This is one of my past work on host-bacterial interactions. results have broad applicability and not specific to plant models used. Please contact me if you need a clean copy.

1. HOST PERCEPTION AND SIGNALING DURING PROGRAMMED CELL DEATH
RESPONSE AND RESISTANCE MEDIATED BY BACTERIAL PATHOGENS
Suresh Gopalan, Ph.D
Work done mid 1995 – mid 1998
DOE Plant Research Laboratory –
Michigan State University
Based on last presentation at:
Prof. Frederick M. Ausubel Lab,
Department of Molecular Biology, MGH & Harvard Medical School
March, 2006

2. Significance
Accomplishments:
1. Identified novel themes in bacterial infection and host immunity.
2. Deduced key concept in mode of action of bacterial disease and immune
effectors (considered pioneering by leaders in the field).
3. Demonstrated common conserved mechanisms across kingdom and species.
4. Identified many cell signaling components of host immunity.
5. Developed several hypothesis using above, later proved correct.
Practical Significance:
Engineering/manipulating disease and resistance mechanisms applicable to a
variety of host-pathogen interactions

3. Perception and signaling of bacterial avirulence proteins in plants:
another facet
Pseudomonas syringae glycinea AvrB – Arabidopsis RPM1 model system
Suresh Gopalan

4. Pathogens
Virulent
Disease (compatible)
Avirulent
Resistance (incompatible)

5. Under ideal conditions Arabidopsis sprayed with appropriate
dose of virulent and avirulent bacterial pathogen will look like this
Adapted from: Arabidopsis Book

6. HYPERSENSITIVE RESPONSE (HR)
A rapid plant cell death at the site of infection of an avirulent pathogen.
Associated with:
1. Restriction of multiplication and spread of pathogen.
2. Coordinate activation of defense related genes.
3. Activation of broad spectrum resistance in uninfected parts of the
plants, termed systemic acquired resistance (SAR)

7. A LABORATORY MANIFESTATION OF HR AND DISEASE
P.s.s.61
(Bean pathogen)
High inoculum, rapid cell death
(about 12 h later)
P.s.tabacum
(Nicotiana pathogen)
Low inoculum
(few days later)

8. GENE-FOR-GENE HYPOTHESIS IN
RACE-CULTIVAR SPECIFICITY
C1
R
avr
C2
+
-
R1
+
HR
D
R2
-
D
D
Single matching gene combination between pathogen and plant can lead to HR

9. Harpin
Avr
Hrp
R gene product
CW
PM
HR/Resistance

11. First cloned pathogen molecule : plant gene product pair
HC-Toxin : HC-Toxin reductase pair C. carbonum :Maize
(John Walton and Steve Briggs group)

12. Properties of some cloned R gene products
NBS
LRRs
LZ
X
Pto
Rps2, Rpm1,
Prf
Kinase
X
X
X
X
Xa21
C
C
X
EC/TM/C
X
C
Cf- 9, Cf-2
X
EC
L6
X
N
X
Putative
location
First kinase type R gene Martin…..Tanksley
First of prototypic LRR containing R gene isolated was RPS2 from Arabidopsis
(recognition specificity avrRpt2): Bent…Staskawicz lab and Mindrinos …Ausubel Lab
RPS4, N for e.g., has homology to DrosophilaToll and mammalian IL1 receptors;
Xa21 for e.g., resembles structure of many RTKs

13. Background and the enigma:
Harpin
Avr
Hrp
CW
PM
R gene product
HR/Resistance
1. Bacteria (these) do not
invade plant cells
2. Hrp genes required for
Avr function
3. Unlike harpins, Avr gene
products do not elicit HR
when injected into
apoplast (intercellular
space)
4. Prevailing notion: Avr
gene products recognized
by R gene products
(receptor-ligand
interaction)
5. Products of cloned R
genes predicted to be
intracellular

14. Some other possibilities
1. Enzymatic action (e.g., AvrD)
2. Another bacterial factor involved (e.g., Harpin)

15. From: Arabidopsis Book

16. Arabidopsis- P. syringae model system used
RPM1
DC3000
DC3000
(+ avrB)
D
HR
rpm1
D
D
Grant…..Dangl with Innes (rps3) – recognizes AvrRpm1 and AvrB

18. Simplified model
rps3 plants

19. Recovery of Arabidopsis (Col - rps3) transgenics
AvrB
+ ss
- ss
+
(symptom)
-
HrpZ
-
+

20. High expression of ss-AvrB in Col - rps3
results in unexpected symptoms
Expression of AvrB in Col (RPM1+) results
in HR cell death and seedling lethality

