1) RPG1 phosphorylates within 5 minutes of spore landing, and phosphorylation is race-specific, only occurring in response to viable spores of matching races. 2) Two effector proteins were isolated from spores that together induce RPG1 phosphorylation and degradation, as well as hypersensitive response in barley with functional Rpg1. 3) The proteins interact with each other and RPG1, suggesting they work cooperatively to trigger defense signaling through RPG1. Protein degradation alone is not sufficient for resistance.

1. Rpg1-mediated durable stem rust
resistance: mechanisms of action
A. Kleinhofs, J. Nirmala,
R. Brueggeman and B. Steffenson

2. Acknowledgments
Multiyear support by USDA/NRI/AFRI to
A. Kleinhofs and B. J. Steffenson and collaboration with
D. von Wettstein and S. Hulbert
past current
Dave Kudrna Jayaveeramuthu Nirmala
Andrzej Kilian Robert Brueggeman
Feng Han
Tom Drader
Arnis Druka
Nils Rostoks
Henriette Horvath
Stephanie Dahl
Research was supported by the United States Department of Agriculture
Cooperative State Research, Education, and Extension Service Grants
#2007-35301-18205 and #2010-65108-20568. to A.K. and B.J.S.

3. Morex/Chevron GP/transgenic
GP vs. Chevron Morex vs. Chevron

4. Novel gene structure
Genomic DNA
Exon 1 2 3 4 5 6 7 8 9 10 11 12 13 14
5’UTR 3’UTR
TATA ATG TGA
cDNA
Protein
1 110 837 a.a.
Pkinase1
Pkinase1 Pkinase2
Pkinase2
TMD
(Brueggeman et al. (2002) PNAS)

5. Rpg1 is constitutively transcribed at low levels in all
tissues tested
Quantification of the Rpg1 mRNA
Percent of actin mRNA
16.00
14.00
12.00 11
9
10.00 8
8.00 6
6.00
4.00 3
2.00 0
0.00
e
f
t
ise
g
f
o
lea
nc
ea
lin
ro
m
el
e
ed
g
sc
g
ro
in
r
in
se
atu
re
nP
dl
dl
lo
ed
e
M
e
e
f
Se
Se
ld
lat
In
Go
io
Et
Tissue type

6. Except the epidermis
90.00
80.00
72.08
70.00
60.00
50.00
43.41
40.00
30.00
20.00
10.00 6.54
3.34
0.31
0.00
Morex epidermis G02-448D Morex G02-448D Morex whole
epidermis mesophyll mesophyll leaf
Tissue type

7. RPG1 protein in leaf epidermis (LE) compared to
leaf after removal of epidermis (LARE)

9. In vivo RPG1 phosphorylates within 5 min.
post inoculation with avirulent Pgt spores.
A: positive control, U : uninoculated control
PS: Phosphoserine, PT: Phosphothreonine

10. Viable but not inviable MCCF spores elicit
RPG1 phosphorylation in cv. Morex
30 min post inoculation
PS PT
Lanes 1 and 3 inviable spores; 2 and 4 viable spores

11. RPG1 phosphorylation is race specific
1 – positive control
2 – uninoculated
3 – 15 min. sample
PS – phosphoserine AB
PT – phosphothreonine AB

12. In vivo phosphorylation of RPG1 is required, but not
sufficient for resistance
Lane 1: Positive control
Lane 2: uninoculated control
Lane 3: 15 min. post inoculation
PS: Phosphoserine
PT: Phosphthreonine

13. Effect of protein kinase inhibitors on
Rpg1- mediated disease reaction.
PKI: Protein kinase inhibitor, PS:
Phosphoserine, PT: Phosphothreonine

16. Isolation and characterization of RGD-binding and VPS9-like proteins

17. RGD-binding and VPS9 proteins together induce in vivo phosphorylation and degradation
of RPG1.
RGD-binding protein alone induced
phosphorylation of RPG1
VPS9 protein alone did not induce phosphorylation
of RPG1
VPS9 failed to induce RPG1 degradation up to 48 h after
application
Application of both proteins together induced
RPG1 phosphorylation within 0.25 h and
degradation prior to 24 h

18. RGD-binding and the VPS9 proteins together induce HR in barley harboring a functional
Rpg1 gene
A B C
Golden Promise
GP-Rpg1/pK1
GP-Rpg1/pK2
GP-Rpg1/T1
RGD+VPS9
RGD+VPS9
ASM170
Steptoe
Morex
VPS9
VPS9
RGD
RGD
Morex Steptoe

19. RGD-binding and VPS9 proteins interact with RPG1 and with each other in yeast
VPS9 interacts with the RGD-binding protein
RPG1 interacts with RGD-binding and VPS9 proteins.
The RPG1 pK1 and pK2 mutants failed to interact with
the RGD-binding and the VPS9 proteins.

20. Summary
*RPG1 phosphorylates in vivo within 5 min
post-spore landing on the leaf surface.
*RPG1 phosphorylation is a highly race specific
event and takes place only with viable spores.
*RPG1 phosphorylation is required for
resistance and probably acts as a very early
signal in pathogen perception.

21. Summary continued
• Two effector proteins isolated from spores
• Both effectors required to mimic spore
function
• Phenotypes tested: RPG1 phosphorylation &
degradation &host hypersensitive response
• RGD-binding and VPS9 proteins interact with
RPG1 and with each other in yeast
• RGD-binding protein may interact with
unknown RGD motif protein(s)

22. Speculation
• RGD-binding protein interacts with RPG1 and
possibly other proteins at the cell membrane
• RPG1 does not have an RGD motif – another
protein may be the receptor
• RPG1 is phosphorylated and signals to initiate
the defense response
• RPG1 degradation may release defense
complex from negative regulation or it may
suppress runaway HR

23. Conserved domains of Rpg1 and
JAK

28. Protein degradation is not sufficient for
disease resistance
0 12 16 20 24 48 0 12 16 20 24 48
G03-448F-3R Chevron
0 12 16 20 24 48 0 12 16 20 24 48
Morex rpr1
0 12 16 20 24 48 0 12 16 20 24 48
Beacon OSU6

30. RPG1 protein elevated in Leaf Epidermis (LE)
compared to rest of the tissue (LARE)
7
RPG1 protein (micromoles of
6 RPG1/g total protein)
5
4
3
2
1
0
LE LARE

31. RPG1 phosphorylation is race specific
PS: Phosphoserine
PT: Phosphothreonine
Lane 1: Positive control
Lane 2: uninoculated control
Lane 3: 15 min. post inoculation