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Model: Arabidopsis - Pseudomonas

Model: Arabidopsis - Pseudomonas. OH. O. O. H. O. H. N. EtO. N. Chemical genetics & Activity-based profiling:. N. H. O. O. novel tools to investigate the Arabidopsis-Pseudomonas interaction. Dr. Renier van der Hoorn Plant Chemetics group, CGC Oktober 28 th 2005.

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Model: Arabidopsis - Pseudomonas

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  1. Model: Arabidopsis - Pseudomonas OH O O H O H N EtO N Chemical genetics & Activity-based profiling: N H O O novel tools to investigate the Arabidopsis-Pseudomonas interaction Dr. Renier van der Hoorn Plant Chemetics group, CGC Oktober 28th 2005

  2. www.cgc.mpg.de groups of 6 Max-Planck Institutes +2 (+3) independent junior groups: Plant Biology MPIZ Cologne Van der Hoorn Org. Chemistry BMC Dortmund Markus Kaiser Chemical Genomics Centre

  3. active inactive enzyme activity profiling substrate biotin- probe inhibitor biotinylated covalent enzyme intermediate locked! detect identify purify mass spec. treatment + - products strep-HRP m/z Activity-based profiling

  4. leaf extract - + E-64: 50 large scale ID by MS: RD21: drought induced 37 CatB-like (new) XCP2: xylem specific aleurain-like (new) 26 AALP: aleurain-like TPE4A-like (new) 19 strep-HRP Profiling papain-like activities OH biotin O O NH O O O 2 H O H N S EtO N N N N E-64 H H H O O HN NH DCG-04 O Arabidopsis: ~30 papain-like proteases labeling reflects activity rather than abundance

  5. Pseudomonas - Arabidopsis what mechanisms occur at protease activity level?

  6. vir avr Arabidopsis cell culture (Rpm1) avrRpm1 100 % cell death 50 avr vir 0 10 24 (hour) 10 24 Infection of plant cell culture + Pseudomonas bacteria disease defence

  7. +Pseudomonas +DCG-04 extract time: 0 10 24 (hr) 14 hour in vivo labeling pI vir Pst avr Pst 3 7 3 7 MW 37 26 anti-biotin RD21 RD21-like differential protease activities RD21: Responsive to Dessiccation-21

  8. induced by avr Pseudomonas biotin pull-down 24 hpi suppressed by vir Pseudomonas ctr avr vir post-translational suppression active RD21 anti-RD21 total RD21 anti-RD21 illustrates potential of activity-based approaches Confirmation of differential RD21 activity RD21 antibody: Ikuko Hara-Nishimura (Kyoto) RD21 activity: role for RD21 in plant-pathogen interactions?

  9. 1. Discover more enzymes with differential activities: using other probes on Arabidopsis - Pseudomonas interaction 2. Determine the function of these enzymes: by traditional reverse genetics... probe-derived chemical library: disease probe - + activity profile: disease phenotype: Plant Chemetics research program ... and by chemical genetics + activity profiling: 3. "Chemetics": use chemicals to identify functions of enzyme activities

  10. 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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