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Genomics of systemic induced defense responses to insect herbivory in hybrid poplar Philippe, Ryan Nicholas 2008

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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 insects. In this study, I buiLd on this base with Large-scaLe profiling of transcriptome responses of 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 1 .2.3 Objective #3: To profile transcriptome response in damaged poplar Leaves on a 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 1.4 A NOTE ON THE SEMANTICS OF ‘DEFENSE’ 5 1.5 REFERENCES 6 2. Poplar Defense against Insect Herbivores 12 2.1 INTRODUCTION 12 2.2 INSECT PESTS IN POPLAR 14 2.3 GENERAL ASPECTS OF POPLAR DEFENSE AGAINST INSECTS 15 2.4 CHEMICAL DEFENSES IN POPLARS 18 2.4.1 Phenotic glycosides 19 2.4.2 Condensed tannins 21 2.5 BIOCHEMICAL DEFENSES IN POPLARS 21 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 2.6 VOLATILE EMISSION AND INDIRECT DEFENSE IN POPLARS 24 2.7 MOLECULAR AND GENOMIC APPROACHES TO POPLAR DEFENSE AGAINSTS INSECTS 25 2.7.1 Emerging results from new genomic research on poplar defense against insects 27 2.8 MANUSCRIPT ACKNOWLEDGEMENTS 29 2.9 REFERENCES 30 3. EST Resource and Microarray Platform Development and their use in a Preliminary Study of Poplar Transcriptome Responses to Insect Herbivory 43 3.1 INTRODUCTION 43 3.2 MATERIALS AND METHODS 46 3.2.1 Plant material and insects 46 3.2.2 cDNA libraries 47 3.2.3 Transformation and colony picking 47 3.2.4 CuLturing and DNA purification of ptasmid clones 49 3.2.5 DNA evaluation 49 3.2.6 DNA sequencing 50 3.2.7 Treatment of trees with FTC 50 111 3.2.8 Microarray fabrication and qualitycontrol.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. General Discussion 193 6.1 ON THE SYSTEMIC DEFENSE RESPONSE IN POPLAR 193 6.1.1 Brief summary of the thesis work 193 6.1.2 MultipLe signaLLing pathways involved in coordinating poplar systemic defense 194 6.2 FUTURE RESEARCH 196 6.2.1 Recommended follow-up work 196 6.2.2 GeneraL considerations for future poplar-insect interaction research 197 6.3 THESIS CONCLUSION 199 6.4 REFERENCES 200 Appendix I: Supplementary Data 202 Appendix II: PubLications 203 V List of Tables Chapter 3 TabLe 3.1 Libraries, tissue sources and species for sequences described in this study 48 TabLe 3.2 Primer sequences used for reaL-time PCR (5’ to 3’ orientation) 54 Table 3.3 PopLar EST summary 56 Table 3.4 PopLar cDNA library summary statistics 58 Table 3.5 Distribution of ESTs in muLtipLe cDNA libraries 59 Table 3.6 Selected forest tent caterpillar-responsive array eLements 67 TabLe 3.7 Selected forest tent caterpillar-responsive transcription factors 71 TabLe 3.8 FoLd-change differences measured using QRT-PCR between five trees subjected to FTC herbivory for 24hrs and five untreated controL trees 75 Chapter 4 Table 4.1 OLigonucLeotide primers used in KPI QRT-PCR experiments 97 TabLe 4.2 PopLar KPI inventory 106 Table 4.3 KPI amino acid sequence identity matrix 108 Table 4.4 KPI microarray data 124 Table 4.5 Pooled repLicate vs. individual replicate microarray data 126 Table 4.6 Fold-change differences in KPI expression measured using QRT-PCR between five trees subjected to mechanical wounding plus FTC OS after 24hrs & five untreated control trees 125 Table 4.7 Induced KPI expression by QRT-PCR 130 Chapter 5 Table 5.1 Primer sequences for QRT-PCR (5’-3’ orientation) 151 TabLe 5.2 QRT-PCR analysis of expression in treated and systemic poplar Leaves 2hrs, 6hrs, and 24hrs foLLowing wounding plus FTC OS 166 Table 5.3 Top 20 strongest up-reguLated transcripts in SSi popLar Leaves in response to mechanicaL wounding pLus FTC OS at 2hrs, 6hrs, and 24hrs, with corresponding expression levels in LSo and SS0 Leaves 169 Table 5.4 Sequence identity matrix of pLant GOLS proteins 177 Table 5.5 QRT-PCR anaLysis of induced galactinol synthases expression 180 vi List of Figures Chapter 2 Figure 2.1 Overview of defenses against insect herbivores in poplars 16 Figure 2.2 Major phenolic gLycosides found in trembling aspen (Populus tremuloides) 20 Chapter 3 Figure 3.1 ReLationship between sequence length of high-quality popLar 3’ ESTs and similarity to the best scoring matches in a variety of comparisons 60 Figure 3.2 Herbivory experiment set-up under greenhouse conditions 63 Figure 3.3 Boxplots showing the distribution of technical and biological variation 64 Figure 3.4 Experiments assessing the performance of the poplar 15.5K cDNA microarray 66 Figure 3.5 QRT-PCR analysis of gene expression in response to forest tent caterpillar herbivory 77 Chapter 4 Figure 4.1 Summary of treatment conditions for the local defense profiLing experiment 92 Figure 4.2 Summary of the hybridization design for the local defense profiling microarray experiments 93 Figure 4.3 Genome organization of the KPI family in popLar 101 Figure 4.4 Phylogenetic tree of poplar KPI sequences 113 Figure 4.5 Sequence alignment of poplar KPI genes 116 Figure 4.6 Phylogenetic tree of plant KPI sequences 121 Figure 4.7 QRT-PCR analysis of gene expression in individual trees in response to mechanical wounding plus forest tent caterpillar OS 127 Figure 4.8 QRT-PCR analysis of induced gene expression of KPI poplar genes in response to insect feeding, mechanical wounding, OS and MeJa treatment 129 Figure 4.9 QRT-PCR analysis of constitutive gene expression for twelve KPI poplar genes 132 Figure 4.10 Potentially intact poplar KPI proteins are found in the insect gut 134 Chapter 5 Figure 5.1 Treated and untreated SSo and SSi leaves were profiled in this study 147 Figure 5.2 Microarray hybridization plan to study temporaL and spatial patterns of poplar herbivore defense response 149 Figure 5.3 Insoluble CWI activity in source and sink leaves of untreated control and FTC-OS-treated poplar trees 2hrs after treatment 155 Figure 5.4 Comparison of variation in microarray element intensity between experiments involving individual replicates versus pooled replicates 157 Figure 5.5 Profiles of differentiaLly expressed (DE) genes from microarray analysis of 15,496 array eLements in poplar systemic response to FTC OS through time 160 vii Figure 5.6 Cluster anaLysis of expression profiles of genes differentialLy expressed (DE) folLowing simulated herbivory with forest tent caterpilLar OS of poplar (eaves 162 Figure 5.7 QRT-PCR analysis for validation of systemic gene expression of selected poplar genes in response to simulated herbivory 164 Figure 5.8 Local suppression of isoprene synthase expression is contrasted with a rapid burst in untreated systemic (eaves 171 Figure 5.9 Chromosome-level organization of Populus trichocarpa GOLS gene family 173 Figure 5.10 Sequence alignment of popLar and other pLant GOLS proteins 175 Figure 5.11 Phylogenetic tree of poplar and other pLant members of the GOLS gene family 176 Figure 5.12 QRT-PCR analysis of systemic gene expression of selected GOLS genes in poplar Leaves in response to simulated herbivory 179 Figure 5.13 GaLactinoL and raffinose metabolite Levels increase following simulated herbivory 181 Figure 5.14 SpecuLative model of systemic defense response in poplar sink Leaves 186 viii List of Abbreviations Ptxd - Populus trichocarpa x deltoides clone Hil -11 4CL - 4-coumarate CoA Ligase ABA - Abscisic acid ABC - ATP-binding cassette ACC - 1-aminocycLopropane-1-carboxyLate oxidase ANOVA - Analysis of variance ATP - Adenosine triphosphate BLAST - Basic Local alignment search toot BLASTN - NucLeotide BLAST TBLASTN - Translated nucLeotide database BLAST (wI protein query) BLASTP - Protein BLAST BLASTX - Translated nucleotide database BLAST (wI translated nucteotide query) bp - Base pair(s) cDNA - CompLementary DNA CDS - Coding sequence CWI - Cell-waLl invertase Ct - QRT-PCR threshoLd cycLe vaLue DE - DifferentialLy expressed; differentiaL expression DNA - DeoxyribonucLeic acid dNTP - Deoxynucleotide triphosphate dGTP - Deoxyguanine triphosphate dTMP - Deoxythymine monophosphate dTDP - Deoxythymine diphosphate dTTP - Deoxythymine triphosphate DMSO - DimethyL suLfoxide DTT - DithiothreitoL E - PCR efficiency E4P - Erythrose 4-phosphate EDTA - EthyLenediaminetetraacetic Acid EPSP - 5-enolpyruvylshikimate 3-phosphate EST - Expressed sequence tag FAC - Fatty acid-amino acid conjugate FC - FoLd-change FDR - False discovery rate FLcDNA - Full-Length complementary DNA ix FPLC - Fast protein Liquid chromatography FTC - Forest tent caterpillar (Malacosoma disstria) FW - Fresh weight GC/MS - Gas chromatography I Mass spectrometry GEO - Gene Expression Omnibus GFP - Green fluorescent protein GOLS - GatactinoL synthase (inositot 3-a-gaLactosyLtransferase; EC 2.4.1.123) GSC - Genome Sciences Centre, Vancouver, BC HPL - Hydroperoxide Lyase HPLC - High performance Liquid chromatography hq - High-quality HSP Heat shock protein(s) ISPS - Isoprene synthase JA - Jasmonic acid JGI - Joint Genome Institute KPI - Kunitz-type protease inhibitor LB - Lysogeny broth; Luria-Bertani broth LC-MS - Liquid chromatography - Mass spectrometry LC-MS/MS- Liquid chromatography - Tandem mass spectrometry LG - Linkage group (chromosome) LLCI - Lower Limit confidence intervaL LOX - Lipoxygenase LPI . Leaf pLastochron index LRR(-RLK)- Leucine-rich repeat (transmembranelreceptor-Like kinase) LSo - LocaL (Treated) source Leaves LTP - Lipid transfer proteins MeJa - MethyL jasmonate MIAME - Minimum Information About a Microarray Experiment mRNA - Messenger RNA MS - Mass spectrometry MW - MoLecuLar weight NaOH - Sodium hydroxide NCBI - NationaL Center for Biotechnology Information nr - Non-redundant OBL - Oblique-banded leaf roLler (Choristoneura roseceana) ORF - Open reading frame OS - OraL secretions x PAL - PhenyLatanine ammonia- Lyase PCR - Potymerase chain reaction PEP - PhosphenoLpyruvate P1 - Protease inhibitor PPO - Potyphenol oxidase PR - Pathogenesis-related QC - Quality controL QRT-PCR - Quantitative reaL-time PCR RFO - Raffinose family/series oligosaccharides RNA - RibonucLeic acid RNAi - RNA inhibition SAM - S-adenosylmethionine SD - Standard deviation SE - Standard error SSi - Systemic (Untreated) sink (eaves SSo - Systemic (Untreated) source leaves TAIR - The Arabidopsis Information Resource TIF5A - TransLation initiation factor 5A TIF - Tagged Image File format TIGR - The Institute for Genomic Research ULCI - Upper Limit confidence interval UTR - Untranslated region UV - Ultraviolet VOC - Volatile organic compound VSN - Variance stabiLizing normaLization VSP - Vegetative storage protein xi Acknowledgements Thanks to alL members of the BohLmann lab 2003-2008, especiaLly Dr. Carsten Kulheim, Dr. Bjoern Hamberger, and Dr. Chris KeeLing, who provided me with excellent advice and guidance. Dr. Steven Ralph (University of North Dakota Dept. of BioLogy) deserves very speciaL mention for his invaLuable mentoring and direction. I thank Ms. Sharon Jancsik for her vitaL technicaL assistance and personal support. I thank Mr. Dana AeschLiman and Mr. Rick White (University of British Columbia Dept. of Statistics) for statistical support, Mr. David Kaplan (UBC FacuLty of Land a Food Systems) for greenhouse support, Dr. Gary Judd (Pacific Agri-Food Research Centre) for access to obLique-banded leaf rolLers, and Mr. Bob McCron (Canadian Forest Service) for access to forest tent caterpillars. Thanks to Dr. Shawn MansfieLd (UBC Forestry), whose expertise and assistance with my anaLysis of soLubLe sugars is greatLy appreciated, as is the use of his experimental resources. I give thanks to NSERC for their continued funding of my post-graduate education, first through a CGS Master’s schoLarship, then with a PGS DoctoraL scholarship. I wouLd also Like to thank the UBC Botany department for their generous financiaL support during my first year. SpeciaL thanks to UBC Dept. of Botany administrators Ms. Penelope BaLakshin and Ms. Veronica Oxtoby for all their support and heLp, as well as to UBC Michael Smith Laboratories administrators Ms. Darlene Crowe, Ms. PaL Bains, and Ms. Catherine Ross. Thanks to Mr. Victor Ling (UBC MSL) for vaLuabLe technical expertise. Thanks to Dr. Rob Guy (UBC Forestry), Dr. Steven Lund (UBC Wine Research Centre), and Dr. Reinhard Jetter (UBC Chemistry & UBC Botany) for their time and commitment as members of my Ph.D. committee. You provided advice and guidance without hesitation, and our discussions of your own experiences in science were invaluable in finding a sense of direction in my research and my life. I would like to thank Dr. Jorg BohLmann (UBC Michael Smith Laboratories) for his