Background on stem rust and Rpg1
AvrRpg1 genes/proteins
RGD peptides and rust control
Summary/Speculations
Stem, leaf sheaths, leaf blade, parts of the
head
Tearing of outer layers of plant tissue/
Shriveled grains
70-90% yield l...
70-90% crop
loss in wheat
and barley
Devastated
the Midwest
barley crop
Confers resistance to several races of
Puccinia graminis f. sp. tritici
Brueggeman et al., PNAS, 2002
Map based cloning fr...
Morex GP GP/Rpg1T1 Horvath et al.,
PNAS 2003
GP: Golden Promise
Expressed constitutively in all organs
Expressed at higher levels in the leaf
epidermis!!!
Does not significantly modulate...
Mostly cytoplasmic, with a minor membrane association
Is a functionally active S/T kinase
pK1 pK2
1 837
catalytically
Inac...
RPG1 Phosphorylation
5 min after spore
landing
Degradation
20-24hai
Highly race specific and required for
resistance!!!
Ni...
Background on stem rust and Rpg1
AvrRpg1 genes/proteins
RGD peptides and rust control
Summary/Speculations
Adhesion pad
Nirmala et al., 2011, PNAS
RGD peptide treated spores Untreated Spores
RGD: Arginine-Glycine-Aspartic acid
Untreated sporesRGD treated spores
9 hours after inoculation
Nirmala et al., 2011, PNAS
RGD treated
Spore
Untreated
Spores
P
U
0.25hai20hai
24
hai48
hai
PS
PT
RPG1
Morex
Steptoe
ASM170
Puccinia graminis f. sp. tritici
urediniospore extract bindi...
3
Fibronectin
Type III
domain
BRCA1 C-terminal
Domain
5
Cell surface
recognition and
attachment
Phosphopeptide binding,
Ph...
VPS9 domain CUE
domain
5 3
Vacuolar
transport
Ubiquitination
Has no known recognition motifs or an obvious signal peptide!...
RPG1
PS
PT
P U 0.25
hai
8
hai
20
hai
22
hai
24
hai
48
hai
PS: Phosphoserine Antibody, PT: Phosphothreonine, P: Positive
co...
RGD
RGDAlone
VPS9alone
RGDAlone
VPS9alone
RGD+VPS9
RGD+VPS9
Morex Steptoe
SOS-VPS9+MyrRGD
Negativecontrol
Positivecontrol
37C
0
25C
0
SD/GLU-UL SD/GLU-UL SD/GAL-UL
37C
0
Nirmala et al., PNAS, 2011
The RPG1 pK1 and pK2 mutants failed to interact with the RGD-binding
and the VPS9 proteins.
A
B C
Positive control
SOS-VPS...
Race Disease
reaction
Spore Sporelings Haustoria Infected
tissue
Comments
MCCF Avirulent + + + + gene intact
SCCLc7a Aviru...
Race Disease
reaction
Spore Sporelings Haustoria Infected
tissue
Comments
MCCF avirulent + + + + gene intact
SCCLc7A aviru...
SCCLc7a MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTIIILYLMASTGYTLAHP 60
MCCF MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTII...
SCCLc7a MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60
MCCF MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEP...
 Source	
   Phosphoryla/on	
   Degrada/on	
   Hypersensi/ve	
  
response	
  
QCCJ	
  RGD+	
  VPS9	
   _	
   _	
   _	
  
92...
Race	
   Muta/on	
   RPG1	
  Phosphoryla/on	
  ac/vity	
  
MCCF	
   P565S	
   Looses	
  ac/vity	
  
MCCF	
   S52P	
   Loos...
Race	
   Muta/on	
   RPG1	
  degrada/on	
  
MCCF	
   A527V	
   Looses	
  ability	
  to	
  degrade	
  RPG1	
  
MCCF	
   S51...
Background on stem rust and Rpg1
AvrRpg1 genes
RGD peptides and rust control
Summary/Speculations
Stem rust Stripe rust Leaf rust
RGDTREATED!
UNTREATED!
RGDTREATED!
UNTREATED!
UNTREATED!
RGDTREATED!
Stem rust Stripe rust Leaf rust
RGDTREATED!
RGDTREATED!
RGDTREATED!
UNTREATED!
UNTREATED!
UNTREATED!
RKQQC
control
RKQQC +
stabilized RGD
RKQQC +
unstabilized RGD
RKQQC +
stabilization control
Background on stem rust and Rpg1
AvrRpg1 genes
RGD peptides and rust control
Summary/Speculations
Phosphorylation of RPG1 acts as the initial signal for
activating the defense response
Two novel effectors work together i...
1)  RGD binding protein interacts with RPG1 and other
proteins at the cell membrane and RPG1 may act as a
receptor
2)  If ...
4) Why are these avirulent forms retained if it is so
easy to change to virulence by mutation?
a) Do they serve some other...
Research was supported by National Research Initiative of the United
States Department of Agriculture Co-operative State R...
Unraveling the Rpg1-mediated Stem Rust Resistance in Barley
Unraveling the Rpg1-mediated Stem Rust Resistance in Barley
Upcoming SlideShare
Loading in …5
×

