Transcriptome Analysis of Conifer Defense Against Bark Beetle- Associated Blue-Stain Fungi and White Pine Weevil

TRANSCRIPTOME ANALYSIS OF CONIFER DEFENSE AGAINST BARK BEETLE- ASSOCIATED BLUE-STAIN FUNGI AND WHITE PINE WEEVIL

NATALIA KOLOSOVA

B.Sc., Novosibirsk State University, Russia, 1997 M.Sc., Purdue University, USA, 2001

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

THE FACULTY OF GRADUATE STUDIES

(Botany)

THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)

November 2010 © Natalia Kolosova, 2010 Abstract

Conifer forests are exposed to a large number of herbivorous insect species and pathogenic fungi, some of which cause extensive epidemics and substantial losses of forest resources. Bark beetles and white pine weevil represent major threats to conifer forest health. Bark beetles vector fungal pathogens, which are involved in killing of the host trees. Conifers employ a variety of defense strategies, including anatomical, chemical and molecular defense mechanisms. Recent development of conifer genomic resources and tools including large EST databases and microarrays have allowed for large-scale analysis of conifer defense. To evaluate transcriptome response of conifer species to fungal pathogens I performed a comparative analysis of the interior spruce (Picea glauca x engelmannii) response to spruce beetle-associated pathogenic blue- stain fungus Leptographium abietinum and the lodgepole pine (Pinus contorta) response to mountain pine beetle-associated pathogenic blue-stain fungus Grosmannia clavigera using a 21,843-clone cDNA spruce microarray platform. In addition, I performed a direct comparison of the interior spruce response to inoculation with the fungus Leptographium abietinum with the response to white pine weevil (Pissodes strobi) herbivory. The microarray analyses revealed substantial changes in the transcriptomes of conifer hosts in response to fungal inoculation or insect feeding with more than a thousand genes significantly differentially expressed in each system and interaction studied. The fungus-induced transcriptomes of spruce and pine shared a large number of similarly responding transcripts with some differences in the dynamics of the induced responses. The transcriptome responses of spruce induced by fungal inoculation and weevil feeding had a large overlap and some treatment-specific trends. Among the most strongly up-regulated transcripts in all interactions were phenylpropanoid pathway transcripts, dirigent protein transcripts, laccases, chitinases and transcripts of the terpenoid pathway. Gene specific expression analysis of selected transcripts confirmed and extended the microarray analysis. Cloning and functional characterization of selected chitinases revealed the presence of chitinolytic activity in two interior spruce and one lodgepole pine class I chitinases. Chitinolytic activity in addition to the strong induction of these chitinases in response to different treatments supported their involvement in conifer defense.

ii Table of contents

Abstract ...... ii Table of contents ...... iii List of tables ...... vi List of figures...... viii List of abbreviations ...... x Conifer and fungal species used in the study ...... xiii Acknowledgements ...... xiv Dedication ...... xvi Co-authorship statement ...... xvii 1 Introduction: conifer defense against insects and pathogens ...... 1 1.1 INSECT PESTS AND FUNGAL PAHTOGENS AFFECTING CONIFER SPECIES ...... 1 1.2 CONSTITUTIVE AND INDUCED ANATOMICAL DEFENSES ...... 2 1.2.1 Conifer anatomical defense strategy ...... 2 1.2.2 Resin ducts ...... 3 1.2.3 Polyphenolic parenchyma, sclerenchyma and stone cells...... 4 1.3 CONSTITUTIVE AND INDUCED CHEMICAL DEFENSES ...... 4 1.3.1 Terpenoid resin ...... 4 1.3.2 Phenolics ...... 6 1.4 MOLECULAR MECHANISMS OF CONIFER DEFENSE RESPONSE ...... 7 1.4.1 Genes involved in the formation of oleoresin ...... 7 1.4.2 Genes involved in the production of phenolics ...... 8 1.4.3 Antimicrobial proteins in conifers ...... 11 1.5 ELICITORS OF CONIFER DEFENSE ...... 13 1.6 ACQUIRED RESISTANCE IN CONIFERS ...... 15 1.7 MICROARRAY ANALYSIS OF CONIFER DEFENSE ...... 17 1.8 THESIS OBJECTIVES ...... 19 1.9 REFERENCES ...... 21 2 Microarray gene expression profiling of interior spruce (Picea glauca x engelmannii) inoculated with Leptographium abietinum and lodgepole pine (Pinus contorta) inoculated with Grosmannia clavigera ...... 32 2.1 INTRODUCTION ...... 32 2.2 MATERIAL AND METHODS ...... 35 2.2.1 Plant material, fungal material and inoculation procedure ...... 35 2.2.2 Anatomical analysis ...... 36 2.2.3 Terpenoid extraction and analysis ...... 36 2.2.4 Microarray hybridization and gene expression data analysis ...... 38 2.2.5 Real-time PCR and gene expression data analysis ...... 41 2.3 RESULTS ...... 42 2.3.1 Induced formation of traumatic resin ducts in interior spruce and lodgepole pine inoculated with L. abietinum and G. clavigera respectively...... 42 2.3.2 Terpenoid accumulation in the bark of interior spruce and lodgepole pine inoculated with blue-stain fungi ...... 44 2.3.3 Inoculation of interior spruce and lodgepole pine with L. abietinum and G. clavigera respectively causes large changes in the transcriptome of both species ...... 46 2.3.4 Cluster analysis of differentially expressed transcripts induced by wounding or fungal inoculation in interior spruce and lodgepole pine ...... 49 2.3.5 Strongly induced transcripts in interior spruce and lodgepole pine by wounding and L. abietinum and wounding and G. clavigera respectively .... 51 iii

2.3.6 Functional categorization of interior spruce and lodgepole pine bark transcriptome response to fungal inoculation with L. abietinum and G. clavigera respectively ...... 57 2.3.7 Induction of phenylpropanoid in interior spruce and lodgepole pine ...... 61 2.3.8 Induction of terpenoid pathways in interior spruce and lodgepole pine...... 68 2.3.9 Induction of chitinases expression in response to G. clavigera and L. abietinum inoculations in interior spruce and lodgepole pine respectively...... 72 2.4 DISCUSSION ...... 75 2.5 REFERENCES ...... 85 3 Comparative analysis of the interior spruce transcriptome response to white pine weevil feeding and fungal inoculation with blue-stain fungus Leptographium abietinum ...... 92 3.1 INTRODUCTION ...... 92 3.2 MATERIAL AND METHODS ...... 94 3.2.1 Plant material and weevil treatment ...... 94 3.2.2 Microarray hybridization and gene expression data analysis ...... 95 3.2.3 Real-time quantitative PCR and gene expression data analysis of selected transcripts in weevil treated interior spruce ...... 96 3.3 RESULTS ...... 98 3.3.1 Weevil feeding induces a large transcriptome response in white spruce bark, similar to the transcriptome changes induced by blue-stain fungal inoculation and wounding ...... 98 3.3.2 Cluster analysis of interior spruce transcriptome responses to weevil feeding and fungal inoculation ...... 101 3.3.3 Comparison of transcripts most strongly induced in interior spruce by weevil feeding or fungal inoculation ...... 103 3.3.4 Comparative functional characterization of induced transcriptomes in interior spruce treated with weevil or fungal inoculation ...... 107 3.3.5 Comparative analysis of the phenylpropanoid pathway response in interior spruce induced by weevil feeding and fungal inoculation ...... 110 3.3.6 Comparative analysis of the effect of weevil feeding and fungal inoculation on terpenoid pathway transcripts in interior spruce ...... 119 3.3.7 Weevil feeding and fungal-treatment induces chitinases in interior spruce ...... 122 3.4 DISCUSSION ...... 124 3.5 REFERENCES ...... 137 4 Cloning and characterization of chitinases from interior spruce and lodgepole pine ...... 144 4.1 INTRODUCTION ...... 144 4.2 METHODS ...... 146 4.2.1 Nucleotide sequence accession numbers ...... 146 4.2.2 Lodgepole pine cDNA library construction and DNA sequencing ...... 146 4.2.3 Subcloning and sequence analysis of chitinase cDNA ...... 146 4.2.4 Heterologous expression and purification of chitinases ...... 147 4.2.5 Enzyme assay ...... 148 4.2.6 Determination of optimum pH and temperature ...... 148 4.2.7 Quantitative real-time PCR (QRT-PCR) ...... 148 4.3 RESULTS ...... 150 4.3.1 Cloning, sequencing and identification of conifer chitinases ...... 150 4.3.2 Heterologous expression and functional characterization of conifer chitinases ...... 153

4.3.3 Gene specific QRT-PCR analysis of the expression of chitinases in interior spruce inoculated with Leptographium abietinum ...... 156 4.3.4 Gene specific QRT-PCR analysis of the expression of chitinases in lodgepole pine inoculated with Grosmannia clavigera ...... 158 4.3.5 Gene specific QRT-PCR analysis of the expression of chitinases in interior spruce inoculated exposed to white pine weevil (Pissodes strobi) feeding ...... 158 4.3.6 Evaluation of antifungal activity ...... 160 4.4 DISCUSSION ...... 161 4.5 REFERENCES ...... 164 5 Concluding discussion ...... 167 5.1 OVERVIEW OF THE THESIS WORK ...... 167 5.2 TRANSCRIPTOME PROFILING OF CONIFER DEFENSE AGAINST FUNGAL PATHOGENS AND INSECTS ...... 168 5.2.1 Response of the conifer transcriptome to fungal pathogens ...... 169 5.2.2 A direct comparison of spruce response to insect feeding and fungal pathogen ...... 172 5.2.3 Biological relevance of the research methodology used to study conifer defense responses ...... 175 5.3 FUNCTIONAL CHARACTERIZATION OF CONIFER DEFENSE RELATED GENES ...... 177 5.4 FUTURE RESEARCH ...... 179 5.4.1 Suggested research directions based on the thesis work...... 179 5.4.2 General future directions...... 180 5.5 REFERENCES ...... 182 Appendix 1. Supplementary data ...... 190 Appendix 2. Evaluation of spruce microarray performance in heterologous hybridizations ...... 198 Appendix 3. Isolation of high-quality RNA from gymnosperm and angiosperm trees ...... 223

List of tables

Table 2.1: Total numbers of differentially expressed transcripts in LP induced by G. clavigera, and in IS induced by L. abietinum...... 47

Table 2.2: Most highly induced transcripts by L. abietinum compared to wounding at different time points in IS bark ...... 52

Table 2.3: Most highly induced transcripts by G. clavigera compared to wounding at different time points in LP bark ...... 54

Table 2.4: Functional annotation of differentially expressed transcripts ...... 58

Table 2.5: Expression of phenylpropanoid pathway transcripts with >5 fold change in at least one species one treatment and q<0.05 ...... 64

Table 2.6: Expression of terpenoid biosynthesis transcripts with >2 fold change in at least one species one treatment and q<0.05 ...... 69

Table 2.7: Expression of chitinase and chitinase-like transcripts present on 21.8K spruce microarray ...... 73

Table 3.1: Overall changes in gene expression induced by weevil feeding, wounding and L. abietinum inoculation in interior spruce bark...... 99

Table 3.2: Transcripts most strongly induced in interior spruce by weevil feeding ...... 104

Table 3.3: Functional annotation of differentially expressed transcripts ...... 108

Table 4.1: Primer sequences used for real-time PCR (5’ to 3’ orientation) ...... 149

Supplemental Table 2.1: Primer sequences used for real-time PCR (5’ to 3’ orientation) ...... 190

Supplemental Table 2.2: Terpenoid composition of constitutive and induced resin in outer stem tissues of interior spruce and lodgepole pine ...... 191

Supplemental Table 2.3: Transcript expression in response to fungal inoculation in lodgepole pine and interior spruce trees determined using a 21.8K cDNA microarray (CD) ...... 192

Supplemental Table 2.4: Distribution of transcripts in clusters (CD) ...... 192

Supplemental Table 2.5: Functional annotation of the spruce microarray ESTs (CD) ...... 192

Supplemental Table 2.6: Fold-change differences of selected gene expression in interior spruce and lodgepole pine measured using RT- PCR of fungal/wounding treatments and untreated controls at 6 hours, 2 days and 2 weeks post- treatment...... 193

Supplemental Table 3.1: Primer sequences used for real-time PCR (5’ to 3’ orientation) ...... 194

Supplemental Table 3.2:Transcript expression in response to weevil feeding in interior spruce trees determined using a 21.8K cDNA microarray (CD) ...... 194

Supplemental Table 3.3: Distribution of transcripts in clusters (CD) ...... 194

Supplemental Table 3.4: Fold-change differences of selected gene expression in interior spruce exposed to weevil feeding measured using qRT- PCR...... 195

Supplemental Table 4.1: Fold-change differences of chitinase expression in interior spruce and lodgepole pine measured using RT- PCR of fungal/wounding treatments and untreated controls at 6 hours, 2 days and 2 weeks post-treatment ..... 196

Supplemental Table 4.2: Fold-change differences of chitinase expression in interior spruce and lodgepole pine measured using RT- PCR of fungal/wounding treatments and untreated controls at 6 hours, 2 days and 2 weeks post- treatment...... 197

Table A2.1: Distribution of ESTs on Spruce 16.7 K microarray by cDNA libraries ...... 204

Table A2.2: Analysis of interspecies 16.7K spruce microarray performance ...... 206

Table A2.3 Correlation of log transformed signal intensity between species used in interspecies hybridizations ...... 209

Table A2.4: Correlation of sequence similarity and average signal intensity for genes with an E value equal or less than E-05 in sequence alignments with the spruce microarray ESTs ...... 211

Table A2.5: Spearman correlation of E value and microarray signal intensity (mean of four replicates) for genes that have E value equal or less than E-05 ...... 213

Table A2.6: Correlation of microarray signal intensity with matched length and sequence identity of EST sequences from tested species to spruce microarray EST sequences. WS ref. – white spruce reference (signal intensity was taken for white spruce samples that were hybridized against reported species) ...... 216 vii

List of figures

Figure 2.1 Microarray experimental design. Connections between treatments indicate samples that were hybridized on one array...... 40

Figure 2.2: Light microscopy of stem cross sections of IS treated with wounding or inoculated with Leptographium abietinum and LP treated with wounding or inoculated with Grosmannia clavigera ...... 43

Figure 2.3: Terpenoid accumulation in the bark of LP untreated, treated with wounding and inoculated with G. clavigera ...... 45

Figure 2.4: Clustering of transcripts based on the pattern of expression through the time course of 6h, 2 days (2d) and 2 weeks (2w) for white spruce and LP treated with wounding and inoculated with the respective fungus ...... 49

Figure 2.5: Expression of transcripts annotated to phenylpropanoid pathway in IS and LP untreated and treated with wounding and inoculated with corresponding fungus ...... 63

Figure 2.6 Quantitative real-time PCR analysis of DIR transcripts levels in IS ...... 66

Figure 2.7: Quantitative real-time PCR analysis of DXS transcripts levels in IS...... 72

Figure 3.1: Microarray experimental design...... 95

Figure 3.3: Expression of transcripts annotated to the phenylpropanoid pathway in interior spruce treated with weevil feeding compared to fungal inoculation...... 111

Figure 3.4: Quantitative real-time PCR analysis of DIR transcripts levels in interior spruce ...... 118

Figure 3.5: Quantitative real-time PCR analysis of DXS transcripts levels in interior spruce ...... 121

Figure 4.1: Alignment of the amino acid sequences of six interior spruce (Pge) and four lodgepole pine (Pc) chitinases ...... 151

Figure 4.2: Schematic structure of chitinases characterized in conifer species so far ...... 152

Figure 4.3: Effect of the amount of protein, pH and temperature on the activity of conifer class I chitinases ...... 155

Figure 4.4: Quantitative real-time PCR analysis of chitinase transcripts levels in interior spruce and lodgepole pine ...... 157

viii

Figure 4.5: Quantitative real-time PCR analysis of chitinase transcripts levels in interior spruce and lodgepole pine ...... 159

Figure A2.1: Spruce microarray performance in interspecies hybridizations. Boxplot of signal intensity (mean of four replicates)...... 207

Figure A2.2: Comparison of the microarray signal intensity distribution among different species...... 208

Figure A2.3: Correlation of the distribution of E values and 16.7K spruce microarray signal intensity (mean of four replicates) for genes with E value

Figure A2.4: Microarray performance correlated with the sequence identity of BLAST aligned sequences (E value < E-05) to spruce sequences present on the 16.7K spruce microarray ...... 214

List of abbreviations

3GT - flavonol 3-O-glucosyltransferase 4CL - 4-coumarate-CoA ligase AACT - acetoacetyl-CoA thiolase ACC - 1-aminocyclopropane -1 carboxylic acid AD - arogenate dehydratase AMP - antimicrobial peptide ANS - anthocyanidin synthase AOS - allene oxide synthase BLAST - basic local alignment search toot BLASTn - nucleotide BLAST BLASTX - translated nucleotide database BLAST (with translated nucleotide query) C3H - p-coumarate-3-hydroxylase C4H - cinnamate-4-hydroxylase CAD - cinnamyl alcohol dehydrogenase CCoAOMT - caffeoyl-CoA 3-O-methyltransferase CCR - cinnamoyl-CoA reductase cDNA - complementary DNA CHI - chalcone isomerase CHS - chalcone synthase STS - stilbene synthase CM - chorismate mutase COMT - caffeic acid O-methyltransferase CYP - cytochrome P450 DAHPS - 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase DFR - dihydroflavonol 4-reductase/leucoanthocyanidin reductase DHQD-SD - 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase DIR - dirigent protein DMAPP - dimethylallyl diphosphate DNA - deoxyribonucleic acid DXR - 1-deoxyxylulose 5-phosphate reductoisomerase DXS - 1-deoxyxylulose 5-phosphate synthase EDTA - ethylenediaminetetraacetic acid EPSPS - 5-enolpyruvylshikimate 3-phosphate synthase EST - expressed sequence tag x F3H - flavanone 3-hydroxylase F3'H - flavonoid 3'-hydroxylase F5H - ferulate-5-hydroxylase FC - fold-change FID - flame ionization detector FLcDNA - full-length complementary DNA FLS - flavonol synthase FPP - farnesyl diphosphate FPPS - farnesyl diphosphate synthase GC - gas chromatography GGPP - geranylgeranyl diphosphate GGPPS - geranylgeranyl diphosphate synthase; GO - gene ontology GPP - geranyl diphosphate GPPS - geranyl diphosphate synthase HCT - hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase HDR - hydroxymethylbutenyl 4-phosphate reductase HMGR - 3-hydroxy-3-methylglutaryl-CoA reductase HMGS - 3-hydroxy-3-methylglutaryl-CoA synthase IPP - isopentenyl diphosphate IPPI - isopentenyl diphosphate: dimethylallyl diphosphate isomerase LAR - leucoanthocyanidin reductase LMCO - laccase multicopper oxidase lytB - 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase MBP - maltose binding protein MeJa - methyl jasmonate MEP - methylerythritol phosphate MEV - mevalonic acid MIAME - minimum information about a microarray experiment MK - mevalonate kinase MPDC - mevalonate diphosphate decarboxylase mRNA - messenger RNA MS - mass spectrometry NCBI - National Center for Biotechnology Information OPR - oxophytodienoate reductases PAL - phenylalanine ammonia-lyase xi PCBER - phenylcoumaran benzylic ester reductase PCR - potymerase chain reaction PLR - pinoresinol-lariciresinol reductase PR - pathogenesis related QRT-PCR - quantitative real-time PCR RNA - ribonucleic acid SE - standard error TAIR - The Arabidopsis Information Resource TIF - tagged image file format TIF5A - translation initiation factor 5A TIGR - The Institute for Genomic Research TPS - terpene synthases UBC - The University of British Columbia

xii Conifer and fungal species used in the study

Binomial Abbreviation Common name Previously used names nomenclature Conifer species Picea glauca Pge, IS interior spruce, - (Moench) Voss x hybrid white spruce engelmannii (Parry ex Engelmann) Pinus contorta Pc, LP lodgepole pine - (Douglas ex Louden)

Fungal species Leptographium L. abietinum - Leptographium abietinum (Peck) Engelmannii, M.J. Wingfield Periconia abietina, Sporocybe abietina, Verticicladiella abietina Grosmannia G. clavigera - Ceratocystis clavigerum, clavigera (Robinson- Ophiostoma clavigerum, Jeffrey & Davidson) Ceratocystis clavigera, Zipfel, de Beer & Europhium clavigerum Wingfield)

xiii Acknowledgements

I owe much gratitude to all members of Bohlmann laboratory for their support in my academic pursuits and for being a great team to be a part of, to work and have fun together. Especially I would like to thank my academic supervisor Dr. Joerg Bohlmann for giving me an opportunity to pursue my PhD studies in his laboratory. I appreciate very much his leadership and academic support throughout my degree. His constructive vision and encouragement helped me to navigate through periods of uncertainty and challenges in my studies. I am especially thankful to Joerg for providing numerous opportunities for my academic development, including participation in conferences, workshops and guest lecturing, which helped and inspired me in pursuing my career interests. Among our lab members I especially want thank Dr. Dawn Hall for her incredible support in helping me to complete my work and for critical reading of my thesis and providing a lot of helpful comments and encouragement. I am also extremely grateful to Dr. Chris Keeling for sharing his expertise and advice, which were very helpful in my scientific projects and professional development. A lot of gratitude is due to Dr. Jeanne Robert whose positive attitude and balanced approach to life lightened up my work and writing process and helped me to have a healthier lifestyle. I would like to thank Britta Hamberger, Lina Madilao, Mack Yuen and Rick White for their technical expertise, advice and help in my work. Special thanks are due to Tristan Gillan and Dr. Alfonso Lara Quesada for their help in taking care of greenhouse trees and insect material. I am thankful too, for moral support of Kyeema Burns, who motivated me to keep going and inspired me with her energy and work ethics. I am also very grateful for all the support of our laboratory manager Karen Reid, for her incredible organizing skills, which made things run very smoothly in the lab, and her genuine care for all lab members. I am very grateful to my committee members Dr. Lacey Samuels, Dr. Carl Douglas and Dr. Xin Li for their support, comments and suggestions for my research and thesis writing. I am also thankful to our collaborator Dr. Colette Breuil and former lab member Dr. Steven Ralph for their advice and research support. Among my university colleagues I would also like to thank Dr. Carol Ritland and Dr. Sunita Chowrira for their great moral support throughout my studies. Teaching and participation in workshops with them have been a bright highlight in my PhD. In addition, I owe my deepest gratitude to Dr. Dennis Danielson for sharing his academic experience with me. By his example he inspired me to see adventurous side xiv of academic pursuits and encouraged me to value sense of meaning, wholeness and harmony in my academic life. Discussions with Dennis renewed my appreciation for beauty and wonder in scientific discovery - the very things that played an important role in my decision to pursue studies in biology. A special gratitude is due to Dr. Olav Slaymaker and Dr. Philip Hill whose advice and support reassured me at different stages of my PhD and provided me with a wiser more wholesome perspective on my studies. A very special mention is due to my friends and fellow UBC PhD students who were my team and my family away from home. I thank Katie Calloway, Sharon Smith, Tim Came and Shinjiro Sueda for being there for me and for sharing this journey together. Finally I want to thank my family, my mom, my brother Michael and his family for always loving me and supporting me in everything I do.