guidance, supervision, and advice throughout this demanding yet extremely rewarding personal and professional experience. I aLso thank him for his constant drive to provide me with the best resources possible, to produce my best work possibLe. My vision and leadership potential owe much to our discussions, and perhaps more to his exampLe. I owe much of my current success to his energy, drive, and passion for science. Thank you for never losing faith in me. FinaLLy, I need to thank Dr. Kate Woods. Without her strength and support, I would tread a Lesser path. Thank you for your patience, understanding, and commitment. My Love for you is eternal. xii Vi Veri Veniversum Vivus Vici. For my parents. Thanks Mom. Thanks Dad. For Everything. xiii Co-Authorship Statement This thesis is the culmination of research from 2003 to 2008. Listed below are papers that have been published or wILL be submitted for pubLication that comprise this thesis. (Chapter 2) Philippe RN and BohLmann J (2007) PopLar Defense against Insect Herbivores. Canadian Journal of Botany, 85: 1111-1126. • The candidate conceived of and wrote the manuscript for an invited review. J. Bohlmann directed manuscript preparation and editing of final version. (Chapter 3) RaLph 5, Oddy C, Cooper D, Yueh H, Jancsik S, KoLosova N, Philippe RN, AeschLiman D, White R, Huber D, RitLand CE, Benoit F, Rigby T, Nantel A, Butterfield YSN, Kirkpatrick R, Chun E, Liu J, Palmquist D, Wynhoven B, Stott J, Yang G, Barber 5, HoLt RA, Siddiqui A, Jones SJM, Marra MA, ELLis BE, Douglas CJ, RitLand K and BohLmann J (2006) Genomics of hybrid popLar (Populus trichocarpa x deltoides) interacting with forest tent caterpilLars (Malacosoma disstria): normalized and fuLl-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar. Molecular Ecology, 15: 1275-1297. • The candidate contributed to the experiments involved with development, fabrication and quality control of 3,317-clone and 15,496-clone spotted poplar cDNA microarray platforms. The candidate was not directly involved in cDNA library generation or EST sequencing. The candidate performed the 15.5K insect-feeding microarray experiment, quantitative reaL-time PCR vaLidation, and data analysis leading to Tables 3.6, 3.7, and 3.8 and Figures 3.2, 3.3, 3.4, 3.5; and assisted with manuscript preparation. Remaining Figures and Tables were prepared by S. Ralph. (Chapter 4) Philippe RN, Ralph SG, Kulheim C, Jancsik 5, White R and Bohlmann J (2008) The Kunitz type protease inhibitor gene family in popLar demonstrates frequent expansion during evolution and widespread activation during insect defense. In preparation. • The candidate conceived of the work together with S. RaLph and J. BohLmann. The candidate performed the experiments and data analysis Leading to Figures 4.1, 4.2, 4.6, 4.7, and 4.8, and Tables 4.1, 4.2, 4.4, and 4.5. S. Ralph (with the assistance of S. Jancsik) performed the experiments and data analysis leading to Figures 4.3, 4.4, and 4.5, and Table 4.3. C. Kulheim performed the experiments and data analysis which led to Figure 4.9. Figure 4.10 and concepts explored therein were developed by the candidate. R. White provided statistical and computational expertise for analysis of the data. The candidate produced aLl of the figures and manuscript with input from S. Ralph. J. BohLmann directed research and coordinated manuscript preparation. xiv (Chapter 5) Philippe RN, Ralph SG, Jancsik S, White R, Mansfield S and BohLmann J (2008) Systemic herbivore defense in poplar involves multiple transcriptional cascades and source-sink resource allocation reorganization. In preparation. • The candidate conceived, designed and performed alt experiments described and wrote the manuscript. R. White provided statistical and computational expertise for analysis of the data. The candidate performed the sugars analysis in S. Mansfield’s lab. J. Bohtmann supervised the work and manuscript preparation. xv 1. Introduction to the Thesis Work 1.1 THESIS GOAL 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, resistance to abiotic stress and pathogens, 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. To sustain productivity and ecosystem health of naturaL and pLanted popLar forests, it is of critical importance to aLso deveLop a better understanding of the moLecuLar mechanisms of defense of poplars against insect pests. Previous research has established a solid foundation of the chemicaL ecoLogy of poplar defense against insects. This thesis seeks to buiLd on this foundation by advancing our knowLedge of the genomics of popLar-insect herbivore interactions and induced defense responses. 1.2 THESIS OBJECTIVES 1.2.1 Objective #1: To establish a comprehensive literature review of poplar-insect interactions Our current understanding of the molecular biology of poplar defense to insect pests is the result of several decades of research, incLuding such early Landmarks as the profiLing of constitutive chemicaL-based phenoLic defenses in popLar (Palo 1984) as weLL as the initial expLoration of popLar induced defense responses (Parsons et at. 1989). Since then, a weaLth of information on the chemical ecoLogy of popLar, its interactions with insect pests, and induced response to insect herbivory has been produced. Previous reviews of poplar defenses against insect herbivores have focused on the variety of insects that attack poplar (Mattson et al. 2001) or on the moLecuLar biology of induced responses (ConstabeL and Major 2005). In recent times, the information in these reviews has been expanded upon with a proLiferation of studies producing and making use of poplar genomics resources. The successfuL design of experiments that produce bioLogicaLLy reLevant resuLts requires cLear understanding of the bioLogicaL system. In order to gain a cLearer understanding of popLar-insect interactions and popLar defense responses, especially as it pertains to rapidly emerging genomics resources and techniques, a comprehensive overview of the fieLd of poplar defense to insect herbivores needed to be produced. The resuLts of the Literature review are presented in Chapter 2. 1.2.2 Objective #2: To develop, validate, and use essential poplar genomics resources This thesis research project was begun alongside, and contributed to, the popLar genome sequencing effort (Tuskan et aL. 2006). Many genomics resources have been produced for popLar, including genetic maps (Cervera et at. 2005; WooLbright et al. 2008; Yin et al. 2004; Zhang et al. 2004) and a physicaL map (Ketteher et at. 2007), Large EST resources (BhaLerao et at. 2003; Brosche et al. 1 2005; Christopher et at. 2004; Kohter et at. 2003; Lawrence et aL. 2006; Nanjo et at. 2004; Ranjan et at. 2004; Rishi et at. 2004; Schrader et at. 2004a; Sterky et at. 2004; Sterky et aL. 1998), high-quaLity fuLl length cDNA resources (Ralph et at. 2008), and platforms for transcriptome analysis (Andersson et at. 2004; Brosche et aL. 2005; Harding et at. 2005; Schrader et at. 2004b), though most of these resources were devetoped with a focus on wood formation and pLant deveLopment (Brunner and NiLsson 2004; Joshi et aL. 2004; PiLate et at. 2004). In order to study the genomic-leveL patterns of response in popLar to biotic stresses, Large and relevant sequence resources emphasizing popLar interactions with insects and pathogens needed to be developed to perform experiments on popLar defense. Results from some of these resource developments are described in Chapter 3. 1.2.3 Objective #3: To profile transcriptome response in damaged popLar leaves on a large scale Insect-induced defense responses identified by microarray transcriptome profiling have been described for a few herbaceous species, such as tobacco (Nicotiana spp.) (e.g. Hatitschke et at. 2003; Heidel and BaLdwin 2004; Hui et al. 2003; Voetckel and Baldwin 2004) and thate cress (Arabidopsis thaliana) (e.g. Cheong et aL. 2002; Ehlting et at. 2008; Reymond et at. 2004; Reymond et at. 2000). These studies have compared transcriptome responses to a variety of insect pests as well as to mechanicaL wounding. Some experiments show that most transcripts that respond to insect herbivore feeding damage - though not aLL - are also responsive to physical wounding aLone (Mithofer et at. 2005; Reymond et at. 2004; Reymond et at. 2000), white others have demonstrated significant differences between the two treatments (Mozoruk et at. 2006). Further, the addition of insect oral secretions (OS) to mechanically wounded plant tissue can result in a response profile even more similar to insect herbivory than to that generated by wounding alone (Mattiacci et at. 1995; Atborn et at. 1997; Halitschke et at. 2001, 2003; Schmelz et at. 2001). Much of our understanding of the genomics of plant-insect interactions has been informed by results from microarray studies on annual plants. In contrast, previous studies of insect-induced responses in poplar, a tong-lived woody perennial, have been focused on a small number of genes (Parsons et at. 1989), such as chitinases (CLarke et at. 1998; Davis et at. 1991a; Lawrence and Novak 2006), vegetative storage proteins (Davis et at. 1993a; Davis et at. 1993b; Lawrence et at. 2001; Lawrence et at. 1997), protease inhibitors (Davis et at. 1991b; Haruta et aL. 2001a; Lawrence and Novak 2001; Miranda et at. 2004), or poLyphenot oxidase (Constabet et at. 2000; Haruta et at. 2001b; Wang and Constabet 2003, 2004a, b), white studies examining the poplar transcriptome response to wounding, herbivory, and insect OS (Christopher et at. 2004; Lawrence et at. 2006; Major and Constabet 2006) have been limited to smaller-scale experiments profiLing a few hundred genes at once. A Large-scale microarray analysis examining transcriptome responses to insect herbivory was needed in order to generate a more comprehensive transcriptome profile of poplar response to insect herbivory. Results from Large-scale transcriptome analysis of poplar treated with insect herbivory, mechanical wounds, insect OS, and methyl jasmonate are described in Chapter 4. 2 1.2.4 Objective #4: To profile a gene family involved in defense response The sequencing and assembly of the poplar genome has reveaLed a pattern of gene family expansion in poplar relative to Arabidopsis thaliana for gene families putativeLy involved in insect defense responses (Tuskan et aL. 2006). It has been proposed that this pattern of expansion is an adaptation of Long-lived woody perenniaLs. Over its Long Lifespan, a perenniaL such as popLar is exposed to more potential insect pests than an annual pLant, and a broad array of defense mechanisms with activities against diverse insect processes wouLd seem advantageous. As outLined in Objective #3, muLtiple genes have been impLicated in poplar defense, incLuding a few members of the Kunitz-type protease inhibitor (KPI) famiLy (Bradshaw et al. 1990; Haruta et al. 2001a; Hollick and Gordon 1993; Major and Contabel 2008). KPIs are thought to inhibit digestive proteases in the insect gut and thereby negatively affect insect growth and development. P. trichocarpa is the first pLant species capabLe of producing KPI defense proteins for which a complete genome sequence is availabLe, and thus provides an excellent opportunity to explore in depth the evolution and genome organization of the KPI gene family. In order to obtain better understanding of the evolution of poplar defenses, characterization of a complete defense gene family such as KPIs from the popLar genome is required. Comparison of this KPI famiLy to the complete Arabidopsis thaUana genome is required to help uncover evoLutionary relationships. The results of a genomic characterization for a defense gene family in popLar are presented in Chapter 4. 1.2.5 Objective #5: To profile systemic transcriptome defense responses in source and sink leaves Though our knowledge of popLar defense responses in treated Leaves is expanding rapidly, our knowledge of the large-scale transcriptional responses in undamaged systemic tissues is comparatively Lacking. This is perhaps surprising, given that work by Arnold and colleagues (ArnoLd et al. 2004; Arnold and Schultz 2002) has cLearLy impLicated source-sink responses in the effective induction of chemicaL defenses in popLar. While Major and Constabel (2006) used their 580-clone macroarray to examine transcriptome responses in undamaged systemic source (SSo) leaves, a profiLe of transcriptional response in untreated systemic sink leaves (SSi) at the developing crown of a poplar tree has not yet been estabLished. In order to obtain a first glimpse of the genomic responses to insect herbivory in undamaged Leaves on a popLar tree, both in mature SSo Leaves and still developing juvenile SSi Leaves, a comprehensive profiling experiment using a 15,496-cLone cDNA microarray examining transcriptome response in both treated and untreated Leaves on a time course of 24hrs was called for. Results from work under this objective are described in Chapter 5. 