lJ Ítìr¡ 1 t¡ The Putative ( 1,3)-9-l-Glucan Synthase Gene Family in Hordeum vulgare Submitted by Michael Scott Schober This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Discipline of Plant and Pest Science School of Agriculture and Wine Faculty of Sciences University of Adelaide,'Waite Campus Glen Osmond, South Australia, 5064, Australia January,2006 Statement of Authorship This thesis contains no material that has been accepted for the award of any other degree or diploma in any university and that, to the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference being made in the text of the thesis. I give consent to this copy of my thesis, when deposited in the University Libraries, being available for photocopying and loan. Michael Scott Schober January 2006 ll Table of Contents STATEMENT or AutgonsulP ll TABLE OF CONTENTS iii ACKNOWLEDGEMENTS vi PUBLTCATIONS vii ABBREVIATIONS viii ABSTRACT ix CHAPTER 1 General Introduction I 1.I INTRODUCTION 2 1.2 (1,3)-p-D-GLUCAN 4 1.2.1 StructuralProperties 4 1.2.2 Cellular Locations and Associated Functions 6 1.2.2.1 Cell Plate Formation 6 1,2.2.2 Plasmodesmata and Sieve Plate Pores 7 1.2.2.3 ReproductiveTissues 9 1.3 STRESSRELATED(1,3)-B-o-GLUCANDEPOSITION ll I .3. 1 Abiotic stress ll l.3.l.l Wounding ll 1.3.1.2 Metaltoxicity t2 1.3.2 Blotic Stress 12 1.3.2.1 Viral infection t2 1.3.2.2 Bacterialinfection 13 1.3.2.3 Nematode infection l3 1.3.2.4 Fungal Infection t4 1.4 (1,3)-B-D-GLUCANSYNTHASES 16 '1.4.1 The Biosynthesis of (1,3)-B-o-Glucan t6 l.4.l.l The Requirement for UDP-Glucose l6 1.4.1.2 The Requirement for Calcium t7 1.4.1.3 Regulation of (1,3)-p-o-Glucan Synthase Activity with GTPases l8 1.4.1.4 Membrane Location of (1,3)-P-D-Glucan Synthase Activity t9 1.4.1.5 Detergent Activation of(1,3)-p-o-Glucan Synthases 20 1.4.1.6 Trypsin Activation of (1,3)-B-o-Glucan Synthases 2l 1.4.1.7 Product Entrapment of (1,3)-p-o-Glucan Synthase Activity 2l 1.4.2 Putative (1,3)-B-o-Glucan Synthase Genes 22 1.4.2.1 Yeast FKS Genes 22 1.4.2.2 Higher Plant GSZ Genes 24 I.5 SUMMARY AND PROJECT AIMS 30 CHAPTER 2 Cloning, Mapping and Sequence Analysis of HvGSL Genes lrom Hordeum vulgare 32 2.1 INTRODUCTION JJ 2.2 MATEzuALS ¡NO METHODS 35 2.2.1 Materials 35 2.2.2 Bioinformatics 36 2.2.3 Total RNA Extraction 36 2.2.4 First Strand cDNA Synthesis for PCR 38 111 2.2.5 PCR Amplification of Single Stranded cDNA 38 2.2.6 Cloning PCR Amplified Fragments into pGEM@-T Easy 40 2.2.7 Transformation of Escherichia coli 4t 2.2.8 PCR Screening of Colonies 44 2.2.9 PlasmidDNAMini-Preparations 44 2.2.10 Restriction Endonuclease Digestion of DNA 45 2.2.11 DNA Sequencing 45 2.2.12 Genetic Mapping of llvGSl Genes 46 2.3 RESULTS 49 2.3.1 Cloning of HvGSLI 49 23.2 Cloning of HvGSL2 cDNA Fragments 49 2.3.3 Cloning of HvGSL3 cDNA Fragments 52 2.3.4 Cloning of a HvGSL4 cDNA Fragment 56 2.3.5 Cloning of a HvGSLS cDNA Fragment 58 2.3.6 Cloning of a HvGSL6 cDNA Fragment 58 2.3.7 Cloning of HvGSLT cDNA Fragments 6t 2.3.8 Cloning of HvGSLS cDNA Fragments 64 2.3.9 Cloning Summary 7l 2.3.10 Genetic Mapping of HvGSLs 7l 2.3.11 Phylogenetic Tree 78 2.3.12 Sequence Alignments 