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

Disease (compatible) Resistance (incompatible)

Pathogens

Avirulent Virulent

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

Avr

R gene product

HR/Resistance

Harpin

CW

PM

Hrp

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

From: Arabidopsis Book

DC3000

DC3000

(+ avrB)

RPM1 rpm1

D D

HR D

Arabidopsis- P. syringae model system used

Grant…..Dangl with Innes (rps3) – recognizes AvrRpm1 and AvrB

Simplified model

rps3 plants

+ ss

- ss

AvrB HrpZ

+

(symptom)

-

- +

Recovery of Arabidopsis (Col - rps3) transgenics

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

AvrB is singly sufficient bacterial component

to cause HR cell death: But…

Where is it recognized?

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)

AvrB

Harpin

Rpm1

Nucleus

HR cell death, resistance

AvrB

PM

Cell wall

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……..

B

C

(B)

(B & C)

(-)

(C)

Interaction

with Rpm1

-

-

+

++

-

-

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)