unraveling the rpg1-mediated stem rust resistance in barley

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 loss

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 from cultivar “Morex”

pK1 pK2

1 837

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 in response to pathogen attack

Rostoks et al., PMPP, 2004

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

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!!!

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 spores RGD treated spores

9 hours after inoculation


RGD treated Spore

Untreated Spores

P U 0.25h

ai20

hai24

hai48

hai

PSPT

RPG1

Morex

Steptoe

ASM

170

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 acid P: Positive control

U: Uninoculated control PS: Phosphoserine antibody

PT: Phosphothreonine antibody

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!!!

VPS9 domain CUE domain

5 3

Vacuolar transport Ubiquitination

Has no known recognition motifs or an obvious signal peptide!!!

RPG1

PSPT

P U 0.25 h

ai

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

RGD

RG

D A

lone

VPS9

alo

ne

RG

D A

lone

VPS9

alo

ne

RG

D+V

PS9

RG

D+V

PS9

Morex Steptoe

SOS-VPS9+Myr RGD

Negative control

Positive control

37 C025 C0

SD/GLU-UL SD/GLU-UL SD/GAL-UL

37 C0

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 controlSOS-VPS9+Myr-RPG1SOS-VPS9+Myr-pK1-RPG1SOS-VPS9+pMyr-pK2-RPG1SOS-RGD+Myr-RPG1SOS-RGD+Myr-pK1-RPG1SOS-RGD+Myr-pK2-RPG1Negative control

SD/GLU-UL SD/GAL-ULSD/GLU-UL

25 C0 37 C037 C0

91.8 kDa

196.2 kDa

4 5 6

206.9 kDa

91.8 kDa

1 2 3


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

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

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




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

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

+ _ _

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

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

Background on stem rust and Rpg1 AvrRpg1 genes RGD peptides and rust control Summary/Speculations

Stem rust Stripe rust Leaf rust

RG

D T

REA

TED

!

UN

TREA

TED!

RG

D T

REA

TED!

UN

TREA

TED!

UN

TREA

TED!

RG

D T

REA

TED!

Stem rust Stripe rust Leaf rust

RG

D T

REA

TED

!

RG

D T

REA

TED

!

RG

D T

REA

TED

!

UN

TREA

TED!

UN

TREA

TED!

UN

TREA

TED!

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 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

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

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

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