3 1.3 THESIS SUMMARY The following manuscript-based thesis is divided into this brief introductory chapter, four manuscript chapters of original work, and a finaL discussion chapter. Chapter One serves to introduce the thesis dissertation and the thesis goal, and describes the thesis objectives. Chapter Two was written as a Literature review on the interactions of popLar and its insect herbivores, providing a summary of knowLedge of poplar insect pests and the constitutive and induced defenses pop(ar uses against their attacks. Included is a synopsis of recent deveLopments in popLar defense research emphasizing emerging genomics work. Chapter Three describes the development of a large poplar EST collection and cDNA microarray genomics pLatform with an emphasis on poplar stress responses, and its appLication in a large-scale profiLing study of the transcriptome responses induced in mature poplar source (eaves by insect herbivory. Results show that whiLe a Large variety of secondary metaboLism genes are invoLved in induced defense responses, even greater response is observed in genes with functions in primary metabolism. Chapter Four explores the evoLution and dynamics of a large gene family involved in popLar defense, the Kunitz-type protease inhibitors (KPIs). Their invoLvement in defense was highLighted by microarray experiments in Chapter Three, and is further extended in this chapter with profiLing of large-scale poplar transcriptome response over a time course of 2hrs to 24hrs after a variety of physicaL and elicitor treatments. KPIs are found to be constitutiveLy expressed and strongly inducible, and abLe to survive intact in the gut of lepidopteran insects. Chapter Five profiles the transcriptome responses to insect feeding in popLar that are activated in a systemic fashion, producing an induced response throughout the entire plant with unique responses observed between source and sink tissues. Responses in sugar metaboLism genes highLight the predominance of primary metabolism in these early defense responses in sink leaves. These responses add to the growing understanding in pLant biology of the mechanisms that aLlow interaction between so-called primary and secondary metabolism for effective growth and defense. Chapter Six provides a synthesis of new information from Chapters Two to Five and previous knowledge of the field, highLights the significance of this research, and presents some prospects for future work. 4 1.4 A NOTE ON THE SEMANTICS OF ‘DEFENSE’ Resistance and tolerance are two principaL forms of pLant defense against insect herbivores. PLant resistance reduces the amount or rate of herbivore damage, while tolerance serves to Lessen the negative consequences of damage. With regard to resistance traits, poplars produce Large quantities of secondary metaboLites, particuLarLy phenolic glycosides and condensed tannins. Poplar phenolic glycosides negatively impact insect performance (Hemming and Lindroth 1995, 1999, 2000; Hwang and Lindroth 1997, 1998; Lindroth et aL. 1988; Osier et at. 2000; Osier and Lindroth 2001). Fewer studies support the roLe of condensed tannins as anti-insect compounds (Ayres et at. 1997), but they do reduce the incidence of herbivory by mammals (BaiLey et al. 2004). Induced proteins such as polyphenol oxidase and protease inhibitors have aLso been implicated in poplar resistance (Major and Constabel 2008; Wang and Constabel 2004a). Poplars should be able to tolerate as weLl as resist herbivory for several reasons. First, their rapid growth rates allow for compensatory growth after damage. Second, poplars have been shown to alter carbon allocation patterns when treated with defense elicitors such as methyl jasmonate (MeJa) (Babst et at. 2005) and divert carbon resources to the stem when attacked by insect herbivores (Stevens et al. 2008). Stored resources in stems and roots could be mobilized to allow regrowth after insect damage. Finally, poplars can be subjected to nearly 100% defoliation by insect feeding, such that resistance responses in Leaves are no longer present to negatively affect the herbivores. Such conditions might promote strong selection for tolerance: Current evidence suggests that resistance and toLerance are complementary, rather than mutually exclusive, defenses in poplars (Stevens et al. 2007). In this work, I refer to all induced responses examined and profiled as ‘defense responses’. This is with the understanding that successful defense against insect herbivores requires that the plant not only induce responses aimed at affecting the insect pests (resistance), but aLso induce responses aimed at reorganizing resource acquisition and aLlocation (‘primary metabolism’) in order to produce these induced ‘secondary metabolism’ responses. Whether this reconfiguration of primary metaboLism is in itself a tolerance response, or is required for successful resistance and thus falLs under its conceptual umbrelLa, is an open question. 5 1.5 REFERENCES Andersson A, KeskftaLo J, Sjodin A, BhaLerao R, Sterky F, WisseL K, Tandre K, Aspeborg H, MoyLe R, Ohmiya Y, BhaLerao R, Brunner A, Gustafsson P, Karlsson J, Lundeberg J, NiLsson 0, Sandberg G, Strauss 5, Sundberg B, Uhlen M, Jansson S and NiLsson P (2004) A transcriptionaL timetabLe of autumn senescence. Genome Biology, 5: Article 4. ArnoLd T, Appet H, PateL V, Stocum E, Kavatier A and SchuLtz J (2004) Carbohydrate translocation determines the phenoLic content of Populus foLiage: a test of the sink-source model of pLant defense. New Phytologist, 164: 157-164. ArnoLd TM and Schultz JC (2002) Induced sink strength as a prerequisite for induced tannin biosynthesis in deveLoping leaves of Populus. Oecologia, 130: 585-593. 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Woolbright SA, DiFazio SP, Yin T, Martinsen GD, Zhang X, ALlan GJ, Whitham TG and Keim P (2008) A dense linkage map of hybrid cottonwood (Populus fremontii x P. augustifolia) contributes to Long- term ecologicaL research and comparison mapping in a model forest tree. Heredity, 100: 59-70. 10 Yin TM, DiFazio SP, Guriter LE, Riemenschneider DE and Tuskan GA (2004) Large-scaLe heterospecific segregation distortion in Populus reveaLed by a dense genetic map. Theoretical and Applied Genetics, 109: 451-463. Zhang D, Zhang Z, Yang K and Li B (2004) Genetic mapping in (Populus tomentosa x Populus bolleana) and P. tomentosa Carr. using AFLP markers. Theoretical and Applied Genetics, 108: 657-662. 11 2. Poplar Defense against Insect Herbivores1 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, resistance to abiotic stress and pathogens, motivated, at [east in part, by the fact that poplars provide an important system for large- scale, short-rotation plantation forestry in the Northern Hemisphere. To sustain productivity and ecosystem health of natural and pLanted popLar forests it is of criticaL importance to aLso 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 insects. This review summarizes some of the reLevant Literature on defense against insect herbivores in popLars with an emphasis on moLecular, biochemical, and emerging genomic research in this important fieLd within forest biotechnology and chemicaL ecoLogy. FoLLowing a generaL introduction, we provide a brief overview of some of the most relevant insect pests of popLars; we then describe some of the generaL defense strategies of poplars along with selected exampLes of their activities. We concLude with a summary of emerging results and perspectives from recent advances in genomics research on poplar defense against insects. 2.1 INTRODUCTION The genus Populus includes the poplars, aspens, and cottonwoods (coLlectiveLy referred to as popLars in this thesis, unless otherwise specified), which can be found throughout the Northern Hemisphere. Individual species can have extensive natural ranges, such as Populus tremuloides Michx. (trembling aspen), which extends through western North America from ALaska to Mexico, or Populus nigra L., which is found across Europe as far as Northern Asia. Many members of the genus Populus are fast-growing, wind-pollinated, early successionaL species that can rapidly colonize disturbed sites, often owing to the ability for asexual reproduction or production of massive numbers of seeds. Poptars are one of the most productive components of riparian ecosystems in the Northern Hemisphere, shaping the ecology of these sensitive environments (Whitham et at. 1996). Poptars also make up the Largest fraction of intensively managed hardwood forest acreage in North America (CoyLe et aL. 2005). Owing to their Long life spans, large sizes, sessiLe lifestyles, and ecoLogical dominance, popLars are subject to interactions with a wide variety of insect herbivores throughout their natural range (Whitham et al. 1996). The ability of poplars to cope over many years with a Large and dynamic community of potential insect herbivores is refLected in a diverse set of constitutive and inducibLe defenses. These defense systems involve chemical defenses (i.e., specialized metabolites also known as secondary metaboLites or natural products), biochemicaL defenses (i.e., proteins or enzymes with 1 A version of this chapter has been pubLished. Philippe RN, and Bohlmann J (2007) Poplar defense against insect herbivores. Canadian JournaL of Botany 85(12): 1111-1126 12 direct effects on the herbivore), physical defenses (i.e., protective anatomical structures), and ecological or indirect defenses (e.g., attraction of predators or parasitoids of the herbivores). Selected aspects of these defense systems wiLL be highLighted in the first part of this review chapter after a brief introduction of some of the most relevant insect pests of poplars. The genomic information, resources, and technoLogies deveLoped in concert with the sequencing and physicaL mapping of the poplar genome (Tuskan et aL. 2006; KeLLeher et aL. 2007), combined with their interesting biology, make popLars a unique system for genomic studies of interactions of Long-Lived trees with their biotic environment. Research on interactions of popLars with insect pests is advancing rapidLy as a result of the implementation of genomics approaches (e.g., Lawrence et aL. 2006; Major and Constabel 2006; RaLph et aL. 2006a; Miranda et aL. 2007). Such research is of criticaL importance for a fundamentaL understanding of the dynamic defenses in Long- lived trees and the further deveLopment and sustainability of popLar as a system for plantation forestry with applications for biomass production and carbon sequestration. There is no doubt that pLantation forestry wiLL eventually face many of the same probLems as modern agriculture with regard to pest management. However, many of the tooLs that are commonly used for pest controL in agricuLture are not available in forestry or are not suitabLe for appLication in forestry. Specifically, Large-scaLe pesticide application (owing to the negative ecologicaL impact) and rapid crop rotation on annuaL or semi-annuaL cycles (owing to the perenniaL biology of trees) are not options in forestry, even under conditions of pLantation forestry. In addition, despite the many opportunities and potentiaL benefits of genetic engineering of pest resistance in trees, forestry has faced considerable obstacLes that have so far prevented the deployment of transgenic trees in most jurisdictions (for review, see Lida et aL. 2004 and references therein). Therefore, sustainabLe management of insect pests in pLanted and naturaL forests wiLL rely on the further development of knowLedge of the naturaL defense and resistance mechanisms of forest trees and on the integration of such knowLedge across muLtipLe scaLes from the molecular leveL to the ecoLogical and Landscape LeveLs (Raffa et aL. 2005) and its appLication in tree breeding. For popLars, a Large foundation of knowLedge aLready exists with regard to their chemicaL, biochemical, and ecoLogical defenses against insects (see following sections for details and references). In addition, recent advances in forestry genomics and proteomics have substantially acceLerated the rate of discovery and functionaL identification of genes for defense and resistance against insects, as welt as the anaLysis of genome-wide patterns of gene and protein expression in response to insect herbivory in angiosperm and in gymnosperm trees (e.g., Huber et aL. 2004; KeeLing and Bohlmann 2006a; Lawrence et aL. 2006; Major and Constabel 2006; RaLph et aL. 2006a, 2006b; Lippert et at. 2007; Miranda et at. 2007). In the second part of this review, we summarize results from the recent research on defense-gene discovery and genomics of popLars interacting with insect pests, concLuding with a perspective for future research. Given the large voLume of Literature on the ecology of poplars interacting with insects, this review is not meant to be comprehensive in aLL its parts. The paper builds to some extent on the 13 foundation provided by a recent review by Constabel and Major (2005) deaLing with the molecuLar biology and biochemistry of induced defenses in popLar. Throughout the paper, we aLso reference seLected pertinent information from studies in other plant systems; however, a comprehensive comparison of plant defenses against insects in popLars with that in other species is beyond the scope of this paper. 