Profiling of Wounding and Diuraphis Noxia Induced Transcripts in Hexaploid Wheat Using Cdna-AFLP Analysis

Profiling of wounding and Diuraphis noxia induced transcripts in hexaploid wheat using cDNA-AFLP analysis BY Thia Schultz Submitted in partial fulfillment of the requirements for the degree Magister Scientiae In the Faculty of Natural and Agricultural Sciences Department of Genetics University of Pretoria Pretoria April 2010 Under the supervision of Prof. AM Botha-Oberholster © University of Pretoria Declaration I, the undersigned, hereby declare that the thesis submitted herewith for the Degree Magister Scientiae to the University of Pretoria, contains my own independent work and has not been submitted for any other degree at any other University. T Schultz 2009 II Table of Contents Table of contents .................................................................................................................... III Acknowledgements.................................................................................................................VI List of Abbreviations...............................................................................................................VII List of Figures .........................................................................................................................IX List of Tables...........................................................................................................................XI Chapter 1 Introduction .......................................................................................................XII Preface..................................................................................................................................XIII Chapter 2 Literature Review 2.1 Wheat................................................................................................................................. 2 2.1.1 Genomics........................................................................................................................ 2 2.2 Resistance systems in plants............................................................................................. 3 2.2.1 Gene-for-Gene hypothesis: Plant innate immunity ......................................................... 3 2.2.2 The wound response ...................................................................................................... 8 2.2.3 Hypersensitive response............................................................................................... 11 2.2.4 Systemic acquired resistance and induced systemic resistance .................................. 14 2.3 The Russian wheat aphid ................................................................................................ 16 2.3.1 Physiology and life cycle............................................................................................... 17 2.3.2 Aphid feeding and saliva............................................................................................... 17 2.3.3 Aphid saliva and the defence response ........................................................................ 19 2.4 Wheat and the Russian wheat aphid ............................................................................... 23 2.4.1 Symptoms of infestation................................................................................................ 23 2.4.2 Existing genetic resistance ........................................................................................... 24 2.5 Newest findings................................................................................................................ 26 2.6 References....................................................................................................................... 29 III Chapter 3 Transcript profiling of wounded and Russian wheat aphid-infested Betta wheat NILs 3.1 Abstract............................................................................................................................ 50 3.2 Introduction ...................................................................................................................... 51 3.3 Materials and Methods 3.3.1 Materials ....................................................................................................................... 54 3.3.2 Treatment of items used during the experiment ........................................................... 54 3.3.3 Total RNA isolation, mRNA purification and cDNA synthesis....................................... 54 3.3.4 cDNA-AFLP and analysis ............................................................................................. 55 3.3.5 Image analysis .............................................................................................................. 56 3.3.6 Cluster analysis............................................................................................................. 57 3.3.7 cDNA-AFLP selection and excision of TDFs ................................................................ 57 3.3.8 Cloning and sequencing ............................................................................................... 58 3.3.9 Assigning putative identity ............................................................................................ 58 3.3.10 Pathway analysis ........................................................................................................ 59 3.3.11 qRT-PCR .................................................................................................................... 59 3.4 Results 3.4.1 RNA extraction.............................................................................................................. 60 3.4.2 cDNA-AFLP primer screening....................................................................................... 62 3.4.3 cDNA-AFLP data analysis ............................................................................................ 63 3.4.4 Cluster analysis............................................................................................................. 77 3.4.5 Identification of biological pathways.............................................................................. 85 3.4.6 qRT-PCR analysis on biological repeat material .......................................................... 87 3.5 Discussion 3.5.1 cDNA-AFLPs as a relevant technique ........................................................................ 102 3.5.2 The wounding response.............................................................................................. 103 3.5.2.1 Wounding response and calcium signalling............................................................. 103 3.5.2.2 Wounding response and the mitochondria .............................................................. 105 IV 3.5.2.3 Wounding response and the chloroplast.................................................................. 106 3.5.2.4 Wounding response and protein anabolism or catabolism ...................................... 107 3.5.2.5 Wounding response and signalling proteins ............................................................ 108 3.5.2.6 Wounding response and the cell wall ...................................................................... 109 3.5.3 RWA infestation and the defence response................................................................ 109 3.5.3.1 Infestation response and ion flux ............................................................................. 110 3.5.3.2 Infestation response and the chloroplast ................................................................. 111 3.5.3.3 Infestation response and transporters ..................................................................... 113 3.5.3.4 Infestation response and ubiquitination ................................................................... 114 3.5.3.5 Infestation response and toxin production ............................................................... 115 3.5.3.6 Infestation response and elicitor recognition............................................................ 115 3.5.4 qRT-PCR ................................................................................................................... 116 3.5.4.1 qRT-PCR fragment 92 ............................................................................................. 116 3.5.4.2 qRT-PCR fragment 104 ........................................................................................... 117 3.5.4.3 qRT-PCR fragment 126 ........................................................................................... 117 3.5.4.4 qRT-PCR fragment 129 ........................................................................................... 118 3.5.4.5 qRT-PCR fragment 235 ........................................................................................... 118 3.5.5 Wounding vs. infestation............................................................................................. 119 3.5.6: The susceptible response .......................................................................................... 121 3.5.7 The resistance response: antibiosis vs. tolerance ...................................................... 121 3.6 References..................................................................................................................... 123 Chapter 4 Summary .......................................................................................................... 139 Appendix Appendix A .........................................................................................................................

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