Unraveling the Rpg1-mediated Stem Rust Resistance in Barley

542 views

Published on

J. Nirmala

Published in: Education, Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
542
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
25
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Unraveling the Rpg1-mediated Stem Rust Resistance in Barley

  1. 1. Background on stem rust and Rpg1 AvrRpg1 genes/proteins RGD peptides and rust control Summary/Speculations
  2. 2. Stem, leaf sheaths, leaf blade, parts of the head Tearing of outer layers of plant tissue/ Shriveled grains 70-90% yield loss
  3. 3. 70-90% crop loss in wheat and barley Devastated the Midwest barley crop
  4. 4. Confers resistance to several races of Puccinia graminis f. sp. tritici Brueggeman et al., PNAS, 2002 Map based cloning from cultivar “Morex” pK1 pK2 1 837
  5. 5. Morex GP GP/Rpg1T1 Horvath et al., PNAS 2003 GP: Golden Promise
  6. 6. Expressed constitutively in all organs Expressed at higher levels in the leaf epidermis!!! Does not significantly modulate in response to pathogen attack Rostoks et al., PMPP, 2004
  7. 7. Mostly cytoplasmic, with a minor membrane association Is a functionally active S/T kinase pK1 pK2 1 837 catalytically Inactive catalytically active Both the domains required for disease resistance!!!! Nirmala et al., PNAS, 2006
  8. 8. RPG1 Phosphorylation 5 min after spore landing Degradation 20-24hai Highly race specific and required for resistance!!! Nirmala et al., 2007, PNAS Nirmala et al., 2010, MPMI Elicitor is probably located on the surface of the spore!!!
  9. 9. Background on stem rust and Rpg1 AvrRpg1 genes/proteins RGD peptides and rust control Summary/Speculations
  10. 10. Adhesion pad Nirmala et al., 2011, PNAS RGD peptide treated spores Untreated Spores RGD: Arginine-Glycine-Aspartic acid
  11. 11. Untreated sporesRGD treated spores 9 hours after inoculation Nirmala et al., 2011, PNAS
  12. 12. RGD treated Spore Untreated Spores
  13. 13. P U 0.25hai20hai 24 hai48 hai PS PT RPG1 Morex Steptoe ASM170 Puccinia graminis f. sp. tritici urediniospore extract binding to and eluted from the RGD affinity column induced RPG1 phosphorylation and degradation in barley cv. Morex Puccinia graminis f. sp. tritici urediniospore extract binding to and eluted from the RGD affinity column induced HR in barley harboring a functional Rpg1 gene RGD: Arginine, Glycine and Aspartic acidP: Positive control U: Uninoculated control PS: Phosphoserine antibody PT: Phosphothreonine antibody
  14. 14. 3 Fibronectin Type III domain BRCA1 C-terminal Domain 5 Cell surface recognition and attachment Phosphopeptide binding, Phosphorylation and protein-protein interaction 1bp 2918 bp Has no known recognition motifs or an obvious signal peptide!!!
  15. 15. VPS9 domain CUE domain 5 3 Vacuolar transport Ubiquitination Has no known recognition motifs or an obvious signal peptide!!!
  16. 16. RPG1 PS PT P U 0.25 hai 8 hai 20 hai 22 hai 24 hai 48 hai PS: Phosphoserine Antibody, PT: Phosphothreonine, P: Positive control, U: Uninoculated control and hai: hours after inoculation
  17. 17. RGD RGDAlone VPS9alone RGDAlone VPS9alone RGD+VPS9 RGD+VPS9 Morex Steptoe
