Functional analysis of the barley stem rust resistance gene Rpg1

1
Functional analysis of the barley stem rust
resistance gene Rpg1
Andris Kleinhofs and Brian Steffenson
Department of Crop and Soil Sciences and
School of Molecular Biosciences, Washington
State University, Pullman, WA, 99164
Department of Plant Pathology, University of
Plant Pathology, University of Minnesota, St.
Paul, MN 55108.
The barley stem rust resistance gene Rpg1
conferring resistance to many races of the stem
rust fungus P. graminis f. sp. tritici was cloned
by a map-based approach. Research to elucidate
the Rpg1-mediated disease resistance pathway in
barley is presented.
Rpg1 protein characterization Rpg1 is a
constitutively expressed and functionally active
novel S/T kinase with dual kinase domains. It is
mostly cytoplasmic. Rpg1 autophosphorylates by an
intramolecular mechanism requiring Mg or Mn
ions, but not Ca. Km and Vmax values of 0.15
and 0.47 nmol/min/mg protein, respectively were
estimated.

• Fig. 1. Rpg1 kinase activity.
• Fig. 2. Rpg1 kinase domain mutants disrupt
• disease resistance.
• Fig. 3. Rpg1 interaction with HvPti1 is
• disrupted by kinase domain mutants.
• Fig. 4 5. Rpg1 is mostly cytoplasmic.
• Fig. 6. Mutations in other genes disrupt
• Rpg1-mediated disease resistance.
• Fig. 7. Yeast two-hybrid analysis identifies
  potential Rpg1 interactors.
• Fig. 8. Rpg1 is specifically degraded upon
• infection with pv. MCC.
• Fig. 9. Microarray analysis identifies genes
• differentially regulated on infection of
• isogenic lines.

Fig. 3. HvPti1 interaction with Rpg1 is disrupted
by mutations in pK1 and pK2. L to R, 1-Positive
control, negative control, and interaction
plates. Wild-type Rpg1 interacted with HvPti1,
while the pK1 and the pK2 mutants failed to
interact, indicating that the Rpg1 interaction
with the HvPti1 protein is important to
Rpg1-mediated disease resistance.
Fig. 2. pK1 and pK2 mutants are highly
susceptible to stem rust pathotype MCC. L to R,
Morex-R. cv., and source of cloned Rpg1 gene
GP-S. cv., and recipient of transformed genes
G03-440-GP transformed with wild-type Rpg1 K1
mutant-Rpg1 with K152N, K153Q K2 mutant-Rpg1
with K461N, K462Q All samples were infected with
stem rust pathotype MCC.
Fig. 1. pK2 and K461 are essential for
autophosphorylation. A, Lane 1 - control, 2 -
pK1 mutant K152N and K153Q, 3 - pK2 mutant K461N
and K462Q, 4 - double mutant. B, Lane 1 - pK2
mutant K461Q, 2 - pK2 mutant K462Q. Top panel,
Coomassie Blue stained SDS-polyacrylamide gel and
bottom panel, autoradiograph. K-lysine,
N-asparagine, Q-glutamine.  

Fig. 7. Yeast two-hybrid system identifies Rpg1
protein interactors. L to R, positive control,
negative control and interaction plates. The
barley Pti proteins are homologous to the
tomato Pto interactors.

• Future Directions
• What is the role of the pK1 domain in disease
  resistance?
• What is phosphorylated in vivo and why?
• What is the role of HvPti1 and other Rpg1
  interactors in disease resistance?
• What is the role of Rpg1 degradation in disease
  resistance?
• What genes/proteins are involved in the Rpg1
  signal transduction pathway? Saturation
  mutagenesis yeast two-hybrid analysis of
  different cDNA libraries microarray analysis and
  verification
• Can we identify the Avr-Rpg1 gene by yeast
  two-hybrid with cDNA library constructed from
  isolated haustoria?

Fig. 4. Rpg1 is mostly cytoplasmic. Lane 89
-100,000g x 2h supernatant and pellet,
respectively. Other fractions are supernatant
and pellet of slower spins.
Fig. 6. Morex mutant rpr1 suppresses
Rpg1-mediated disease resistance. Top - resistant
cv. Morex Bottom - susceptible rpr1 mutant in
Morex genotype. Three deletions are associated
with this mutation. Mutants in two additional
genes that are required for Rpg1 function have
been identified.
Fig. 5. EM immunolocalization of Rpg1
Some of the above questions will be answered with
already employed technologies such as yeast
two-hybrid, in vitro mutagenesis, in vivo
mutagenesis, microarray analysis and stable
transformation. Others will require development
of new technologies such as the Virus Induced
Gene Silencing (VIGS) and transient Agrobacterium
transformation.
Fig. 9. Microarray experiment design. Comparison
of the highly isogenic transgenic and Golden
Promise lines identified 16 differentially
regulated genes while comparison of the
transgenic line infected with MCC vs. QCC
identified 34 differentially regulated genes.
Fig. 8. Rpg1 is degraded in resistant and
susceptible lines within 20-48h upon infection
with pv. MCC. Top to bottom, highly resistant
transgenic line, resistant cv. Morex,
susceptible mutant rpr1, susceptible wild barley
line OSU6.
This project was supported by the National
Research Initiative of the USDA Cooperative State
Research, Education and Extension Service, grant
number 2004-35301-14635.