Dedication

“Et lux in tenebris lucet…” Ioannes 1:5 Dedicated to my Dad.

xvi Co-authorship statement

(Chapter 2) Kolosova N, White R, Ralph S, Breuil C and Bohlmann J. Conifer defense against blue-stain fungi: microarray gene expression profiling of interior spruce inoculated with Leptographium abietinum and lodgepole pine inoculated with Grosmannia clavigera. In preparation. The experimental design was developed by the author together with J Bohlmann and R White. The research, most of the data analysis (except the statistical analysis) and manuscript preparation was done by the author. R White performed statistical analysis of the data and provided computational expertise. S Ralph provided microarray analysis expertise, primers for the real time PCR analysis of selected genes and methods section for microarray hybridization conditions. C Breuil provided fungal strains used in the research and expertise in fungus handling and application. J. Bohlmann supervised the work and manuscript preparation.

(Chapter 3) Kolosova N, White R, Ralph S, Breuil C and Bohlmann J. Comparative analysis of interior spruce transcriptome response to white pine weevil feeding and fungal inoculation with blue-stain fungus Leptographium abietinum. In preparation. The experimental design was developed by the author together with J Bohlmann and R White. The research, most of the data analysis (except the statistical analysis) and manuscript preparation was done by the author. R White performed statistical analysis of the data and provided computational expertise. S Ralph provided microarray analysis expertise and primers for the real time PCR analysis of selected genes. C Breuil provided fungal strains used in the research and expertise in fungus handling and application. J. Bohlmann supervised the work and manuscript preparation.

(Chapter 4) Kolosova N, Breuil C and Bohlmann J. Cloning and characterization of a set of novel chitinases from interior spruce and lodgepole pine. In preparation. The research for the paper was designed, performed and analyzed by the author. R White performed statistical analysis of the data. C Breuil provided fungal strains used in the research and expertise in fungus handling and application. Manuscript preparation was done by the author. J. Bohlmann supervised the work and manuscript preparation.

xvii 1 Introduction: conifer defense against insects and pathogens

1.1 INSECT PESTS AND FUNGAL PAHTOGENS AFFECTING CONIFER SPECIES

Conifer trees (order Coniferales) are long lived plants that successfully populate large areas with diverse climate and growth conditions. The Pinaceae family includes some of the most economically important conifer species such as pine species (Pinus spp.) and spruce species (Picea spp.). Their long life span and extreme diversity of growth conditions exposes conifer trees to a large number of herbivorous insect species and pathogenic fungi. For example, a number of specialized insects such as bark beetles, weevils and budworms are causing substantial losses to both natural and plantation forests, in particular during outbreaks that affect vast forest areas. Bark beetles have caused large disturbances in conifer forests of North America in the last 10 years. The current mountain pine beetle (Dendroctonus ponderosae) epidemic has affected more than 13 million ha of western Canada’s forests and is spreading further east by breaching geoclimatic barriers and affecting new species of pine, potentially endangering the transcontinental boreal forests (Raffa et al., 2008). Bark beetles can kill healthy and weakened tress by mass attack, followed by tunneling in phloem, cambium and outer sapwood and the assistance of vectored pathogenic blue-stain fungi that are ubiquitous associates of bark beetles (Paine et al., 1997). Among weevils, one of the most destructive species is the white pine weevil (Pissodes strobi) that causes extensive damage to spruce and pine species in forests of North America (Alfaro et al., 2002). Weevils cause most of the damage to conifer trees during the developing larvae stage, during which larvae feeds on the inside of the tree apical shoot and destroys it. Among conifer defoliators, the western spruce budworm (Choristoneura occidentalis) is one of the most destructive herbivore in North American forests affecting mostly Douglas-fir and spruce species (Alfaro et al., 1982; Nealis et al., 2009). Repeated defoliation of buds and young shoots over several years results in tree deformity and a reduction in growth. In addition to insect attacks, conifers are exposed to a large array of pathogenic fungi. Among fungi transferred by bark-beetles, some of the most pathogenic and widely spread are the ophistomatoid fungi such as the mountain pine beetle-associate Grosmannia clavigera that can rapidly colonize the phloem and sapwood, eventually killing the tree if inoculated at a high enough dosage (Yamaoka et al., 1995). Among fungi that infect conifer species independently, Heterobasidion annosum is one of the 1 highly destructive pathogens that can affect most conifer species and cause major forestry losses (Asiegbu et al., 2005a). Heterobasidion annosum infects tree roots and spreads up into the stem, which ultimately results in the tree withering and death. Another threat to conifer forests is pitch canker disease caused by Fusarium circinatum that infects pine species throughout the world (Gordon, 2006) Conifers are often attacked simultaneously by insect and fungal pathogens. Association of blue-stain fungi with bark beetles provides the fungi with the opportunity not only to move from tree to tree, but also to breach conifer tree defense barriers such as thick bark tissues that protect conifers from pathogen invasion. In return, the blue- stain fungi assist beetles in colonizing the tree. Pathogenic blue-stain fungi weaken tree defenses, cause tree dehydration, and accelerate tree death (Paine et al., 1997). The presence of blue-stain fungi is necessary for successful reproduction of the mountain pine beetle (Six and Paine, 1998). In addition to mutualistic insect-fungi attacks, chronic infections by root pathogenic fungi predisposes weakened trees to bark beetle attacks, which may lead to an eruption of bark beetle populations and consequently endanger extensive areas of healthy trees (Raffa et al., 2008). Despite exposure to a large variety of herbivores and pathogens, conifer trees are resistant to most attacks due to effective defense mechanisms that protect them from insect and pathogen invasion. These defenses include the production of chemicals such as terpenoids and phenolics that can be toxic to beetles and fungi, the formation of special anatomical structures to store and transport these chemicals, and the activation of pathogenesis-related genes (Franceschi et al., 2005; Keeling and Bohlmann, 2006a; Bohlmann, 2008).

1.2 CONSTITUTIVE AND INDUCED ANATOMICAL DEFENSES

1.2.1 Conifer anatomical defense strategy The primary defensive structures of conifers include the outer periderm, which consists of mostly dead lignified and suberized cells, and serves as a first barrier to insect and fungal pathogen penetration. The periderm is followed by the cortex and secondary phloem, which contain a variety of defense related structures including sclerenchyma, consisting of lignified cells, calcium oxalate crystals, which provide mechanical defense; polyphenolic parenchyma, which may be involved in the production and storage of phenolics; and resin ducts, blisters or resin cells, which store terpenoids. Constitutive resin ducts in the conifer xylem also contribute to defense (Franceschi et al., 2005). 2 Upon wounding, fungal infection or insect attack, conifer trees respond with lesion formation, which is commonly referred to as the hypersensitive response involving cell death and the accumulation of constitutive and induced phenolics and terpenoids in the affected areas (Lieutier, 2002). The hypersensitive response and the release of toxic chemicals may restrict and possibly kill invading insects and fungal pathogens. Formation of the wound periderm localizes the damage. The induced activation of polyphenolic parenchyma and traumatic resin ducts, which are formed in response to the attacks, further enhance conifer defense capacity against the current threat and additional attacks (Franceschi et al., 2005).

1.2.2 Resin ducts Resin ducts play an important role in the defense of conifers. In the Pinaceae, species of the genus Pinus and Picea have well-developed, constitutive resin duct systems in the bark and, in some cases, in the xylem. Axial resin ducts in the bark and xylem are connected by radial resin ducts forming a three-dimensional resin network (Franceschi et al., 2005). Once disrupted by wounding or insect damage, resin ducts release resin stored under pressure that may repel insects and pathogens. In response to wounding, pathogen invasion, herbivore attack or the defense elicitor methyl jasmonate, the formation of new resin ducts is observed in the xylem of several members of the Pinaceae, including species of Pinus, Picea, Larix and Pseudotsuga (Franceschi et al., 2000; Martin et al., 2002; Hudgins et al., 2003, 2004; Huber et al., 2005; Miller et al., 2005; Zulak and Bohlmann, 2010). Induced formation of traumatic resin ducts has been studied in much detail in Norway spruce. Traumatic resin ducts in Norway spruce (Picea abies) xylem are formed in response to mechanical wounding (Nagy et al., 2000), inoculation with blue-stain fungus (Franceschi et al., 2000), inoculation with root rot fungus (Krekling et al., 2004), and in response to treatment with methyl jasmonate (Franceschi et al., 2002; Martin et al., 2002). Formation of traumatic resin ducts was also reported for wound-induced and fungus-inoculated Austrian pine (Pinus nigra) (Luchi et al., 2005), and for fungus-infected western white pine (Pinus monticola) (Hudgins et al., 2005). It was shown that wounding and fungal infection leads to the formation of traumatic resin ducts in spruce and pine, not only at the site of infection but to some degree also systemically (Christiansen et al., 1999; Nagy et al., 2000; Krekling et al., 2004; Luchi et al., 2005). Traumatic resin ducts contribute to increased resin production and the newly formed resin can be different in chemical composition compared to constitutive resin (Martin et al., 2002; Faldt et al., 2003; Miller et al., 2005, Zulak et al., 2009). 3 The number and density of constitutive cortical resin ducts in white spruce (Picea glauca) bark was positively associated with resistance to white pine weevil (Alfaro et al., 1997) and the traumatic resin ducts induced by white pine weevil attack contribute additional resin which can lead to mortality of weevil eggs and larvae (Alfaro, 1995).

1.2.3 Polyphenolic parenchyma, sclerenchyma and stone cells Many conifer species possess phloem polyphenolic parenchyma (PP) cells that are localized in the bark and are likely to be involved in the production of phenolics (Franceschi et al., 1998). Wounding, fungal infection and methyl jasmonate induce the formation of additional layers of PP cells (Franceschi et al., 2000; Franceschi et al., 2002; Krokene et al., 2003; Hudgins et al., 2004; Krekling et al., 2004). Comparison of two clones of Norway spruce selected for their resistance to blue-stain fungi demonstrated a correlation between resistance to blue-stain fungi and the density of phenolic parenchyma cells (Franceschi et al., 1998). Norway spruce pre-treated with sub-lethal doses of blue-stain fungus provided convincing evidence for a role of traumatic resin ducts and phenolic parenchyma in conifer resistance. This pretreatment induced the formation of traumatic resin ducts and swelling and proliferation of polyphenolic parenchyma cells. Trees pretreated from three to nine weeks before fungal mass inoculation were 60 to 90 percent more resistant than untreated trees (Krokene et al., 2003). In addition to polyphenolic parenchyma, phenolics such as lignin participate in the formation of sclerenchyma, which consists of cells with lignified cell walls (sclereids, stone cells) and contributes to defense due to its mechanical strength. Sclereids may either occur in continuous stretches or individually (Franceschi et al., 2005). Analysis of phenolics present in individual stone cells revealed that, in addition to lignin, these cells also accumulate low-molecular weight phenolics such as stilbenes and flavonoids (Li et al., 2007). This observation suggests that stone cells may be involved in chemical defense, in addition to providing physical strength. In spruce bark, increased sclereid cell density is associated with higher resistance to weevil (King and Alfaro, 2009).

1.3 CONSTITUTIVE AND INDUCED CHEMICAL DEFENSES

1.3.1 Terpenoid resin An important group of chemicals involved in conifer defense are terpenoid-based resins. Constitutive terpenoid resin production is considered to be one of the most important defenses of conifers against the initial invasion of pests and fungi (Paine et al., 4 1997; Langenheim, 2003; Raffa et al., 2005). When bark integrity is breached by wounding or insect invasion, resin, which is usually contained under pressure, flushes out or traps invading insects or pathogens and seals the wound. Conifer resin contains few abundant terpenoids and many additional terpenoids of small or trace quantities. The mixture of terpenoids includes approximately equal amount of monoterpenes (C10) and diterpenes (C20) and small quantities of sesquiterpenes (C15). Resin analysis revealed the presence of over thirty different identified terpenoids (with higher numbers of identified monoterpenoids) in spruce species (Martin et al., 2002; Miller et al., 2005; Zeneli et al., 2006) and similar variety of terpenoids was detected in other conifer species (Huber et al., 2005; Manninen et al., 2002). Monoterpenes and sesquiterpenes are volatile compounds that evaporate from resin exposed at wound sites, whereas the less volatile diterpenes accumulate and crystallize, ultimately forming a solid wound seal (Phillips and Croteau, 1999; Trapp and Croteau, 2001; Huber et al., 2004; Keeling and Bohlmann, 2006a). Several oleoresin terpenoids have been shown to have antibacterial properties (Himejima et al., 1992), antifungal properties, including toxicity against blue-stain fungi (Delorme and Lieutier, 1990; Paine and Hanlon, 1994; Kopper et al., 2005) and toxicity and repellent properties towards bark beetles (Paine et al., 1997) and weevils (Nordlander, 1990; Tomlin et al., 1996; Alfaro et al., 2002).

Induced terpenoid defenses The production of resin in a number of conifer species is induced by wounding, insect damage, pathogen infection or treatment with methyl jasmonate (Keeling and Bohlmann, 2006a). Induced resin can have a different terpenoid composition, as compared to constitutive resin (Martin et al., 2002; Miller et al., 2005; Zulak et al., 2009). A higher ratio of monoterpenoids to diterpenoids in induced terpenoid resin is observed in response to wounding, which simulates weevil damage, making resin less viscous and likely more effective in flooding the wound site and killing weevil eggs and larva (Tomlin et al., 2000). Bark beetle-associated blue-stain fungus induced a nearly one hundred-fold increase in total mono- and sesquiterpene levels at the inoculation site in Norway spruce (Viiri et al., 2001). The blue-stain fungus Grosmannia clavigera induced the formation of monoterpenes and diterpenes in two-year-old lodgepole pine (Pinus contorta) saplings (Croteau et al., 1987) and monoterpene formation in mature (about 80 years old) lodgepole pines (Miller et al., 1986). Induced resin production in Norway spruce was correlated with resistance to blue-stain fungus Ceratocystis polonica (Zeneli et al., 2006). The importance of induced terpenoid resin production against bark beetle 5 attack was demonstrated in a study which correlated induced resin production in lodgepole pine inoculated with Grosmannia clavigera with the survival of these trees during mass mountain pine beetle attack (Raffa and Berryman, 1982)

1.3.2 Phenolics Phenolics, including stilbenes, lignans, flavonoids, proanthocyanidins and tannins, are abundant in the bark of conifers (Pan and Lundgren, 1996; Viiri et al., 2001; Franceschi et al., 2005). Stilbenes and flavonoids were shown to have an antifeedant effect on bark beetles (Faccoli and Schlyter, 2007) and antifungal properties (Woodward and Pearce, 1988; Evensen et al., 2000; Celimene et al., 2001; Vargas-Arispuro et al., 2005). In particular, the stilbene resveratrol inhibited the growth of beetle-associated ophistomatoid fungi (Salle et al., 2005). A higher constitutive concentration of certain phenolics such as taxifolin glycoside and tannins in conifer bark was associated with conifer resistance to fungal pathogens (Brignolas et al., 1995; Bois and Lieutier, 1997; Brignolas et al., 1998). In addition to soluble phenolics, lignin is considered to be one of the key defense-related constituents of tree bark (Pearce, 1996). Increased lignin amount in the bark was associated with spruce resistance to weevils (King and Alfaro, 2009), bark beetles and beetle-associated fungi (Wainhouse et al., 1990; Wainhouse et al., 1997; Wainhouse et al., 1998). The antifungal effect of lignin against bark beetle- associated blue-stain fungi was also confirmed in vitro with inhibition studies of fungal growth (Bonello et al., 2003).

Induced phenolics Wounding and fungal infection induces the production of phenylpropanoids in conifer trees (Franceschi et al., 2005). Norway spruce trees that were wounded and inoculated with the blue-stain fungus had higher concentrations of (+)-catechin, the flavonoid taxifolin, and the stilbene trans-resveratrol when compared to controls (Evensen et al., 2000). Fungal infection induced the accumulation of higher quantities of phenolics than wounding alone. Infection of Austrian pine with Sphaeropsis sapinea, a fungus that causes shoot blight and cankers, induced the production of soluble and cell- wall bound phenolics, including increased lignin deposition (Bonello and Blodgett, 2003). The ability to accumulate phenolics in response to wounding and fungal inoculation, including the accumulation of flavonoids in Norway spruce, was correlated with fungal resistance in conifer species (Brignolas et al., 1995; Brignolas et al., 1998), the accumulation of stilbenes and flavonoids in Scots pine (Pinus sylvestris) (Bois and

6 Lieutier, 1997) and the accumulation of stilbenes and lignin in Austrian pine (Wallis et al., 2008).

1.4 MOLECULAR MECHANISMS OF CONIFER DEFENSE RESPONSE

1.4.1 Genes involved in the formation of oleoresin The current knowledge of molecular mechanisms involved in the formation of oleoresin terpenoids have been described in recent reviews (Keeling and Bohlmann, 2006a, 2006b; Zulak and Bohlmann, 2010). In brief, terpenoids are synthesized from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are used by prenyltransferases to form geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP). GPP, FPP and GGPP are the substrates for monoterpene synthases, sesquiterpene synthases and diterpene synthases, respectively (Keeling and Bohlmann, 2006a). IPP and DMAPP are produced through the mevalonic acid (MEV) and methylerythritol phosphate (MEP) pathways. The MEP pathway is thought to be the primary route for biosynthesis of monoterpenoids and diterpenoids of conifer resin (Cordoba et al., 2009). Several conifer genes involved in the MEP pathway were recently characterized from Norway spruce (Phillips et al., 2007) and Japanese Red Pine (Pinus densiflora) (Kim et al., 2009) including members of the 1-deoxyxylulose 5- phosphate synthase (DXS), 1-deoxyxylulose 5-phosphate reductoisomerase (DXR) and hydroxymethylbutenyl 4-phosphate reductase (HDR) gene families. Increased expression of MEP pathway genes was associated with increased resin production in conifers (Phillips et al., 2007; Kim et al., 2009; Zulak et al., 2009). IPP and DMAPP are the substrates for the prenyltransferases GPP synthases, FPP synthases and GGPP synthases. Several prenyltransferases have been characterized in conifers (Keeling and Bohlmann, 2006a; Schmidt and Gershenzon, 2007, 2008), including a bifunctional GGPP synthase that produces GGPP and GPP (Schmidt et al., 2009). Terpene synthases (TPS) use DMAPP, GPP, FPP and GGPP as substrates and are represented by large gene families in angiosperms and gymnosperms. TPSs have been functionally characterized in a few conifer species including at least twenty different monoterpene synthases, nine sesquiterpene synthases and eleven diterpene synthases, with the majority of the enzymes being characterized from grand fir (Abies grandis), Norway spruce, loblolly pine (Pinus taeda) and Taxus species (Keeling and Bohlmann, 2006a, 2006b). The functional characterization of the TPS revealed the 7 ability of many terpene synthases to produce multiple products and demonstrated that TPS play a central role in generating the structural diversity of terpenoids in conifer defense. It was shown that the expression of TPS genes is upregulated in response to wounding, insect attack and methyl jasmonate treatment (Keeling and Bohlmann, 2006a). Wounding of grand fir induced the expression of all three classes of terpene synthases (Steele et al., 1998). Monoterpene synthases were induced within two hours of wounding but the induction of sesqui- and diterpene synthases was only detectable three days after wounding. Additionally, wounding and weevil attack of Sitka spruce resulted in increased transcript levels of monoterpene synthases (McKay et al., 2003; Miller et al., 2005). The level of transcript induction was comparable between wounding and weevil attack, although insect attack resulted in a more rapid transcript upregulation. A detailed study of the effect of weevil attack on Sitka spruce revealed a slower induction of diterpene synthases as compared to the induction of monoterpene synthases, with only weak induction of sesquiterpene synthases (Miller et al., 2005). Blue-stain fungal inoculation also resulted in elevated enzyme activity of monoterpene and diterpene synthases in lodgepole pine (Croteau et al., 1987). Methyl jasmonate treatment induced gene expression and enzyme activities of mono-, sesqui-, and diterpene synthases in Sitka spruce and Norway spruce (Martin et al., 2002; Martin et al., 2003; Miller et al., 2005; Zulak et al., 2009). Increased transcript and protein levels of terpene synthases correlates well with the increased production of resin terpenoids in conifers (Zulak et al., 2009). The consistent and strong defense-related inducibility of terpene synthase expression and correlated increased resin production supports a central role of these enzymes in conifer defense. In conifers, diterpenes are oxidized by cytochrome P450 enzymes (Ro et al., 2005; Hamberger and Bohlmann, 2006; Ro and Bohlmann, 2006). Two of the cytochrome P450 enzyme involved in oxidation of selected resin diterpenoids, were functionally characterized: PtCYP720B1 in loblolly pine (Ro et al., 2005) and SsCYP720B4 in Sitka spruce (Hamberger and Bohlmann, 2006). Expression CYP720B1 was induced by methyl jasmonate treatment, supporting the involvement of this gene in induced resin production (Ro et al., 2005).

1.4.2 Genes involved in the production of phenolics A large variety of phenylpropanoids are synthesized from phenylalanine through a complex grid of biosynthetic pathways. Genes involved in the production of lignin, flavonoids and other phenylpropanoid phytoalexins have been characterized in 8 numerous angiosperm species (Dixon et al., 2002; Boerjan et al., 2003). Several genes involved in the phenylpropanoid pathway of conifers and their involvement in defense mechanisms have also been characterized.

The first step of the phenylpropanoid biosynthesis: phenylalanine ammonia lyase The first committed step in phenylpropanoid biosynthesis is the conversion of phenylalanine to cinnamic acid by phenylalanine ammonia lyase (PAL). PAL was characterized in loblolly pine (Whetten and Sederoff, 1992), and a family of PAL genes has been characterized in jack pine, Pinus banksiana (Butland et al., 1998). Expression of PAL genes was upregulated in jack pine cell cultures treated with a fungal elicitor extracted from the ectomycorrhizal fungus, Thelephora terrestris (Butland et al., 1998). In Scots pine seedlings, the expression of PAL transcripts was upregulated by endophytic and pathogenic Rhizoctonia fungi with higher levels of PAL transcript being induced by the pathogenic fungi two days post-inoculation (Gronberg et al., 2009). In Norway spruce, PAL protein was localized to phenolic parenchyma cells, supporting a role in the biosynthesis of defense-associated phenolics (Franceschi et al., 1998).

Flavonoid biosynthesis: chalcone synthase Chalcone synthase catalyses the first pathway-specific step in flavonoid biosynthesis. Chalcone synthase mRNA accumulated in white spruce (Picea glauca) needles treated with wounding both locally and systemically (Richard et al., 2000). In another study, an increase in chalcone synthase transcript level was correlated with resistance of Norway spruce clones to blue-stain fungi. Chalcone synthase transcript levels peaked several days earlier in a Norway spruce clone that had higher resistance to blue-stain fungus as compared to the less resistant clone (Nagy et al., 2004). A rapid induction of chalcone synthase expression in Norway spruce correlated with the increased resistance to bark beetle-associated blue-stain fungus suggests that the increased activation of flavonoid biosynthesis confers improved resistance of conifers to fungal pathogens (Brignolas et al., 1995; Nagy et al., 2004).

Stilbene biosynthesis One of the most common stilbenes in conifers is pinosylvin. Accumulation of pinosylvin and an increase of pinosylvin synthase activity were observed in seedlings of Scots pine treated with the fungus Botrytis cinerea (Gehlert et al., 1990). Several stilbene synthases were cloned (Fliegmann et al., 1992; Schwekendiek et al., 1992) and the multigene family of elicitor-responsive stilbene synthases involved in pinosylvin 9 production has been characterized in Scots pine (Preisig-Muller et al., 1999). Analysis of Scots pine pinosylvin synthase promoters using a tobacco system revealed inducibility in response to fungal treatment (Preisig-Muller et al., 1999). Other studies of pinosylvin synthase and pinosylvin methyltransferase revealed that expression of these genes is upregulated in wounded and fungus-inoculated Scots pine trees (Chiron et al., 2000). Inoculation of Scots pine seedlings with endophytic and pathogenic Rhizoctonia fungi species resulted in increased stilbene synthase transcript levels, with increased induction occurring two days after inoculation with pathogenic fungus suggesting a defense related role for the gene (Gronberg et al., 2009). Biochemical characterization of three stilbene synthases and a chalcone synthase from Japanese red pine, Pinus densiflora, indicated the potential interaction of stilbenoid and flavonoid biosynthesis through inhibition of chalcone synthase by pinosylvin (Kodan et al., 2002).

