Chemical Genomics Approach to Endocytic Pathways in Arabidopsis thaliana using

Nicotiana tabacum pollen

R.J. Washington, M. Brown, N.V. Raikhel

NSF-REU Program 2008

Source: News in Science, 8 January 2002www.abc.net.au/science/news/stories/s453641.htm

ERGolgi

vacuolePVC

TGN

EndosomesPlasma

membrane

PIN2

RIP1

AUX1BRI1

A closer look at the endomembrane system

EXPERIMENTAL APPROACHClassical genetics vs Chemical Genomics

Functional protein

Genetic mutation Chemical interaction

Non-functional protein

Classical genetics Chemical Approach

Cl

Cl

Pollen tip polarized activity

Germination: Three tier activity

– Zone 1- Apical: New materials deposited at apical end via secretory vesicles

– Zone 2- Sub-apical: actin filaments, mitochondria and EM organelles

– Zone 3- Shank: composed of actin filaments

All zones constantly participate in concert to propel tube growth via the dynamic endocytic/exocytotic processes

Model from Cole and Fowler (2006) Current Opinion Plant Biol. 9:1-10

Primary Chemical Screen

WT Tobacco Pollen

BD Pathway microscope

Chemical Library

Inhibition of pollen germination

384 well plate

Aberrant pollen morphology

+

Secondary Chemical Screen

RIP1:GFP marker Tobacco Pollen

Mislocalization of marker

96 well plate

Aberrant pollen morphology

Examine with BD Pathway microscope

Primary Screen Inhibitors serially diluted

A B

+

Library Number of Compounds Primary Hits Primary

Percentage Secondary HitsSecondary Percentage

Diverset 20,000 218 1.09% 22/176 12.5%

Novacore 10,000 18 0.18% 0/18 0%

TimTec 10,000 33 0.33% 0/30 0%

LATCA 3650 89 [76] 2.4% 16/73 21.9%

CLICK 2768 15 0.54% 0/7 0%

TOTALS 46418 360 .78% 38/302 12.6%

Summary of library progress of pollen screens

ANTIMICROBIAL

3

 

 ANTIFUNGAL

1

ANTICARCINOGEN

3

ANTIPARASITICAL

4

ANTIBACTERIAL 

3

CYTOSKELETAL EFFECTS

2

 

TRANSPORT PROTEINS 

4

OTHER EFFECTS

5

Bioactivity of secondary hit compounds from database screen inquiry

Based on structure database searchScreening database used in inquiry: Science Finder Scholar Program

Future Work

Chemical Screening of Arabidopsis seedlings− Root and vegetative tissues phenotype effects categorized for

each test compound− Marker lines imaged via confocal microscopy and analyzed to

determine localization effects

Plasma membrane TGN Bri-overexpression PIN2

Acknowledgements• Principal Investigator: Dr. Natasha V. Raikhel

• Mentor: Michelle Brown

• Lab members:Glenn Hicks, Marci Surpin, Mien van de Ven, Stephanie RobertJulie Sohn, Georgia Drakakaki, April Agee, Abel Rosado Rey

• Support Staff and Facilities: Dr. David Carter (CEPCEB Academic Coordinator, Microscopy/Imaging) Dr. Thomas Girke (CEPCEB Academic Coordinator, Bioinformatics) Institute for Integrative Genome Biology (IIGB)-Chemical Libraries Plant Transformation Research Center

• Support Funding: National Science Foundation