2.2 INSECT PESTS IN POPLAR In the foLlowing section, we provide a brief overview of some the most important insect herbivores that feed on poptars. For further information, the reader is referred to book chapters and reviews on insect pests of poplar (Mattson et al. 2001; Coyte et aL. 2005). Poptars are preyed upon by a large variety of herbivorous insect pests, with at [east 300 species of insects and mites commonly found on the various species in the genus Populus in North America and aLmost doubLe that with approximateLy 525 species in Europe (Mattson et at. 2001). Amongst these numbers are a wide variety of defoLiators, shoot feeders, and stem borers, and yet onLy a few species are responsible for substantial leveLs of damage in natural forests. Even though defoliators form the Largest proportion of insect pests on poplars, most defoLiators are not considered a great threat to tree survival because poplars can tolerate the Loss of Large amounts of their leaves (Robison and Raffa 1994; Reichenbacker et al. 1996; Kosota et aL. 2001). However, widespread defoliation is known to substantialLy decrease biomass production of popLar trees (Reichenbacker et at. 1996). DefoLiators that can be responsible for substantial damage incLude the cottonwood Leaf beetLe (Chrysomela scripta Fabricius), a major insect pest of natural and pLanted poplar throughout most of North America (Mattson et aL. 2001; CoyLe et at. 2005) and the most important poplar defoLiator in the eastern USA (Burkot and Benjamin 1979). Both the Larvae and adults feed on leaves, causing growth Loss and destruction of Leaders and shoots (CaLdbeck et at. 1978; Bassman et at. 1982; Coyle et at. 2002). The forest tent caterpiLlar (FTC) (Malacosoma disstria Hübner) is another major defoLiator of poplars in North America (Prentice 1963; Stehr and Cook 1968). FTC are capabLe of causing widespread or complete defoliation during outbreaks, which recur approximateLy every 10 years and Last 2-5 years, and yet seldom cause mortaLity of trees; however, repeated exposure to FTC defoliation can result in reduced growth (Hindahl and Reeks 1960) and can make individual trees more susceptible to the impact from other forms of stress (Churchill et al. 1964). In the extreme, when repeated defoliation is combined with poor climatic conditions, large-scale dieback of poplar forests can occur (Gregory and Wargo 1986; Hogg et al. 2002). The large aspen tortrix (Choristoneura conflictana WaLker) feeds primarily on aspen forests north of the range of FTC (Mattson et al. 2001), whiLe the white-marked tussock moth (Orgyia leucostigma Smith) (Baker 1972) and the gypsy moth caterpiLLar (Lymantria dispar L.) (McManus and McIntyre 1981) feed on a range of trees including poplars. Poplar stem boring insects incLude such pests as the poplar borer (Saperda calcarata Say) (Solomon 1995), the poplar gaLl saperda (Saperda inornata Say) (Nord et aL. 1972a), the poplar branch 14 borer (Oberia schaumii LeConte) (Nord et at. 1 972b), and the poplar and wiLLow borer (Cryptorhynchus lapathi L.) (Schoene 1907; Harris and Coppel 1967). These insects lay their eggs under the bark, where the Larvae hatch and tunnel into the wood, decreasing wood quality, creating wounds for pathogen infections, and increasing the chance of wind breakage of the weakened stems. PopLar shoot feeders such as the spotted poplar aphid (Aphis maculatae Oestlund) and the cottonwood twig borer (Gypsonoma haimbachiana Kearfott) prey on the tips of growing shoots and often cause dieback of infested tips, resuLting in muLtiple Leaders, which Leads to stunted trees with maLformed stems (Mattson et al. 2001). While these insect herbivores in natural settings rarely cause Lasting devastation of poplar or aspen forests, unLess combined with other deLetenous biotic or abiotic environmental factors (Hogg et aL. 2002), damage from these pests can be responsible for widespread economic Loss in popLar plantations (HarreLl et al. 1981; Coyle et aL. 2002) and can also increase the risk of infestation by fungal pathogens (Klepzig et al. 1997). Densely packed popLar pLantations, which are often of very limited genetic diversity or even represent cLonat populations, can create the spatiaLly uniform, Low biodiversity environments amenabLe to devastating insect outbreaks (Neuvonen and Niemela 1983; NiemeLa and Neuvonen 1983; Mattson et aL. 1991; Haack and Mattson 1993). In addition to the pests mentioned above, a variety of other insects can proLiferate in the resource-rich conditions presented by high-density poplar plantations (Coyle et aL. 2005). TraditionaL techniques of chemicaL and bio rational control for crop pests have proven somewhat effective in poplar plantations (Abrahamson et aL. 1977; CoyLe et al. 2000). A detailed knowledge of the interactions of poplars with their insect pests, including a better understanding of the molecular mechanisms of these interactions, is necessary to guide pest control efforts that are compatible with the ecosystems of naturaL forests and pLantation forests. 2.3 GENERAL ASPECTS OF POPLAR DEFENSE AGAINST INSECTS Plant defenses against insect herbivores are costly (BaLdwin 1998; Mauricio 1998; Koricheva 2002; Strauss et al. 2002) and invoLve a fine balance of resource aLlocation between growth, deveLopment, reproduction, and defense. While plants that alLocate resources primarily towards growth and development may be limited in their ability to defend against insect herbivores or pathogens (Simms and Rausher 1987; Herms and Mattson 1992), constitutive deployment of defenses may cause faster-evolving insects to deveLop strategies to tolerate or overcome host defenses. The presence or absence of defenses can effectively shape communities of herbivores that can cope with various degrees of host defenses (Kessler et al. 2004; PaschoLd et al. 2007). Defense systems against insects that are multigenic and flexible may aLlow plants to cope with dynamic communities of herbivores, and muLtiLayered defenses appear to be of particuLar importance in long-lived trees that cannot escape their herbivore environments with short vegetative periods or short generation times (BohLmann 2008). In general, many plant species, including popLar, rely on combinations of a variety of 15 Sink Leaves Source to Sink Transition Source Leaves Figure 2.1: Overview of defenses against insect herbivores in poplars. 16 constitutive and induced defenses against insects to cope with the possible trade-offs between pLant growth and defense and the possible adaptation of herbivore communities to pLant defenses (Figure 2.1). Induced defenses may be Locally restricted to the site of the herbivore’s actual attack (Local defense), or they can be activated systemicaLly in distant parts of the pLant or throughout the entire plant (systemic defense). Some of the constitutive and induced defenses act directLy against the herbivore (direct defenses) or invoLve the ecological interactions with other organisms such as attraction of predators of the herbivore (indirect or muttitrophic defenses). The defenses of popLars involve chemical defenses in the form of specialized metabotites (mainly phenolics), biochemical defenses with proteins, or enzymes that directly effect the herbivore (e.g., anti-digestive proteins), and physical defenses in form of protective anatomical structures. Even in the absence of an insect infestation, poptars devote energy to a suite of physical and chemical defense systems to provide a primary Level of constitutive protection against insect pests. In addition to a group of weLl-characterized phenolic defenses (see below), poplars have some obvious physical means of protection, such as thick bark tissues, as a first barrier against stem-boring insects and leaf trichomes, which may protect against foliage feeding insects. Work with other plants has demonstrated that trichomes can be involved in insect defense as physicaL barriers to attack or by accumulating high LeveLs of toxic compounds (Wagner 1991; Mauricio 1998; Simmons and Gurr 2005). WhiLe a defensive function has yet to be demonstrated for trichomes in poptars, Leaf trichome density has been correlated to insect avoidance and mortality in the wilLow (Salix) and alder (Alnus) (Soetens et at. 1991; Gange 1995). Current research into the potential rote of trichomes in poplar defense uses activation-tagged mutant lines (Regan 2007). Unlike the combined physical and chemical protection of many conifer trees (Keeling and Bohlmann 2006a, 2006b), the bark of poplars lacks massive terpenoid oLeoresin chemicaL defenses, but may contain phenolic chemical defenses (Thamarus and Fumier 1998). Induced defenses divert resources away from primary processes such as growth and development only when chattenged by the presence of insect pests (Mattson and Palmer 1988; CLausen et at. 1989; Robison and Raffa 1997; HavitI and Raffa 1999). Induced defenses in poplars are effective in protecting the pLant from insect damage from FTC (Robison and Raffa 1997), gypsy moth larvae (HaviLt and Raffa 1999), and white-marked tussock moth (GLynn et at. 2003). The signaLs that activate Locally or systemically induced defenses against insects are not welL characterized in popLars, but based on studies with other plant systems they are likeLy to invoLve octadecanoids, ethylene, or smalL peptides (Ryan and Pearce 2003; Howe 2004; KessLer et at. 2004; SchiLmitLer and Howe 2005) and couLd potentially also involve airborne votatiles (Frost et al. 2007; Heit and SiLva Bueno 2007). The induction of local and systemic defense gene expression in poptars foLLowing external application of methyl jasmonate (MeJa) supports the notion that octadecanoids are involved in defense signalling in popLars (Havill and Raffa 1999; Constabel et at. 2000; Haruta et at. 2001a, 2001b; Arimura et at. 2004) and in the activation of induced resistance as shown with protection against gypsy moth (Havitl and Raffa 1999). Wounding or herbivore feeding of popLar Leaves also elicits systemic up-regulation of transcripts 17 for genes coding for enzymes in the octadecanoid pathway such as Lipoxygenases, attene oxide synthase, and atlene oxide cyctase (Arimura et at. 2004; Lawrence et aL. 2006; Major and ConstabeL 2006; see Chapter 3 and Chapter 5). In the context of activation of systemicaLly induced defenses, the importance of physiologicaL source-sink relationships in poplar Leaves has been highlighted. Sink strength can be induced by insect feeding or jasmonic acid treatment, which resuLts in an increase in the aLLocation and rate of resource import and effects an increase in Levels of phenoLic defense compounds (ArnoLd and SchuLtz 2002; ArnoLd et at. 2004; Babst et aL. 2005). Induced sink strength is aLso elicited by simuLated insect attack, specifically the application of FTC oral secretions to mechanicaLLy wounded poplar Leaves (see Chapter 4 and Chapter 5). Using the Leaf plastochron index (LPI) deveLoped by Larson and Isebrands (1971) as a reference system to standardize physioLogicaL characterization of popLar (eaves (the youngest Leaf with a Lamina length of 2 cm is designated LPI 0), a transition between sink status (net importer of resources) and source status (net exporter of resources) occurs in poplar Leaves between LPI 5 and LPI 7 under noninduced conditions. Based on the vascuLar architecture of phtoem connections in poptars, each leaf is serviced by three vascular bundLes, and the degree of connectivity of any given pair of two Leaves is determined by their reLative position aLong and around the stem axis (Larson 1979). Each leaf shares a direct phtoem connection with the fifth Leaf over (e.g., LPI 2 and LPI 7), which is the orthostichous Leaf found directLy above or beLow it on the stem. The fact that vascuLar connectivity, aLong with assimilate movement, pLays a key role in determining patterns of systemic induction of defenses in poplars has been demonstrated by Davis et at. (1991a), with maximum systemic up- regulation of the win3 protease inhibitor gene in the Leaves directLy connected to the treated Leaf. Poplar vascuLar architecture has aLso been Linked to induction of phenolic defenses (Arnold et at. 2004). The impact of vascuLar architecture on induction of systemic defenses is aLso observed in other plant species such as tomato (Lycopersicon esculentum MiLL) (Orians et at. 2000), tobacco (Nicotiana attenuata Torr. ex S. Wats.) (Schittko and BaLdwin 2003), and bittersweet nightshade (Solanum dulcamara L.) (Viswanathan and ThaLer 2004) and has recentLy been reviewed by Orians (2005). 