Lignans and lignin Lignans and lignin are synthesized from monolignols which undergo oxidation and random or directed radical coupling. Lignin is mainly synthesized from p-coumaryl, coniferyl and sinapyl alcohols. These alcohols are incorporated into hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin, respectively (Boerjan et al., 2003). Gymnosperm lignins are mostly composed of G units with a minor amount of H units. S units are considered to be unique to angiosperms, although S lignin was detected in few gymnosperm species as an exception (Weng and Chapple, 2010). Laccases and peroxidases are involved in the production of the phenoxy radicals (Boerjan et al., 2003). Laccases are represented in conifers by large gene families (Sato et al., 2001; Ralph et al., 2006a; Koutaniemi et al., 2007) and laccase gene expression was induced in the bark of Sitka spruce by wounding, weevil feeding and budworm herbivory (Ralph et al., 2006a). Peroxidases are also present in conifers as multigene families (Koutaniemi et al., 2007) and likely have multiple roles in lignification and the defense related production of reactive oxygen species (Passardi et al., 2005). A conifer peroxidase was first cloned from Norway spruce (Fossdal et al., 2001), and the activity of this peroxidase was upregulated in Norway spruce infected with the blue-stain fungus, Ceratocystis polonica. The speed of peroxidase activation was correlated with increased resistance to fungal inoculation (Nagy et al., 2004). Two peroxidase transcripts, which were associated with lignification, were induced in Norway spruce bark by Heterobasidion annosum (Koutaniemi et al., 2007) and similarly, peroxidase transcript levels were induced in the roots of Scots pine infected with Heterobasidion annosum (Adomas et al., 2007). 10 Dirigent proteins are involved in the directed stereospecific coupling involved in the biosynthesis of lignans and perhaps lignin as well (Davin et al., 1997; Davin and Lewis, 2000; Boerjan et al., 2003). The involvement of dirigent proteins in lignin biosynthesis has yet to be established (Hatfield and Wilfred, 2001) as it was suggested based on indirect evidence, including linkage specificity in lignin and localization of dirigent proteins in secondary cell wall (Davin and Lewis, 2000). A large family of dirigent proteins has been recently characterized in Sitka spruce (Ralph et al., 2006b; Ralph et al., 2007a). Several dirigent protein genes were induced by wounding, weevil herbivory and budworm herbivory in Sitka spruce (Ralph et al., 2006b; Ralph et al., 2007a).

1.4.3 Antimicrobial proteins in conifers

In addition to plant specialized metabolites (e.g. terpenes, phenolics, alkaloids) that serve as chemical barriers and toxins to pathogens, plants produce a large number of antimicrobial proteins that may have additional roles in defense (De Lucca et al., 2005). Many of these proteins are also classified as pathogenesis-related (PR) proteins based on their induction by pathogen attack and their lack of constitutive expression. Seventeen families of PR proteins have been described in angiosperm plants (Van Loon and Van Strien, 1999; Sels et al., 2008). Several antimicrobial proteins have been characterized in conifer species, including chitinases (Wu et al., 1997; Hietala et al., 2004; Liu et al., 2005), -1,3-glucanases (Sharma et al., 1993; Asiegbu et al., 1995), peroxidases (Fossdal et al., 2001; Adomas et al., 2007), defensins (Sharma and Lonneborg, 1996; Kovalyova and Gout, 2008), thaumatin-like proteins (Piggott et al., 2004), PR-10 proteins (Liu et al., 2003) and antimicrobial peptides (Asiegbu et al., 2003; Asiegbu et al., 2005b). Chitinases are able to digest chitin and degrade the fungal cell wall, resulting in the direct inhibition of fungal growth, and the release of fungal cell wall elicitors that contribute to the induction of plant defense (Collinge et al., 1993). In addition to antifungal properties, chitinases can affect insects by damaging the chitin containing peritrophic matrix (Kramer and Muthukrishnan, 1997). Chitinases may also play a role in programmed cell death in angiosperms and conifers (Kasprzewska, 2003; Wiweger et al., 2003). Plant chitinases are represented by multigene families that are divided into seven classes based on gene structure (Meins et al., 1994; Neuhaus, 1999). Class I, II and IV chitinases have been cloned from conifer species (Wu et al., 1997; Davis et al., 2002; Wiweger et al., 2003; Liu et al., 2005; Schmidt et al., 2005). In conifers, chitinase 11 expression is induced by wounding, fungal inoculation, herbivory, methyl jasmonate, salicylic acid or chitosan (Wu et al., 1997; Davis et al., 2002; Schmidt et al., 2005; Ralph et al., 2006a). In Douglas-fir (Pseudotsuga menziesii) infected with the root rot fungus Phellinus suphurascens, chitinase was localized to the fungal hyphae, consistent with a role in the degradation of fungal cell walls (Islam et al., 2009). Increased expression of chitinases was associated with Norway spruce resistance to Heterobasidion annosum (Fossdal et al., 2006), and different patterns of chitinase protein expression was observed in resistant and susceptible western white pine (Pinus monticola) infected with Cronartium ribicola (Liu et al., 2005). Beta-1,3-glucanases are often induced along with chitinases and contribute to the antifungal efficiency of chitinases by digesting -1,3-glucan fibers that are part of the fungal cell wall (Mauch et al., 1988; Collinge et al., 1993). Induction of -1,3-glucanases and chitinases was observed in Norway spruce roots infected with the parasitic oomycete Pythium sp. (Sharma et al., 1993). Further studies revealed the localization of spruce chitinase and -1,3-glucanase proteins on the hyphal walls of Heterobasidion annosum in infected Norway spruce roots supporting a role for these enzymes in the digestion of fungal cell walls (Asiegbu et al., 1995). In addition to the antimicrobial proteins with known biochemical function there are a number of plant antimicrobial proteins with unknown biochemical function such as thaumatin-like proteins, defensins, PR-10 proteins and antimicrobial peptides. Expression of the thaumatin-like protein gene from western white pine was induced by wounding and blister rust pathogen (Cronartium ribicola) infection (Piggott et al., 2004) and transcripts annotated as thaumatin were among the most strongly upregulated in Scots pine inoculated with Heterobasidion annosum (Adomas et al., 2007). The function of this protein is unknown, although it may participate in the degradation of the fungal cell wall based on the function of a related thaumatin-like protein in barley (Zareie et al., 2002; Piggott et al., 2004). It is also suggested that thaumatin-like proteins may disrupt hyphal and spore membranes by forming transmembrane pores (De Lucca et al., 2005). A thaumatin-like protein accumulated in Douglas-fir root infected with root rot fungus and was localized extracellularly on the host cell membrane, supporting a role in plant antifungal defense (Islam et al., 2009). Several defensin genes have been characterized from conifer species (Sharma and Lonneborg, 1996; Kovalyova and Gout, 2008). Defensin gene expression was induced by wounding, methyl jasmonate and fungal treatment in white spruce and the antifungal activity of selected conifer defensins was established in vitro (Pervieux et al., 2004; Gout and Kovalyova, 2008; Kovalyova and Gout, 2008). Transgenic tobacco and 12 Norway spruce overexpressing a Norway spruce defensin-like gene were more resistant to bacterial and fungal pathogens respectively (Elfstrand et al., 2001). PR-10 proteins were cloned from several conifer species including maritime pine, western white pine, white spruce, and Douglas-fir. Several PR-10 genes are induced by wounding, fungal inoculation and methyl jasmonate in western white pine (Ekramoddoullah et al., 2000; Dubos and Plomion, 2001; Liu et al., 2003; Mattheus et al., 2003). Angiosperm PR-10 proteins are known to have ribonuclease properties and antifungal activity (De Lucca et al., 2005) and PR-10 proteins were localized on fungal cell walls in western white pine infected with blister rust fungus, supporting involvement in antifungal defense (Liu et al., 2003). A variety of additional unique antimicrobial peptide (AMP) groups were characterized in angiosperm plants (De Lucca et al., 2005). A family of AMPs (Sp-AMP) was characterized in Scots pine (Asiegbu et al., 2003). Based on homology with plant and yeast proteins it was suggested that these AMPs may inhibit fungal and bacterial cell wall biosynthesis. Expression of an AMP transcript was induced by Heterobasidium annosum in Scots pine and the AMP was accumulated on the host cell surface, indicating its direct participation in antifungal defense (Asiegbu et al., 2003; Asiegbu et al., 2005; Adomas et al., 2007).

1.5 ELICITORS OF CONIFER DEFENSE

Research into the signaling mechanisms involved in plant defense in angiosperms revealed three common types of low molecular weight signaling molecules that affect the downstream expression of defense-related genes: octadecanoids, including jasmonates; ethylene; and salicylic acid, (Koornneef and Pieterse, 2008). Pathways activated by these signal molecules may interact with each other to produce various patterns of disease and herbivore-induced responses. While defense-associated signaling has been extensively studied in angiosperms using Arabidopsis thaliana and other model species (Thomma et al., 2001; Kunkel and Brooks, 2002; Koornneef and Pieterse, 2008; Wu and Baldwin, 2009), very little is known about defense signaling in conifers.

Jasmonates induce defense responses in conifers In conifers, application of methyl jasmonate (MeJa) induces anatomical and biochemical changes similar to those induced by wounding, insect herbivore attack, and pathogen invasion (Huber et al., 2004; Miller et al., 2005; Zulak et al., 2009). At the 13 anatomical level, polyphenolic parenchyma activation and traumatic resin duct development is initiated and stimulated by methyl jasmonate treatment (Franceschi et al., 2002; Martin et al., 2002; Hudgins et al., 2003). At the biochemical level, MeJa induces accumulation of terpenoids and terpene synthase activity (Martin et al., 2002;; Martin et al., 2003). At the transcript level, MeJa induces genes involved in early steps of terpenoid biosynthesis (DXS, DXR and HDR) (Phillips et al., 2007) and TPS gene expression (Faldt et al., 2003, Miller et al., 2005). A comparison of MeJa induction and white pine weevil (Pissodes strobi) feeding on Sitka spruce showed that both treatments produced remarkably similar anatomical and biochemical responses in Sitka spruce (Miller et al., 2005). Both treatments induced the formation of traumatic resin ducts, accumulation of terpenoids and strong upregulation of TPS expression. The application of methyl jasmonate induces pathogenesis-related protein gene expression, including increased chitinase transcript levels in slash pine (Pinus elliottii) seedlings (Davis et al., 2002), and enhanced expression of PR-10 transcripts in wound- induced western white pine (Liu et al., 2003). In addition, methyl jasmonate application induced chalcone synthase in white spruce (Richard et al., 2000). Gene expression analysis of Sitka spruce in response to white pine weevil feeding revealed an increased expression of genes putatively involved in the octadecanoid pathway (Miller et al., 2005; Ralph et al., 2006a). The ability of methyl jasmonate to induce defense responses similar to those elicited by wounding, insect feeding, and fungal inoculations is consistent with a role for octadecanoid signaling in induced conifer defense.

Ethylene is involved in the wound- and jasmonate induced defense responses in conifers The involvement of ethylene in the induction of histological changes and the correlation between ethylene and jasmonate signaling has recently been demonstrated in Douglas-fir and in giant redwood, Sequoiadendron giganteum (Hudgins and Franceschi, 2004). The application of methyl jasmonate and ethylene induced the formation of traumatic resin ducts and activated of polyphenolic parenchyma in both species. Inhibition of ethylene signaling resulted in a decreased histological response to wounding and methyl jasmonate treatment of Douglas-fir, indicating that the methyl jasmonate response in conifers is mediated by ethylene. Ethylene production was correlated with monoterpene biosynthesis following fungal inoculation of slash pine and loblolly pine (Popp et al., 1995), and application of an ethylene releaser, ethrel, increased monoterpene synthase activity in grand fir (Katoh and Croteau, 1998). 14 Several ACC (1-aminocyclopropane -1 carboxylic acid) synthases, enzymes involved in ethylene biosynthesis, were cloned and characterized in white spruce, interior spruce (Picea glauca x Picea engelmannii) and Douglas-fir (Ralph et al., 2007b). Gene expression of selected ACC synthases was induced by wounding and white pine weevil feeding in Sitka spruce and ACC synthase protein accumulated in Douglas-fir induced by wounding (Ralph et al., 2007b). Another enzyme involved in ethylene biosynthesis, ACC oxidase, was cloned from Sitka spruce, white spruce and Douglas-fir (Hudgins et al., 2006). Both methyl jasmonate and wounding treatments increased the accumulation of ACC oxidase protein in Douglas-fir (Hudgins and Franceschi, 2004; Hudgins et al., 2006). In Douglas-fir bark, both ACC synthase and ACC oxidase were localized to resin duct epithelial cells, polyphenolic parenchyma cell, which are involved in conifer defense, and ray parenchyma cells, which connect phloem, cambium and xylem and may serve in spreading of defense related signaling (Hudgins et al., 2006; Ralph et al., 2007b). The effect of ethylene in inducing conifer defense responses in addition to defense-related increased expression and localization of the enzymes involved in ethylene biosynthesis supports the role of ethylene in conifer defense signaling.

Salicylic acid and conifer defense responses The role of salicylic acid as defense elicitor in conifers is not well established. Salicylic acid accumulated in Norway spruce seedlings treated with the pathogen Pythium irregulare and MeJa (Kozlowski and Metraux, 1998; Kozlowski et al., 1999). Treatment of Norway spruce with MeJa resulted in the induced emission of methyl salicylate (Martin et al., 2003). Application of salicylic acid induced the expression of chitinases in slash pine seedlings (Davis et al., 2002). However when applied to Douglas-fir and giant redwood saplings, salicylic acid failed to induce any histological responses, while MeJa and ethylene treatment induced the activation of phenolic parenchyma and the formation of traumatic resin ducts (Hudgins and Franceschi, 2004). Wound-induced expression of PR-10 protein in western white pine was enhanced by methyl jasmonate but was suppressed by salicylic acid (Liu et al., 2003), suggesting different involvement of these elicitors in conifer defense.

1.6 ACQUIRED RESISTANCE IN CONIFERS

Acquired resistance can develop in plants after they have been exposed to infection, insect attack or wounding and results in higher resistance of plant tissues to 15 subsequent infection or insect attack (Kiraly et al., 2007). Local acquired resistance develops in tissues surrounding the infection or damage site and systemic acquired resistance develops in distant tissues. These responses have been extensively studied in angiosperms (Kiraly et al., 2007; Walters, 2009). Successful induction of Norway spruce resistance against the beetle-associated blue-stain fungus Ceratocystis polonica is possible by prior inoculation of the tree with a sublethal dose of the same fungus (Christiansen et al., 1999; Krokene et al., 1999; Krokene et al., 2003). Pretreatment with a sublethal dose of fungus induced the formation of traumatic resin ducts and increased the number of polyphenolic parenchyma cells (Krokene et al., 2003). Pretreatment with fungus was significantly more effective in inducing resistance when compared to wounding alone (Christiansen et al., 1999; Krokene et al., 1999). In addition, the timing of pretreatment is important for successful resistance to fungi. Trees that were pretreated with fungus one week prior to mass inoculation were not significantly more resistant to subsequent fungal inoculations, whereas trees pretreated three to nine weeks prior to mass inoculation had reduced disease symptoms when inoculated in the pretreated stem areas (Krokene et al., 2003). In these studies, pretreatment and the subsequent inoculations were done on the same part of the stem, supporting the involvement of local acquired resistance. However, only small areas of the stems were pretreated (e.g. 5%) (Christiansen et al., 1999) and increased resistance was observed in the entire stem section indicating the spread of resistance from the inoculation cites. Systemic induction of traumatic resin ducts was observed in spruce (Christiansen et al., 1999; Krekling et al., 2004) and pine (Luchi et al., 2005). Systemically acquired resistance was observed in Monterey pine (Pinus radiata) pretreated with the pitch canker pathogen Fusarium circinatum, and resulted in the increased resistance against the pathogen for at least a year following initial inoculation (Bonello et al., 2001; Bonello et al., 2006). A similar increase in resistance was observed in Austrian pine inoculated with canker pathogens, and a resistance increase was observed above and below the inoculation sites, indicating bidirectional signaling (Blodgett et al., 2007). Systemically induced resistance was associated with increased lignin content and may involve the accumulation of soluble phenolics (Bonello and Blodgett, 2003; Blodgett et al., 2007; Wallis et al., 2008). In addition, cross induction of systemic acquired resistance by a canker pathogen and insect Neodiprion sertifer (European pine sawfly) was observed in Austrian pine (Eyles et al., 2007). Locally and systemically acquired resistance seems to be a common and important contributing factor to conifer defense (Bonello et al., 2006).

16 Pretreatment of conifers with methyl jasmonate A long term, effective increase in the resistance of pretreated conifers provides the potential to use pretreatment techniques as protective measures. The ability of methyl jasmonate to induce defense responses in conifers combined with its easy application supports its use in the development of conifer pretreatment strategies. The effect of methyl jasmonate treatment without wounding of mature Norway spruce was similar to that of the fungal pretreatment, in that the formation of traumatic resin ducts was induced, resin flow was increased, and phenolic parenchyma were activated resulting in the increased resistance of Norway spruce to the blue-stain fungi Ceratocystis polonica (Franceschi et al., 2002; Zeneli et al., 2006; Krokene et al., 2008) and resistance to colonization by the bark beetle Ips typographus (Erbilgin et al., 2006). Treatment of Norway spruce seedlings with methyl jasmonate also increased seedling resistance to Pythium ultimum (Kozlowski et al., 1999), reducing the mortality rate of spruce seedlings up to 50 percent. Treatment of Maritime pine (Pinus pinaster) seedlings with methyl jasmonate increased their resistance to large pine weevil (Hylobious abietis) (Moreira et al., 2009). Acquired resistance develops in methyl jasmonate pretreated trees within three weeks to a month and is likely to be sustained for several months because of the formation of stable anatomical structures (Erbilgin et al., 2006). Methyl jasmonate treatment does not seem to a have phytotoxic effect but may result in moderated reduction of conifer sapwood growth (Krokene et al., 2008). However, these reduced growth may be transient due to temporal nature of methyl jasmonate action (Gould et al., 2008).

1.7 MICROARRAY ANALYSIS OF CONIFER DEFENSE

Spruce and pine genomics resources have become available with the development of the extensive EST databases and microarrays (Allona et al., 1998; Kirst et al., 2003; Stasolla et al., 2003; Ralph et al., 2006a; Ralph et al., 2008). Transcriptome profiling of the Sitka spruce defense response induced by mechanical wounding, spruce budworm (Choristoneura occidentalis) and white pine weevil (Pissodes strobi) using a cDNA microarray containing 9.7K cDNA elements revealed a large reorganization of the transcriptome, with 25 to 36% of the studied transcriptome being differentially expressed in Sitka spruce induced by these treatments (Ralph et al., 2006a). Similarly, a smaller scale defense-related pine transcriptome profiling revealed altered expression of about 10% of transcripts in Scots pine induced 17 by the pathogenic fungus Heterobasidion annosum using a 2.1K loblolly pine microarray (Adomas et al., 2007). These studies revealed, on the transcriptome level, strong induction of several branches of the phenylpropanoid pathway, antimicrobial proteins such as chitinases, thaumatins and others by wounding, herbivory and pathogen treatment in conifers. In addition, microarray studies of Sitka spruce induced by wounding and insect feeding showed a high induction of the terpenoid pathway and the induction of transcripts annotated to octadecanoid and ethylene signaling pathways (Ralph et al., 2006a). The defense response in conifers included the reorganization of primary metabolic processes such as the downregulation of photosynthesis by wounding, insect feeding and fungal inoculation (Ralph et al., 2006a; Adomas et al., 2007). These microarray studies provided a variety of new candidate genes, including a number of transcripts annotated to the phenylpropanoid pathway, antimicrobial proteins, transcription factors and transcripts involved in signaling as targets for further study into their involvement in conifer defense. The development of conifer EST resources allowed the application of proteomics to evaluate the Sitka spruce bark proteome response to wounding and weevil feeding (Lippert et al., 2007). Among over a hundred proteins, which were differentially expressed in induced Sitka spruce, only a small number were also identified as similarly differentially expressed by the spruce microarray, supporting the necessity of complementary genomics and proteomics approaches to the study of conifer defense. In particular, several proteomics studies highlighted the induction of heat shock proteins in conifer species by wounding, pathogen attack and herbivory (Smith et al., 2006; Wang et al., 2006; Lippert et al., 2007). Observed differences in high protein induction levels and only moderate or no induction on the transcript level for some of the heat shock proteins suggested regulation of expression on the post-transcriptional level (Lippert et al., 2007). Heat shock proteins were shown to be involved in angiosperm plant defense and disease resistance, and they likely play a role in stabilizing defence related proteins (Byth et al., 2001; Hubert et al., 2003; Lu et al., 2003; Kanzaki et al., 2003; Maimbo et al., 2007). Thus proteomics studies may help to identify additional candidate genes with potential importance in conifer defense or may point to additional levels of gene expression control in conifer defense responses (Lippert et al., 2007).

18 1.8 THESIS OBJECTIVES

In this thesis project I aimed at addressing a gap in the characterization of molecular mechanisms of conifer defense response against bark beetle fungal associates on the transcriptome level. In addition, the goals of this thesis research included a first direct comparative evaluation of the specificity of conifer defense responses to fungal inoculation and insect attack on the transcriptome level. To achieve these goals I performed comparative studies of spruce and pine responses to bark beetle associated blue stain fungi and spruce response to weevil herbivory. Functional characterization of chitinases, selected based on the transcriptome studies, was aimed at better understanding of the role of these genes in conifer defense.

The thesis goals were pursued by research described in chapters 2, 3 and 4, which address three major objectives:

Objective 1: To perform a large scale comparative analysis of the transcriptome response of interior spruce (Picea glauca x engelmannii) inoculated with spruce beetle (Dendroctonus rufipennis) associated blue-stain fungus Leptographium abietinum and lodgepole pine (Pinus contorta) inoculated with mountain pine beetle (Dendroctonus ponderosae) associated blue-stain fungus Grosmannia clavigera. (Chapter 2). The following questions were addressed:  What is the scope and how similar are the spruce and pine transcriptome responses to blue-stain fungus?  Which pathways are involved in the spruce and pine transcriptome responses to fungus?  Which genes are potentially important in the defense responses and should be further characterized?

Work described in chapter 2 required the development of an efficient and reproducible method for RNA isolation from conifer tissues (Appendix 3) and evaluation of spruce array performance with other species of Pinaceae (Appendix 2).

Objective 2: To perform a transcriptome microarray analysis of interior spruce in response to white pine weevil feeding and compare this response with the response of interior spruce to inoculation with a blue-stain fungal pathogen (Leptographium abietinum) (Chapter 3). 19 The following questions were addressed:  What is the scope and how similar is the spruce transcriptome response to weevil as compared to blue-stain fungus?  What is the difference in the involvement of different pathways and gene families in the spruce response to weevil and fungus?  Which genes are specific to the spruce weevil response and which genes are involved in spruce response to both weevil and fungus?