  18. 18. SOS-VPS9+MyrRGD Negativecontrol Positivecontrol 37C 0 25C 0 SD/GLU-UL SD/GLU-UL SD/GAL-UL 37C 0 Nirmala et al., PNAS, 2011
  19. 19. The RPG1 pK1 and pK2 mutants failed to interact with the RGD-binding and the VPS9 proteins. A B C Positive control SOS-VPS9+Myr-RPG1 SOS-VPS9+Myr-pK1-RPG1 SOS-VPS9+pMyr-pK2-RPG1 SOS-RGD+Myr-RPG1 SOS-RGD+Myr-pK1-RPG1 SOS-RGD+Myr-pK2-RPG1 Negative control SD/GLU-UL SD/GAL-ULSD/GLU-UL 25 C 0 37 C 0 37 C 0 91.8 kDa 196.2 kDa 4 5 6 206.9 kDa 91.8 kDa 1 2 3 Nirmala et al., 2011, PNAS
  20. 20. Race Disease reaction Spore Sporelings Haustoria Infected tissue Comments MCCF Avirulent + + + + gene intact SCCLc7a Avirulent + + + + gene intact HKHJ Avirulent + + + + gene intact 92-MN-90 Virulent + + + + gene intact (SNPS) QCCJ Virulent + + + + gene intact (SNPS) Ug99 Virulent + + ? + gene (SNPS) qRT-PCR, RNA seq data and Illumina microarray
  21. 21. Race Disease reaction Spore Sporelings Haustoria Infected tissue Comments MCCF avirulent + + + + gene intact SCCLc7A avirulent + + + + gene intact HKHJ avirulent + + + + gene intact 92-MN-90 Virulent + + + + 3 alleles SNPS QCCJ Virulent + + + + 2 alleles SNPS UG99 virulent + + ? + 2 alleles SNPS qRT-PCR, RNA seq and Illumina microarray data
  22. 22. SCCLc7a MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTIIILYLMASTGYTLAHP 60 MCCF MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTIIILYLMASTGYTLAHP 60 HKHJ MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTIIILYLMASTGYTLAHP 60 92-MN-90 MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTIIILYLMASTGYTLAHP 60 QCCJ MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTIIILYLMAPTGYTLAHP 60 Ug99 MTGSFGGKSGFEKAGLFTDSFHSPNFTGQFSGTGTDIEDLEDTIIILYLMASTGYTLAHP 60 ***************************************************.******** SCCLc7a DQPHVPTPPKFSAALSDILKENVDSPAENDSSPSGHKPMEFPNPMSHILSDRPESTPNGD 540 MCCF DQPHVPTPPKFSAALSDILKENVDSPAENDSSPSGHKPMEFPNPMSHILSDRPESTPNGD 540 HKHJ DQPHVPTPPKFSAALSDILKENVDSPAENDSSPSGHKPMEFPNPMSHILSDRPESTPNGD 540 92-MN-90 DQPHVPTPPKFSAALSDILKENVDSPAENDSSPSGHKPMEFPNPMSHILSDRPESTPNGD 540 QCCJ EQPHVPTPPKFSAALSDILKENVDSPAENDSSPSGHKPMEFPNPMSHILSDRPESTPNGD 540 Ug99 DQPHVPTPPKFSAALSDILKENVDSPAENDSSPSGHKPMEFPNPMSHILSDRPESTPNGD 540 .*********************************************************** SCCLc7a DKGAEEEAEPRSTHPTSSPPNNKLPGQTSGLAEQSQPSRGIVDDGEDDPFSAASPTISNP 600 MCCF DKGAEEEAEPRSTHPTSSPPNNKLPGQTSGLAEQSQPSRGIVDDGEDDPFSAASPTISNP 600 HKHJ DKGAEEEAEPRSTHPTSSPPNNKLPGQTSGLAEQSQPSRGIVDDGEDDPFSAASPTISNP 600 92-MN-90 DKGAEEEAEPRSTHPTSSPPNNKLSGQTSGLAEQSQPSRGIVDDGEDDPFSAASPTISNP 600 QCCJ DKGAEEEAEPRSTHPTSSPPNNKLSGQTSGLAEQSQPSRGIVDDGEDDPFSAASPTISNP 600 Ug99 DKGAEEEAEPRSTHPTSSPPNNKLSGQTSGLAEQSQPSRGIVDDGEDDPFSAASPTISNP 600 ************************.*********************************** SCCLc7a EEKQEIPEVDRSVGRDQPAAPSPPSPTALDSLASPSKTIGENKPHANGGSSSEVFDEISI 720 MCCF EEKQEIPEVDRSVGRDQPAAPSPPSPTALDSLASPSKTIGENKPHANGGSSSEVFDEISI 720 HKHJ EEKQEIPEVDRSVGRDQPAAPSPPSPTALDSLASPSKTIGENKPHANGGSSSEVFDEISI 720 92-MN-90 EEKQDIPEVDRSVGRDQPAAPSPPSPTALDSLASPSKIIGENKPHANGGSSSEVFDEISI 720 QCCJ EEKQEIPEVDRSVGRDQPAAPSPPSPTALDSLASPSKIIGENKPHANGGSSSEVFDEISI 720 Ug99 EEKQEIPEVDRSVGRDQPAAPSPPSPTALDSLASPSKIIGENKPHANGGSSSEVFDEISI 720 ****:******************************************************* SCCLc7a