78 2.3.13 Membrane Topology Prediction of the HvGSLS Protein 84 2.3.14 Analysis of the Proposed Catalytic Site 87 2.4 DISCUSSION 88 CHAPTER 3 Transcript Profiling o1 HvGSL Genes 94 3.1 INTRODUCTION 95 3.2 MATERIALSANDMETHODS 96 3.2.1 Materials 96 3.2.2 Bio-lnformatics 96 3.2.3 MicroarrayAnalysis 96 3.2.4 Quantitative Real-Time PCR 98 3,3 RESULTS 104 3.3.1 EST Data Analysis 104 3.3.2 MicroarrayAnalysis t04 3.3.3 Q-PCR Analysis l16 3.3.4 Co-ordinate Transcription of flvGSZ Genes 132 3.4 DISCUSSION 133 3.4.1 Conclusions t42 CHAPTER 4 Transient Post-Transcriptional Gene Silencing of Putative (1,3)-p-o-Glucan Synthases in Horcleum vulgtre 145 4.1 INTRODUCTION 146 4.2 MATERIALS AND METHODS ts4 4.2.1 Materials 1s4 4.2.2 Identification of HvGSLT and HvGSLS Gene-Specific Regions 154 4.2.3 GeneralMolecularTechniques 160 4.2.4 Construction of the HvGSLZ dsRNAi Vector 160 4.2.5 Construction of lhe HvGSLd dsRNAi Vector r62 4.2.6 Transiently-Induced Post-Transcriptional Gene Silencing 164 4.3 RESULTS 168 4.3.1 Experimental Control for Transient Gene Silencing 168 lv 4.3.2 The Effect of Transiently Silencing HvGSLT on Papillary Structure 168 4.3.3 The Effect of Transiently Silencing HvGSLS on Papillary Structure 172 4.4 DISCUSSION t76 4.5 CONCLUSIONS 180 CHAPTER 5 General Discussion 181 5.I SUMMARY OF EXPERIMENTAL RESULTS r82 5.2 FUTURE WO 188 APPENDIX A 194 APPENDIX B 196 APPENDIX C 197 REFERENCES 200 v Acknowledgements I would like to thank my principal supervisor, Professor Geoff Fincher, for providing me with a scholarship and funding through an Australian Research Council grant. I would also like to thank him for providing guidance and direction throughout the duration of my project and also for his support and patience during difficult periods of my research. I also wish to thank my co-supervisors, Dr Rachel Burton and Dr Andrew Jacobs for their friendship, patience and guidance. Both Rachel and Andrew have taught me many molecular biological techniques and have provided useful discussions. I would like to thank Dr Andreas Schreiber and Dr Andrew Harvey for their assistance with bioinformatics. I thank Dr Neil Shirley for providing his quantitative real-time PCR expertise. Thanks to Dr Paul Gooding for his molecular biology advice and friendship. I would like to thank Associate Professor Maria Hrmova for her intellectual conversation and for showing that good science can arise from determination and dedication. Thanks to Mrs Margaret Pallotta for providing assistance with gene mapping. I would also like to thank the many people that have contributed by interesting scientific discussions, advice and by making my research enjoyable. In particular, I thank Professor Bruce Stone, Dr David Gibeaut, Dr Jing Li, Dr Christina Lunde, Dr Qisen Zhang, Dr Brendon King, Mr Ahmad Wardak and Mrs Ursula Langridge and the current and past students in the Fincher laboratory including Mr Naser Farroki, Ms Jacinda Rethus, Ms Vanessa Richardson and Mr Damien Drew. I would also like to thank the past and present members of the Fincher laboratory. I wish to thank both my family and the Rathjen family for their