Objective 3: To perform functional characterization of chitinases genes involved in spruce and pine defense response (Chapter 4). This study was aimed at cloning chitinase genes involved in spruce and pine defense responses and performing first biochemical evaluation of chitinases genes function in conifer species. The following questions were addressed:  Do spruce and pine genes annotated as chitinases have chitinolytic activity?  Which chitinase gene expression is induced by weevil feeding and fungal inoculation in interior spruce and by fungal inoculation in lodgepole pine?  Do spruce and pine chitinases have measurable antifungal activity?

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31 2 Microarray gene expression profiling of interior spruce (Picea glauca x engelmannii) inoculated with Leptographium abietinum and lodgepole pine (Pinus contorta) inoculated with Grosmannia clavigera 1

2.1 INTRODUCTION

Bark beetles in association with pathogenic blue-stain fungi are among the most destructive forest pests. Pathogenic blue-stain fungi are transferred by bark beetles and seem to be required for successful beetle colonization and tree death (Paine et al., 1997; Six and Paine, 1998). Blue-stain fungi colonize the phloem and sapwood of the attacked trees, cause interruption of water and nutrient transport and may contribute to depletion of tree defenses. Even though the presence of pathogenic fungi induces tree defenses, which may be effective against fungi and beetles, mass attack by beetles and accompanied by mass inoculation with beetle associated pathogenic fungi are aimed at exhausting tree defenses. The exhaustion of tree defenses is achieved by massive girdling action of beetles and fungal growth which stresses and kills tree tissues eventually resulting in tree mortality, which is necessary for successful colonisation. In addition, larvae and emerging new generation of beetles feed on blue-stain fungi mycelium, which is abundant in the pupal chambers (Paine et al., 1997). The spruce beetle (Dendroctonuc rufipennis) attacks several species of spruce across a wide geographical range in North America and is associated with the highly pathogenic fast growing blue-stain fungus Leptographium abietinum (Ohsawa et al., 2000; Six and Bentz, 2003). Inoculation of spruce seedling with Leptographium abietinum resulted in phloem necrosis and drying, rapid tree wilting and death within a month (Ohsawa et al., 2000). The extent of Leptographium abietinum establishment in mature spruce correlated with spruce beetle attack success (Reynolds, 1992). The mountain pine beetle (Dendroctonus ponderosae) is currently causing one of the largest outbreaks of bark beetles ever recorded in North America, affecting over 13 million hectares of lodgepole pine (Pinus contorta) forest (Raffa et al., 2008). The mountain pine beetle attacks several species of pine and is associated with the highly pathogenic blue-stain fungus Grosmannia clavigera (Yamaoka et al., 1995). Grossmannia clavigera grows rapidly in sapwood and can kill mature lodgepole pine within one year (Yamaoka et al., 1995). Association of Grosmannia clavigera with

1 A version of this chapter will be submitted for publication. Kolosova N, White R, Ralph S, Breuil C and Bohlmann J. Conifer defense against blue-stain fungi: microarray gene expression profiling of interior spruce inoculated with Leptographium abietinum and lodgepole pine inoculated with Grosmannia clavigera. 32 mountain pine beetle was required for successful beetle brood development in lodgepole pine (Six and Paine, 1998). Conifer trees employ a number of different constitutive and induced defense strategies against fungal pathogens. Constitutive structural defenses include constitutive resin ducts and polyphenolic parenchyma that store terpenoids and phenolics. These chemicals can be toxic to beetles and to fungi. In addition, conifers employ induced defenses that include the formation of traumatic resin ducts, the activation of cortical resin ducts and polyphenolic parenchyma and the additional accumulation of terpenoids and phenolics, as well as the activation of protein based defenses such as pathogenesis related genes (Huber et al., 2004; Franceschi et al., 2005; Raffa et al., 2005). Induced formation of traumatic resin ducts, activation of polyphenolic parenchyma, and activation of pathogenesis related genes such as chitinases and peroxidases was shown may be associated with increased resistance of conifers to pathogenic blue-stain fungi inoculation (Franceschi et al., 1998; Krokene et al., 2003; Nagy et al., 2004a, 2004b; Fossdal et al., 2006). Despite the economic importance of conifers and the devastating effect of blue-stain fungi associated with bark beetles, the molecular mechanisms of conifer defense against blue-stain fungi are not well understood. The application of large-scale DNA sequencing and microarray technology to forest trees opened the possibility for substantial transcriptiome analysis in conifers. Large scale transcriptome profiling of the Sitka spruce defense response induced by mechanical wounding, spruce budworm (Choristoneura occidentalis) and white pine weevil (Pissodes strobi) revealed differential expression of up to 25 to 36% of genes represented on a 9.7K cDNA array (Ralph et al., 2006b). Smaller scale transcriptome profiling of Scots pine induced by pathogenic fungus Heterobasidion annosum used a 2.1K loblolly pine microarray (Adomas et al., 2007); and of slash pine (Pinus elliottii) induced by Fusarium circinatum used a microarray containing 311 unique cDNAs (Morse et al., 2004). These studies revealed altered expression levels of about 10% of the studied transcriptomes. These studies of transcriptome defense response revealed strong induction of several branches of the phenylpropanoid pathway by herbivore and pathogen treatment (Ralph et al., 2006b; Adomas et al., 2007). Another group of genes induced in pines by fungal treatments included a few genes with antimicrobial properties such as thaumatin-like proteins, antimicrobial peptide (AMP) and chitinases (Morse et al., 2004; Ralph et al., 2006b; Adomas et al., 2007). The defense response of lodgepole pine to date has not been studied on a gene transcript level and none of the genes involved in defense response have been characterized. 33 Successful application of an interspecies array opened new opportunities of utilizing genomics resources developed for spruce for transcriptome studies in other conifer species. Interspecies application of loblolly pine (Pinus taeda) 2.2K microarray was evaluated with other species of pine and spruce (van Zyl et al., 2002) and was successfully applied to developmental studies of white spruce somatic embryos (Stasolla et al., 2003). The goal of this study was to characterise molecular mechanisms of interior spruce (Picea glauca x engelmannii) response to Leptographium abietinum and lodgepole pine response to Grossmannia clavigera on the transcriptome level. We performed the first large scale comparative transcriptome response analysis of interior spruce inoculated with L. abietinum and lodgepole pine inoculated with G. clavigera using 21K spruce microarray during a time course study of 2 hours, 2 days and 2 weeks. Comparative transcriptome analysis allowed us to observe the activation of phenylpropanoid pathway, terpenoid pathway, and pathogenesis related genes in both species. We also detected differences in the responses of the two species to the corresponding pathogens on a transcriptome level. Expression of selected transcripts from highly induced pathways was further analyzed using gene specific expression analysis.

34 2.2 MATERIAL AND METHODS

2.2.1 Plant material, fungal material and inoculation procedure Interior spruce (Picea glauca x engelmannii, clone I1026) seedlings were propagated by somatic embryogenesis and generously provided by Dr. David Ellis (CellFor Inc., Victoria, Canada). Seedlings were grown to three years of age outside at the University of British Columbia (UBC) under natural light and environmental conditions. One week prior to treatment, the seedlings were transferred to the UBC greenhouse with greenhouse temperature fluctuating between 20 and 24oC and an average humidity of 45%. Three year old seedlings of lodgepole pine (Pinus contorta), grown from seeds collected in the interior British Columbia, were purchased from Surrey Nursery, BC. Lodgepole pine seedlings were grown outside of the UBC green house and transferred to the UBC greenhouse one week prior to treatments. Interior spruce was inoculated with Leptographium abietinum fungus, strain 2PG6P-La (Alamouti et al., 2007). Lodgepole pine was inoculated with Grosmannia clavigera (strain SLKw1407) (DiGuistini et al., 2007). Fungal inoculation was performed using a modified method of Croteau et al. (1987). Fungal mycelia were grown on malt agar covered with porous cellophane (Amersham) at 24oC for one week and collected in the active state of fungal growth. Fungal mycelium was cut into pieces of 0.5 x 1 cm. For inoculation, saplings were wounded with a sterile needle at the lower part of the stem. Four puncture wounds (~ 3 mm deep and 3-4 mm wide) were introduced on two opposing sides of a 10-12 cm stem section (equally spaced along the section). For each wound, fungus from a single 0.5 x 1 cm2 film section was applied to the wound surface. Wounding treatment was identical to the type of wounding that was used in fungal inoculations with no fungus introduced. Control trees did not receive any treatment. Successful establishment of fungus in treated saplings was confirmed by reisolation of fungus. Bark tissue was harvested from the lower part of the stems into liquid nitrogen from the treated and control seedlings 6 hours, 2 days, 2 weeks and 6 weeks post- treatment (for treated trees this included all bark around the treated area from 10-12 cm treated section with the exception of a small part of the treated stem that was used for anatomical analysis and fungus reisolation and tissue was harvested from the similar stem location from the control trees). Harvested bark tissue was stored at -80oC. Three biological replicates were used per time point and treatment for the clonal interior spruce inoculation experiment, and four biological replicates were used per time point and treatment for the lodgepole pine inoculation experiment. 35

2.2.2 Anatomical analysis Interior spruce and lodgepole pine stem disks were collected from the treated area of the stems and fixed immediately in fixative solution 2% (v/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde in 50mM PIPES buffer, pH 7.2, Tween 20 0.05% (v/v). Stem discs were washed twice with 50mM PIPES buffer, pH7.2 and sections were cut using a microtome (Model 860 American Optical Company). Sections from treated seedlings were cut close to wounding and inoculation sites (1-2mm from the treatment sites). Cross-sections were stained in saturated (aqueous) copper acetate overnight for resin duct anatomical analysis. After the staining, cross sections were washed in water and mounted on glass slides using the method previously described (Wang and Aitken, 2001). For resin duct counting lodgepole pine cross-sections were stained with 0.5% (w/v) aqueous safranin for 5 min, then rinsed with distilled water and processed as described previously (Wang and Aitken, 2001). Digital images were taken of cross sections using a scanner (Kodak, RFS 2035 Plus Film). Four individual trees per treatment (control, wounding, fungal) were analysed. For statistical analysis a linear model was fit treating condition as a factor. The pairwise differences between conditions were estimated and statistical significance was accesed using t statistics. For finer microscopy, lodgepole pine stem disks were cut into 2-3 mm thick cross-section blocks and left in fixative overnight at 4oC. Samples were rinsed with 50 mM PIPES and dehydrated in an ethanol series through consecutive steps of 70%, 80%, 90%, 95% and three 100% ethanol changes with at least 30 minutes incubation between changes. Samples were consecutively infiltrated with pure LR White acrylic resin (with fresh LR White resin at every step) for 16, 2 and 6 hours of infiltration. Samples were placed in Eppendorf tubes containing fresh LR White resin and polymerized for 40 hours at 60oC. Sections 1µm thick were cut with a microtome (Reichart Ultracut E) and dried from a drop of water onto glass slides and stained with 1% Toluidine Blue (w/v). A photomicroscope (Axioplan 2 Zeiss, Germany) was used to visualize and photograph the samples.

2.2.3 Terpenoid extraction and analysis Terpenoid extraction was done as described previously (Martin et al., 2002). For resin analysis three separate bark samples were collected from three interior spruce trees and four lodgepole pine trees per treatment per time point. Samples of approximately 1 cm in length and 0.3 cm wide were extracted with 1.5 ml tert-butyl methyl ether (Aldrich, Milwaukee, WI) and were spiked with 100µg/mL isobutyl benzene 36 and 200 µg/ml dichlorodehydroabietic acid (Helix Biotech, Richmond Canada) as internal standards. Diterpenoids were methylated as described previously (Martin et al., 2002). Dry weights were measured after drying tissues at 55oC for 72 hours. Qualitative and quantitative analysis of terpenoids was performed on GC (Agilent 6890A, Agilent Technologies) with an autosampler (Agilent 7683) and flame ionization detector (FID). FID hydrogen flow was 40 ml/min, air flow 450 ml/min and makeup flow 50 ml/min. The flow of hydrogen carrier gas was 1 ml/min. The injection volume was 1 µl. The split ratio was 20:1. Monoterpenoids were analyzed using DB-WAX column (30m x 0.25 mm i.d. x 0.25µm film thickness; J&W Scientific, Folsom, CA). The injector temperature was 250oC, and the FID temperature was 250oC. The oven was programmed with an initial oven temperature of 40oC for three minutes, a temperature increase of 3oC/min to 110 oC, followed by a 10oC/min increase to 180oC and 20oC/min increase to 250oC with a 15 min hold at 250oC. Diterpenoids were analyzed using an AT – 1000 column (30m x 0.25mm i.d. x 0.25µm film thickness Altech, Deerfield, IL). The injector temperature was 240oC, and FID temperature was 300 oC. The oven was programmed with an initial temperature 180oC for 1 min, a temperature increase of 1oC/min to 220oC and 20oC/min to 240oC with 15 min hold at 240oC. GC-FID chromatograms were analyzed using Agilent Chemstation software (Agilent technologies) For additional identification of terpenoids, samples were analyzed using GC- mass spectrometry on an Agilent 6890A GC equipped with Agilent 7683 Series autosampler and 5973N mass selective detector, using the same GC columns as for GC-FID. The injection volume was 0.1µl with splitless mode. The carrier gas was helium with constant flow of 1.0 ml/min. Similar to GC-FID method of analysis for monoterpenoids, the oven was programmed with an initial oven temperature of 40oC for three minutes, a temperature increase of 3oC/min to 110oC, followed by a 10oC/min increase to 180oC and a 15oC/min increase to 240oC with a 15 min hold at 240oC. For the analysis of diterpenoids the oven was programmed with an initial temperature of 150oC for 1 min, a temperature increase of 1.5oC/min to 220oC and of 20oC/min to 240oC with a 12 min hold. Identification of terpenoids was based on comparison of retention times and mass spectra with those of authentic standards and based on matching mass spectra in the Wiley275 Mass Spectra Library (Agilent Technologies) and HP2205 Library (Agilent Technologies). Terpenoid concentration was calculated using integration of GC-FID peak area and normalization against peak areas of internal standards.

37 2.2.4 Microarray hybridization and gene expression data analysis Total RNA was extracted using a previously published protocol (Kolosova et al., 2004). Isolated total RNA was qualified and quantified by spectrophotometer. The quality of RNA was checked by agarose gel and reverse transcription with incorporation of 32P or by using a 2100 Bioanalyzer (Agilent Technologies). For microarray experiments total RNA was isolated from the bark of spruce trees inoculated with L. abietinum, treated with wounding, or from untreated control for three time points (6h, 2 days and 2 weeks) with three independent biological replicates for each treatment and time point. RNA was isolated from the bark of lodgepole pine inoculated with O.clavigerum, treated with wounding, or untreated control for three time points (6h, 2days and 2 weeks) with three independent biological replicates for each treatment and each time point. All microarray experiments were designed to comply with MIAME guidelines (Brazma et al., 2001). The 21.8K cDNA spruce microarray platform represented cDNAs from several developmental stages of xylem, phloem, bark and roots, as well as elicitor-treated bark. Hybridizations were performed using the Genisphere Array900 kit (Genisphere, Hatfield, USA) following manufacturer’s instructions. Ten micrograms total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo d(T18) primers with a 5' unique sequence overhang specific to either the Cy3 or Cy5 labeling reactions. The RNA strand of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65°C for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0). Following pooling of the appropriate cDNAs, samples were precipitated with linear acrylamide and resuspended in a 45 µL hybridization solution consisting of 0.25M NaPO4, 0.5% SDS, 1x SSC, 2x Denhardt’s solution, 1 mM EDTA, 2.75 µL LNA d(T) blocker, 2 µg sheared salmon testes DNA (Invitrogen) and 0.3 µL of Cy5-labeled GFP cDNA (Cy5-dUTP and Ready-To-Go labeling beads, Amersham Pharmacia Biotech). Immediately prior to use, arrays were pre-washed 2 in 0.1% SDS at room temperature for 5 min each, followed by two washes in MilliQ-H2O for 2 min each, 3 min at 95°C in MilliQ-H2O, and dried by centrifugation (3 min at 2000 rpm in an IEC Centra CL2 centrifuge with rotor IEC 2367-00 in 50 mL conical tube). The cDNA probe was heat denatured at 80°C for 10 min, then maintained at 65°C prior to adding to a microarray slide heated to 55°C, covered with a 22  60  1.5 mm glass coverslip (Fisher Scientific, Waltham, USA), and incubated for 16 h at 60°C. Arrays were washed in 2 SSC, 0.2% SDS at room temperature for 5 min to remove the coverslip, followed by 15 min at 65°C in the same solution, then three washes of 5 min in 2 SSC at room

38 temperature, and three washes of 5 min in 0.2 SSC at room temperature, and dried by centrifugation. The Cy3 and Cy5 3DNA capture reagent (Genisphere) were then hybridized to the bound cDNA on the microarray in a 45 µL volume consisting of 0.25M

NaPO4, 0.5% SDS, 1x SSC, 2x Denhardt’s solution, 1 mM EDTA, 2.5 µL Cy3 capture reagent and 2.5 µL Cy5 capture reagent. The 3DNA capture reagent is bound to its complementary cDNA capture sequence on the Cy3 or Cy5 oligo d(T) primers. The second hybridization was performed for 3 h at 60°C, then washed and dried as before. Fluorescent images of hybridized arrays were acquired by using ScanArray Express (PerkinElmer, Foster City, USA). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm, respectively. All scans were performed at the same laser power (90%), but with the photomultiplier tube settings for the two channels adjusted such that the ratio of the mean signal intensities was ~1, and the percentage of saturated array elements was < 1% but > 0%, while minimizing background fluorescence. Fluorescent intensity data were extracted by using the ImaGene 5.5 software (Biodiscovery, El Segundo, USA). The design of microarray hybridizations was the same for interior spruce and lodgepole pine. A loop design with dye balance was chosen to allow comprehensive analysis of transcriptome changes between treatments at three time points (Figure 2.1).

39 F 3 C 3 W 6h 6h 6h 1 3 1 1 1 F C W 1 3 3 1 2d 2d 2d 1 3 4 1 0 F C W 3 3 3 2w 2w 2w 0 2 3 0 1 Figure 2.1 Microarray experimental design. 0Each connection between two different samples (indicated by treatment and time point) specifies hybridization of the two 0 samples against each other performed on one microarray. The number above the connection indicates the number of replicates0 for the particular hybridization of two connected samples against each other. Treatments: C-control, W-wounding, F-fungal . inoculation. Treatment time course included 6 hours (6h), 2 days (2d) and 2 weeks (2w). 5

0 Three hybridizations were performed for. each comparison of different treatments (fungal, wounding, control) within each time point4 and one hybridization was performed for the comparison of the same treatments between0 time points. The experiment was designed to be Cy3 Cy5 dye balanced. Raw and. processed data and Tiff images are available at the Gene Expression Omnibus (www.ncbi.n3 lm.nih.gov/geo/). EST sequence data for the microarray ESTs is available 0 in National Center for Biotechnology Information (NCBI) databases and is searchable. by EST IDs. Data analysis was done as described previously (Holliday et al., 2008). To 2calculate changes in gene expression, a linear mixed-effect model was used for 36 slides0 (hybridizations) for the experiment with each studied species. P values were calculated. for each EST-by-treatment effect (including wounding vs. control, fungus vs.1 control, and fungus vs. wounding comparisons for each time point) and q values0 were calculated to correct for false discovery rate for each p value (Storey and Tibshirani,C 2003). o t 40 e m p 2.2.5 Real-time PCR and gene expression data analysis Total RNA (5µg per treatment) was pretreated with DNaseI (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions to remove genomic DNA. cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen) with an oligo dT18 primer. cDNA synthesis was evaluated using gel electrophoresis. Gene specific primers were designed using interior spruce EST sequences available at the NCBI database and lodgepole pine EST sequences produced as a result of the sequencing project reported in this paper. Guidelines used for the primer design were previously described in (Ralph et al., 2006b). Primer specificity was confirmed by running individual RT-PCR products on a 2% agarose gel, melting curve analysis and by sequencing the resulting product. Primer sequences are provided in Supplemental Table 2.1. Real-time PCR conditions and data analysis were performed as described previously (Ralph et al., 2007). A minimum of three biological replicates were used for each quantification and each cDNA source. Transcript abundance was normalized to translation initiation factor 5A (TIF5A, IS0013_F24, GenBank: DR448953). For statistical analysis a linear model was fit (treating each condition at each time point as a factor) for each gene using data from three biological replicates for each treatment at three time points (6 hours, 2 days and 2 weeks). The pairwise differences between conditions at three time points were estimated and statistical significance was assessed using t statistics.

41 2.3 RESULTS

2.3.1 Induced formation of traumatic resin ducts in interior spruce and lodgepole pine inoculated with L. abietinum and G. clavigera respectively Visual examination revealed lesion sizes about 0.5 cm for spruce trees and 1 cm for lodgepole pine trees at inoculation sites at 2 weeks after inoculation with fungus. We observed some yellowing of needles around inoculation sites in lodgepole pine only at 6 weeks post-treatment. Lodgepole pine trees left until 18 weeks post-treatment had increased yellowing of needles which in some cases spread to the parts of tree above inoculation site and in some cases exhibited large visible lesions (up to 10 cm). Spruce trees left until 18 weeks post-inoculation did not have these symptoms. These observations suggested that fungal infection was successful in both species. Anatomical changes were assessed in stem cross sections of interior spruce and lodgepole pine inoculated with blue-stain fungus, L. abietinum and G. clavigera, respectively. Copper acetate stained stems cross sections were examined using light microscopy for 6 weeks after treatment. In untreated control interior spruce saplings, axial resin ducts were present in bark with very few scattered resin ducts in xylem (Figure 2.2A).

Figure 2.2: Light microscopy of stem cross sections of interior spruce treated with wounding or inoculated with Leptographium abietinum and lodgepole pine treated with wounding or inoculated with Grosmannia clavigera. A. Cross section of control interior spruce. B. Cross section of wounding treated interior spruce 2 weeks post-treatment. C. Cross section of L. abietinum treated interior spruce 2 weeks post- treatment. D. Cross section of control lodgepole pine. E. Cross section of wounding treated lodgepole pine 6 weeks post-treatment. F. Cross section of G. clavigera inoculated lodgepole pine 6 weeks post-treatment. G. Lodgepole pine constitutive resin ducts in untreated tree. H. Lodgepole pine traumatic resin ducts 6 weeks after inoculation with G. clavigera. I. Statistical analysis of the number of resin ducts in lodgepole pine untreated (control), treated with wounding and inoculated with G. clavigera 6 weeks post-treatment. Values represent mean  standard deviation (calculated using four biological replicates). Resin ducts are indicated by arrows.