21. AvrB is singly sufficient bacterial component
to cause HR cell death: But…
Where is it recognized?

22. Presence of AvrB and RPM1 inside the
same plant cell results in cell death
(Biolistic Bombardment)
RPM1/GUS
RPM1/GUS/AvrB
rpm1/GUS
rpm1/GUS/AvrB
(construct without signal sequence used)
Under similar conditions ss-hrpZ used in similar test was not effective

23. AvrB
Harpin
Cell wall
PM
AvrB
Rpm1
Nucleus
HR cell death,
resistance

25. Yeast Two Hybrid (YTH) Based Interaction Analysis
AvrB
BD
GAL1 UAS Promoter
AD
X
Reporter (His/lacZ)
AD
Y
GAL1 UAS Promoter
Reporter (His/lacZ)
AD
AvrB
X
BD
GAL1 UAS Promoter
Reporter (His/lacZ)

26. YTH screen of AvrB-AvrC chimeras with defined specificities in soybean
Interaction
with Rpm1
B
-
C
-
(B)
+
(B & C)
+
(-)
(C)
Chimeras constructed by Tamaki, Kobayashi, Keen, NT (1991)
-
-

27. Yeast Two Hybrid (YTH) Complementation Results
-Trp/-Leu/-His
-Trp/-Leu
V
I
B
(-)
(B)
(C)
Complementation of His auxotrophy
C

28. Yeast Two Hybrid (YTH) Marker Enzyme Activation
-Trp/-Leu/-His
-Trp/-Leu
V
I
B
(-)
(B)
(C)
Activation of lacZ reporter
C

29. YTH screen of an Arabidopsis library using the chimera with AvrB specificity
Among others:
Rubisco (lot of..), a MAPK, Myb-related transcription factor

30. Immuno-precipitation (IP) Based Interaction Analysis
IP based identification of AvrB interacting
Arabidopsis proteins

31. Does AvrB interact with a plant kinase?
Avr -FLAG
+ Arabidopsis protein
FLAG antibody (IP)
Kinase
Antibody

32
P-ATP- Kinase reaction
*
*
Denature, SDS-PAGE, Western blot, Autoradiography
Kinase
Avr
Avr
Western Blot
X-ray film

32. AvrB is phosphorylated by an Arabidopsis kinase
(IP followed by phosphorylation analysis)
AvrB - FLAG
AvrC - FLAG
Col (Rpm1+)
Nd-0 (Rpm1-)
AvrC
AvrB
??
+
+
+
+
+
+
+
+
+
+ +
+

33. AvrB is phosphorylated on serine and threonine
residues by the plant kinase(s)
S
T
Y
Phospho – amino acid analysis

34. Identification of regions in AvrB that are phosphorylated
P
P
P
Phospho-AvrB
P
Proteolytic digestion
P
P
*
*
*
HPLC
SENSOR/COLLECTOR
PROTEIN SEQUENCING

35. AvrB is phosphorylated by an Arabidopsis kinase
(radioactive peptides identified unambiguosly)
MGCVSSKSTTVLSPQTSFNEASRSFRALPGPSQRQLE
VY -- DQCLIGAARWPDDSSKSNTPENRAYCQSMYNSIRSA
G -- DEISRGGITSFEELWGRATEWRLSKLQRGEPLYSAFAS
ERTS -- DT-- DAVTPLVKPYKSVLARVV -- DHE -- DAH -DEIMQ -- DNLFG -- DLNVKVYRQTAYLHGNVIPLNTFRVAT -DTEYLR -- DRVAHLRTELGAKALKQHLQRYNP -- DRI -DHTNASYLPIIK -- DHLN -- DLYRQAISS -- DLSQAELISLIART
HWWAASAMP -- DQRGSAAKAEFAARAIASAHGIELPPFRN
GNVS -- DIEAMLSGEEEFVEKYRSLL -- DS -- DCF
Aspartate protease was used

36. Visual difference…. the untold observation….
-Trp/-Leu/-His
-Trp/-Leu
V
I
B
(-)
(B)
(C)
Activation of lacZ reporter
C

37. Quantitative b-gal assays of interaction
of the chimeras and Rpm1
4
3.5
3
2.5
2
1.5
1
0.5
0
B
(B)
(B&C)
(-)
(C)
C

38. One logical inference for quantitative difference
in the interaction of chimeras with RPM1…..
Interaction
with Rpm1
B
-
C
-
(B)
+
(B & C)
++
(-)
-
(C)
-