emotional and intellectual support. Finally and most importantly, I would like to thank my beautiful wife, Judy, for her love and patience. v1 Publications Seminars "The (1,3)-B-o-glucan synthase gene family in Hordeum vulgare" lTth September,2003 University of Adelaide, Discipline of Plant and Pest Science Postgraduate Symposium "The (1,3)-B-o-glucan synthase gene family in Hordeum v.tlgare" 24Th May,2004 Functional Genomics Program Collaboration with Department of Botany, Melbourne University Conference Proceedings Schober MS, Jacobs AK, Burton RA, Fincher GB. "The (1,3)-p-n-glucan synthase gene family in Hordeum vulgare" ComBio2004, Sept 26-30 2004, Burswood Resort Convention Centre, Perth, WA, Australia. Jacobs AK, Lipka V, Burton RA, Schober MS, Panstruga R, Strizhov N, Schulze- Lefert P, Fincher GB. "The callose synthase gene family in Arabidopsis and barley" Plant and Animal Genome Conference 12, Jan 10-14 2004. Town and Country Convention Centre. San Diego, California USA vll Abbreviations 3'RACE . .3' rapid amplification of cDNA ends 3'UTR .....3' untranslated region A .....Absorbance aa .....Amino acid(s) ABF .....Aniline blue fluorochrome bp .....Base pair(s) CaMV .....Cauliflower mosaic virus CHAPS .. ...3-[(cholamidopropyl)-dimethylammonio]- I -propane-sulphate cDNA ,.....Complementary DNA Da ,.. ... Dalton dCTP ,..... Deoxycytidine triphosphate DNA ,.. ... Deoxyribose nucleic acid DNase ,.....DNA hydrolase dNTP ......Deoxynucleotide triphosphate DOP ...... Degree of polymerisation DTT ... ... Dithiothreitol EDTA ... ...Ethylene diamine tetra-acetic acid EST ......Expressed sequence tag û b ......Unit of relative centrifugal force GDP ......Guanosine diphosphate GFP ......Green fluorescent protein GSL ... .. Glucan synthase-like GTE ......Glucose tris EDTA GTP ......Guanosine triphosphate GUS ...... p-glucuronidase gene hr ......hour(s) kb ... ... Kilobase pairs LB ... ... Luria-Bertani LBA ... ... Luria-Beftani agar M ......Molar MCS ......Multiple cloning site min ......Minute(s) mRNA ......messenger RNA MOPS ......3-(N-morpholino)propanesulfonic acid PCR ......Polymerase chain reaction PEG ... ... Polyethylene glycol QTL ......Quantitative trait loci RNA ...... Ribonucleic acid RNase ... ... RNA hydrolase rpm ...... Revolutions per minute RT ......Room temperature RT ......Reverse transcriptase SDS ,......Sodium dodecyl sulphate SDS-PAGE ......SDS-polyacyrlamide gel electrophoresis sec , ... ... Second(s) SSC , ... ... Standard saline citrate TE .......Tris EDTA T,n .......Melting temperattre tris .......Tris[hydroxylmethyl] amino methane UDP ....... Uridine diphosphate UGT ....,..UDP-glucose transferase UV .......Ultraviolet vl11 Abstract (1,3)-B-o-Glucan, which is often referred to as callose, is deposited in numerous locations and at various stages during normal growth and development in higher plants, as well as in response to abiotic antl biotic stresses. For examplc, (1,3)-B-o- glucan is the main polysaccharide component of the forming cell plate during mitosis, and is deposited at various stages during the meiotic events of micro- and megasporogenesis.