43 Two weeks after wounding, few traumatic resin ducts had developed in the xylem of interior spruce (Figure 2.2B). A much stronger traumatic resin duct response was induced by L. abietinum (Figure 2.2C). A continuous ring of traumatic resin ducts was detectable by copper acetate that stained resin duct lumens filled with terpenoid resin. In control lodgepole pine constitutive axial resin ducts are present in the bark with a number of smaller constitutive resin ducts dispersed in xylem (Figure 2.2D). Formation of traumatic resin ducts was not detected in lodgepole pine xylem two weeks after wounding or fungal treatment (data not shown). Six weeks post-treatment, formation of new resin ducts was observed in G. clavigera treated lodgepole pine. The presence of constitutive resin ducts in pine xylem makes it difficult to detect formation of new resin ducts (Figure 2.2E and 2.2F). No changes were apparent in cross sections of lodgepole pine six weeks after wounding treatments (Figure 2.2E). Newly formed resin ducts were detected in the xylem of lodgepole pine due to the presence of new distinct patterns of the resin ducts distribution as compared to the distribution of constitutive resin ducts in control tissues (Figure 2.2F and 2.2H). Constitutive resin ducts in control xylem are evenly scattered in the xylem yearly rings (Figure 2.2D, G), whereas the additional formation of traumatic resin ducts in fungus treated lodgepole pine resulted in the formation of tangential stretches of connected resin ducts (Figure 2.2H). To quantify the resin duct response in lodgepole pine to wounding and fungal treatment we calculated the number of resin ducts per square unit in untreated lodgepole pine seedlings and in lodgepole pine seedlings six weeks after wounding treatment and fungal inoculation (Fig 2.2 I). The average number of resin ducts was slightly, but not statistically significantly, higher in wounded trees as compared to control tissues (p - 0.21). The number of resin ducts in lodgepole pine treated with fungus was significantly higher compared to control (p < 0.001) and to wounding (p - 0.002) indicating the presence of traumatic resin duct formation in the xylem of lodgepole pine seedling in response to G. clavigera inoculation. The formation of traumatic resin ducts in interior spruce and lodgepole pine in response to fungal inoculation, indicated distinct reaction of the seedlings of both species to fungal inoculation and confirmed effectiveness of used fungal inoculation procedure.

2.3.2 Terpenoid accumulation in the bark of interior spruce and lodgepole pine inoculated with blue-stain fungi The terpenoid content and composition of control, wound-treated and fungus- inoculated interior spruce and lodgepole pine bark (including bark, phloem and 44 cambium) at 6 hours, 2 days, 2 weeks and 6 weeks post-treatment was evaluated by GC/FID and GC/MS (Figure 2.3, Supplemental Table 2.2A, B).

70 160 60 A Control 140 B Control 50 Wounding 120 Wounding Fungus 100 Fungus 40 80 30

DiterpenoidsDiterpenoids Monoterpenoids

Monoterpenoids 20 40

mg/g tissue dry wtwt dry dry mg/gmg/g tissue tissue

mg/g tissue dry wtwt dry dry mg/gmg/g tissue tissue 10 20 0 0 6h 2d 2w 6w 6h 2d 2w 6w Time point Time point

Figure 2.3: Terpenoid accumulation in the bark of lodgepole pine untreated, treated with wounding and inoculated with G. clavigera. Terpenoids were analyzed in bark that was collected 6h, 2 days, 2 weeks and 6 weeks post-treatment. Values represent mean±SE from four samples per treatment per time point. A. Accumulation of monoterpenoids in lodgepole pine bark. B. Accumulation of diterpenoids in lodgepole pine bark.

An increase in monoterpenoids (Figure 2.3A, Supplemental Table 2.2B) and diterpenoids (Figure 2.3B, Supplemental Table 2.2B) was found in samples from G. clavigera treated lodgepole pine seedlings two weeks after treatment. Fungal inoculation with G. clavigera caused a 3.1-fold increase in the amount of monoterpenoids in lodgepole pine bark as compared to control tissues; and a 2.4-fold increase in the amount of lodgepole pine bark monoterpenoids as compared to tissues treated with wounding. Diterpenoid amounts in bark of lodgepole pine inoculated with G. clavigera increased 4.9-fold and 2.7-fold as compared to wounding treatment and untreated controls respectively, 6 weeks post treatment. Analysis of change in accumulation of monoterpenoids and diterpenoids in interior spruce inoculated with L. abietinum or wounded was not conclusive due to high variability of data in control and treated samples throughout the time course of the study (Supplemental Table 2.2A).

45 2.3.3 Inoculation of interior spruce and lodgepole pine with L. abietinum and G. clavigera respectively causes large changes in the transcriptome of both species

We performed comparative transcriptome analysis of the effect of L. abietinum and G. clavigera on interior spruce and lodgepole pine respectively. The performance of the spruce microarray was evaluated for its use in lodgepole pine studies (Appendix 2). In brief, 80% of elements on the spruce microarray yielded reliable data in hybridizations with lodgepole pine. A time course study was designed based on previous work with white spruce (Picea glauca) seedlings where inoculations with L. abietinum caused wilt symptoms 6 to 28 days after inoculation (Ohsawa et al., 2000). With lodgepole pine seedlings, yellowing of needles was observed 2-3 weeks after inoculation with G. clavigera. To observe the dynamics of the transcriptional response, comparative bark transcriptome analysis of host trees was performed with samples harvested 6h, 2d and 2 weeks after fungal inoculation. In addition to no-treatment controls, wounding treatment was used to test fungus-specific effects. Identical hybridization designs were used for the expression studies in both conifer species (Figure 2.1). Genes were considered differentially expressed if transcript abundance exhibited more than 2x fold change with p < 0.05 and q < 0.05 (Storey and Tibshirani, 2003). A complete list of microarray results is available in Supplemental Table 2.3. Only 11 transcripts were differentially expressed in untreated lodgepole pine over the time course. Of the 144 transcripts that were differentially expressed in untreated interior spruce over the time course (Supplemental Table 2.3), none were differentially expressed in the comparison of samples collected at 2 days and 2 weeks. Most differentially expressed transcripts (121) in untreated interior spruce were identified in comparison of samples collected at 6h with those collected at 2 days. In untreated lodgepole pine 10 of the 11 differentially expressed transcripts were detected in comparison of samples collected at 6 h and at 2 weeks. Some of these changes in expression in untreated trees may be associated with diurnal rhythms. Substantial transcriptome changes were induced by fungal inoculation of interior spruce and lodgepole pine (Table 2.1).

Table 2.1: Total numbers of differentially expressed transcripts in lodgepole pine induced by G. clavigera, and in interior spruce induced by L. abietinum Interior spruce Common Common Common W/C W/C and F/C F/C F/C and F/W F/W W/C and F/W Time point up down up down up down up down up down up down

6h 589 53 520 28 884 65 49 0 60 1 21 0

2d 343 77 325 74 858 566 216 78 222 100 120 6

2w 0 0 0 0 61 10 1 0 1 0 0 0

Lodgepole pine Common Common Common W/C W/C and F/C F/C F/C and F/W F/W W/C and F/W Time point up down up down up down up down up down up down

6h 468 36 399 13 491 23 1 0 1 0 0 0

2d 93 1 89 1 857 350 363 50 383 65 14 0

2w 13 1 13 1 675 275 300 65 320 78 3 0

Overlap between interior spruce and lodgepole pine W/C F/C F/W Time point up down up down up down

6h 235 4 302 1 0 0

2d 53 0 378 91 105 10

2w 0 0 38 1 1 0 C-control, W-wounding, F-fungal treatment. Numbers in table indicate number of genes with fold change >2, p<0.05, q<0.05

The total number of differentially expressed transcripts over the entire time course in interior spruce was 2052 and in lodgepole pine was 1868 (Supplemental Table 2.3). In interior spruce inoculated with L. abietinum, 884 transcripts were upregulated already at 6h in fungal treatment vs. control. Similarly, expression of 858 transcripts was upregulated in fungus treated interior spruce bark at 2 days post inoculation with a significant decrease in response at 2 weeks post inoculation (61 transcripts significantly induced). The response of lodgepole pine to G. clavigera inoculation was quite strong at 6h post-treatment with 491 transcripts significantly upregulated in fungal treatment vs. control treatment comparison, it reached a peak at 2 days post inoculation (857

47 transcripts significantly induced) and remained strong at 2 weeks post-inoculation (675 transcripts significantly induced). In both species, the bark transcriptome response to wounding was significant at 6 hours post-treatment with 589 transcripts significantly induced in interior spruce and 468 transcripts significantly induced in lodgepole pine and decreased afterwards. A number of transcripts induced by both wounding and fungal treatment compared to control was similar to the number of transcripts induced by wounding in both species at 6h of treatment (Table 2.1). This indicates that the response to fungal treatment includes most of the response to wounding treatment, and additional transcripts, which have significant expression in comparison of fungal treatment to wounding. The transcriptome response of interior spruce and lodgepole pine bark to fungal inoculation or to wounding includes more significantly upregulated genes than downregulated genes (Table 2.1). Comparison of fungal treatment to wounding treatment allowed the elucidation of the fungus-specific transcriptome response, including over two hundred transcripts specifically induced by fungal treatment in interior spruce and close to 400 transcripts in lodgepole pine at the point of strongest response – 2 d post-treatment. In order to evaluate the number of unique transcripts induced by fungal treatment, we calculated the overlap of transcripts differentially expressed in wounding vs. control comparison and fungus vs. wounding comparison (Table 2.1). In interior spruce bark 21 transcripts were induced only by fungus among 60 transcripts induced by fungus compared to wounding at 6h and 120 transcripts among 222 transcripts at 2 d of treatment (Table 2.1, Supplemental Table 2.3). This indicates that there are a number of transcripts induced by wounding treatment that are further induced by fungal treatment. In lodgepole pine, very few transcripts induced in the fungus vs. wounding comparison (14 at 2d and 3 at 2w, Table 2.1) were also induced by wounding vs. control, indicating a presence of fungus-specific response. Comparison of transcriptome responses to fungal and wounding treatments in interior spruce and lodgepole pine bark revealed that about 50% of the induced response was the same in most post-treatment time points especially at time points with strong response, and much fewer downregulated transcripts were consistent between interior spruce and lodgepole pine (Table 2.1).

48 2.3.4 Cluster analysis of differentially expressed transcripts induced by wounding or fungal inoculation in interior spruce and lodgepole pine A cluster analysis was performed to identify overall temporal patterns of wound- or fungus-induced transcripts in lodgepole pine and interior spruce. There are 27 possible clusters of response dynamics based on upregulation (U), downregulation (D) or no significant response (-) over three time points for each transcript. However, only few of the possible clusters were realised (Figure 2.4, Supplemental Table 2.4) since certain clusters are more biologically relevant than the others.

Figure 2.4: Clustering of transcripts based on the pattern of expression through the time course of 6h, 2 days (2d) and 2 weeks (2w) for white spruce and lodgepole pine treated with wounding and inoculated with the respective fungus. The line passes through mean expression calculated on the logarithmic scale. Thickness of each line corresponds to the square root of the number of genes in each group shown (the numbers of transcripts are shown for each in the right bottom corner of each graph). IS – interior spruce, LP – lodgepole pine, C – control, W- wounding, F – fungal treatment.

Figure 2.4

50 Analysis of the dynamics patterns demonstrated that most of the genes induced by wounding in interior spruce and lodgepole pine are up-regulated as early as 6 hours (cluster U--) and expression of these genes returns to baseline level at 2 days. The magnitude of response to wounding at 6 hours in both species is similar, with 419 transcripts induced in interior spruce bark and 418 transcripts induced in lodgepole pine. Lodgepole pine and interior spruce have 170 differentially expressed transcripts in common in the (U--) clusters. In interior spruce, more transcripts were induced by fungus vs. wounding (51) in the (U--) cluster compared to only one transcript in lodgepole pine at 6h of treatment. The lodgepole pine response to fungus was activated mostly at 2 days to 2 weeks (-U-), (-UU), (--U). Only 32 transcripts overlap in the cluster (-U-) in two species of the 212 induced transcripts in interior spruce and of the 240 induced transcripts in lodgepole pine. A lower overlap of the response as compared to the overlap observed in the overall changes (Table 2.1) for genes induced at 2 days post inoculation indicates the difference in the dynamics of the responses. The lodgepole pine response included 143 genes induced in fungus vs. wounding comparison at 2 days as well as at 2 weeks (cluster –UU) and 177 genes significantly induced only at 2 weeks (cluster--U) post-inoculation, whereas interior spruce had only one gene with a fungus specific response at 2 weeks. The dynamics of downregulation of expression had some similarities in both species with the difference of downregulation of transcripts in wounding treatment compared to control in cluster (-D-) and in fungal treatment compared to wounding in cluster (--D).

2.3.5 Strongly induced transcripts in interior spruce and lodgepole pine by wounding and L. abietinum and wounding and G. clavigera respectively The most strongly induced transcripts in by fungal treatment compared to wounding in interior spruce and lodgepole pine are shown in (Table 2.2), and (Table 2.3) respectively.

Table 2.2: Most highly induced transcripts by L. abietinum compared to wounding at different time points in interior spruce bark. C-control, W-wounding, F-fungal treatment. Dynamic of expression is shown as three sign sequences indicating change in expression at 6h, 2 days and 2 weeks, U – upregulated, (-) – no change, D – downregulated transcript expression. *- values marked with asterisk indicate transcript fold changes that did not meet the criteria of differentially expressed genes. All not marked fold change numbers in the table indicate fold change of differentially expressed transcripts with fold change > 2 and p,q values < 0.05. Clone ID E-value AGI BLASTX vs. Arabidopsis IS IS IS IS IS LP LP F/W F/C W/C F/W W/C F/W W/C 6hours WS00914_E10 n.a. No significant hit 20.00 5.87 0.31 U-- D------WS0101_I09 n.a. No significant hit 11.11 4.89 0.42* U------WS00933_K09 1E-14 At1g12060 Apoptosis regulator Bcl-2 protein 9.09 3.95 0.44* U------WS00923_H21 n.a. No significant hit 7.69 1.71* 0.22 U-- D-- --- U-- WS0013_B21 3E-12 At2g20550 DnaJ-like protein 5.00 1.72* 0.34 U-- D------WS02612_E01 5E-21 At5g47830 expressed protein 4.00 2.18 0.54* U------WS0064_B23 5E-54 At3g25800 serine/threonine protein phosphatase 2A 4.00 1.14* 0.28 U-- D------WS00926_L05 n.a. No significant hit 3.57 2.15 0.61* U------WS0093_N10 n.a. No significant hit 3.57 2.37 0.67* U------WS00924_A13 2E-69 At5g64370 beta-alanine synthase 3.45 3.35 0.98* U------WS0078_H18 n.a. No significant hit 3.45 3.37 0.98* U------WS00924_K06 n.a. No significant hit 3.23 12.97 4.02 UU- U-- -U- U-- WS0062_O09 2E-51 At3g24500 ethylene-responsive transcriptional 3.23 0.89* 0.27 U-- D------coactivator WS01030_L05 7E-45 At3g19950 Zinc finger, C3HC4 type 3.23 1.25* 0.39 U-- D------WS00915_E23 n.a. No significant hit 3.03 1.97* 0.65* U------WS01018_L23 n.a. No significant hit 3.03 7.07 2.34 U-- U-- --- U-- WS0264_K16 2E-09 At1g23980 Zinc finger, C3HC4 type 2.94 2.44 0.82* U------U --- WS00940_L04 n.a. No significant hit 2.86 14.06 4.95 UU- U-- -U- U-- WS00911_C21 7E-65 At3g13110 serine O-acetyltransferase (SAT-1) 2.86 2.57 0.89* U------WS00918_I17 1E-11 At1g28190 expressed protein 2.86 3.74 1.33* U------IS0011_M10 n.a. No significant hit 2.86 1.15* 0.40 U-- D------WS00819_L21 n.a. No significant hit 2.70 3.21 1.18* U------WS0013_C09 n.a. No significant hit 2.63 4.72 1.78* U------WS00836_G19 n.a. No significant hit 2.63 2.89 1.10* U------WS0045_F06 n.a. No significant hit 2.56 3.66 1.42* U------52

Table 2.2 (cont): Most highly induced transcripts by L. abietinum compared to wounding at different time points in interior spruce bark. Clone ID E-value AGI BLASTX vs. Arabidopsis IS IS IS IS IS LP LP F/W F/C W/C F/W W/C F/W W/C 2 days WS0091_I07 n.a. No significant hit 8.33 18.63 2.21 -U- UU------WS01027_K23 n.a. No significant hit 6.67 14.21 2.14 -U- UU- -UU --- WS0063_E19 n.a. No significant hit 6.67 27.79 4.07 -U- UU- -U- --- WS00811_N22 n.a. No significant hit 6.25 14.93 2.44 -U- -U------WS00924_F07 2E-68 At2g43590 chitinase 6.25 35.97 5.58 -U- -U- --U --- WS0063_D22 n.a. No significant hit 5.88 22.86 3.81 -U- -U- -UU --- WS00917_K11 n.a. No significant hit 5.88 16.23 2.8 -U- UU- -UU U-- WS00715_B10 2E-43 At1g19320 thaumatin-like protein 5.88 25.24 4.17 -U- -U- -UU --- WS01013_N08 3E-24 At2g39980 PF02458 transferase 5.56 6.84 1.24* -U- --- -U- --- WS0064_G14 n.a. No significant hit 5.56 18.91 3.36 -U- -U- -UU --- WS0071_H13 3E-37 At5g54160 caffeic acid O-methyltransferase like 5.56 13.55 2.43 -U- -U- -UU --- WS00713_G19 2E-12 At3g04720 hevein-like protein 5.26 30.14 5.74 -U- -U- -UU --- WS00923_J11 n.a. No significant hit 5.26 15.44 2.87 -U- -U- -UU D-- WS0104_J07 1E-22 At5g54160 caffeic acid O-methyltransferase like 5.26 9.59 1.82* -U- --- -U- --- WS0064_H09 3E-52 At1g77120 alcohol dehydrogenase (ADH) 5.26 16.04 3.02 -U- UU- -U- --- WS00934_O14 n.a. No significant hit 4.76 3.27 0.7* -U- --- -UU --- WS00921_G15 6E-36 At1g03230 extracellular dermal glycoprotein 4.76 20.33 4.18 -U- -U------WS01028_A20 n.a. No significant hit 4.76 12.78 2.64 -U- -U- -U- --- WS01035_I24 3E-31 At1g61720 dihydroflavonol 4-reductase 4.55 19.77 4.39 -U- -U- --U --- WS0043_N10 n.a. No significant hit 4.35 1.63* 0.38 -U- -D- -UU --- WS00912_L05 1E-13 At1g74950 expressed protein 4.35 21.14 4.95 -U- UU- -UU UU- WS00931_D04 4E-56 At4g11650 osmotin-like protein 4.17 31.75 7.7 -U- -U- -UU --- WS01035_C15 n.a. No significant hit 4.17 34.63 8.33 -U- -U- -UU --- WS0262_F02 9E-17 At4g22710 cytochrome P450 4.17 10.7 2.6 -U- -U------IS0012_L15 4E-24 At4g22710 cytochrome P450 4.17 35.29 8.42 -U- -U------2 weeks WS0048_G23 7E-55 At4g11650 osmotin-like protein 3.84 3.62 0.95* -UU -U- -UU -U-

Table 2.3: Most highly induced transcripts by G. clavigera compared to wounding at different time points in lodgepole pine bark. C-control, W-wounding, F-fungal treatment. Dynamic of expression is shown as three sign sequences indicating change in expression at 6h, 2 days and 2 weeks, U – upregulated, (-) – no change, D – downregulated transcript expression. *- values marked with asterisk indicate transcript fold changes that did not meet the criteria of differentially expressed genes. All not marked fold change numbers in the table indicate fold change of differentially expressed transcripts with fold change > 2 and p,q values < 0.05 Clone ID E-value AGI BLASTX vs. Arabidopsis LP LP LP LP LP IS IS F/W F/C W/C F/W W/C F/W W/C 6 hours WS00924_D22 5E-77 At1g26910 60S ribosomal protein L10 (RPL10B) 2.17 2.33 1.08* U------2 days WS00934_O14 n.a. No significant hit 14.29 19.97 1.34* -UU --- -U- --- WS0043_N10 n.a. No significant hit 14.29 19.08 1.30* -UU --- -U- -D- WS0045_H07 n.a. No significant hit 10.00 16.74 1.75* -UU ------U- WS01040_C03 7E-06 At1g48800 terpene synthase 10.00 10.79 1.12* -UU ------WS01012_J14 2E-06 At1g02820 late embryogenesis abundant protein 10.00 19.69 1.99* -U- U-- -U- U-- WS00926_A07 2E-76 At3g48850 mitochondrial phosphate transporter 10.00 14.14 1.47* -U- U-- --- U-- WS00931_D04 4E-56 At4g11650 osmotin-like protein 9.09 21.31 2.32* -UU --- -U- -U- WS0102_K16 n.a. No significant hit 9.09 15.03 1.62* -UU --- -U- -U- WS00934_P05 2E-22 At2g31230 ethylene-responsive factor 9.09 11.98 1.28* -UU --- -U- --- WS00917_K11 n.a. No significant hit 8.33 29.01 3.37* -UU U-- -U- UU- WS0018_O07 2E-13 At2g43590 chitinase 8.33 6.54 0.79* -UU --- -U- -U- WS00715_B10 2E-43 At1g19320 thaumatin 7.69 16.01 2.11* -UU --- -U- -U- WS00913_L16 4E-21 At4g36470 SAM:jasmonic acid carboxyl methyltransferase 7.69 7.91 1.05* -UU ------WS00111_I09 3E-22 At1g77330 ACC(JMT) oxidase 7.69 8.86 1.13* -U- --- -U- UU- WS00930_G18 5E-59 At3g54420 chitinase (classIV) 7.14 21.63 3.00* -UU --- -U- -U- WS0071_H13 3E-37 At5g54160 caffeic acid O-methyltransferase like 7.14 14.77 2.04* -UU --- -U- -U- WS0048_G23 7E-55 At4g11650 osmotin-like protein (OSM34) 6.67 26.60 4.00 -UU -U- -UU -U- WS01041_D19 6E-29 At4g36470 SAM:jasmonic acid carboxyl methyltransferase 6.67 9.16 1.38* -UU ------WS00923_I02 1E-62 At4g11650 osmotin(JMT) -like protein (OSM34) 6.67 8.75 1.36* -UU ------U- WS00916_D08 n.a. No significant hit 6.67 9.59 1.42* -UU ------U- WS01033_J22 6E-10 At1g70080 terpene synthase 6.67 8.42 1.29* -UU ------WS0063_L03 1E-65 At3g25420 serine carboxypeptidase S10 6.25 10.36 1.66* -UU --- -U- -U- WS00917_O06 n.a. No significant hit 6.25 7.75 1.22* -UU U------