2.4 CHEMICAL DEFENSES IN POPLARS Phenotics are the major cLass of specialized metabolites (traditionally referred to as secondary metabolites or naturaL products) in the Salicaceae (Palo 1984; Tsai et at. 2006). The topic of phenolics in poplar defense has been comprehensively treated by Tsai et at. (2006) in the context of a recent genome anaLysis, and the reader is referred to this paper for an annotation of the gene content and transcriptome of popLar devoted to phenotic defenses. Plant phenoLics are products of the phenyLpropanoid pathway that gives rise to thousands of known chemicaL structures incLuding simpLe phenolics, ftavonoids, stilbenes, coumarins, Lignans, and the structural poLymer, Lignin (HahLbrock and ScheeL 1989; Dixon and Paiva 1995; Boerjan et at. 2003). Phenotics with known or putative roLes in poplar defense incLude the phenoLic gtycosides, hydroxycinnamate derivatives, and condensed tannins 18 (Tsai et at. 2006). Highlighting the importance of the compLex phenytpropanoid pathway in Populus, the genomic anaLysis of genes for phenotic defenses by Tsai et at. (2006) provided evidence for expanded gene famiLies for ftavonoid metaboLism in Populus trichocarpa (Torr. & Gray.) reLative to Arabidopsis thaliana ((L.) Heynh.). As for other specialized metaboLites in popLar defense, votatiLes in the form of Low moLecuLar weight phenotics, benzene cyanide, and various mono-, sesqui- and homo terpenoids may aLso contribute to direct or indirect defense in popLars (Arimura et aL. 2004). The nitrogen-containing alkaLoids do not appear to have a roLe in popLar defense. 2.4.1 Phenolic glycosides The biochemistry of phenolic glycosides has been reviewed by Pierpoint (1994), and the genes for their biosynthesis in popLar are described in Tsai et at. (2006). Phenotic glycosides have been weLl studied in P. tremuloides and incLude four major compounds, saticin, saticortin, tremutoidin, and tremutacin (Figure 2.2), adding to a totaL of at Least 20 of such compounds identified in the Salicaceae (Tsai et aL. 2006). The amounts of these compounds found in aspen bark and foLiage are highly geneticaLly variabLe, as weLt as differentiaLLy responsive to resource avaiLabiLity and feeding damage (Osier and Lindroth 2004, 2006; Stevens and Lindroth 2005). PhenoLic gtycosides are known to deter generalist herbivores, and have been shown to negatively impact Larval growth and development in a variety of insects (Tahvanainen et at. 1985; Lindroth et at. 1988; Ctausen et at. 1989; Lindroth and Hwang 1996; Lindroth and Kinney 1998; Osier et at. 2000; Osier and Lindroth 2001; Osier and Lindroth 2004). Different phenoLic glycosides vary in bioLogicaL activity (Figure 2.2) (Lindroth et at. 1988). Upon ingestion by insects, some phenotic gtycosides may be metaboLized to more reactive products (CLausen et at. 1990) that have the potential to induce oxidative stress or bind covatentty with proteins, potentiaLly disrupting effective digestions (Fetton et at. 1992; AppeL 1993; Summers and FeLton 1994). Phenolic glycosides appear to play a rote in shaping the community of insect herbivores of aspens. For example, the performance of both FTC and gypsy moth larvae is influenced by variations in LeveLs of phenoLic gLycosides (Lindroth and Hemming 1990; Lindroth and BLoomer 1991; Lindroth and Weisbrod 1991; Hemming and Lindroth 1995; Hwang and Lindroth 1997; DonaLdson and Lindroth 2007) and differences in Levels of phenolic gtycoside can account for a greater amount of variation in performance in gypsy moth Larvae than in FTC (Hemming and Lindroth 1995; Hwang and Lindroth 1997). In contrast to gypsy moth, performance of FTC appears to be affected more by fotiar nitrogen (Hemming and Lindroth 1995; Hemming and Lindroth 1999). Phenolic gtycosides are formed as constitutive defenses in aspen. Whether or not they aLso function as induced defenses or in a delayed induced resistance has been more difficult to establish. Ctausen et at. (1989) and Lindroth and Kinney (1998) reported modest induction of phenotic gtycosides foLLowing insect feeding or artificiaL wounding, but subsequent experiments did not support an immediate induction of phenoLic glycosides (Osier and Lindroth 2001; Kao et at. 2002). NevertheLess, when Stevens and Lindroth (2005) examined aspen trees affected by insect damage at a Late time point 19 HOo H H Salicin OH HOo HO Qo Tremuloidin OH HO Qo TremuIaci: Figure 2.2: Major phenotic gLycosides found in trembLing aspen (Populus tremuloides). The four saLicyLate-derived compounds are ordered by increasing anti-insect activity (Lindroth et aL. 1988). Salicortin 20 (after 8 weeks) during the same growing season, they found that previousLy damaged trees had accumulated increased Loads of phenotic gtycosides in their Leaf tissues. With these findings in mind, it is cLear that induced defenses and their possibLe contributions to resistance in Long-Lived trees have to be tested over much Longer periods of time (i.e., over the entire growing season and ideaLly over multiple growing periods) than what is commonly done with short-Lived pLants in moLecuLar anaLyses under Laboratory conditions. 2.4.2 Condensed tannins Condensed tannins are oligomeric or polymeric fLavonoids, aLso known as proanthocyanidins, with diverse structures and ecological functions (Bavage et aL. 1997; MarLes et aL. 2003). The biosynthesis of condensed tannins and related compounds in poplars has been thoroughLy treated by Tsai et at. (2006) in the context of a genome annotation and expression analysis of the relevant genes. Condensed tannins are widespread in the pLant kingdom (Porter et at. 1986), and their LeveLs are known to be highly variabLe in different poplar species and in different genotypes of the same species (Greenaway et at. 1991, 1992), ranging from 0.5% up to 20% Leaf dry mass (Swain 1979; SaLminen et aL. 2004). The bioLogicaL activities of condensed tannins depend Largely on the nature and ratio of the flavonoid subunits and on their degree of polymerization and configuration in the polymers, as weLL as on the biochemical conditions found in different insect digestive systems (Zucker 1983; Appel 1993; Barbehenn and Martin 1994; Ayres et at. 1997). LeveLs of condensed tannins have been correLated with negative impacts on the performance of gypsy moth Larvae and FTC (Hemming and Lindroth 1995; Hwang and Lindroth 1997). In addition to functions as feeding deterrents and protein-complexing anti- nutrients, they aLso function as antimicrobial agents, protectants against ultravioLet Light, and possibLy toxins (Swain 1979; Hagerman and ButLer 1991; McALLister et at. 2005). ALong with their effects on forage quaLity and Litter digestibiLity of poplar Leaves, condensed tannins pLay a substantiaL roLe in nutrient cycLing in ecosystems dominated by popLars (SchimeL et aL. 1996; Schweitzer et aL. 2004; Madritch et aL. 2006). The formation of condensed tannins is induced by insect attack and severaL enzymes invoLved in poplar ftavonoid and condensed tannin biosynthesis are induced by wounding and herbivory (Osier and Lindroth 2001; Kao et at. 2002; Peters and ConstabeL 2002; Tsai et at. 2006). 2.5 BIOCHEMICAL DEFENSES IN POPLARS The constitutive and induced formation of chemicaL defenses invoLves the activity of many enzymes aLong the corresponding biosynthetic pathways. In addition, popLars use proteins or enzymes with direct defense activities against insect herbivores. The Kunitz protease inhibitors (KPI), endochitinases, and poLyphenot oxidases (PPO) are well-studied defense-related proteins in poplar with possible anti-digestive functions. Reduced digestion of Leaf forage owing to anti-digestive proteins may result in starvation of insect Larvae or may slow down insect deveLopment and thereby increase the time of exposure to natural enemies, which may be attracted by the simultaneous herbivore-induced 21 local or systemic emission of volatile organic compounds (VOCs). Other popLar proteins that have not yet been studied in the context of defense could aLso function directly against insects, once ingested by the herbivore. 2.5.1 Kunitz protease inhibitors Protease inhibitors (PIs) are a group of small proteins that function in herbivore defense by binding to digestive enzymes in the insect gut and inhibiting their activity. With the reduced effectiveness of protein digestion, the insect can experience a shortage of amino acids Leading to sLowed deveLopment or starvation (Broadway and Duffey 1986; Ryan 1990). KPIs are encoded by a Large gene famiLy in popLars and they are among the most strongly up-regulated defense genes in response to wounding or herbivore feeding (Bradshaw et at. 1990; Haruta et at. 2001a; Christopher et aL. 2004; Lawrence et at. 2006; Major and ConstabeL 2006; Ratph et at. 2006a; Miranda et at. 2007). WhiLe the effectiveness of KPIs against poptar pests has yet to be demonstrated, the win3-encoded poplar KPI protein, when produced in tobacco and tomato, ted to decreased Larval mass of feeding tobacco budworm (Heliothis virescens Fabricius) (Lawrence and Novak 2001). In some cases, as a resuLt of an arms race between insects and plant defenses, insects have adapted to Pis by up-regulating alternative digestive enzymes that are tess sensitive to inhibition (Jongsma et at. 1995). FolLowing earLier work in poptar that identified a smalt famiLy of five different KPI genes (Haruta et at. 2001 a; Christopher et at. 2004), our recent anaLysis of the poplar genome sequence, as wett as the avaitabLe poplar EST and fuLl Length cDNA sequences, revealed a substantialty larger gene famity of nearly 30 different KPI5 in the poplar genome (see Chapter 4). Atthough most of the poptar KPIs are up-reguLated in response to wounding or insect herbivory, their degree of induction varies as determined by quantitative reaL-time PCR (QRT-PCR). The large suite of KPIs may allow poplar trees to deal with muLtiple evolving generations of insects by providing a genetic storehouse of varied Pis. Indeed, Ingvarsson (2005a, 2005b) and Tatyzina and Ingvarsson (2006) found some evidence for rapid evolution in this gene family in poplar. 2.5.2 Endochitinases Chitinases were among the first putative defense genes identified in poplar, when win6 and win8, two distinct genes sharing about 50% amino acid sequence identity and having similarities to basic endochitinases, were identified as strongly and systemically up-regulated in popLar leaves in response to wounding (Parsons et al. 1989; Davis et at. 1991b; CLarke et at. 1998). These two genes represent a small fraction of this Large gene family, which contains seven different classes in plants (Graham and Sticklen 1994; Kasprzewska 2003). These chitin-degrading enzymes may have a variety of functions in poplar, such as invoLvement in deveLopment and growth, wound repair, nonspecific stress responses, or defense against fungal pathogens or insect pests. In addition, work by Davis et at. (2002) with inducible chitinases and chitinase-like genes in pine suggests that some of these genes may in fact 22 have functions that do not invoLve chitinase activity. Exploring the currently avaiLabLe poplar EST, full Length cDNA, and genome sequence information we found win8 represented as a single-copy gene, while winó is part of a multi-gene family (R.N. Philippe and J. Bohlmann, unpublished resuLts). Two other chitinase-like genes, both unreLated to win6 or win8, were identified in hybrid popLar. At least one of these chitinase-Like genes is wound-inducible (Christopher et at. 2004). Chitinases can function in pLant defense against pathogens (Cotlinge et at. 1993; Neuhaus 1999), and transgenic poplar expressing a fungal endochitinase has been shown to possess enhanced resistance to leaf rust pathogen (Noel et al. 2005). Induced poplar chitinases may also have a function against insect herbivores. For example, Colorado potato beetle (Leptinotarsa decemlineata Say) feeding on tomato plants producing the win6 encoded endochitinase from P. trichocarpa x deltoides Bartr. experienced slowed development (Lawrence and Novak 2006). The strong up-regulation of win6 and win8 in both local and systemic leaves of poplar saplings treated by mechanical wounding and application of FTC oral secretions support a rote in insect defense (see Chapter 3 and Chapter 5). A potential target for plant chitinases in defense against insects is the peritrophic membrane, which contains chitin and forms a protective barrier around the ingested food contents of the gut lumen (Richards and Richards 1977; Chapman 1985). 