Université de Strasbourg Thèse présentée à la FACULTÉ DES SCIENCES DE LA VIE ET DE LA TERRE pour obtenir le titre de DOCTEUR DE L’UNIVERSITÉ de STRASBOURG Domaine : Biologie Moléculaire Végétale par Michael Hartmann Titre du sujet de thèse: Inhibition and regulation of isoprenoid biosynthetic pathways in plants Inhibition et régulation des voies de biosynthèse des isoprénoïdes chez les végétaux Soutenue le 14 septembre 2010 devant la Comission d’Examen : Thomas J. BACH Directeur de thèse Université de Strasbourg, Strasbourg Francis KARST Rapporteur interne Institut National de la Recherche Agronomique, Colmar Michael H. WALTER Rapporteur externe Leibniz-Institut für Pflanzenbiochemie, Halle/Saale (GER) Benoît ST-PIERRE Rapporteur externe Université François Rabelais, Tours Rüdiger HELL Examinateur Heidelberg Institute for Plant Science, Heidelberg (GER) Michel ROHMER Examinateur Université de Strasbourg, Strasbourg This doctoral thesis has been supported by a full grant of the „Région ALSACE” (Bourse Régionale de Recherche 2005: Convention de thèse n° d’enregistrement 05/908/702 – HARTMANN Michael). Table of Contents Table of contents: 1 INTRODUCTION ............................................................................................. 11 1.1 Isoprenoids 11 1.1.1 Isoprenoids – a diverse class of metabolites ............................................................................. 11 1.1.2 Biosynthesis of isoprenoids in plants ....................................................................................... 14 1.1.3 Plants have evolved two different pathways for the synthesis of isoprenoids .......................... 14 1.1.4 Biosynthesis of higher isoprenoids ........................................................................................... 24 1.2 Non-mevalonate pathway as a target for the development of antimicrobial drugs and herbicides 26 1.2.1 Microbial targets (eubacteria) ................................................................................................... 29 1.2.2 Antimalarial targets .................................................................................................................. 30 1.2.3 Herbicides ................................................................................................................................. 31 1.3 How to study the effects of potential inhibitors of the MEP pathway? 32 1.4 Goal of this work 34 2 RESULTS AND DISCUSSION ........................................................................ 36 2.1 Development of a reporter system to screen for inhibitors that interfere with protein geranylgeranylation 36 2.1.1 The prenylation of proteins in plants ........................................................................................ 36 2.1.2 The thesis of Esther Gerber (2005) – Development of a bioassay based on a visual marker ... 41 2.1.3 The beginning of my work ....................................................................................................... 50 2.1.4 Impact of the inhibition of the sterol biosynthetic pathway on the localization of the H6-GFP-DB- CVIL fusion protein in BY-2 cells ....................................................................................... 63 2.1.5 Post-prenylation inhibitors and transport of GFP-DB-CVIL to the plasma membrane ............ 77 2.1.6 Testing and evaluating novel inhibitors of the MEP pathway: from qualitative to quantitative analysis .............................................................................................................................................. 82 2.2 Development of an image-based chemical screening system for inhibitors of the plastidial MEP pathway 104 2.2.1 Introduction: State of the art of modern drug screening ......................................................... 104 2.2.2 Biological assay development ................................................................................................ 108 2.2.3 Image acquisition and analysis ............................................................................................... 110 2.2.4 Optimization of the assay ....................................................................................................... 120 2.3 Generation of transgenic tobacco BY-2 cell lines 145 I Table of Contents 3 CONCLUSION AND PERSPECTIVES ....................................................... 157 4 MATERIAL AND METHODS ...................................................................... 165 4.1 Instruments and software 165 4.1.1 Instruments ............................................................................................................................. 165 4.1.2 Software, algorithms and databases ........................................................................................ 166 4.2 Enzymes, chemicals and consumable materials 168 4.2.1 Enzymes and Molecular Biology Kits .................................................................................... 168 4.2.2 Chemicals ............................................................................................................................... 170 4.2.3 Consumable Materials ............................................................................................................ 170 4.3 Living Material 170 4.3.1 Plant material .......................................................................................................................... 170 4.3.2 Bacteria ................................................................................................................................... 171 4.3.3 Antibiotics .............................................................................................................................. 172 4.4 Biochemical Methods 173 4.4.1 Protein extraction for BY-2 proteomics.................................................................................. 173 4.4.2 Protein quantification ............................................................................................................. 174 4.4.3 SDS-PAGE ............................................................................................................................. 174 4.5 General Techniques in Molecular Biology 175 4.5.1 Polymerase Chain Reaction (PCR) ......................................................................................... 175 4.5.2 Preparation of XL1-Blue competent cells .............................................................................. 176 4.5.3 Transformation of E.coli chemical-competent cells ............................................................... 176 4.5.4 Isolation of plasmid DNA from Escherichia coli ................................................................... 177 4.5.5 Stable transformation of BY-2 cells by agroinfection ............................................................ 178 4.5.6 Preparation of A. tumefaciens chemical competent cells ........................................................ 180 4.5.7 Preparation of A.tumefaciens electro-competent cells ............................................................ 180 4.5.8 Transformation of A.tumefaciens chemical competent cells by heatshock ............................. 181 4.5.9 Transformation of A.tumefaciens electro-competent cells by electroporation ........................ 181 4.5.10 Restriction of DNA fragments ................................................................................................ 181 4.5.11 Ligation of DNA fragments .................................................................................................... 182 4.5.12 Cloning of blunt end DNAs into a sequencing vector using the Digestion – Ligation method (DIG- LIG) .................................................................................................................................... 182 4.5.13 Agarose Gel electrophoresis of DNA and RNA ..................................................................... 182 4.5.14 Glycerol stocks ....................................................................................................................... 183 4.5.15 Preparation of genomic DNA from bacteria ........................................................................... 183 4.5.16 Isolation of total RNA from plant tissue ................................................................................. 184 4.5.17 Reverse transcription .............................................................................................................. 184 II Table of Contents 4.5.18 Spectrophotometric analysis of nucleic acids ......................................................................... 185 4.5.19 Particle bombardment of TBY2 cells ..................................................................................... 186 4.5.20 Microscopy ............................................................................................................................. 187 4.6 Bioinformatical Analysis 188 4.6.1 Molecular Biological Software ............................................................................................... 188 4.6.2 Algorithms and software for prediction .................................................................................. 188 4.6.3 Database search ...................................................................................................................... 189 4.6.4 Image acquisition, analysis and processing software ............................................................. 189 5 REFERENCES ...............................................................................................