GSDSNQLPDDEDHEVSDSKGESGSGDTFFSTASVPLQPSHPDPTLHSDLIQPHNPALLSD 780 MCCF GSDSNQLPDDEDHEVSDSKGESGSGDTFFSTASVPLQPSHPDPTLHSDLIQPHNPALLSD 780 HKHJ GSDSNQLPDDEDHEVSDSKGESGSGDTFFSTASVPLQPSHPDPTLHSDLIQPHNPALLSD 780 92-MN-90 GSDSIQLPDDEDHEVSDSKGESGSGDTFFSTASVPLQPSHPDPTLHSDLIQPHNPALLSD 780 QCCJ GSDSNQLPDDEDHEVSDSKGESGSGDTFFSTASVPLQPSHPDPTLHSDLIQPHNPALLSD 780 Ug99 GSDSNQLPDDEDHEVSDSKGESGSGDTFFSTASVPLQPSHPDPTLHSDLIQPHNPALLSD 780 ****:******************************************************* SCCLc7a LVHPNLQQQHQPTDLITSDLVEHSSPSSSPPPPNHLAD 818 MCCF LVHPNLQQQHQPTDLITSDLVEHSSPSSSPPPPNHLAD 818 HKHJ LVHPNLQQQHQPTDLITSDLVEHSSPSSSPPPPNHLAD 818 92-MN-90 LVHPNLQQQHQPTDLITSDLVEHSSPSSSPPPPNHLAD 818 QCCJ LVHPNLQQQHQPTDLITSDLVEHSSPSSSPPPPNHLAD 818 Ug99 LVHPNLHQQHQPTDLITSDLVEHSSPSSSPPPPNHLAD 818 ************************************** Avr Avr Avr Vir Vir Vir Vir Vir Avr Avr Avr Vir Avr Avr Avr Vir Vir Vir Avr Avr Avr Vir Vir Vir Avr Avr Avr Vir Vir Vir Avr Avr Avr Vir Vir Vir
  23. 23. SCCLc7a MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60 MCCF MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60 HKHJ MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60 QCCJ(D) MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDH----- 60 QCCJ(F) MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60 Ug99(F) MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60 UG99(D) MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDH----- 60 92MN90 (1) MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60 92MN90 (2) MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDHSHTQQ 60 92MN90 (D) MSTATIQPTIEDHQQHQTPPLIDLELASPSLAEEQEQEQQEQQEPEQQQQNSVDH----- 60 ************************************************************ SCCLc7a EEFEYNVEQAIVSLPRSPVIENIKASSGGFSAFTQGASSSNSNMGGAGGEAGGTGGGGER 540 MCCF EEFEYNVEQAIVSLPRSPVIENIKASSGGFSAFTQGASSSNSNMGGAGGEAGGTGGGGER 540 HKHJ EEFEYNVEQAIVSLPRSPVIENIKASSGGFSAFTQGASSSNSNMGGAGGEAGGTGGGGER 540 QCCJ(D) ------------------------------------------------------------ QCCJ(F) EEFEYNVEQAIVSLPRSPVIENIKASSGGFSAFTQGASSSNSNMGGVGGEAGGTGGGGER 540 Ug99(F) EEFEYNVEQAIVSLPRSPVIENIKASSGGFSAFTQGASSSNSNMGGVGGEAGGTGGGGER 540 UG99(D) ------------------------------------------------------------ 92MN90(1) EEFEYNVEQAIVSLPRSPVIENIKASSGGFSAFTQGANSSNSNMGGVGGEAGGTGGGGER 540 92MN90(2) EEFEYNVEQAIVSLPRSPVIENIKASSGGFSAFTQGASSSNSNMGGVGGEAGGTGGGGER 540 92MN90(D) ------------------------------------------------------------ *************************************:********:************* Avr Avr Avr Vir Vir Vir Vir Vir Vir Vir Avr Avr Avr Vir Vir Vir Vir Vir Vir Vir
  24. 24.  Source   Phosphoryla/on   Degrada/on   Hypersensi/ve   response   QCCJ  RGD+  VPS9   _   _   _   92MN90  RGD+VPS9-­‐1   _   _   _   92MN90  RGD+  VPS9-­‐2   _   _   _   Ug99  RGD+  VPS9   _   _   _   MCCF  RGD+  QCCJ  VPS9   +   _   _   MCCF  RGD+92MN90   VPS9-­‐1   +   _   _   MCCF  RGD+    92MN90     VPS9-­‐2   +   _   _   MCCF  RGD+   Ug99  VPS9-­‐2   +   _   _  