2.5.3 PoLyphenol oxidases Much of the Literature dealing with polyphenol oxidase (PPO) has previously been reviewed by Steffens et al. (1994) and more recently by Mayer (2006) and by Marusek et al. (2006). PPOs catalyze the oxidation of ortho-diphenolic compounds to quinones, and are found throughout the plant kingdom (Vaughn and Duke 1984). The quinones produced by PPOs upon tissue damage are highly reactive and rapidly cross-link proteins leading to characteristic tissue browning (Duffey and Felton 1991). A variety of physiological roles are proposed for PPOs (Steffens et al. 1994; Mayer 2006) which, in poplars, have been shown to play a role in defense against insect herbivores (Wang and Constabel 2004a). In hybrid poplar and aspen, wounding or insect herbivory of leaves induces systemic expression of PPO genes and PPO enzyme activity (ConstabeL et al. 2000; Haruta et al. 2001b). Three PPOs have previously been cloned in hybrid poplar, and two are wound-responsive in leaf, stem, or root tissues, while the third is constitutively expressed in roots (Constabel et at. 2000; Wang and Constabel 2003, 2004b). PPOs are proposed to possess anti-herbivore activity in the gut of insects, where PPO-generated quinones can cross-link proteins and amino acids during feeding, resulting in decreased absorption of amino acids (Felton et al. 1989; Felton et al. 1992). Using transgenic plants, Wang and Constabel (2004a) have shown that PPO-overexpressing poplar trees reduce larval mass gain. Given that these results were obtained with FTC egg masses stored in the laboratory at -2 °C for more than 6 months past spring hatching, the biological relevance of this possible PPO-based defense in nature remains to be determined. However, consistent with a possible anti-insect function, Wang and Constabel (2004a) also showed that PPO not only resists proteolysis in the FTC gut, but the protein is activated beyond its 23 Latent form found in Leaves (Constabel et aL. 2000), suggesting that Limited proteoLysis in the insect gut is responsibLe for activating PPO. This process may resemble the activation of tomato threonine deaminase in the midgut of Manduca sexta L., which has its regulatory domain proteoLyticaLLy cLeaved from the catalytic domain during ingestion or partiaL digestion in the insect gut (Chen et aL. 2005). Activation in the insect gut of anti-insect proteins perhaps provides a mechanism whereby the pLant protects its tissues from the catalytic activities of defensive proteins until they are ingested by the insect. 2.5.4 Other putative defense proteins arid proteins that respond to insect attack Recent proteomics work by Gregg Howe and co-workers in the tomato - Manduca sexta system has beautifulLy ilLustrated that proteins known for their role in primary pLant metabolic processes may also have direct defense activities once ingested by an insect (Chen et aL. 2005, 2007). Genome and proteome expression profiLing of popLar defense responses upon insect attack (RaLph et al. 2006a; Miranda et al. 2007; S. RaLph, D. Lippert, R. PhiLippe, and J. Bohlmann, unpublished resuLts) have identified a myriad of proteins that should be tested further for their stabiLity, immediate activity, or proteoLytic activation in the environment of an insect gut. For example, at Least some of the KPI proteins mentioned above appear to be resistant to digestion in the insect gut of some popLar herbivores (see Chapter 5). On the other hand, not every gene or protein that shows an increase in abundance upon herbivore attack has an effect on the herbivore, be it through the formation of chemical defenses or as a protein with anti-insect activity. A Large number of genes and proteins that may be annotated as defense-related based on patterns of induced abundance may be part of an overall metabolic rearrangement in the plant under biotic stress. For example, substantiaL down-regulation has been found for transcripts of photosynthetic processes in FTC attacked popLar leaves (Ralph et at. 2006a) and these changes may in turn effect up- and down-reguLation of compensatory processes. Also, earLier studies have shown a strong, wound- and insect-induced increase of win4-encoded vegetative storage protein (VSP) in poplar leaves both Locally and systemically (Parsons et at. 1989; Davis et aL. 1993), but no direct or indirect role in defense has been identified. The win4 gene is expressed at Low Levels in the growing shoot apex and increases in response to nitrogen fertilization (van CLeve and ApeL 1993; Coleman et at. 1994; Lawrence et at. 1997; Lawrence et at. 2001; Cooke and Weih 2005), in agreement with a roLe in nitrogen storage. Re-allocation of nitrogen reserves could weLl be the primary roLe of up regulation of win4 in poplar defense. 2.6 VOLATILE EMISSION AND INDIRECT DEFENSE IN POPL&RS The herbivore-induced response of poplars includes the Local and systemic formation and emission of VOCs, including mono-, sesqui-, and homo-terpenoids, simple phenoLics, and benzene cyanide (Arimura et aL. 2004). Herbivore-induced VOCs in poplar may directLy act as repettents of the 24 insect pests and (or) act indirectLy as attractants of predators and parasitoids of the insect herbivore in multitrophic ecological defense (Mondor and RoLand 1997, 1998; Havill and Raffa 2000). WhiLe VOC emissions may serve as semiochemicaL cues for indirect defense, such as in the attraction of parasitic wasps to gypsy moth-damaged poplar Leaves (Havilt and Raffa 2000), they may aLso serve as host Location cues in the attraction of insect herbivores (Kendrick and Raffa 2006). There is aLso evidence that induced airborne VOCs may act in plant-pLant signalling in poplar (Baldwin and Schultz 1983), where the eavesdropping on the defense of neighboring plants may activate or prime defenses before an insect infestation is acute. The topic of pLant-plant defense signalling with airborne voLatiles has recently been reviewed (Baldwin et aL. 2006 and references therein), and the concept has been extended to include the possibility of within-plant defense signalling mediated by VOCs (Frost et aL. 2007; HeiL and Silva Bueno 2007). Although popLars were among the first plants for which the idea of VOC defense signaling was tested, most of the research on emission of VOCs in poplars of the Last 10 or 15 years has focused on the emission of the hemiterpene isoprene for its ecophysiologicat role in abiotic stress tolerance (i.e., thermotolerance or protection against oxidative stress) (e.g., Behnke et al. 2007 and references therein). Whether the massive emission of isoprene by poplars also has an effect on defense against insects is not known. At the biochemicaL and molecular levels, Arimura et al. (2004) identified an insect-induced, rhythmic diurnal and systemic emission of VOCs from leaves of poplar saplings attacked by FTC larvae. The sesquiterpenoid (—)-germacrene D is a major component of these FTC-induced VOCs in hybrid poplar and its emission is controlled by systemic expression of the corresponding terpenoid synthase gene that was biochemically characterized (Arimura et al. 2004). The systemic induction of the terpenoid synthase gene expression proceeds in an acropetal direction from the base to the tip of young trees in a source-sink fashion, but apparently not in the opposite direction. The (—)-germacrene D synthase was also identified as one of the strongest up-reguLated genes in the systemic response of hybrid poplar upon reaL and simulated feeding by FTC as detected by microarray gene expression profiling (see Chapter 5), but is only one of more than 50 terpenoid synthase genes identified in the popLar genome (Tuskan et al. 2006). Similar to the comprehensive genome analysis of phenyLpropanoid pathway genes in poplar defense (Tsai et al. 2006), the avaiLable poplar genome resources can now afford a detaiLed anaLysis of genes of terpenoid VOC metabolism in poplar defense. 2.7 MOLECULAR AND GENOMIC APPROACHES TO POPLAR DEFENSE AGAINSTS INSECTS A series of pioneering studies in the early 1990s led by MiLton Gordon and co-workers estabLished that poplars possess a diverse suite of LocaLLy and systemicalLy wound- and insect responsive genes. These studies Led to the cloning and moLecular characterization, for example, of the KPI and endochitinase defense proteins (e.g., Parsons et al. 1989; Bradshaw et aL. 1991; Davis et aL. 1993); as welL, they provided the basis for the targeted molecuLar characterization of poplar defense against insects in the following years. Clearly, this foundation also provides much of the background for 25 ongoing genomics research of popLar defense against insects, which has been acceLerated with the sequencing, assembly and annotation, and physical mapping of the popLar genome (Tuskan et aL. 2006; KetLeher et at. 2007) and with the development of other Large-scaLe functional genomic resources such as ESTs, full-Length cDNAs, and microarrays more specificaLLy aimed at research on popLar interactions with herbivores (Ralph et aL. 2006a). Prior to the pubLication of many of the large-scaLe poplar EST, fuLL-Length cDNA, and genome sequences, Christopher et at. (2004) developed a 5’-EST database containing several hundred nonredundant genes from a cDNA library that was made from (eaves of hybrid popLar treated by mechanicaL wounding (Constabel et at. 2000). This library, enriched for wound-responsive transcripts, confirmed the induced expression of genes identified by Gordon and co-workers, whiLe aLso uncovering new genes invoLved in the popLar wound response. In a similar vein, Lawrence et at. (2006) used differential display of RNA to identify popLar genes that respond within a few hours after gypsy moth- infestation or wounding. They identified 57 insect- and wound-responsive defense genes, incLuding many genes not previousLy associated with poplar defense responses; they demonstrated wounding- induced up-regulation for transcripts of the octadecanoid pathway; and they analyzed the 5’ upstream putative promoter region of 15 wound-induced poplar genes, noting that these regions are enriched for DRE box, W box, and H box motifs. Considering that Lawrence et at. (2006) compared transcript abundance in wounded tissues and untreated control tissues from the same tree, and given that popLar trees can respond systemicaLLy to wounding, it is possible that this screening method may have favoured the discovery of LocaLLy as opposed to systemicaLLy responding genes. The first array-based gene expression anaLysis of poplar defense induced by insects was reported by Major and Constabet (2006), who used a 580-clone cDNA macroarray to profile the Leaf transcriptome response 24hrs after wounding and treatment with FTC oral secretions, both in treated and systemic tissues. As observed with gypsy moth oraL secretions (Havitl and Raffa 1999), FTC oraL secretion was found to induce a strong defense response in poplar Leaves, probably owing to the presence of elicitors that are simiLar or identicaL to the volicitin fatty acid - amino acid conjugate (ALborn et at. 1997; Major and Constabel 2006). Most of the genes responsive to oraL secretion are included in the set of wound-responsive genes, highLighting that whiLe there are differences in the magnitude of induction, the transcriptional response to the two treatments is quaLitativeLy