54 WS00932_P22 4E-53 At5g01210 transferase PF02458 6.25 10.83 1.75* -UU --- -U- U--

WS0092_L20 5E-50 At2g43590 chitinase 6.25 7.92 1.28* -UU ------U-

Table 2.3 (cont): Most highly induced transcripts by G. clavigera compared to wounding at different time points in lodgepole pine bark. Clone ID E-value AGI BLASTX vs. Arabidopsis LP LP LP LP LP IS IS F/W F/C W/C F/W W/C F/W W/C 2 weeks WS00930_G18 5E-59 At3g54420 chitinase (class IV) 33.33 40.97 1.42* -UU --- -U- -U- WS0063_L03 1E-65 At3g25420 serine carboxypeptidase S10 20.00 25.64 1.35* -UU --- -U- -U- WS00712_K20 7E-53 At3g54420 chitinase (class IV) 16.67 23.13 1.38* -UU --- -U- -U- WS0064_M21 2E-38 At1g05680 UDP-glucoronosyl/UDP-glucosyl transferase 16.67 16.55 1.07* -UU --- -U- -U- WS00930_B11 6E-44 At1g47980 expressed protein 16.67 46.06 2.83* --U ------WS00931_D04 4E-56 At4g11650 osmotin-like protein (OSM34) 14.29 33.75 2.30* -UU --- -U- -U- WS0048_G23 7E-55 At4g11650 osmotin-like protein (OSM34) 12.50 35.71 2.72* -UU -U- -UU -U- WS00937_E11 n.a. No significant hit 12.50 17.42 1.32* -UU --- -U- -U- WS0061_A12 n.a. No significant hit 12.50 16.34 1.34* -UU --- -U- -U- WS00927_N15 2E-95 At3g12500 endochitinase 12.50 20.18 1.57* -UU UU- --- -U- WS0046_G17 n.a. No significant hit 12.50 20.26 1.57* --U UU- --- -U- WS0102_K16 n.a. No significant hit 11.11 26.55 2.27* -UU --- -U- -U- WS0046_M24 7E-38 At3g12500 endochitinase 11.11 14.91 1.34* -UU UU- --- UU- WS00923_O16 n.a. No significant hit 11.11 19.03 1.68* --U ------WS00716_E14 4E-07 At2g43610 chitinase (class I) 10.00 9.79 1.00* -UU --- -U- --- WS00926_I24 1E-23 At5g48540 secretory protein-related 10.00 16.26 1.70* -UU ------U- WS0045_H07 n.a. No significant hit 10.00 21.25 2.08* -UU ------U- WS01028_O15 1E-42 At1g71695 peroxidase 12 (PER12) 10.00 13.62 1.37* --U -U- --- UU- WS00922_B21 2E-88 At3g12500 endochitinase 10.00 15.54 1.62* --U UU- --- -U- WS00924_F07 2E-68 At2g43590 chitinase 9.09 12.84 1.41* --U --- -U- -U- WS00715_B10 2E-43 At1g19320 thaumatin 9.09 11.99 1.36* -UU --- -U- -U- WS00930_M06 2E-26 At1g73050 (R)-mandelonitrile lyase 9.09 53.5 5.82 --U U-U -U- UU- WS00113_G22 1E-34 At1g73050 (R)-mandelonitrile lyase 9.09 39.76 4.45* -UU U-- -U- UU- WS00929_K15 2E-78 At3g12500 endochitinase 9.09 15.27 1.63* --U UU- --- -U- WS00913_L16 4E-21 At4g36470 SAM:jasmonic acid carboxyl methyltransferase 9.09 14.35 1.51* -UU ------

(JMT)

The tables are organized to allow a comparison of the effect of wounding and fungal treatment at different time points within each species and between the two species using the dynamic group identification based on the patterns of expression as discussed above. These tables point to the limitation of conifer gene annotation, however they still allow the initial analysis of interior spruce and lodgepole pine responses to fungal inoculation. Most transcripts that were up-regulated by the fungal treatment as compared to wounding in interior spruce at 6 hr returned to base level at 2d and 2 weeks of treatment and included transcripts putatively involved in signalling (Table 2.2). Only one transcript was responsive to fungal treatment as compared to wounding in lodgepole pine at 6 hr post-treatment. Most of the genes highly induced by the fungus compared to wounding were induced in both species at 2 days post-treatment. The highly induced transcripts in interior spruce and lodgepole pine at 2 days post-treatment included PR proteins such as chitinases, thaumatin or thaumatin-like proteins, osmotin-like proteins (Table 2.2 and Table 2.3). The highly induced PR proteins transcripts by fungal treatment compared to wounding treatment in interior spruce were also induced by wounding treatment, whereas only one of these transcripts was induced by wounding treatment in lodgepole pine. Another category of transcripts highly induced by fungal treatment as compared to wounding in both species included representatives of the phenylpropanoid pathway (Table 2.2, 2.3). The list of highly induced transcripts in lodgepole pine also included terpene synthases (that were not differentially expressed in interior spruce) and representatives of signaling pathways. The overall response of interior spruce to wounding treatment and fungal inoculation returned to control levels 2 weeks post- treatment, with only one gene, the osmotin-like protein, being significantly induced by fungal treatment compared to wounding treatment (Table 2.2). This gene was also highly induced in lodgepole pine (Table 2.3). In fungus-induced lodgepole pine, chitinase transcripts were highly represented in the most highly induced transcripts (7 out of 25, in comparison of fungal treatment with wounding) at 2 weeks of treatment and these transcripts were also induced in interior spruce by either wounding or fungal treatment or both at 2 days post-treatment (Table 2.3). Many of the same categories of transcripts were among most downregulated transcripts by fungal treatment in both species. Many of the downregulated transcripts represented primary metabolism, including photosynthesis (Supplemental Table 2.3). Overall, the extent of downregulation evaluated by the fold change of expression between fungal treated and control tissues or fungal treated and wounding treated 56 tissues was much lower as compared to induced genes. The highest fold change observed in downregulated expression was 5 fold, with most values being close to 2 fold as compared to values reaching 10-40 fold in the case of induced genes (Table 2.2, Table 2.3, Supplemental Table 2.3).

2.3.6 Functional categorization of interior spruce and lodgepole pine bark transcriptome response to fungal inoculation with L. abietinum and G. clavigera respectively The available functional annotation for conifer species is very limited and often inferred from angiosperm annotations. In order to gain additional insight into functional characterization of interior spruce and lodgepole pine transcriptome responses to fungal inoculation as compared to wounding and control treatment, we used available annotation and functional characterization from the Arabidopsis TAIR database (TAIR; http://www.arabidopsis.org) and the available annotation from conifer specific functional analysis of selected pathways developed in our inventory (Ralph and Bohlmann, unpublished results). The transcriptome response of interior spruce and lodgepole pine bark was characterized by segregating differentially expressed transcripts into functional groups using relevant Arabidopsis GO (Gene Ontology) categories (http://www.arabidopsis.org) and selected conifer annotated pathways (Table 2.4a,b; Supplemental Table 2.5).

Table 2.4: Functional annotation of differentially expressed transcripts A. Upregulated transcripts IS LP Functional category1 W/C F/C F/W W/C F/C F/W GO:Response to abiotic and biotic stimulus (875)2 63 108 17 41 121 60 GO:Defense response (336) 34 50 13 33 61 28 *Phenylpropanoid pathway (340) 62 96 20 49 118 38 *Terpenoid pathway (82) 18 17 3 3 25 8 GO:Jasmonic acid biosynthesis and response (78) 15 26 1 16 28 17 GO:Ethylene biosynthesis and response (36) 9 14 4 8 15 11 GO:Salicylic acid biosynthesis and response (34) 3 5 4 0 4 4 GO:Primary metabolism (3948) 172 332 48 130 347 115 GO:Electron transport and energy pathways (417) 26 51 8 20 42 18 GO:Photosynthesis (58) - 1 - - - -

B. Down-regulated transcripts IS LP Functional category1 W/C F/C F/W W/C F/C F/W GO:Response to abiotic and biotic stimulus (875) 19 40 2 2 52 13 GO:Defense response (336) 1 4 1 - 12 5 *Phenylpropanoid pathway (340) 1 21 4 - 2 - *Terpenoid pathway (82) - 1 - - - - GO:Jasmonic acid biosynthesis and response (78) - - - - 3 - GO:Ethylene biosynthesis and response (36) 1 - - - - - GO:Salicylic acid biosynthesis and response (34) ------GO:Primary metabolism (3948) 21 91 11 5 99 28 GO:Electron transport and energy pathways (417) 7 18 1 1 12 6 GO:Photosynthesis (58) 4 17 1 2 14 4 C-control, W-wounding, F-fungal treatment. 1- Functional categories marked with GO included spruce microarray transcripts annotated to functionally characterized Arabidopsis genes to respective GO category in TAIR database (TAIR; http://www.arabidopsis.org), *- functional categories marked with an asterisk were assembled based on characterization of conifer secondary metabolism by Ralph and Bohlmann (unpublished data). 2 Numbers in paranthesis indicate the number of transcripts from a given functional category on the spruce microarray.

The analysis of the GO category response to biotic and abiotic stimulus revealed a number of transcripts that are induced by fungal treatment as compared to control, with fewer transcripts induced by wounding treatment as compared to control and by fungal treatment as compared to wounding in interior spruce (Table 2.4A, Supplemental Table 2.3, 2.5). More genes were specifically induced by fungal treatment compared to wounding in lodgepole pine as compared to interior spruce (Table 2.4A, Supplemental Table 2.3, 2.5). Most induced transcripts by fungal treatment compared to control in both species included PR proteins, transcripts from phenylpropanoid pathway and ethylene signalling. Several PR protein transcripts were specifically induced by fungal treatment compared to wounding in both species. Many of the downregulated transcripts from this category in both species were genes involved in primary metabolism (Table 2.4B, Supplemental Table 2.3, 2.5). Evaluating the GO category of defense response is of particular interest due to transcripts in this category being not only “triggered in response to the presence of a foreign body or the occurrence of an injury,” but also with this expression change resulting in “restriction of damage to the organism attacked or prevention/recovery from the infection caused by the attack” (TAIR, http://www.arabidopsis.org). A fairly high number of transcripts from this category were induced in interior spruce and lodgepole pine by fungal treatment with very few representatives being downregulated in both species (Table 2.4A, B). Most strongly induced transcripts in both species by fungal treatement compared to control included phenylalanine ammonia-lyase 1 (PAL1), dirigent proteins, allene oxide synthase (AOS), lipoxygenase, several PR proteins, with chitinases being among the most highly induced transcripts, and transcript annotated to (R)-mandelonitrile lyase (Supplemental Table 2.3, 2.5). Most of these transcripts were also induced by fungal treatement compared to wounding. To evaluate the involvement of three plant signaling pathways in the interior spruce and lodgepole pine response to wounding and fungal inoculation, we examined differentially expressed transcripts from the GO categories of jasmonic acid, salicylic and ethylene biosynthesis and response. A number of genes from the jasmonate and ethylene signaling and response categories were induced by wounding and fungal treatment in interior spruce and lodgepole pine. Few transcripts from salicylic acid signaling and response were induced by wounding and fungal treatment in interior spruce and induced by fungal treatment in lodgepole pine (Table 2.4A). Very few transcripts associated with the signaling pathways were downregulated (Table 2.4B).

Among 78 transcripts associated with jasmonic acid biosynthesis and response, 26 transcripts were induced by fungal treatment compared to control in interior spruce and 22 of these transcripts were also induced in lodgepole pine. Many of these transcripts were also induced by wounding in interior spruce and the induction was more fungus specific in lodgepole pine. Some of the induced transcripts in this category in both species included transcripts putatively involved in jasmonate acid biosynthesis such as 12-oxophytodienoate reductases (OPR1, OPR2 and OPR3), allene oxide synthase (AOS), lipoxygenase. Among 36 transcripts from the GO category ethylene biosynthesis and response present on the microarray, 14 transcripts were induced by fungal treatment compared to control in interior spruce and 13 of these transcripts were also induced in lodgepole pine (Supplemental Table 2.3, 2.5). Induction of more of these transcripts was fungus specific in lodgepole pine. The transcripts in these category induced by fungal treatment in both species included 1-aminocyclopropane-1-carboxylate synthase (ACC) synthases and ACC oxidase, with ACC synthase being only induced by fungal treatment and not wounding in both species. Among 34 transcripts from the salicylic acid biosynthesis and response category present on the microarray, 5 transcripts were induced in interior spruce and 3 of these transcripts were also induced in lodgepole pine. None of the induced transcripts were annotated as involved in salicylic acid biosynthesis and included transcripts known to respond to salicylic acid. Thus, this data gives only indirect indication of possible limited involvement of salicylic acid signaling in interior spruce and lodgepole pine response to fungal treatment. We evaluated the effect of fungal inoculation on transcripts annotated to GO category: primary metabolism. Among 3948 transcripts annotated to primary metabolism present on the microarray, a large number of transcripts were induced by fungal treatment compared to control in interior spruce (332) and lodgepole pine (347), with 187 of these transcripts induced in both species (Table 2.4a). Smaller number of transcripts was induced by wounding treatment compared to control in both species. A number of transcripts were downregulated by fungal treatment and a much smaller number of genes were downregulated by wounding treatment in both species (Table 2.4a). Analysis of the GO category: electron transport and energy pathways revealed the induction of 51 transcripts in interior spruce and 42 transcripts in lodgepole pine by fungal treatment with 21 transcripts induced in both species (Table 2.4A, Supplemental Table 2.3, 2.5). Fewer transcripts in this category were downregulated by fungal 60 treatment in both species and most of these transcripts were annotated as involved in photosynthesis (Table 2.4b, Supplemental Table 2.3, 2.5). Seventeen transcripts in this category were downregulated in interior spruce and 14 were downregulated in lodgepole pine by fungal treatment (Table 2.4b, Supplemental Table 2.3, 2.5). Few transcripts were downregulated by wounding treatment in both species. Only one transcript of was induced in interior spruce by fungal treatment as compared to control with no transcripts being induced in lodgepole pine.

2.3.7 Induction of phenylpropanoid in interior spruce and lodgepole pine The phenylpropanoid pathway begins with the conversion of phenylalanine to trans-cinnamic acid catalyzed by the enzyme L-phenylalanine ammonia-lyase (PAL). A complex metabolic grid proceeds to the formation of diverse phenylpropanoids, including monolignols/lignin, coumarins, benzoic acid, stilbenes and flavonoids/isoflavonoids (Dixon et al., 2002). Based on the strong induction of members of the phenylpropanoid pathway in interior spruce and lodgepole pine by wounding and fungal treatments, we examined the effect of these treatments on all phenylpropanoid transcripts present on the 21K array based on annotation of conifer ESTs developed by Ralph and Bohlmann (unpublished results). Among 3124 ESTs annotated to phenylpropanoid pathway, 340 were represented on the 21K spruce microarray (Supplemental Table 2.5). A high number of the transcripts from phenylpropanoid pathway was induced in interior spruce by wounding (62) and fungal treatment compared to control (96) with a number of genes (20) induced by fungal treatment compared to wounding treatment (Table 2.4A). Similar high numbers of phenylpropanoid pathway transcripts were induced in lodgepole pine by fungal (118) and wounding (49) treatment compared to control and by fungal treatment compared to wounding (38) (Table 2.4A). Many fewer transcripts from the phenylpropanoid pathway were downregulated by fungal treatment in both species (Table 2.4B). Among 340 spruce microarray transcripts annotated to the phenylpropanoid pathway 104 transcripts had significant change in expression (more than 2 fold, p,q<0.05) by wounding vs. control or fungus vs. wounding at any of the three time points (6 h, 2 days and 2 weeks) in either interior spruce or lodgepole pine or both. This subset of 104 genes allowed us to examine all transcriptional changes occurring in the phenylpropanoid pathway induced by wounding and fungal treatment in both species. To visualize expression data for the phenylpropanoid pathway, we used heat maps that were organized using hierarchical cluster analysis with average linkage using TIGR MeV 61 software version 3.1 (Saeed et al., 2003). Among 104 differentially expressed phenylpropanoid pathway transcripts, 38 were differentially expressed in both species induced either by wounding or specifically by fungus at one or more time points (Figure 2.5A), 34 transcripts were differentially expressed only in interior spruce (Figure 2.5B) and 32 transcripts were differentially expressed only in lodgepole pine (Figure 2.5C). All of the differentially expressed transcripts in both species (Figure 2.5A) are induced and none are downregulated. The heat map of these transcripts demonstrates the induction of most of the members of this group by wounding treatment compared to control and expression of many of the genes is further induced by fungal treatment in both species. The transcripts induced in both species include most of highly induced transcripts as compared to transcripts induced in each species separately (Figure 2.5A, Table 2.5).

Fold change (log2)

-2.0 0.0 2.0

.C6h .C2d .C2w

.C6h .C2d .C2w .C6h .C2d .C2w

.C6h .C2d .C2w

.C6h .C2d .C2w .C6h .C2d .C2w

.W6h .W2d .W2w

.W6h .W2d .W2w .W6h .W2d .W2w

.W6h .W2d .W2w

.W2d .W2w .W6h .W2d .W2w

A .W6h B C

isWvs isWvs isWvs isFvs isFvs isFvs lpWvs lpWvs lpWvs lpFvs lpFvs lpFvs

isWvs isWvs isWvs isFvs isFvs isFvs lpWvs lpWvs lpWvs lpFvs lpFvs lpFvs isWvs isWvs isWvs isFvs isFvs isFvs lpWvs lpWvs lpWvs lpFvs lpFvs lpFvs

Figure 2.5: Expression of transcripts annotated to phenylpropanoid pathway in interior spruce and lodgepole pine untreated and treated with wounding and inoculated with corresponding fungus. Transcripts with significant fold change (fold change >2, p,q<0.05) in at least one species and one treatment were clustered. A. Phenylpropanoid pathway transcripts with significant expression changes in at

63 least one treatment one time point in interior spruce and lodgepole pine. B. Phenylpropanoid pathway transcripts with significant expression

changes in at least one treatment one time point only in interior spruce. C. Phenylpropanoid pathway transcripts with significant expression changes in at least one treatment one time point only in lodgepole pine.

Table 2.5: Expression of phenylpropanoid pathway transcripts with >5 fold change in at least one species one treatment and q<0.05 IS IS IS IS IS IS IS IS IS LP LP LP LP LP LP LP LP LP W/C W/C W/C F/C F/C F/C F/W F/W F/W W/C W/C W/C F/C F/C F/C F/W F/W F/W Clone ID Annotation E value 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w WS00914_H24 DIRa 8E-22 17.9 9.5 1.7 21.6 21.0 3.3 1.2 2.2 1.9 4.2 21.9 9.7 2.8 33.8 9.8 0.7 1.5 1.0 WS01032_M02 DIRa 1E-117 17.5 6.9 1.8 18.9 16.9 3.2 1.1 2.4 1.8 2.9 15.7 6.9 1.8 29.8 6.9 0.6 1.9 1.0 WS00825_K05 DIRa 1E-132 8.5 3.6 1.1 9.8 7.5 1.6 1.1 2.1 1.4 2.5 7.6 3.7 2.0 11.9 3.4 0.8 1.6 0.9 WS01011_J07 DIRa 4E-48 4.7 2.6 1.1 6.3 6.3 1.9 1.4 2.4 1.7 2.9 17.5 6.8 2.0 30.3 6.6 0.7 1.7 1.0 WS0071_H13 COMT like 3E-45 1.1 2.4 2.0 1.1 13.6 2.0 1.0 5.6 1.0 0.6 2.0 0.9 0.7 14.8 3.7 1.1 7.1 4.2 WS0104_J07 COMT like 2E-22 1.2 1.8 1.6 1.2 9.6 1.5 1.0 5.3 1.0 0.8 1.3 0.9 0.9 4.6 1.7 1.0 3.7 1.9 WS00923_N05 LMCO 0 9.7 3.3 1.6 9.7 5.0 1.3 1.0 1.5 0.8 7.5 6.1 4.4 5.9 6.8 6.8 0.8 1.1 1.6 WS00730_B15 LMCO 2E-79 5.5 2.5 1.2 6.2 3.1 1.2 1.1 1.2 1.0 5.6 6.0 2.8 3.4 7.0 3.8 0.6 1.2 1.3 WS0016_N04 LMCO 7E-67 4.8 2.1 1.4 4.5 2.5 1.4 0.9 1.2 1.0 6.7 5.2 2.8 5.3 5.9 3.4 0.8 1.1 1.2 WS01021_E17 LMCO 1E-58 3.5 1.6 1.6 3.2 1.8 1.5 0.9 1.1 0.9 5.9 5.6 3.1 4.2 7.3 4.3 0.7 1.3 1.4 WS0105_M20 LMCO 7E-71 2.9 1.2 1.8 2.2 1.0 1.6 0.7 0.8 0.9 5.5 4.1 3.0 3.8 5.4 3.7 0.7 1.3 1.3 WS0044_O08 PAL 2E-22 5.1 4.0 1.0 6.9 8.5 1.2 1.4 2.1 1.2 4.9 1.2 1.3 4.0 5.1 2.0 0.8 4.3 1.5 WS0264_J18 ANS 4E-58 3.1 5.2 1.5 1.7 7.3 1.2 0.5 1.4 0.8 1.1 2.5 1.7 0.9 2.2 2.1 0.8 0.9 1.2 WS0045_K01 ANS 2E-39 3.7 5.1 1.5 2.2 5.6 1.5 0.6 1.1 1.0 1.2 2.1 1.4 0.7 2.1 1.5 0.6 1.0 1.1 WS00912_H11 ANS 5E-37 3.4 1.5 1.0 5.2 1.4 1.0 1.5 1.0 0.9 5.1 1.3 1.0 6.7 2.0 1.3 1.3 1.5 1.3 WS00931_C11 C4H 4E-79 4.5 2.3 1.2 5.5 4.8 1.5 1.2 2.1 1.3 8.2 4.4 2.4 9.0 7.8 5.2 1.1 1.8 2.2 IS0012_L15 C3H 5E-32 1.7 8.4 1.1 1.5 35.3 1.8 0.9 4.2 1.7 1.0 1.2 1.0 1.0 0.9 1.0 1.0 0.7 1.1 WS00931_G15 F3H 2E-38 15.2 1.8 1.1 14.0 4.2 1.6 0.9 2.3 1.4 4.6 1.2 1.9 3.1 1.8 1.3 0.7 1.5 0.7 WS00916_A12 F3'H 4E-44 8.7 1.6 0.9 9.1 1.8 0.9 1.1 1.1 1.0 1.4 0.8 1.3 1.4 0.6 0.7 1.0 0.8 0.5

Numbers in the table indicate expression levels fold change in specified treatment comparisons (C, control; W, wounding; F, fungal treatments). Colour scale from yellow to red correlates with fold-change expression and was applied only to fold change >2 values with q<0.05. DIR, dirigent protein; COMT, caffeic acid O- methyltransferase; LMCO, laccase multicopper oxidase; PAL, phenylalanine ammonia-lyase; ANS, anthocyanidin synthase/leucoanthocyanidin dioxygenase, C4H, cinnamate-4-hydroxylase; C3H, p-coumarate-3-hydroxylase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3'-hydroxylase. Fold-change: ≥ 5.00 > 1.50 ≥ 10.00 < 10.00 < 5.00

The most highly induced transcripts by fungal treatment compared to control in interior spruce were dirigent proteins (DIR), caffeic acid-O-methyltransferase like (COMT like) ESTs, laccases (LMCO), phenylalanine ammonia-lyase (PAL), anthocyanidin synthase (ANS), cinnamate-4-hydroxylase, with p-coumarate-3-hydroxylase (C3H), flavonone-3-hydroxylase (F3H) and flavonoid 3’-hydroxylase (F3’H). All of these transcripts were induced by wounding treatment vs. control in interior spruce and most were induced in lodgepole pine with several transcripts exhibiting increased induction by fungal treatment in both species. Two COMT like and two ANS transcripts were specifically induced by fungal treatment in lodgepole pine (Table 2.5). Overall this data indicates significant induction of the phenylpropanoid pathway by fungal treatment in both species with many of the transcripts induced by wounding treatment and enhanced induction by the presence of fungus.

Gene specific analysis of dirigent proteins family expression using real-time PCR Dirigent proteins (DIR) represented the most strongly induced family from the phenylpropanoid pathway in both species inoculated with blue-stain fungus. This family is represented by 30 DIR transcripts on the 21K microarray. To further characterize the response of the DIR transcripts to fungal treatment in interior spruce and lodgepole pine, we performed real-time PCR analysis of DIR and DIR-like expression in interior spruce bark based on the previous characterization of the 35 member spruce dirigent protein family (Ralph et al., 2007). Real-time PCR primers used in this study were designed previously based on the full-length sequences of representatives of DIR-a, DIR-b/d and DIR-f dirigent protein sub-families (Ralph et al., 2007). We performed real time PCR analysis of gene-specific transcript levels using 12 primer pairs designed for members of three subfamilies of dirigent family (Figure 2.6). These primer pairs represent 18 dirigent and dirigent-like transcripts (6 primer pairs that coamplified closely related dirigent and dirigent-like sequences, including 4 primer pairs representing 6 dirigent proteins from DIR-a subfamily, 2 primer pairs representing 4 DIR-b/d subfamily members and 6 primer pairs representing 8 DIR-f subfamily members (Ralph et al., 2007). Real-time PCR data is represented as relative expression normalized to the transcript levels of the housekeeping gene levels of eukaryotic translation initiation factor TIF5A (Figure 2.6), and as fold induction relative to control treatment in the case of wounding treatment; and to control and wounding treatment in case of fungal treatment (Supplemental Table 2.6). The real time PCR data was statistically analyzed and considered significant with a fold change >2 and p < 0.05.