  25. 25. Race   Muta/on   RPG1  Phosphoryla/on  ac/vity   MCCF   P565S   Looses  ac/vity   MCCF   S52P   Looses  ac/vity   MCCF   D481E   Looses  ac/vity   MCCF   E665D   Looses  ac/vity   MCCF   N725I   Looses  ac/vity   MCCF   Q787H   Looses  ac/vity   QCCJ   P52S   Does  not  regain  ac/vity   QCCJ   E481D   Does  not  regain  ac/vity   92MN90   I725N   Does  not  regain  ac/vity   92MN90   D665E   Does  not  regain  ac/vity   Ug99   H787Q   Does  not  regain  ac/vity   QCCJ   S565P   Does  not  regain  ac/vity   92MN90   S565P   Does  not  regain  ac/vity   Ug99   S565P   Does  not  regain  ac/vity   QCCJ   P52S,  E481D   Does  not  regain  ac/vity   92MN90   I725N,  D665E   Does  not  regain  ac/vity   Ug99     H787Q,  S565P   Regains  ac/vity   QCCJ   P52S,  E481D,  S565P   Regains  ac/vity   92MN90   I725N,  D665E,  S565P   Regains  ac/vity  
  26. 26. Race   Muta/on   RPG1  degrada/on   MCCF   A527V   Looses  ability  to  degrade  RPG1   MCCF   S518N   Looses  ability  to  degrade  RPG1     QCCJ   V527A   Regains  Ability  to  degrade  RPG1   Ug99   V527A   Regains  ability  to  degrade  RPG1   92MN90-­‐2   V527A   Regains  ability  to  degrade  RPG1   92MN90-­‐1   V527A   Does  not  regain  ability  to  degrade  RPG1     92MN90-­‐1   N518S   Does  not  regain  ability  to  degrade  RPG1   92MN90-­‐1   V527A/N518S   Regains  ability  to  degrade  RPG1     RPG1 phosphorylation was induced with MCCF-RGD binding protein
  27. 27. Background on stem rust and Rpg1 AvrRpg1 genes RGD peptides and rust control Summary/Speculations
  28. 28. Stem rust Stripe rust Leaf rust RGDTREATED! UNTREATED! RGDTREATED! UNTREATED! UNTREATED! RGDTREATED!
  29. 29. Stem rust Stripe rust Leaf rust RGDTREATED! RGDTREATED! RGDTREATED! UNTREATED! UNTREATED! UNTREATED!
  30. 30. RKQQC control RKQQC + stabilized RGD RKQQC + unstabilized RGD RKQQC + stabilization control
  31. 31. Background on stem rust and Rpg1 AvrRpg1 genes RGD peptides and rust control Summary/Speculations
  32. 32. Phosphorylation of RPG1 acts as the initial signal for activating the defense response Two novel effectors work together in activating RPG1 They are present on the surface of the spores and the recognition probably occurs at the surface of the plant cells Mutational analysis reveals that single amino acid substitutions alters effector recognition and activation Stabilized RGD peptides can be exploited to control rust
  33. 33. 1)  RGD binding protein interacts with RPG1 and other proteins at the cell membrane and RPG1 may act as a receptor 2)  If so RPG1 might be a integrin like molecule mediating inside-out signaling! 3)  RPG1 does not have a RGD motif in the right orientation-therefore might need another protein/integrin which acts as a receptor, which might phosphorylate RPG1 and initiate defense signaling
  34. 34. 4) Why are these avirulent forms retained if it is so easy to change to virulence by mutation? a) Do they serve some other function? 5) Exploit the stabilized RGD peptides to control the rusts
  35. 35. Research was supported by National Research Initiative of the United States Department of Agriculture Co-operative State Research, Education, and Extension Service Grant No. 2007-35301-18205 and National Institute of Food and Agriculture Grant No. 2010-65108-20568. Durable rust resistance in wheat Dr. Andris Kleinhofs Dr. Diter Von Wettstein Dr. Scot Hulbert Dr. Xianming Chen Dr. Brian Steffenson Dr. Pablo Olivera Dr. Mathew Rouse Dr. Les Szabo

×