simiLar. OveralL, Major and ConstabeL (2006) found very simiLar sets of genes in the LocaL and systemic tissues. Among the strongly induced genes, they found candidate DNA binding proteins containing the ZIM (or JAZ) motif (Major and ConstabeL 2006), which couLd be involved in transcriptionaL reguLation of the herbivore-induced and jasmonate-mediated defense response (Chini et at. 2007; Thines et at. 2007). To aLLow for the first large-scale transcriptome anaLysis of the popLar response to insect feeding, Ralph et at. (2006a) deveLoped a database of more than 139,000 high-quaLity popLar ESTs representing over 35,000 putativeLy unique transcripts from cDNA libraries incLuding herbivore-, wound-, and eLicitor-induced tissues. This resource was used to produce a 15,496 cLone (15.5 k) cDNA microarray, 26 encompassing approximately 25% of the annotated popLar genome, and to profile defense responses in Local source Leaves that had been fed upon by FTC (Ralph et al. 2006a). After 24hrs of FTC feeding, 1,191 genes were found to be up-regulated (77% of the transcriptome monitored) and 537 were down- regulated (3.6%), demonstrating a substantiaL impact of insect feeding on the popLar Leaf transcriptome. The responding transcripts were categorized by function and formed a large set of induced genes with known function in pLant defense (e.g., KPI and endochitinases), aLong with a variety of genes invoLved in defense signaLLing (octadecanoid and ethylene signaLLing), transport, secondary metaboLism, and transcriptional reguLation (Ralph et al. 2006a). Many differentially expressed poplar genes are annotated with functions in primary metabolism (many of them being down-reguLated in response to FTC feeding) with no previous function in defense ascribed to them. In addition, a diverse group of 40 different transcription factors were shown to be responsive to FTC feeding at 24hrs, including members of the zinc finger C3H type, AP2-EREBP ethylene-responsive, MYB or WRKY transcription factor famiLies (Ralph et aL. 2006a). 2.7.1 Emerging results from new genomic research on poplar defense against insects Large amounts of new data are currentLy emerging from ongoing work on the genomics and proteomics of poplar defense against insects. In the folLowing section we briefly summarize unpublished results from our Laboratory (R.N. Philippe, S. Ralph, and J. Bohlmann, unpublished resuLts, 2007). There is no doubt that more data will also arise from the work of others. In general, our current research objectives in genomics and proteomics of poplar defense against insects are to delineate the temporal and spatial patterns of insect-induced transcripts and proteins in poplar Leaves, to test which genes and proteins respond specificaLLy to herbivory, and to test induced poplar proteins for activity in insects. Our work on temporal and spatiaL patterns of expression considers the effect of herbivory on Local and systemic responses, and the effect of source-sink relationships in locaL and systemic Leaves. To test the specificity of herbivore response genes, we reLy on comparative analyses of leaves treated with FTC feeding, mechanicaL wounding, mechanical wounding combined with the appLication of FTC oral secretions, and treatment with MeJa. In addition, with regard to the specificity of the response to herbivory, we have compared the transcriptome of poplar leaves affected by FTC feeding with that of a pathogen-induced transcriptome response (Miranda et aL. 2007). Proteomic work on induced defenses is making use of iTRAQ proteome profiling and the tracking of target proteins in insect guts using multipLe-reaction-monitoring (MRM) tools. To develop temporaL and spatial profiles of transcriptional response to insect herbivory, we subjected hybrid popLar sapLings to FTC feeding, mechanical wounding, or mechanical wounding combined with the application of FTC oral secretions, and collected Leaves at 2, 6, and 24hrs after treatment. We collected the treated source leaves, the adjacent untreated source Leaves, and the developing untreated sink leaves and profiled gene expression using the 15.5 k cDNA microarray platform described in Ralph et aL. (2006a). When profiling the transcriptome response to MeJa, we 27 found substantial overlap with the response to FTC feeding, but also many additional transcripts that were not induced by FTC in our experiments. The strong response of popLar Leaves to external MeJa application compared with FTC feeding might refLect a dose-response effect, but it couLd also be due to Lack of fine-tuning of the defense response after MeJa treatment or to a possible lack of suppression of gene expression as may be caused by a real insect attack. In the response to FTC oraL secretions, a number of leucine-rich repeat (LRR) receptor kinases were strongly induced both in LocaL and systemic tissues. While LRR receptor proteins are best known for their role in pathogen recognition and disease resistance (Dangl and Jones 2001), such proteins can also be invoLved in the binding of small peptide signal moLecuLes in plants, such as the binding of systemin by the LRR receptor-Like protein SRi 60 in Lycopersicon peruvianum (L.) Miller (Scheer and Ryan 1999; Scheer and Ryan 2002), the binding of AtPepl in Arabidopsis in signalLing of an ampLified defense response against pathogen attack (Yamaguchi et aL. 2006), or the roLe of WPKI in jasmonate-mediated signaLling in corn (He et al. 2005). The poplar LRR receptor-Like proteins induced by FTC oral secretions couLd potentialLy function in the recognition of herbivore-induced defense signaLs. Our analysis of the spatial distribution of the differentiaLLy expressed transcriptome in treated local (eaves and in untreated systemic (eaves identified many of the same genes responding in the LocaL source Leaves, systemic source Leaves, and systemic sink Leaves, aLthough with different temporaL patterns in these different Locations, which became apparent when comparing the response at 24hrs after treatment. At the earLy time points (i.e., 2hrs and 6hrs), we observed rapid changes in transcript abundance associated with primary metaboLism in systemic sink tissues, which may suggest reaL(ocation of resources for defense. SpecificalLy, transcripts annotated with sugar metabolism and phLoem transport are among the most strongly up-regulated in systemic sink Leaves 2hrs after treatment, correLating with rapid changes in the sugar profiLes of sink and source leaves. Systemic source leaves, on the other hand, were slower to respond, peaking at 24hrs with a weaker reflection of the 24hrs transcriptional response observed in the treated source (eaves and the systemic sink (eaves. OveralL, the large-scale transcriptome profi(ing is reveaLing unexpected compLexities in the defense response of poplar Leaves involving a cascade of responses in source and sink transcriptome profiles that are rapidly changing over time after treatment. Beyond the expected response of previous(y known defense genes, the gene expression profiles obtained in our experiments highlighted substantial changes of a portion of the transcriptome that is most Likely associated with an induced change of resource allocation, which may be essential for long-Lived, sessite trees to tolerate periodic attack by insects. 28 2.8 MANUSCRIPT ACKNOWLEDGEMENTS The authors wouLd Like to thank two anonymous reviewers and Dr. Janice Cook for their indispensabLe comments. 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EST Resource and Microarray Platform Development and their use in a Preliminary Study of Poplar Transcriptome Responses to Insect Herbivory2 As part of a genomics strategy to characterize inducible defenses against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA Libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing popLar genome sequence and Poputus ESTs by focusing on herbivore- and eLicitor-treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normaLized or full-Length Libraries we generated 139,007 3’- or 5’-end sequenced ESTs, representing more than one-third of the ca. 385,000 pubLicLy available Populus ESTs. Clustering and assembly of 107,519 3’-end ESTs resulted in 14,451 contigs and 20,560 singLetons, altogether representing 35,011 putative unique transcripts, or potentially more than three-quarters of the predicted Ca. 45,000 genes in the poplar genome. Using this EST resource we developed a cDNA microarray containing 15,496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (FTC, Malacosoma disstria Hübner). After 24hrs of feeding, 1,191 genes were cLassified as up-regulated, compared to onLy 537 down-reguLated. Functional classification of this induced gene set reveaLed genes with roles in plant defense (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signaling (e.g. Lipoxygenase, allene oxide synthase, 1 -aminocycLopropane-1 -carboxyLate oxidase), transport (e.g. ABC proteins, catreticutin), secondary metabolism (e.g. poLyphenot oxidase, isoflavone reductase, (—)-germacrene D synthase) and transcriptional regulation [e.g. Leucine-rich repeat transmembrane kinase, severaL transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome-scale approach to characterize insect-induced defenses in a woody perennial providing a soLid platform for functional investigation of pLant-insect interactions in popLar. 3.1 INTRODUCTION The genus Populus, consisting of Ca. 40 species of poplars and aspen distributed in diverse habitats throughout the northern hemisphere, has been firmLy established as a system for genomic research of angiosperm tree biology (TayLor 2002; BhaLerao et aL. 2003; Brunner et aL. 2004). With an estimated size of 485+10Mb, the genome of Populus is only 4. 5x Larger than the Arabidopsis genome, 2 A version of this chapter has been published. Ralph S, Oddy C, Cooper D, Yueh H, Jancsik S, Kolosova N, Philippe RN, Aesch(iman D, White R, Huber D, Rittand CE, Benoit F, Rigby T, NanteL A, Butterfield YSN, Kirkpatrick R, Chun E, Liu J, PaLmquist D, Wynhoven B, Stott J, Yang G, Barber 5, HoLt RA, Siddiqui A, Jones SJM, Marra MA, Ellis BE, DougLas CJ, RitLand K and Bohlmann J (2006) Genomics of hybrid poplar (Populus trichocarpa x deltoides) interacting with forest tent caterpiLlars (Malacosoma disstria): normalized and fuLL-length cDNA Libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in popLar. Molecular Ecology 15(5): 1275-1297 43 and is roughly 40x smaLler than genomes of members of the pine family (Pinaceae), which includes many of the economically important gymnosperm tree species. The genome of a femaLe Populus trichocarpa tree (NisquaLty-1) has recentLy been shotgun sequenced to a depth of 7.5x coverage (http: / /genome.jgi-psf.org/Poptrl /Poptrl . home. htmt), with the assembly and annotation, and generation of supporting physical and genetic maps, being contributed by members of the InternationaL Poplar Genome Consortium (www.ornl.gov/ipgc/). CompLementary to complete genome sequencing, the large-scale sequencing of expressed genes permits analysis of the transcriptome of an organism. Sampling of the transcriptome can be performed using high-throughput singLe pass sequencing of cDNA libraries constructed from different tissues and developmental stages, or from plants subjected to different environmentaL conditions or stress treatments to generate expressed sequence tags (ESTs; Adams et at. 1993). The application of normalization techniques to reduce the frequency of highly expressed genes can increase the rate of gene discovery, permitting the identification of rare transcripts (Soares et al. 1994; Bonaldo et at. 1996). When our poplar EST project was initiated in the spring of 2002, other large-scale poplar EST sequencing efforts were aLready established that focused primarily on wood formation, dormancy and floral development (Sterky et at. 1998; Bhaterao et at. 2003; Schrader et at. 2004; Sterky et at. 2004). In addition, other small-scate gene discovery activities have devetoped poplar cDNA Libraries and EST sequences focusing on wood formation (Dejardin et al. 2004), root development (Kohler et at. 2003), and stress response (Christopher et al. 2004; Nanjo et at. 2004; Rishi et al. 2004). In order to maximize gene discovery both within the large-scale EST program described here, and relative to the Ca. 247,000 Populus ESTs in the public domain (May 27th, 2005 dbEST retease of GenBank) we have focused our efforts on normalized cDNA libraries and inctuded a variety of insect-induced and biotic eticitor induced tissues with the goat to