GENOMICS OF SYSTEMIC INDUCED DEFENSE RESPONSES TO INSECT HERBIVORY IN HYBRID POPLAR by RYAN NICHOLAS PHILIPPE B.Sc. (Hon.), the University of British Columbia, 2003 A THESIS SUBMITTED 1N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA September 2008 © Ryan Nicholas Philippe, 2008 Abstract The availability of a poplar (Populus trichocarpa Torr & A. Gray, bLack cottonwood) genome sequence is enabling new research approaches in angiosperm tree biology. Much of the recent genomics research in popLars has been on wood formation, growth and deveLopment, and abiotic stress tolerance, motivated, at Least in part, by the fact that popLars provide an important system for Large scale, short-rotation pLantation forestry in the Northern Hemisphere. Given their widespread distribution and long lifespan, poplar trees are threatened by a Large variety of insect herbivore pests, and must deal with their attacks with a successfuL defense response. To sustain productivity and ecosystem health of natural and planted poplar forests, it is of critical importance to develop a better understanding of the molecular mechanisms of defense and resistance of poplars against insect pests. Previous research has established a soLid foundation of the chemical ecology of poplar defense against base with Large-scaLe profiling of transcriptome responses of insects. In this study, I buiLd on this popLar trees to insect herbivory. A 15,496-clone cDNA microarray was developed and used to anaLyse transcriptome responses through time to a variety of insect, mechanicaL, and chemicaL eLicitor treatments in treated source leaves, as well as in undamaged systemic source and sink leaves of hybrid poplar (Populus trichocarpa x deltoides). Comparing mechanical wounding with insect feeding and chemical eLicitor treatment with methyl jasmonate demonstrated that qualitatively similar profiles of transcriptome response were eLicited with differences in the timing of induction. Transcriptome anaLysis in undamaged systemic (eaves of treated trees uncovered distinct early changes in primary metabolism (e.g. sugar metabolism) and general stress responses (e.g. heat shock proteins) prior to the activation of insect herbivory response genes (e.g. Kunitz-type protease inhibitors). Source-sink reLationships are maintained and strengthened by insect damage on source Leaves, emphasizing changes in resource aLlocation patterns as being important for poplar defense. OveraLl, a model of popLar defense begins to emerge where a cascade of transcriptome profiLes through space and time Lead to reorganization of metabolism for tolerance and induction of defense. 11 Table.of Contents Abstract Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations ix Acknowledgements Xii Dedication xiii Co-Authorship Statement xiv 1. Introduction to the Thesis Work 1 1.1 THESIS GOAL I 1.2 THESIS OBJECTIVES 1 1.2.1 Objective #1: To establish a comprehensive literature review of poplar-insect Interactions 1 1.2.2 Objective #2: To develop, validate, and use essential poplar genomics resources 1 response in damaged poplar Leaves on a 1 .2.3 Objective #3: To profile transcriptome large scale 2 1.2.4 Objective #4: To profile a gene family involved in defense response 3 1.2.5 Objective #5: To profile systemic transcriptome defense responses in source and sink leaves 3 1.3 THESIS SUMMARY 4 5 1.4 A NOTE ON THE SEMANTICS OF ‘DEFENSE’ 1.5 REFERENCES 6 2. Poplar Defense against Insect Herbivores 12 2.1 INTRODUCTION 12 14 2.2 INSECT PESTS IN POPLAR 15 2.3 GENERAL ASPECTS OF POPLAR DEFENSE AGAINST INSECTS 18 2.4 CHEMICAL DEFENSES IN POPLARS 2.4.1 Phenotic glycosides 19 2.4.2 Condensed tannins 21 21 2.5 BIOCHEMICAL DEFENSES IN POPLARS 2.5.1 Kunitz protease inhibitors 22 2.5.2 Endochitinases 22 2.5.3 Polyphenol oxidases 23 2.5.4 Other putative defense proteins and proteins that respond to insect attack 24 24 2.6 VOLATILE EMISSION AND INDIRECT DEFENSE IN POPLARS 25 2.7 MOLECULAR AND GENOMIC APPROACHES TO POPLAR DEFENSE AGAINSTS INSECTS 2.7.1 Emerging results from new genomic research on poplar defense against insects 27 29 2.8 MANUSCRIPT ACKNOWLEDGEMENTS 2.9 REFERENCES 30 of Poplar 3. EST Resource and Microarray Platform Development and their use in a Preliminary Study Transcriptome Responses to Insect Herbivory 43 43 3.1 INTRODUCTION 46 3.2 MATERIALS AND METHODS 3.2.1 Plant material and insects 46 47 3.2.2 cDNA libraries 3.2.3 Transformation and colony picking 47 3.2.4 CuLturing and DNA purification of ptasmid clones 49 49 3.2.5 DNA evaluation 3.2.6 DNA sequencing 50 3.2.7 Treatment of trees with FTC 50 111 3.2.8 Microarray fabrication control. and quality 51 3.2.9 Microarray hybridization and analysis 52 3.2.10 Quantitative reaL-time PCR (QRT-PCR) 54 3.3 RESULTS AND DISCUSSION 56 3.3.1 Sequencing and assembLy of popLar ESTs 56 3.3.2 Quality and complexity of cDNA libraries and gene discovery 57 3.3.3 Comparison