39. Informatics and insight
MGCVSSKSTTVLSPQTSFNEASRSFRALPGPSQRQLE
VYDQCLIGAARWPDDSSKSNTPENRAYCQSMYNSIRSA
GDEISRGGITSFEELWGRATEWRLSKLQRGEPLYSAFAS
ERTSDTDAVTPLVKPYKSVLARVVDHEDAHDEIMQDNLF
GDLNVKVYRQTAYLHGNVIPLNTFRVATDTEYLRDRVAH
LRTELGAKALKQHLQRYNPDRIDHTNASYLPIIKDHLNDLY
RQAISSDLSQAELISLIARTHWWAASAMPDQRGSAAKAEF
AARAIASAHGIELPPFRNGNVSDIEAMLSGEEEFVEKYRSL
LDSDCF
Which is consistent with……

40. Recall results of phospho-peptide analysis……..
MGCVSSKSTTVLSPQTSFNEASRSFRALPGPSQRQLE
VY -- DQCLIGAARWPDDSSKSNTPENRAYCQSMYNSIRSA
G -- DEISRGGITSFEELWGRATEWRLSKLQRGEPLYSAFAS
ERTS -- DT-- DAVTPLVKPYKSVLARVV -- DHE -- DAH -DEIMQ -- DNLFG -- DLNVKVYRQTAYLHGNVIPLNTFRVAT -DTEYLR -- DRVAHLRTELGAKALKQHLQRYNP -- DRI -DHTNASYLPIIK -- DHLN -- DLYRQAISS -- DLSQAELISLIART
HWWAASAMP -- DQRGSAAKAEFAARAIASAHGIELPPFRN
GNVS -- DIEAMLSGEEEFVEKYRSLL -- DS -- DCF

41. Interaction
with Rpm1
B
-
C
-
(B)
+
(B & C)
++
(-)
-
(C)
-

42. So far….
1. AvrB is singly sufficient to elicit RPM1 dependent cell death in plants
when expressed inside the plant cell.
2. AvrB possibly interacts with RPM1, and the interaction is probably
affected by one or more phosphorylation sites
3. AvrB is phosphorylated by a plant kinase of serine/threonine specificity
What plant kinase???????

43. In-gel kinase assay to detect AvrB phosphorylating protein
AvrB
Casein
32
Renature, in-gel kinase assay with  - P ATP
Autoradiography
Non-specific
(auto-phosphorylation)

44. AvrB is phosphorylated by an Arabidopsis kinase
qualitatively independent of Avr-R interaction
1
~ 50 kDa
2
Under identical conditions
no phospho protein was
detected in casein impregnated gel
1. Columbia treated with DC3000 – 4.5 h
2. Columbia treated with DC3000 (avrB) – 4.5 h

45. So far….
1. AvrB is singly sufficient to elicit RPM1 dependent cell death in plants
when expressed inside the plant cell.
2. AvrB possibly interacts with RPM1, and the interaction is probably
affected by one or more phosphorylation sites
3. AvrB is phosphorylated by a plant kinase of serine/threonine
specificity
One more piece of the puzzle…..

46. Interaction
with Rpm1
B
-
C
-
(B)
+
(B & C)
++
(-)
-
(C)
-
The (B) and (B &C) chimeras do not elicit cell death in
Arabidopsis, even in the presence of RPM1, but…
THERE IS RESTRICTION OF BACTERIAL GROWTH

47. CONCLUSIONS based these data
1. AvrB is singly sufficient to elicit RPM1 dependent cell death in plants
when expressed inside the plant cell.
Such intracellular site of action seems to be common property for most bacterial
avirulence proteins and other Type III effectors
2. Transgenic plants revealed a previously unknown and possibly RPM1
independent function of AvrB.
3. AvrB possibly interacts with RPM1, and the interaction is probably
affected by one or more phosphorylation sites
4. Chimeras with AvrB specificity interacts with other proteins with the same
specificity as RPM1 (including a MAPK, myb-related transcription factor, rubisco)
Are some of these targets of virulence function of AvrB?????
5. AvrB is phosphorylated by a plant kinase of serine/threonine specificity
that is not dependent on activation by AvrB-RPM1 interaction
6. Phosphorylation of AvrB is important for its HR cell death elicitation??????

48. ACKNOWLEDGEMENTS
Work done at: DOE – Plant Research Laboratory, Michigan State University
Sheng Yang He Laboratory
Sheng Yang He
Laura Muncie (Undergraduate Assistant)
Anne Jones (Lab tech)
Alan Collmer (collaborator and AvrB - FLAG)
Noel Keen (AvrB - AvrC chimeras)
ABRC – rps3 mutant and library for YTH screen
Other students of S. Y. He laboratory
Partial funding: NSF (S. Y. He and S. Gopalan)