host perception and signaling during bacterial infections
DESCRIPTION
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.TRANSCRIPT
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
HOST PERCEPTION AND SIGNALING DURING PROGRAMMED CELL DEATH
RESPONSE AND RESISTANCE MEDIATED BY BACTERIAL PATHOGENS
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
Perception and signaling of bacterial avirulence proteins in plants:
another facet
Pseudomonas syringae glycinea AvrB – Arabidopsis RPM1 model system
Suresh Gopalan
Adapted from: Arabidopsis Book
Under ideal conditions Arabidopsis sprayed with appropriate
dose of virulent and avirulent bacterial pathogen will look like this
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)
P.s.s.61
(Bean pathogen)
P.s.tabacum
(Nicotiana pathogen)
High inoculum, rapid cell death
(about 12 h later)
Low inoculum
(few days later)
A LABORATORY MANIFESTATION OF HR AND DISEASE
GENE-FOR-GENE HYPOTHESIS IN
RACE-CULTIVAR SPECIFICITY
R avr
+
-
+ -
HR D
D D
C1 C2
R1
R2
Single matching gene combination between pathogen and plant can lead to HR
First cloned pathogen molecule : plant gene product pair
HC-Toxin : HC-Toxin reductase pair C. carbonum :Maize
(John Walton and Steve Briggs group)
RPS4, N for e.g., has homology to DrosophilaToll and mammalian IL1 receptors;
Xa21 for e.g., resembles structure of many RTKs
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
Properties of some cloned R gene products
NBS LRRs LZ Kinase Putative
location
Rps2, Rpm1,
Prf
Pto
N
Cf - 9, Cf - 2
L6
X
X X X
X X
X
X
Xa21 X X
C
C
EC/TM/C
C
EC
NBS LRRs LZ Kinase Putative
location
Rps2, Rpm1,
Prf
Pto
N
Cf - 9, Cf - 2
L6
X
X X X
X X
X
X
Xa21 X X
C
C
EC/TM/C
C
EC
Avr
R gene product
HR/Resistance
Harpin
CW
PM
HrpAvr
R gene product
HR/Resistance
Harpin
CW
PM
Avr
R gene product
HR/Resistance
Harpin
CW
PM
Hrp
Background and the enigma:
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
1. Enzymatic action (e.g., AvrD)
2. Another bacterial factor involved (e.g., Harpin)
Some other possibilities
DC3000
DC3000
(+ avrB)
RPM1 rpm1
D D
HR D
Arabidopsis- P. syringae model system used
Grant…..Dangl with Innes (rps3) – recognizes AvrRpm1 and AvrB
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
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
Under similar conditions ss-hrpZ used in similar test was not effective
(construct without signal sequence used)
BD
AvrB
AD
X
GAL1 UAS Promoter Reporter (His/lacZ)
GAL1 UAS Promoter Reporter (His/lacZ)
Y
BD
AvrB
GAL1 UAS Promoter Reporter (His/lacZ)
X
AD
AD
Yeast Two Hybrid (YTH) Based Interaction Analysis
YTH screen of AvrB-AvrC chimeras with defined specificities in soybean
Chimeras constructed by Tamaki, Kobayashi, Keen, NT (1991)
B
C
(B)
(B & C)
( - )
(C)
Interaction
with Rpm1
-
-
+
+
-
-
V
I B
(-)
(B)
(C) C
-Trp/-Leu/-His -Trp/-Leu
Complementation of His auxotrophy
Yeast Two Hybrid (YTH) Complementation Results
V
I B
(-)
(B)
(C) C
-Trp/-Leu/-His -Trp/-Leu
Activation of lacZ reporter
Yeast Two Hybrid (YTH) Marker Enzyme Activation
Among others:
Rubisco (lot of..), a MAPK, Myb-related transcription factor
YTH screen of an Arabidopsis library using the chimera with AvrB specificity
IP based identification of AvrB interacting
Arabidopsis proteins
Immuno-precipitation (IP) Based Interaction Analysis
+ Arabidopsis protein
FLAG antibody (IP)
Avr -FLAG
AntibodyKinase
P-ATP- Kinase reaction32
Denature, SDS-PAGE, Western blot, Autoradiography
*
*
Avr AvrKinase
Western Blot X-ray film
Does AvrB interact with a plant kinase?
AvrB is phosphorylated by an Arabidopsis kinase
(IP followed by phosphorylation analysis)
AvrB AvrC
AvrB - FLAG
AvrC - FLAG
Col (Rpm1+)
Nd-0 (Rpm1-)
+
+ +
+
+
+
+
+
+ +
+
+
??
AvrB is phosphorylated on serine and threonine
residues by the plant kinase(s)
S T Y
Phospho – amino acid analysis
* * *
PROTEIN SEQUENCING
P P P
Phospho-AvrB
Proteolytic digestion
HPLC
SENSOR/COLLECTOR
P P P
Identification of regions in AvrB that are phosphorylated
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
AvrB is phosphorylated by an Arabidopsis kinase
(radioactive peptides identified unambiguosly)
V
I B
(-)
(B)
(C) C
-Trp/-Leu/-His -Trp/-Leu
Activation of lacZ reporter
Visual difference…. the untold observation….
0
0.5
1
1.5
2
2.5
3
3.5
4
B (B) (B&C) (-) (C) C
Quantitative b-gal assays of interaction
of the chimeras and Rpm1
B
C
(B)
(B & C)
(-)
(C)
Interaction
with Rpm1
-
-
+
++
-
-
One logical inference for quantitative difference
in the interaction of chimeras with RPM1…..
MGCVSSKSTTVLSPQTSFNEASRSFRALPGPSQRQLE
VYDQCLIGAARWPDDSSKSNTPENRAYCQSMYNSIRSA
GDEISRGGITSFEELWGRATEWRLSKLQRGEPLYSAFAS
ERTSDTDAVTPLVKPYKSVLARVVDHEDAHDEIMQDNLF
GDLNVKVYRQTAYLHGNVIPLNTFRVATDTEYLRDRVAH
LRTELGAKALKQHLQRYNPDRIDHTNASYLPIIKDHLNDLY
RQAISSDLSQAELISLIARTHWWAASAMPDQRGSAAKAEF
AARAIASAHGIELPPFRNGNVSDIEAMLSGEEEFVEKYRSL
LDSDCF
Which is consistent with……
Informatics and insight
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
Recall results of phospho-peptide analysis……..
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???????
Renature, in-gel kinase assay with - P ATP32
Autoradiography
AvrBCasein
Non-specific
(auto-phosphorylation)
In-gel kinase assay to detect AvrB phosphorylating protein
~ 50 kDa
1 2
1. Columbia treated with DC3000 – 4.5 h
2. Columbia treated with DC3000 (avrB) – 4.5 h
AvrB is phosphorylated by an Arabidopsis kinase
qualitatively independent of Avr-R interaction
Under identical conditions
no phospho protein was
detected in casein impregnated gel
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…..
So far….
B
C
(B)
(B & C)
(-)
(C)
Interaction
with Rpm1
-
-
+
++
-
-
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
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??????
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)