0.12 0.012 Dir 2/32 - DIRa Dir 22-DIRf * * * 0.09 0.009

0.06 0.006

0.03 * 0.003

0.00 0.000 0.08 0.052 Dir 5/15 - DIRa * DIR 23/24-DIRf

0.06 * 0.039 Relative Relative expression level Relative Relative expression level 0.04 0.026

0.02 0.013 * * * 0.00 0.000 0.060 0.04 Dir 6-DIRa * Dir 25-DIRf 0.045 0.03 * 0.030 * 0.02

0.015 0.01 * * 0.000 0.00 0.0020 0.100 Dir 16 - DIRa DIR 26-DIRf

0.0015 0.075

Relative Relative Relative expression expression level level 0.0010 0.050

0.0005 0.025

0.0000 0.000 0.028 0.002 Dir 1/20-DIRb/d Dir 27-DIRf 0.021 0.015

0.014 0.010

0.007 0.005 * 0.000 0.000 0.20 DIR 12/21-DIRb/d * 0.28 Dir 28/29-DIRf 0.15

* 0.21 Relative Relative expression level Relative Relative expression level * 0.10 0.14

0.05 0.07

0.00 0.00 C C C W W W F F F C C C W W W F F F 6h 2d 2w 6h 2d 2w 6hr 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w Figure 2.6: Quantitative real-time PCR analysis of DIR transcripts levels in interior spruce. Relative expression levels of 12 representative transcripts of DIR family in interior spruce bark untreated, treated with wounding and inoculated with L. abietinum during the time course of 6h, 2 days (2d) and 2 weeks (2w). Values represent mean + standard error of the mean. Transcript abundance is shown relative to TIF5A transcript in the same sample. * - indicated statistically significant difference compared to control, a dot indicated statistically significant difference compared to wounding.

A strong induction of representatives of DIR-a subfamily DIR2/32, DIR5/15 and DIR6 by wounding and fungal treatment occurred throughout the time course. DIR2/32 were induced similarly by wounding treatment (111.6 fold change (FC)) and by fungal treatment as compared to control (131.8 FC) at 6 hours post treatment with this induction decreasing with wounding at 2 day post treatment (23.3 FC), and being maintained with fungal treatment (83.9 FC). Levels of DIR2/32 returned to control levels by 2 weeks post-treatment. Similarly, DIR6 was induced at 6 hr post treatment by wounding (24.9 FC) and fungal treatment (50.1 FC) with less induction in both treatments at 2 weeks post-treatment. DIR5/15 expression was also induced at 6 hr post-treatment by wounding (9.5 FC) and fungal treatment (8.7 FC), and reached the highest level at 2 days post-treatment in the case of wounding (13.2 FC) and fungal treatments (18.8 FC), returning to control levels by 2 weeks post-treatment. Another representative of the DIR-a subfamily, DIR16, did not exhibit differential expression throughout the time course in the bark of interior spruce treated with wounding and fungal treatment and levels of DIR16 transcript were much lower as compared to other representatives of DIR-a subfamily. The representatives of DIR-b/d and DIR-f subfamilies exhibited less pronounced patterns of gene expression change. DIR12/21 from DIR-b/d subfamily was moderately induced by wounding (3.7 FC) and fungal (4.6 FC) treatments compared to control with no significant induction was present at later time points. DIR1/20 was downregulated by fungal treatment at 2 days post-treatment (0.22 FC) compared to control. Most representatives of DIR-f subfamily did not show a significant change in expression levels in wounding and fungal treated interior spruce. Expression of DIR23/24 was decreased in the case of fungal treatment at 2 days post- inoculation compared to control (0.20 fold change) and compared to wounding treatment (0.29 FC). Overall this data was consistent with the microarray data that showed a strong induction of representatives of the DIR-a subfamily by wounding and fungal treatment 6h and 2 days post-treatment, and much weaker induction for two transcripts of DIR-b subfamily representatives with downregulation of the expression for the other two transcripts of the subfamily in interior spruce (Table 2.5, Supplemental Table 2.6). Previously described alignment of dirigent proteins demonstrated very high sequence similarity between members of dirigent proteins within subfamilies (Ralph et al., 2007) with sequence similarity ranging from 61 to 98% on protein level in DIR-a subfamily, 50 to 98% within DIR-b subfamily and 32 to 99% within DIR-f subfamily with long stretches of identical sequences in many comparisons. High sequence similarity can result in cross hybridization of transcripts within subfamilies, thus these microarray results need to be interpreted on the subfamily levels 67 for the dirigent family and inference of the expression pattern for specific members can only be made based on transcript specific analysis by real-time PCR.

2.3.8 Induction of terpenoid pathways in interior spruce and lodgepole pine Microarray analysis of the terpenoid pathway was performed based on previously developed annotation of spruce ESTs (Ralph and Bohlmann, unpublished results), and annotation of previously characterized terpenoid synthases included on the microarray. Among 680 ESTs annotated to terpenoid pathway, 84 spruce ESTs were represented on the 21K spruce microarray. Further analysis of 20 cytochromes P450 present on 21K microarray revealed that only four cytochromes P450 are involved in terpenoid biosynthesis with other cytochrome P450 being putatively involved in primary metabolism or with unknown function (Hamburger B, personal communication). In addition, 13 previously characterized conifer terpene synthases and one additional EST (WS01028_M14) annotated to 1-deoxy-D-xylulose 5-phosphate synthases (DXS) were included in the analysis (Supplemental Table 2.5). Microarray data analysis indicated the induction of terpenoid pathway by wounding and fungal treatment in interior spruce and lodgepole pine bark (Table 2.4A). Among 82 transcripts annotated to the terpenoid pathway, 35 had significant changes in expression (more than 2 fold, p,q<0.05) by wounding vs. control or fungus vs. wounding at any of the three time points (6 hours, 2 days and 2 weeks) in either interior spruce or lodgepole pine or both (Table 2.6). None of the transcripts from the terpenoid pathway were downregulated in lodgepole pine and only one transcript was downregulated in interior spruce.

Table 2.6: Expression of terpenoid biosynthesis transcripts with >2 fold change in at least one species one treatment and q<0.05. Numbers in the table indicate expression levels fold change in specified treatment comparisons (C, control; W, wounding; F, fungal treatments). Colour scale from yellow to red correlates with fold-change expression and was applied only to fold change values >2.00 with q<0.05. IS, interior spruce; LP, lodgepole pine; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; lytB, 1-hydroxy-2-methyl-2-(E)-butenyl 4- diphosphate reductase; AACT, acetoacetyl-CoA thiolase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3- methylglutaryl-CoA reductase; MK, mevalonate kinase; MPDC, mevalonate diphosphate decarboxylase; IPPI, isopentenyl diphosphate: dimethylallyl diphosphate isomerase; FPPS, farnesyl diphosphate synthase; GPPS, geranyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; TPS, terpene synthase; CYP, Cytochrome P450. ≥ 5.00 Fold-change: <0.50 > 1.50 ≥ 10.00 < 5.00 < 10.00

Table 2.6: (cont.) Expression of terpenoid biosynthesis transcripts

IS IS IS IS IS IS IS IS IS LP LP LP LP LP LP LP LP LP Clone ID BLAST E value W/C W/C W/C F/C F/C F/C F/W F/W F/W W/C W/C W/C F/C F/C F/C F/W F/W F/W 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w MEP pathway WS00930_F08 DXS 1E-86 19.3 1.3 1.3 13.1 2.3 1.6 0.7 1.7 1.3 7.9 1.4 1.8 4.0 1.8 1.7 0.5 1.3 0.9 WS01028_M14 DXS 7E-27 1.1 0.9 1.3 0.7 0.8 1.4 0.6 0.9 1.1 1.4 1.6 1.3 1.2 1.7 2.2 0.9 1.0 1.7 WS01030_N10 lytB 1E-175 2.2 1.7 1.3 1.8 1.4 1.5 0.8 0.8 1.1 1.3 1.8 1.2 1.0 1.5 1.4 0.8 0.8 1.1 WS00815_M18 lytB 1E-175 2.3 1.9 1.3 1.9 1.7 1.5 0.8 0.9 1.1 1.1 1.6 1.1 0.8 1.5 1.3 0.7 0.9 1.2 MEV pathway WS00732_A08 AACT 1E-75 1.0 0.9 1.0 1.1 1.0 0.9 1.1 1.2 0.9 2.1 1.3 1.3 2.1 3.3 2.3 1.0 2.6 1.8 IS0014_G10 AACT 1E-100 1.1 0.8 1.0 1.2 1.1 1.1 1.1 1.3 1.1 1.9 1.3 1.3 1.8 2.9 2.1 0.9 2.3 1.6 WS00820_M23 HMGS 9E-65 1.6 1.1 0.9 1.9 1.6 1.0 1.2 1.4 1.1 1.5 1.2 1.3 1.5 2.0 1.6 1.0 1.7 1.3 WS00930_B21 HMGR 5E-48 1.0 1.0 1.0 1.1 2.1 1.2 1.1 2.2 1.1 0.9 1.1 1.3 0.8 6.7 3.6 0.9 6.3 2.8 WS0072_I23 HMGR 6E-36 1.0 1.0 1.1 1.0 1.3 1.0 1.0 1.3 1.0 0.9 0.9 1.2 0.9 1.9 1.6 1.0 2.1 1.4 WS01025_B12 HMGR 4E-23 1.1 0.6 1.2 0.9 0.5 1.1 0.9 0.7 1.0 0.8 0.9 0.8 0.7 1.0 0.8 0.9 1.1 1.1 WS00925_M22 MK 8E-72 1.2 1.3 1.0 1.3 1.7 1.1 1.1 1.3 1.1 1.3 1.4 1.2 1.2 2.4 1.6 0.9 1.7 1.4 WS0104_E21 MPDC 7E-60 1.5 1.7 1.1 1.7 2.9 1.3 1.1 1.8 1.2 1.2 1.3 1.3 1.2 4.8 2.3 1.0 3.6 1.7 Prenyl transferases WS01029_I02 IPPI 1E-112 2.3 1.7 1.0 2.3 3.0 1.1 1.0 1.8 1.1 1.4 1.1 1.2 1.1 1.9 1.6 0.8 1.7 1.4 WS0074_I12 FPPS 2E-74 1.6 1.3 1.0 2.2 2.1 1.0 1.3 1.6 1.0 1.4 1.2 1.2 1.5 2.2 1.9 1.1 1.9 1.7 WS01039_D11 GPPS 3E-50 1.1 1.4 1.0 1.0 1.6 1.1 0.9 1.2 1.1 1.1 1.3 1.1 1.1 2.3 1.3 1.0 1.8 1.2 WS00911_G14 GGPPS 1E-116 4.6 1.1 1.5 3.4 1.4 1.3 0.7 1.3 0.8 2.4 1.0 1.0 2.1 1.0 1.0 0.9 1.0 1.0 Terpene synthases WS0019_A03 TPS 4E-89 5.3 3.4 1.2 4.0 8.7 2.2 0.8 2.6 1.8 1.3 1.4 1.6 1.1 1.2 1.2 0.8 0.9 0.8 WS0063_F08 TPS 2E-78 7.3 4.3 1.4 4.9 8.2 2.7 0.7 1.9 1.9 1.4 1.9 2.1 1.1 2.6 3.0 0.8 1.4 1.5 WS00723_E14 TPS 3E-90 7.3 4.1 1.5 6.3 5.9 2.1 0.9 1.4 1.5 2.3 1.4 1.2 3.2 1.4 1.1 1.4 1.0 0.9 WS0094_F18 TPS 1E-100 6.3 2.7 1.4 6.9 4.9 1.9 1.1 1.8 1.4 1.3 1.9 1.4 1.5 1.7 1.0 1.1 0.9 0.7 WS00724_C19 TPS 2E-97 5.9 2.3 1.5 5.2 4.4 2.0 0.9 1.9 1.4 1.8 1.8 1.1 2.5 1.2 0.9 1.4 0.7 0.8 WS00923_A21 TPS 4E-51 3.3 3.8 1.9 2.5 4.0 2.2 0.8 1.1 1.2 1.5 2.3 1.5 1.1 4.6 1.4 0.8 2.0 0.9 WS0092_L05 TPS 6E-94 6.7 2.6 1.5 6.9 3.9 1.6 1.0 1.5 1.1 1.5 2.3 1.7 1.1 2.1 1.5 0.8 0.9 0.9 WS00926_E08 TPS 1E-108 2.6 1.9 1.2 2.3 3.9 1.4 0.9 2.0 1.1 1.1 1.5 1.5 1.2 4.6 1.8 1.0 3.0 1.2 WS0092_I21 TPS 3E-86 3.4 2.5 1.6 2.9 3.5 1.6 0.8 1.4 1.0 1.1 2.2 1.7 1.1 4.2 1.5 1.0 1.9 0.9 WS0063_I21 TPS 0 3.0 2.1 1.3 1.7 2.2 1.7 0.6 1.0 1.3 2.0 3.4 1.7 1.2 3.5 1.8 0.6 1.0 1.0 WS0022_E04 TPS 3E-71 1.7 1.4 1.2 1.1 1.7 1.5 0.6 1.2 1.3 2.0 2.3 1.7 1.1 2.4 1.6 0.5 1.0 1.0 WS00819_E12 TPS 2E-97 1.8 1.1 1.1 1.7 1.3 1.3 0.9 1.1 1.2 1.6 1.6 1.3 2.9 1.3 1.0 1.8 0.8 0.8 WS0078_K20 TPS 8E-52 9.7 3.1 1.4 7.2 5.9 1.4 0.7 1.9 1.1 1.2 1.6 1.3 0.9 1.5 1.0 0.8 0.9 0.8 JF16 TPS N/A 2.1 1.7 1.2 1.3 1.6 1.4 0.6 1.0 1.2 1.5 1.5 1.5 1.1 1.6 1.5 0.7 1.1 1.0 WS01040_C03 TPS 1E-43 1.2 1.1 1.0 1.2 1.1 1.1 1.0 1.1 1.1 0.9 1.1 1.4 0.8 10.8 6.8 1.0 10.0 5.0 WS01033_J22 TPS 3E-60 1.1 0.9 1.1 1.1 1.0 1.0 1.0 1.1 0.9 1.1 1.3 1.0 0.9 8.4 3.1 0.8 6.7 3.2 WS0086_N12 TPS 1E-56 1.6 1.2 1.2 1.1 1.1 1.5 0.7 0.9 1.2 1.1 1.8 1.4 0.8 3.6 3.7 0.7 2.0 2.7 JF67 TPS N/A 1.1 1.1 1.2 1.3 1.2 1.0 1.1 1.1 0.9 1.2 1.6 0.9 1.0 2.2 1.2 0.9 1.4 1.3 Cytochrome P450

70 WS00810_K17 CYP720B 1E-176 2.1 1.3 1.1 1.7 1.0 1.3 0.8 0.7 1.3 1.2 1.7 1.2 1.0 1.5 1.1 0.8 0.9 1.0

Early steps of isoprenoid biosynthesis demonstrated strong upregulation for 1- deoxy-D-xylulose 5-phosphate synthase (DXS) transcript (WS00930_F08) by wounding and fungal treatment as compared to control in interior spruce and lodgepole pine at 6 hr post-treatment. Lack of the DXS induction by fungal treatment as compared to wounding treatment revealed that this induction is predominantly due to wounding in both species. The mevalonate (MEV) pathway was predominantly induced in lodgepole pine by fungus with only one transcript acetoacetyl-CoA thiolase (AACT) being induced by wounding treatment as well. In interior spruce two transcripts were induced by fungus only two days post-treatment (HMGR and MPDC) and one of the HMGR transcripts was downregulated by fungal treatment. Three of four prenyltransferases were induced in interior spruce and lodgepole pine by fungal treatment (Table 2.6). Among 39 known and putative terpene synthases on the 21K microarray, 11 were induced in interior spruce by fungal and wounding treatments. Induction of terpene synthases exhibited somewhat different pattern in lodgepole pine induced by wounding and fungal treatments. None of the terpene synthases were significantly induced by wounding treatment and 14 terpene synthases were induced by fungal treatment as compared to control. Eight of these terpene synthases were overlapping with terpene synthases induced by fungal treatment in interior spruce. The microarray analysis revealed differences in the expression patterns of different terpene synthases in interior spruce and lodgepole pine bark, however, high sequence similarity among different members of terpene synthase family (Martin et al., 2004) limits the ability of gene specific microarray analysis due to the possibility of cross hybridization. Microarray analysis of the expression of cytochromes P450 (CYP450) involved in the final step in terpenoid biosynthesis revealed that among 4 cytochromes P450 only one transcript was induced by wounding treatment (FC 2.1) in interior spruce bark and none are induced by wounding or fungal treatment in lodgepole pine.

Gene specific analysis of DXS family expression using real-time PCR To confirm microarray results with real-time PCR, the DXS transcript (WS00930_F08) was chosen due to its strong induction by fungal and wounding treatments in both species. Three DXSs transcripts are present on the 21K microarray. They represent the family of three known spruce DXS enzymes (Phillips et al., 2007). To confirm the specific induction of one of the members of DXS family by wounding and fungal treatment, we performed real-time PCR analysis of the three DXS transcripts in interior spruce induced by wounding and fungal treatment. Real-time PCR data are 71 presented as relative expression normalized to housekeeping gene levels of the eukaryotic translation initiation factor TIF5A (Figure 2.7), and as fold induction relative to control treatment in the case of wounding treatment, and to control and wounding treatment in the case of fungal treatment (Supplemental Table 2.6).

0.8 DXS WS00930_F08 0.6 * Figure 2.7: Quantitative real-time PCR * analysis of DXS transcripts levels in 0.4 interior spruce. Relative expression 0.2 levels of three transcripts of DXS family 0.0 * in interior spruce bark untreated, treated with wounding and inoculated with L. 0.048 DXS WS0097_H02 abietinum during the time course of 6 0.036 hours (6h), 2 days (2d) and 2 weeks

0.024 (2w). Values represent mean + standard error of the mean. Transcript abundance 0.012 is shown relative to TIF5A transcript in

0.000 the same sample. * - indicated Relative Relative expression level Relative Relative expression level statistically significant difference 0.060 DXS WS01028_M14 compared to control 0.045

0.030

0.015

0.000 C C C W W W F F F 6h 2d 2w 6h 2d 2w 6h 2d 2w

The real time PCR data was statistically analyzed and considered significant with >2 fold change and p< 0.05. The real-time PCR analysis confirmed the strong induction of DXS WS00930_F08 at 6h post-treatment by wounding (FC 94.4) and fungal (FC 73.7) treatments. Levels of the DXS returned to control levels at 2d and 2 weeks post- treatment in wounding treated and fungus-inoculated interior spruce. The other two DXS transcripts WS0097_H02 and WS01028_M14 were not induced by either wounding or fungal treatment in interior spruce during the time course of the study.

2.3.9 Induction of chitinases expression in response to G. clavigera and L. abietinum inoculations in interior spruce and lodgepole pine respectively Chitinases were among the most strongly induced transcripts by fungal treatment in interior spruce and lodgepole pine bark (Table 2.2, Table 2.3). Analysis of the expression of the 21 chitinases and chitinase-like transcripts present on the 21.8K microarray revealed that the majority of the chitinases are induced by wounding and fungal treatment throughout the time course of the experiment in both interior spruce and lodgepole pine (Table 2.7). 72

Table 2.7: Expression of chitinase and chitinase-like transcripts present on 21.8K spruce microarray. Color scale from yellow to red correlates with fold-change expression and was applied only to fold change values with q<0.05. Numbers in the table indicate expression levels fold change in specified treatment comparisons (C, control; W, wounding; F, fungal treatments). IS IS IS IS IS IS IS IS IS LP LP LP LP LP LP LP LP LP NCBI W/C W/C W/C F/C F/C F/C F/W F/W F/W W/C W/C W/C F/C F/C F/C F/W F/W F/W Clone ID BlastX* E value 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w 6h 2d 2w chitinases WS00924_F07 AAT094261 7E-128 1.0 5.6 1.5 1.0 36.0 1.8 1.0 6.3 1.2 1.6 3.2 1.4 5.1 19.3 12.8 3.2 5.9 9.1 WS00930_G18 AAQ100932 7E-66 1.2 4.7 1.6 1.9 14.1 1.9 1.7 3.0 1.2 1.2 3.0 1.4 3.4 21.6 41.0 2.9 7.1 33.3 WS0018_O07 AY6395873 2E-24 1.1 4.9 0.8 1.0 10.1 2.0 0.9 2.0 2.6 0.6 0.8 1.7 1.2 6.5 6.6 2.0 8.3 3.8 WS0046_M24 BAF460124 1E-49 2.1 5.4 0.8 2.7 9.4 0.9 1.3 1.7 1.2 5.6 7.4 1.3 4.9 29.9 14.9 0.9 4.0 11.1 WS00712_K20 BAD779325 1E-70 1.9 2.6 1.5 2.3 8.6 1.8 1.2 3.2 1.2 1.3 1.8 1.4 2.2 10.6 23.1 1.6 5.9 16.7 WS00927_N15 BAD028246 7E-100 1.7 5.2 0.9 2.2 8.4 0.9 1.3 1.6 1.0 5.4 6.5 1.6 4.8 22.9 20.2 0.9 3.4 12.5 WS0092_L20 AAQ170507 7E-93 1.2 4.0 0.9 1.4 8.0 2.0 1.2 2.0 2.2 0.5 1.3 1.9 0.9 7.9 6.2 1.6 6.3 3.3 WS00929_K15 CAA101898 2E-87 1.6 3.3 0.8 2.1 6.6 1.0 1.4 2.0 1.3 5.8 6.8 1.6 4.7 21.6 15.3 0.8 3.1 9.1 WS00716_E14 AAV317439 8E-11 1.2 2.0 1.1 1.4 5.5 1.1 1.2 2.8 1.1 0.9 1.5 1.0 1.0 5.6 9.8 1.1 3.7 10.0 WS01032_J21 AAV3174210 2E-11 0.9 2.8 0.7 0.8 4.3 1.9 0.9 1.5 2.5 0.6 0.9 1.3 0.7 3.8 3.4 1.3 4.5 2.6 WS00922_B21 CAC8181211 8E-100 1.8 3.5 0.9 2.3 3.7 1.0 1.3 1.0 1.2 5.4 9.0 1.6 4.8 18.3 15.5 0.9 2.0 10.0 IS0014_F01 AAV3174212 4E-88 1.1 2.4 0.8 1.1 3.6 1.4 1.0 1.5 1.7 0.6 0.8 1.2 1.0 3.7 3.8 1.6 4.5 3.1 WS01018_O22 BAD7793213 8E-51 1.2 1.6 1.2 1.1 2.7 1.4 1.0 1.7 1.2 0.7 1.3 1.7 0.9 3.4 8.8 1.2 2.6 5.3 WS0045_M18 ABD9281914 1E-75 0.8 1.6 0.8 1.4 1.3 0.8 1.7 0.8 1.0 1.3 1.3 0.8 1.1 2.5 1.7 0.8 1.9 2.1 WS0106_P21 AAT0942715 7E-76 0.8 1.1 1.0 1.2 1.3 1.2 1.5 1.1 1.2 3.5 4.6 1.4 2.8 15.3 10.0 0.8 3.3 7.1 WS0061_N18 BAF4601216 2E-18 1.1 1.0 1.0 0.8 0.9 1.1 0.7 0.9 1.2 1.0 0.7 1.0 1.2 0.9 1.0 1.2 1.3 0.9 WS00924_E01 AAT0942717 3E-104 0.9 1.0 1.0 0.9 0.8 0.9 1.0 0.9 0.9 1.1 1.0 1.0 1.0 0.9 0.9 1.0 0.9 0.9 chitinase like WS0261_D24 NP_17207618 8E-08 1.2 0.9 1.0 0.7 1.2 0.9 0.8 1.0 1.1 1.0 0.8 1.0 1.0 0.8 0.7 1.1 0.9 0.7 WS0031_C03 NP_18831718 3E-53 1.1 1.3 0.9 0.8 1.1 1.0 0.9 1.1 1.0 1.1 1.0 1.4 1.1 1.0 1.2 1.0 1.0 0.8 WS00712_A06 NP_17207618 7E-27 1.0 0.8 1.0 0.7 1.0 0.9 1.0 0.7 1.0 1.0 0.7 1.0 0.9 0.5 0.7 0.8 0.7 0.7 WS02610_C03 NP_17207618 6E-04 1.0 1.0 1.1 1.1 1.0 0.8 0.8 1.0 1.1 0.8 0.8 0.9 0.8 0.8 0.9 1.0 1.0 1.1 *Chitinases were annotated based on the best annotated sequences match using blastX and NCBI database. Annotation IDs represent the following species: 1- Picea abies, 2- Vitis vinifera, 3- Picea abies, 4- Chamaecyparis formosensis, 5- Cryptomeria japonica, 6- Taxodium 7 8 9 10 11 12 13 14 73 distichum, - Picea abies, - Cicer arietinum, - Picea abies, - Picea abies, - Musa acuminate, - Picea abies, - Cryptomeria japonica, - 15 16 17 18

