF149114
  • 16. Comparison of LRR domainclone 4B clone 4ExxLxxLxxLxxLxL/I xxLxxLxxLxxLxL/IQTRSLPSKIRRLSL QSSNEEDVQPM CKIRRLSHVRSLPTMSSLSHVRSLTV FSKDLSLL TV FSKDFSLLSALSGFLVLRALDL SGCEEVGNHHM SELSSFLVLRALDL SGCTEVRDHNLKDICNLFHLRYLSL EGTSITEIP KDICNLFHLRYMSL QGGCITEIPKEISNLRLLQLLVI RSTKMKKFPSTF KEISNLQLLQVLDI RSTEIEKFPSTVVQLGQLVFIDMGNR EVSRLLL VELRQLVFIDMGNG MVSASLLKSMSTLPSLSSLAI GIGELREEDL KLMSTLPSLSSLAI GLTGLREEDLQILGSMPSLHDLSI DVGYWERGRD QILGSMSSLRDLSL IVVHWGKVTGKRLVIDSGSPFRSL TRFSIKGCGFIDF KRLVIGNGCPFQSL TRFNIKSIDGTGFMFAEGTLQKLQILE-LSI FGKAIKDRFGDFQ GTLQKLQILE-LDL YVEKAKDQLGDFQFGLENLSSLEHVYV DARGRGIIPSQE FGLENLSSLEHVNV VARGGGSEGELINALE KELDINPNKPTLTV KKVTPCSFTFVH
  • 17. Expression of genomic sequences 4E specific 4E specific forward reverse 4E 4E specific primersclone Fragment size = 524 bp LRR TGA LRR start LRR start TGA Size Amplified4B = 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 primers4B 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 4EClone 4E was very closely linked to the resistancephenotype in a random subsample of 55 lines ofthe BC2F3 and BC3F3 segregating population as 42lines (76%) were identified as resistant on thegenotype and 43 (78%) resistant on the phenotype.
  • 21. Fluorescent in situ hybridization of Morometaphase 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 highlyconserved 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 -2Relative 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 striperust 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 LRRCC γK2a γSLR γCLR
  • 29. Phenotypic changes in Moro leaves infected with P. striiformis f. sp. tritici. A B C D E F GMock 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 dpiA B C hr h hrD E F h h h
  • 32. 14 dpiG H IJ 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 sameTransgenesis functional properties as Yr10 silencing 4B in Moro resultedBSMV-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 AlbertaAgricultural Research Institute and AAFC A-base projects to ALand 111 Project from the Ministry of Education of China (GrantNo.B07049) to ZK.

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