against public Populus ESTs, the popLar genome, and Arabidopsis thaliana 59 3.3.4 DeveLopment of a popLar cDNA microarray 61 3.3.5 Microarray transcriptome profiling of FTC herbivory of popLar Leaves 62 3.3.6 Genes of unknown functions affected by FTC 65 3.3.7 Genes of general metabolism affected by FTC 65 3.3.8 Photosynthesis genes affected by FTC 68 3.3.9 Transport genes affected by FTC 68 3.3.10 Transcriptional regulation and signaLing affected by FTC 70 3.3.11 Octadecanoid and ethyLene pathway genes affected by FTC 72 3.3.12 Stress response genes affected by FTC 73 3.3.13 Secondary metabolism genes affected by FTC 73 3.3.14 Oxidative stress genes affected by FTC 75 3.3.15 Refined gene-specific expression using QRT-PCR 75 3.4 MANUSCRIPT ACKNOWLEDGEMENTS 78 3.5 REFERENCES 79 4. FLcDNA CLoning and Genome Mining of Poplar Kunitz-type Protease Inhibitors ReveaLs a Rapidly Diverging Family of Insect Gut-Resistant Proteins w/ Tissue-Specific Stress-Inducible Expression 88 4.1 INTRODUCTION 88 4.2 MATERIALS AND METHODS 90 4.2.1 Plant material, insects and rearing, and oral secretion collection 90 4.2.2 Herbivory/Wounding/OS/MeJa treatments and tissue harvest 91 4.2.3 RNA isoLation, microarray hybridization and analysis 91 4.2.4 Isolation of poplar full-Length KPI cDNA clones 95 4.2.5 Sequence and phylogenetic analyses 95 4.2.6 Quantitative real-time PCR analyses 96 4.2.7 Liquid chromatography - tandem mass spectrometry (LC-MS/MS) anaLysis of protein In insect gut contents 98 4.3 RESULTS 100 4.3.1 PopuLus trichocarpa NisqualLy-1 KPI genome anaLysis 100 4.3.2 The poplar KPI inventory 104 4.3.3 Phytogenetic analysis of poplar KPI gene family 111 4.3.4 Poplar KPI sequence alignment 115 4.3.5 Poplar KPI evolution 119 4.3.6 Microarray profiLing highlights up-regulation of KPIs in poplar defense response 123 4.3.7 AnaLysis of the response in individual trees confirms vaLidity of results obtained with pooLed replicates 125 4.3.8 Validation of microarray results and refined gene expression analysis 128 4.3.9 Constitutive KPI expression levels in different organs of popLar 131 4.3.10 KPI proteins are found in the insect gut 133 4.4 DISCUSSION 135 4.4.1 Status of the poplar genome sequence assembLy informed from genome and FLcDNA analysis of the KPI family 135 4.4.2 Poplar KPI diversity 135 4.4.3 Insect-related initial suppression of KPI genes 136 4.4.4 KPI proteins may be intact in the insect gut 137 4.5 MANUSCRIPT ACKNOWLEDGEMENTS 137 4.6 REFERENCES 138 iv 5. Induced Systemic Defense in Poplar to Simulated Herbivory InvoLves a Cascade of Transcriptional & Metabolic Responses with Changes in Source-Sink ReLationships and Resource Allocation Patterns 143 5.1 INTRODUCTION 143 5.2 MATERIALS AND METHODS 146 5.2.1 PLant materiaL and insects 146 5.2.2 CoLLection of oral secretions and treatment of trees 146 5.2.3 Invertase assay 148 5.2.4 RNA isolation 148 5.2.5 Microarray hybridization and gene expression data anaLysis 148 5.2.6 Quantitative real-time PCR (QRT-PCR) and gene expression data analysis 151 5.2.7 Isolation of poplar full-length galactinol synthase (GOLS) cDNA clones 152 5.2.8 GOLS sequence and phyLogenetic anaLysis 152 5.2.9 SoLubLe sugars and starch analyses 153 5.3 RESULTS 154 5.3.1 Invertase activity demonstrates source-sink reLationship between leaf groups 154 5.3.2 Individual trees confirm validity of results using pooLed replicates 156 5.3.3 Large-scale patterns of systemic transcriptome responses to FTC OS treatment in hybrid poplar reveal common late-response profiles across leaf types, contrasted with varying early-response profiles 158 5.34 CLustering analysis demonstrates source/sink and treated/untreated distinctions in transcriptome response to OS treatment 161 53.5 QRT-PCR validation of systemic microarray experiment 163 5.3.6 Systemic sink leaves have a unique pattern of early response to FTC OS 167 5.3.7 Isoprene synthase (ISPS) expression burst in systemic leaves contrasts suppression in wounded Leaves 170 5.3.8 PopLar gaLactinoL synthase (GOLS) gene family 172 5.3.9 Galactinol synthases form a well-conserved gene family in angiosperms 174 5.3.10 Galactinol synthase expression demonstrates source- or sink-specific induction in response to simuLated herbivory 178 5.3.11 Simulated herbivory induces galactinol a raffinose biosynthesis in popLar Leaves ...178 5.4 DISCUSSION 182 5.4.1 Simulated insect feeding on poplar results in enhanced source-sink relationships ....182 5.4.2 Expression profiLing reveals unique responses in systemic sink tissues 182 5.4.3 Isoprene synthase gene expression differs between treated local and untreated systemic Leaves 183 5.4.4 GalactinoL and galactinol synthase are involved in systemic induced insect defense 183 5.4.5 Multiple signaLs potentially involved in activation of systemic defense response in poplar 184 5.4.6 ConcLusions 185 5.5 MANUSCRIPT ACKNOWLEDGEMENTS 185 5.6 REFERENCES 187 6.