comptement previous large-scate poptar EST activities. ESTs are also the starting reagents for the construction of cDNA microarrays for transcriptome profiling studies (Schena et al. 1995). A major emphasis of our program in forest heatth genomics is to generate and utilize genomics resources to investigate how tree genomes respond to attack by herbivorous insects, which is relatively poorly understood in contrast to plant responses to abiotic stress or pathogens. Insect-induced defense responses identified by microarray transcript profiting have recently been described for a few herbaceous species, such as the wild tobacco Nicotiana attenuata (Hui et at. 2003; Heidet and Baldwin 2004; Kessler and Baldwin 2004; Voelckel and Batdwin 2004), Arabidopsis thaliana (Reymond et al. 2000, 2004), and Sorghum bicolor (Zhu-Satzman et al. 2004). In contrast, insect-induced responses in poplar, which provides a unique system to study genomics of plant-insect interactions in a tong-lived woody perenniat, have onty been studied for a small number of genes. The newty devetoped poptar genomics resources now provide the first opportunity for genome-wide transcriptome anatysis of insect-induced defense systems in an angiosperm tree. 44 Forest insect pests pose a chaLLenge to the sustainabiLity of both naturaL and pLanted forests. The risk of forest insect pest epidemics, which cannot be addressed with short-term crop rotation or pesticide appLication, as is possibLe in agricuLture, is increasing with the introduction of exotic pest species and with global cLimate changes. The Larvae of several insect herbivores [forest tent caterpillar (Malacosoma disstria), gypsy moth (Lymantria dispar), aspen blotch Leafminer (PhyUonorycter tremuloidiella), large aspen tortrix (Choristoneura conflictana)] can cause extensive defoliation to stands of Populus species, particuLarLy trembling aspen (P. tremuloides), during outbreak periods. Other insects, such as the Larvae of the wiLLow weevil (Cryptorhynchus lapathi) affect stem tissues of popLar trees. Forest tent caterpillars (FTCs) are distributed throughout North America and Eurasia. Larvae hatch in earLy spring and immediately begin to feed on the Leaves of their hosts. By their finaL instar, Larvae grow to over 1,000 times their mass at hatching and consume more than 15,000 times their initiaL body weight in leaf tissue (FitzgeraLd 1995). During population outbreaks, FTCs commonLy defoLiate trees occurring over miLLions of hectares, with a density as high as 20,000 caterpilLars per tree (Stairs 1972; FitzgeraLd 1995). Defoliated trees have reduced photosynthetic capacity and may produce Less wood, but onLy in extreme cases are trees kiLled directLy due to repeated episodes of defoLiation by FTC Larvae (Gregory and Wargo 1986). However, repeated and proLonged attack by FTCs may result in an increased incidence of fungal disease and infestation by other insects (ChurchiLL et aL. 1964; Hogg et al. 2002). The first Lines of defense against insect herbivores are constitutive chemicaL and physicaL barriers; however, if these barriers are breached, active, inducible defenses are of centraL importance in reducing herbivory (AgrawaL 1998; Karban and BaLdwin 1997). In popLar trees, the new foLiage that FTCs feed upon undergoes profound physicaL and chemicaL changes that render maturing Leaves increasingLy less acceptabLe to the caterpilLars. Slow growth, and even population coLLapse, may resuLt if caterpillars faiL to synchronize their deveLopment with that of the host tree (FitzgeraLd 1995; Parry et aL. 1998). Compared to young, emerging poplar leaves, mature Leaves contain Lower water and nitrogen content, a higher content of non-nutritive fiber, possess increased toughness, and increased LeveLs of phenoLic compounds, which combined deters caterpiLLar feeding, reduces the digestibiLity of Leaf protein, and Leads to reduced caterpiLLar growth (Fitzgerald 1995). Constitutive LeveLs of phenotic compounds in aspen Leaves, incLuding phenolic glucosides such as saLicortin and tremuLacin, and to a lesser extent condensed tannins, are strongLy influenced by genotype and nutrient avaiLabiLity, and have been demonstrated to negatively impact growth and performance of FTCs and other herbivores (Hwang and Lindroth 1997; Osier and Lindroth 2001, 2004). In addition to constitutive defenses, herbivores trigger at least two types of inducibLe defense responses in poplars: direct defenses that can result in the inhibition of insect growth or deveLopment and indirect defenses consisting of voLatiLes emitted from pLants that can serve as airborne signals that deter herbivores or attract predators and parasites of herbivores. Inducible direct defenses in popLars invoLve a broad range of proteins (e.g. protease inhibitors, oxidative enzymes) and phytochemicals (e.g. 45 phenolics) (ConstabeL 1999; Huber et at. 2004). In herbaceous plant-herbivore defense systems, constitutive and induced defense mechanisms appear to be tightLy regulated, permitting economy when active defense is not required, and presenting a shifting defense profiLe when herbivores are present (Karban and BaLdwin 1997; Kessler and Baldwin 2002). It is therefore a priority to identify the signaling systems and the transcriptional and other insect-induced changes that regulate defense responses. Relatively few studies of the induced defense response have been conducted in Populus species at the moLecular leveL. To date, targeted studies have identified induced genes encoding trypsin protease inhibitors (Bradshaw et aL. 1990; Holtick and Gordon 1993; Haruta et at. 2001a), endochitinases (Parsons et aL. 1989; Davis et at. 1991), vegetative storage proteins (Davis et aL. 1993), polyphenol oxidases (ConstabeL et aL. 2000; Haruta et aL. 2001 b), dihydrofLavonoL reductase (Peters and Constabel, 2002) and genes of terpenoid metaboLism, including a sesquiterpene synthase involved in FTC-induced systemic volatiLe emissions (Arimura et al. 2004). In addition, a small-scale array consisting of 569 cDNA clones identified a set of 85 cDNAs that were differentiaLLy and systemicalLy expressed in Leaves 24 hours after appLying mechanical wounding (Christopher et al. 2004). We have recently established a program targeted at genome-wide discovery and expression profiling of insect-induced defense genes in poplar. We describe here resuLts from the deveLopment of 15 standard, normaLized and full-length cDNA (FLcDNA) libraries that were sequenced from the 5’ and 3’ ends of cDNA clones to generate 139,007 ESTs from poplar. AssembLy of high-quality (hq) 3’-end sequences has identified 35,011 putative unique transcripts. We demonstrate greatly enhanced gene discovery by focusing on normaLized, rather than standard cDNA libraries. Using this EST resource we have constructed a cDNA microarray consisting of 15,496 non-redundant ESTs, which has been applied to an initial study of the transcriptional response in poplar Leaves to feeding by FTC Larvae. 3.2 MATERIALS AND METHODS 3.2.1 Plant material and insects Popu(us trichocarpa Torr. & Gray x P. deltoides Bartr. (Salicaceae), Hi 1-11 genotype, was grown on the University of British Columbia South Campus farm. Cuttings of 30-100 cm were taken in February of 2003 from previous year shoots, placed in soiL (35% peat, 15% perlite, 50% pasteurized mineral soil, 250 gm3 OsmocoteTM 13-13-13 plus micronutrients) in two-gal. pots (Stuewe and Sons Inc.), and watered daily. Trees were maintained in a greenhouse under constant summer conditions where a constant 16/8-hour photoperiod was provided by high-pressure sodium lamps. Trees of 150 to 170 cm in height were used in experiments in August 2003. Average greenhouse temperature during the month was 23.8°C (21.3°C minimum and 28.9°C maximum), with an average relative humidity of 62.7%. Forest tent caterpillars, Malacosoma disstria Hübner (Lepidoptera: Lasiocampidae), were from the Great Lakes Forestry Centre (NRCan, SauLt Ste. Marie, Canada). FTC were reared and maintained on artificiaL diet (Addy, 1969) at 27°C, 50 to 60% reLative humidity, 16/8-hour photoperiod. 46 3.2.2 cDNA libraries For a description of pLant materiaLs used in the construction of cDNA Libraries pLease see TabLe 3.1. TotaL RNA was isoLated according to the protocol of Kotosova et at. (2004), folLowed by poLy(A) RNA purification with oLigo d(T) ceLLulose using the PoLy(A) Pure kit (Ambion), foLLowing manufacturer’s instructions. TotaL RNA was quantified and quality checked by spectrophotometer and agarose get. RNA was aLso evaluated for integrity and the presence of contaminants using reverse-transcription with Superscript II reverse transcriptase (Invitrogen) with an oligo d(T18) primer and aP32 dGTP incorporation. After removal of unincorporated nucLeotides using get fiLtration coLumns (Microspin S 300 HR coLumns, Amersham Pharmacia Biotech) the resuLting cDNA smear was resoLved using a verticaL 1% agarose aLkaLine get and visuaLized using a Storm 860 phosphorimager (Amersham Pharmacia Biotech). Standard cDNA Libraries were directionaLly constructed (5’ EcoRl and 3’ XhoI) using 5pg of poty(A) RNA and the pBtuescript II XR cDNA Library construction kit, foLLowing manufacturer’s instructions (Stratagene) with modifications. BriefLy, first strand synthesis was performed using Superscript II reverse transcriptase (Invitrogen) and an anchored oLigo d(T) primer (5’- GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTmTTTrTTrTmTrvN-3’). Size fractionation was performed on XhoI-digested cDNA immediately prior to Ligation into the vector using a 1% NuSieve GTG Low meLting point agarose get (BioWhittaker MoLecuLar Applications) and B-agarase (New England BioLabs) to isoLate cDNAs ranging from 300 bp to 5 kb. Select cDNA Libraries were normaLized to Cot = 5 by using the Soares method (Soares et at. 1994; BonaLdo et at. 1996). The average insert size of cDNA Libraries was routineLy determined by performing coLony PCR on 48 randomLy seLected bacteriaL coLonies from the amplified Library using -21M13 forward (5’-TGTAAAACGACGGCCAGT-3’) and M13 reverse (5’-CAGGAAACAGCTATGAC-3’) primers. PCR ampLicons were resoLved on 1% agarose geLs and visually compared to DNA size markers HindlII and I kb Ladder (Invitrogen). FLcDNA Libraries were constructed according to the methods of Carninci et aL. (1996), with modifications, and wilL be described in detaiL elsewhere. UnLess otherwise mentioned, aLL other reagents and soLvents were from Fischer Scientific, Sigma-ALdrich, EM Science or Invitrogen. 3.2.3 Transformation and colony picking A 1 pL aLiquot of Ligation mix from each cDNA Library was transformed by eLectroporation into 4OpL of E. coil DH1OB Ti resistant ceLls (Invitrogen). Transformed celLs were recovered using imL of SOC medium (Invitrogen) and pLated onto 22cm x 22cm agar pLates (Genetix) containing 2xYT agar and lOOpg/pL AmpiciLlin. Agar plates were incubated overnight at 37°C for 16h. BacteriaL coLonies were picked from the agar pLates and arrayed into 384-weLl microtiter pLates (Genetix) containing 6OpL of 2xYT medium + 7.5% gLycerol (made in house) using the Genetix QPIX automated colony picker (Genetix). Plates were incubated at 37°C for 1 8h then each microtiter plate was inspected for weLLs that contained no growth. 47 Table 3.1. Libraries, tissue sources and species for sequences described in this study cDNA Library Tissue/Developmental Stage Species (genotype) c d Populus trichocarpaPT-X-FL-A-1 Outer xylem (Nqualiy-1) c d P. trichocarpaPT-P-FL-A-2 Phloem and cambium ..._____ Young and mature leaves, along with green shoot P. trichocarpa .___,.._RL_..._..__.___._._.__________ Local and systemic (above region of feeding) mature leaves harvested after continuous feeding by forest P. trichocarpa x PTxDlLFLA4c tent caterpillars, Malacosoma disstria. Local tissue deltoides was collected 4,8 and 24hrs post-treatment and (Hil-il) systemic tissue 4, 12 and 48hrs posttreatmente. Local mature leaves harvested after continuous P. trichocarpa x pTXDILA5a feeding by M. disstria. Tissue was collected 2, 12 and deltoides -______ Bark (with phloem and cambium attached) harvested p1XNlBA6a after continuous feeding by willow weevil, P. trichocarpa x nigra Cryptorhynchus lapathi. Tissue was collected 2, 6 (NxM6) and 48hrspptreatmente. PT DX A 7a Outer xylem harvested bi-weekly between April and P. trichocarpa .ZZ____ .JY1.-125) Three month old sapling trees grown in aerated hydroponic media in growth chambers. Roots were P. trichocarpa x PTxD-NR-A-82 harvested from trees grown in media without