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Model: Arabidopsis - Pseudomonas

Dr. Renier van der HoornPlant Chemetics group, CGC

Oktober 28th 2005

Chemical genetics &Activity-based profiling:

novel tools to investigate theArabidopsis-Pseudomonas interaction

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www.cgc.mpg.de

Chemical Genomics Centre

groups of 6 Max-Planck Institutes+2 (+3) independent junior groups:

Plant BiologyMPIZ CologneVan der Hoorn

Org. ChemistryBMC DortmundMarkus Kaiser

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activityprofilingsubstrate

enzyme

Activity-based profiling

covalentintermediate

products

active inactive

probebiotin-inhibitor

biotinylatedenzyme

treatment

purifydetect

strep-HRP

+ -mass spec.

identify

m/z

locked!

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Profiling papain-like activities

leaf extract

strep-HRP

19

26

37

50

E-64: - +

RD21: drought inducedCatB-like (new)XCP2: xylem specificaleurain-like (new)AALP: aleurain-likeTPE4A-like (new)

large scale ID by MS:

E-64

Arabidopsis: ~30 papain-like proteases

biotin

NH

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O O

O

S

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NH

NH2

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O

labeling reflects activityrather than abundance

DCG-04

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what mechanisms occur at protease activity level?

Pseudomonas - Arabidopsis

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Infection of plant cell culture

vir

avrRpm1

avrArabidopsiscell culture

(Rpm1)

+ Pseudomonas bacteria

defence disease

vir

10

100

50

024 10 24(hour)

% cell death

avr

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differential protease activities

RD21: Responsive to Dessiccation-21

14 hour in vivo labeling

0time: 10 24(hr)

+Pseudomonas +DCG-04 extract

vir Pst

anti-biotin

MW

pI

avr Pst

26

37

3 37 7

RD21 RD21-like

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RD21 activity: induced by avr Pseudomonas suppressed by vir Pseudomonas post-translational suppression

Confirmation of differential RD21 activityRD21 antibody: Ikuko Hara-Nishimura (Kyoto)

role for RD21 in plant-pathogen interactions? illustrates potential of activity-based approaches

activeRD21

biotin pull-down

ctr

anti-RD21

avr vir24 hpi

totalRD21

anti-RD21

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Plant Chemetics research program

3. "Chemetics": use chemicals to identify functions of enzyme activities

2. Determine the function of these enzymes: by traditional reverse genetics...

1. Discover more enzymes with differential activities: using other probes on Arabidopsis - Pseudomonas interaction

probe-derived chemical library:

disease phenotype:

probe disease- +

activityprofile:

... and by chemical genetics + activity profiling:

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Team & Collaborations

Group members:Takayuki Shindo (started 1-10-2005)

Zhe-Ming Wang (starts 1-12-2005)

Farnusch Kaschani (starts 1-1-2006)

Current collaborations:Markus Kaiser (CGC, Dortmund)

Matt Bogyo (Stanford University, USA)

Hermen Overkleeft (Leiden University, NL)

Benjamin Cravatt (Scripps Institute, USA)

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