Limonium bicolor, -Picea abies, - Chamaecyparis formosensis, - Picea abies, - Arabidopsis thaliana. Fold change: ≥ 5.00 > 1.50 ≥ 10.00 < 5.00 < 10.00

Thirteen chitinases were induced by fungal treatment in interior spruce at 2 days post-treatment with 12 of these also being induced by wounding treatment. Six chitinases were induced by fungal treatment compared to wounding treatment at 2 days post-treatment and 4 chitinases were induced specifically by fungal treatment at 6 hr, with one chitinase induced by wounding and fungal treatment at 6 hr post-treatment. Expression of chitinases in interior spruce decreased to control levels at 2 weeks post- treatment. A similar pattern of most of the chitinases being induced was observed in lodgepole pine exposed to wounding or fungal treatment with more transcripts being induced to a higher degree as compared to interior spruce. Fifteen chitinase transcripts were induced by fungal treatment at 2 days post-treatment in lodgepole pine. The 4 upregulated chitinase transcripts at 6 hr post-treatment were induced similarly by fungal and wounding treatment. The up-regulated expression of the chitinase transcripts induced by wounding was maintained at 6h and 2 days post-treatment and returned to control levels at 2 weeks post-treatment in lodgepole pine. Conversely, fungus-induced expression of chitinases was maintained at 2 weeks post-treatment with 14 transcripts being specifically induced by fungal treatment. Expression of chitinase-like transcripts was not affected by wounding or fungal treatment in both species throughout the time course (Table 2.7). The ability of the microarray study to distinguish differences in the expression of different chitinase transcripts may be limited due to the possibility of cross- hybridization of different members of chitinase family. Gene specific expression analysis confirmed strong induction of several chitinases in interior spruce and lodgepole pine by fungal and wounding treatments (Chapter 4). Chitinolytic activity was confirmed in two strongly induced interior spruce chitinases and one lodgepole pine chitinase (Chapter 4).

2.4 DISCUSSION

In this study we performed a comparative characterization of transcriptome response in the bark of interior spruce inoculated with pathogenic blue-stain fungus L. abietinum and in the bark of lodgepole pine inoculated with pathogenic blue-stain fungus G. clavigera. It was previously shown, and confirmed here, that microarrays can be used for gene expression profiling across spruce, pine boundaries (van Zyl et al., 2002; Stasolla et al., 2003). Including wounding treatment in the comparisons allowed us to elucidate the fungal specific component of the transcriptome responses. In addition, understanding the response of interior spruce and lodgepole pine to wounding contributes to our understanding of the defense response of interior spruce to spruce beetle, and lodgepole pine to mountain pine beetle because both of these bark beetles cause a wounding damage as they bore through the bark to reach inner phloem and developing xylem. Induction of the defense response by fungal treatment in this study was confirmed by anatomical analysis of fungus and wounding treated interior spruce and lodgepole pine used in the microarray study. The anatomical analysis revealed, for the first time, formation of traumatic resin ducts in interior spruce inoculated with L. abietinum and lodgepole pine inoculated with G. clavigera (Figure 2.2). Spruce formation of traumatic resin ducts is indicative of the defense related response in all studied spruce species upon exposure to wounding, methyl jasmonate, insect or fungal treatment (Alfaro, 1995; Christiansen et al., 1999; Martin et al., 2002; McKay et al., 2003; Hudgins et al., 2004; Krekling et al., 2004). The only reports of the formation of traumatic resin ducts in pine xylem are available for Austrian pine (Pinus nigra Arn.) inoculated with fungus Diplodia scorbiculata that causes shoot blight and canker on conifers (Luchi et al., 2005), and for Pinus monticola inoculated with white pine blister rust (Cronartium ribicola) (Hudgins et al., 2005). In our case study, the interior spruce traumatic resin duct formation was complete by 2 weeks post-treatment in fungus- treated trees, whereas formation of additional resin ducts in lodgepole pine was visible only at 6 weeks post-treatment. This slower response in lodgepole pine is consistent with the presence of a number of constitutive resin ducts in the xylem, as compared to the absence of constitutive resin ducts in the xylem of interior spruce. Wounding treatment induced a weaker traumatic resin duct formation than fungal-treatment in interior spruce, and no additional resin ducts formation was detected in lodgepole pine following wounding. This may indicate different defense response strategies of the two species, with interior spruce employing fast responding induced defenses in addition to 75 constitutive defenses (e.g. terpenoid resin in bark) while lodgepole pine depends more on constitutive responses early on with slower activation of induced defenses. The hypothesis of a more important role of constitutive defense strategies in pines compared to more involved induced defense strategies in spruce was suggested by Christiansen et al. (1987) in consideration of pine being exposed to more frequent bark beetle attacks in hotter drier climates compared to spruce species that prefer cooler and more humid environments with less frequent exposure to bark beetles (Christiansen et al., 1987). Overall, our microarray results demonstrated the large scale gene expression reorganization of interior spruce and lodgepole pine bark in response to inoculation with L. abietinum and G. clavigera respectively. Close to 10% of the analyzed transcriptome was differentially expressed in fungal treated interior spruce (2052 transcripts) and in lodgepole pine (1868 transcripts) even with stringent criteria for differentially expressed transcripts (Fold change >2; p,q<0.05). In previous work, similar scope of conifer response to fungal inoculation was demonstrated in Scots Pine (Pinus sylvestris) root inoculated with the pathogenic necrotrophic fungus Heterobasidion annosum (Adomas et al., 2007), in Scots pine inoculated with the ectomycorrhizal fungus Laccara bicolor (Heller et al., 2008) and in a smaller study of the effect of the necrotrophic pathogen Fusarium circinatum on slash pine (Pinus elliottii) (Morse et al., 2004). Large rearrangement of the plant transcriptome in response to biotic stress was also demonstrated for spruce induced by insect feeding (Ralph et al., 2006b). The magnitude of the response of spruce and pine to wounding treatment was less than half of their response to fungal inoculation, suggesting recognition of fungal pathogens and activation of additional defense responses. This transcriptome response is consistent with the increased anatomical changes in interior spruce and lodgepole pine induced by fungal treatment as compared to wounding and also with previously demonstrated increased anatomical changes and chemical changes in Norway spruce (Krekling et al., 2004; Viiri et al., 2001) and chemical (Figure 2.3; Croteau et al., 1987) and biochemical changes in lodgepole pine (Croteau et al., 1987) induced by fungal inoculation compared to wounding alone. Overlap of transcriptome responses of interior spruce and lodgepole pine to fungal inoculation included about 50% of the evaluated transcriptome and analysis of the dynamics of the transcriptome response revealed different timing of the transcriptome response in these species (Figure 2.4). Interior spruce and lodgepole pine transcriptome responses were initiated as early as 6h post-treatment, with almost twice as many transcripts induced by fungal treatment in interior spruce compared to lodgepole pine, consistent with the heightened initial induced response in interior 76 spruce. The rapid induction of the interior spruce transcriptome is consistent with the rapid anatomical response in interior spruce compared to lodgepole pine. The transcriptome response to wounding was comparable in both species at 6 h post- treatment, however the wounding response diminished but remained high at 2 days post-treatment in interior spruce while decreasing much faster in lodgepole pine. This data is consistent with a more intense inducible defense response in spruce with lodgepole pine only maintaining a strong response in the case of a serious threat of pathogen establishment. At 18 weeks post-inoculation lodgepole pine trees exhibited yellow needles and large open bark lesions that were indicative of desiccation and fungal spread, while the interior spruce seedlings did not have these symptoms. Based on microarray data and the evaluation of the symptoms it seems that spruce induced response was successful in localizing and stopping the pathogen spread already at two weeks post-treatment, which is consistent with a very small transcriptome response remaining, whereas lodgepole pine response to fungus was still strong at two weeks post-treatment, being consistent with the presence of continuous threat from fungal pathogen. The observed differences in the dynamics of responses between two species detected by the microarray may reflect different defense strategies suggested by Christiansen et al. (Christiansen et al., 1987) but they may also result from differences in the studied biological systems including different fungal species, which may trigger different responses in spruce and pine due to the difference in fungal elicitors and rate of fungal growth as well as differences in resistance of the studied trees to the fungal pathogens. Interior spruce and lodgepole pine transcriptome responses to fungal inoculation had more transcripts induced than downregulated (Table 2.1). Most of the differentially expressed transcripts representing defense-related genes were upregulated (Table 4A, B) similar to the pattern observed in Scotts pine inoculated with Heterobasidion annosum (Adomas et al., 2007). This is consistent with the necrotrophic nature of the pathogens (Leptographium abietinum and Grosmania clavigera) used in this study, as it was previously demonstrated that the suppression of host defense is characteristic of invasion by biotrophic pathogens (Mendgen and Hahn, 2002; Voegele and Mendgen, 2003; Miranda et al., 2007). The strong induction of transcripts involved in ethylene and jasmonic acid signalling (Table 2.2, 2.3, 2.4A) in interior spruce and lodgepole pine inoculated with the corresponding necrotrophic pathogens is consistent with the involvement of these signalling pathways observed in angiosperm responses to necrotrophs (McDowell and Dangl, 2000). The microarray data supported the importance of methyl jasmonate and ethylene signalling in conifer defense against fungi, 77 which had been previously suggested based on similar responses of conifers to fungal inoculation as compared with methyl jasmonate and ethylene treatments (Liu et al., 2003; Hudgins and Franceschi, 2004). The microarray analysis indicated the substantial rearrangement of primary metabolism, with the downregulation of transcripts involved in photosynthesis in interior spruce and lodgepole pine inoculated with fungus being consistent with the changes observed in Scots pine inoculated with fungus (Adomas et al., 2007). A similar rearrangement of primary metabolism in defense related responses was observed in fungus treated poplar (Miranda et al., 2007), and in spruce (Chapter 3; Ralph et al., 2006b) and poplar exposed to herbivory (Ralph et al., 2006a). The rearrangement of primary metabolism and the downregulation of photosynthesis may be involved in the reallocation of energy resources to the biosynthesis of defense compounds, but may also protect the photosynthetic apparatus against oxidative damage (Bolton, 2009). Strong induction of secondary metabolism, in particular phenylpropanoid and terpenoid pathways, was observed in interior spruce and lodgepole pine transcriptome responses to wounding treatment and fungal inoculation (Table 2.4A, 2.5, 2.6; Figure 2.5) and is consistent with previous studies of conifer response to bark beetle attack, wounding and fungal inoculations (Franceschi et al., 2005).

Phenylpropanoid pathway Annotation of 340 transcripts represented on the microarray to the phenylpropanoid pathway was done based on known genes in Arabidopsis and conifers (Ralph and Bohlmann, unpublished data). While most of the annotated transcripts likely have an assigned function and are involved in the phenylpropanoid pathway in conifers, some of the transcripts, such as those annotated to COMT and F5H, may have a different function, as these genes are involved in the biosynthetic pathway leading to synapyl alcohol and were only identified in angiosperms (Weng and Chapple, 2010). In addition, all transcripts annotated as laccases and dirigent proteins were included in the set of phenylpropanoid pathway transcripts. Some of the laccases and dirigent proteins may have different functions as functional characterization is available only for few genes from these diverse multigene families (Davin et al., 1997; Kim et al., 2002; Boerjan et al., 2003; McCaig et al., 2005; Sato and Whetten, 2006). Close to 30% of the transcripts annotated as involved in the phenylpropanoid pathway showed differential expression, with most of the transcripts being upregulated in wound- or fungus treated interior spruce and lodgepole pine (Table 2.4A, Figure 2.5). This result is consistent with the involvement of this pathway in Scots pine defense 78 against Heterobasidium annosum (Adomas et al., 2007). The phenylpropanoid transcripts that were induced in both spruce and pine were among the most highly induced transcripts, and many of these transcripts were induced by wounding and augmented by fungal treatment (Table 2.2, 2.3, 2.5). This trend, in addition to the previously demonstrated induction of the phenylpropanoid pathway in spruce exposed to herbivory (Ralph et al., 2006b) highlights common response of the phenylpropanoid pathway to biotic stresses in conifer. Similar to a previous study of phenylpropanoid pathway induction by wounding, weevil and budworm treatment in Sitka spruce (Ralph et al., 2006b), strongly induced phenylpropanoid transcripts by wounding and fungal treatment in interior spruce and lodgepole pine included PAL and C4H, the enzymes that control the metabolic flux into the phenylpropanoid pathway (Ro and Douglas, 2004), as well as dirigent proteins and laccases. Among transcripts annotated to flavonoid biosynthesis, ANS transcripts were highly induced by wounding and fungal treatment in both interior spruce and lodgepole pine and F3H and F3’H were induced by wounding and fungal treatment in interior spruce but not in lodgepole pine (Table 2.5). Strong induction of enzymes involved in flavonoid biosynthesis by wounding and fungal treatment may indicate the involvement of flavonoids in the spruce and lodgepole pine defense responses. Accumulation of flavonoids was observed previously in conifers affected by pathogenic fungi (Brignolas et al., 1998; Evensen et al., 2000; Bonello and Blodgett, 2003). Accumulation of the flavonoid (+)-catechin was associated with resistance of Norway spruce to Ceratocystis polonica (Brignolas et al., 1998). In addition, the strong induction of the flavonoid biosynthetic pathway was also observed in poplar defense responses induced by leaf rust fungal pathogen (Miranda et al., 2007). Flavonoids may serve as antioxidants in protection of cells from oxidative damage (Michalak, 2006) and possibly have antifungal properties, as was demonstrated for flavonoid-derived compounds (Dixon, 2001).

Dirigent proteins and laccases Dirigent proteins (DIR) and laccases were among the most highly induced transcripts in response to wounding and fungal treatment in interior spruce and lodgepole pine (Table 2.5). These enzymes are proposed to be involved in lignan and possibly lignin biosynthesis in which laccases are involved in the oxidation of monolignols (Davin et al., 1997; Davin and Lewis, 2000; Kim et al., 2002; Boerjan et al., 2003; Sato and Whetten, 2006). These monolignols may then undergo random or directed coupling (involving dirigent proteins), leading to the formation of lignans and possibly lignin (Davin et al., 1997; Davin and Lewis, 2000). Lignans are known to have 79 antifungal properties (Vargas-Arispuro et al., 2005) and the additional production of lignin is known to be associated with pathogen attack in conifer trees (Bonello and Blodgett, 2003; Wallis et al., 2008), and could contribute to the strengthening of cells walls. In addition, in vitro studies established a negative effect of lignin on growth of bark beetle-associated fungi (Bonello et al., 2003). Gene specific analysis of the expression of selected DIR family members in interior spruce revealed a strong induction of the representatives of the DIR-a subfamily (DIR2/32, DIR5/15 and DIR6) (Figure 2.6) by wounding and fungal treatment. A previous expression study of the dirigent protein family in Sitka spruce treated with mechanical wounding, weevil and budworm also demonstrated a strong induction of selected members of the DIR-a subfamily (Ralph et al., 2006c; Ralph et al., 2007). Dirigent DIRa-2/32 was strongly induced by these treatments in Sitka spruce and this transcript is also one of the most strongly induced dirigent protein transcripts by L. abietinum treatment, and by wounding treatment in interior spruce, indicating the general nature of inducible expression of DIRa-2/32. DIRa- 6 is induced by fungal treatment and wounding treatment in interior spruce and was also induced by wounding and weevil feeding, but was not significantly induced by budworm feeding in the Sitka spruce study (Ralph et al., 2007). DIRa-5/15 were also induced by fungal and wounding treatments in interior spruce but was downregulated by budworm feeding in Sitka spruce (Ralph et al., 2007). Unique expression profiles of these dirigent protein transcripts in response to different treatments indicate the specificity of dirigent proteins involvement in the conifer defense response. Representatives of the DIR-b subfamily did not show a strong response to either wounding or fungal treatment in interior spruce. DIRb/d 12/21 was moderately induced by wounding and fungal treatment in interior spruce and was induced by wounding treatment and downregulated by weevil feeding in Sitka spruce (Ralph et al., 2007). DIRb/d 1/20 was downregulated by fungal treatment in interior spruce and was downregulated by weevil and budworm feeding in Sitka spruce (Ralph et al., 2007). Five of the six studied members of the DIR-f subfamily did not show any change in expression patterns in interior spruce treated with wounding and fungus. DIRf 23/24 was downregulated by fungal treatment in interior spruce and was similarly downregulated by weevil and budworm feeding in Sitka spruce (Ralph et al., 2007). Among the other DIR-f subfamily members, DIRf-26 was induced by budworm feeding but not by weevil feeding in Sitka spruce, DIRf-27 was downregulated by weevil but not by budworm feeding, DIRf-22 was downregulated by both weevil and budworm treatment in Sitka spruce, and the expression of DIRf-25 and DIRf-28/29 was not affected by either weevil or budworm treatment (Ralph et al., 2007). Similar to the Sitka spruce DIR family analysis (Ralph et 80 al., 2007), our study suggested that the differential response of DIR genes may indicate different participation of representatives of different DIR subfamilies in defense response with representatives of DIRa subfamily potentially involved in general and selective defense responses. In conclusion, our data demonstrate the strong induction of a variety of different transcripts annotated to the phenylpropanoid pathway common to wounding and fungal treatment in both species. The strong response to wounding may indicate adaptation of interior spruce and lodgepole pine to beetle attacks that start with wounding of the tree by the beetle mass attack and follows with fungal colonization. Induced biosynthesis of phenylpropanoids with antifungal properties would increase the ability of the attacked tree in resisting fungal attack by allowing wound induced trees to combat fungal growth that follows beetle attack.

Terpenoid pathway Close to half of the transcripts annotated to the terpenoid pathway were induced in interior spruce or lodgepole pine by wounding or fungal treatment during the time course of the study, with a considerable overlap of induced transcripts by fungal treatment in both species within the gene set represented on the microarray (Table 2.6). The analysis of resin terpenoids in the bark of interior spruce and lodgepole pine inoculated with fungus revealed increase in the amount of monoterpenoids and diterpenoids in lodgepole pine bark two weeks post-treatment and no consistent change in the bark of interior spruce (Figure 2.3). Increased amount of monoterpenoids and diterpenoids was previously reported in lodgepole pine inoculated with blue-stain fungus (Miller et al., 1986; Croteau et al., 1987) and increased activity of terpene synthases was also detected as a result of fungal inoculation (Croteau et al., 1987). Our inability to detect changes of terpenoid amounts in interior spruce bark may be related to saturation of bark with constitutive resin similar to previously reported studies (Martin et al., 2002). However, increased production of resin and accumulation of terpenoids induced by blue- stain fungus was detected in bark of mature Norway spruce trees (Baier et al., 2002). Formation of traumatic resin ducts observed in interior spruce and lodgepole pine inoculated with fungi (Figure 2.2) contributes to the increase in terpenoid accumulation. Enhanced resin flow was correlated with Norway spruce resistance to blue-stain fungus Ceratocystis polonica (Zeneli et al., 2006) and increased induced production of terpenoid resin in lodgepole pine inoculated with Grosmannia clavigera was correlated with the survival of lodgepole pine trees during an exposure to high mountain pine beetle population (Raffa and Berryman, 1982). These studies and the reported toxicity 81 or deterrent properties of terpenoids toward bark beetles and beetle-associated fungi (Keeling and Bohlmann, 2006) indicate the relevance of induced terpenoid production in defense responses of conifers to bark beetle attack. Microarray analysis revealed the induction of transcripts from most parts of the isoprenoid biosynthetic pathway including the MEP and MEV pathways, prenyltransferases and terpene synthases, in both species, by wounding and fungal treatment compared to control (Table 2.6). These results are consistent with previous studies that demonstrated the induction of the expression of genes involved in terpenoid pathway in conifers in response to wounding and fungal pathogens (Keeling and Bohlmann, 2006). Wounding treatment induced similar terpenoid responses to fungal treatment in interior spruce, whereas the lodgepole pine wounding treatment significantly induced only few transcripts, with the majority of the response being fungus specific (Table 2.6). This result could indicate a common response of different conifer species to biotic stimulus, with interior spruce relying more on induced defenses in the cases of wounding and fungal treatment, and lodgepole pine relying more on constitutive terpenoid defenses in the case of wounding stress and activating induced defenses in the case of more severe biotic stresses. Among the transcripts annotated to the terpenoid biosynthetic pathway, one of the three 1-deoxy-D-xylulose-5-phosphate synthase (DXS) transcripts present on the microarray was strongly induced by wounding and fungal treatment compared to control in interior spruce and also induced in lodgepole pine (Table 2.6). The DXS enzyme catalyzes the initial step of the MEP pathway leading to the production of isopentenyl diphosphate and was shown to be a rate limiting enzyme in plastidic isoprenoid biosynthesis in Arabidopsis (Estevez et al., 2001). Previous characterization of three Norway spruce PaDXS enzymes demonstrated that two type-II PaDXS (PaDXS2A and PaDXS2B) genes were induced in bark of wounding and fungal treated Norway spruce saplings and type-I PaDXS was constitutively expressed and not induced by wounding or fungal treatment (Phillips et al., 2007). Gene specific expression analysis of three DXS transcripts orthologous to the PaDXS genes confirmed the strong induction of DXS WS00930_F08 (orthologus to PaDXS2B) in the bark of interior spruce treated by wounding and fungal inoculation (Figure 2.7), and demonstrated constitutive levels of expression for other 2 DXS transcripts (DXS WS01028_M14 and DXS WS0097_H02 orthologous to PaDXS2A and PaDXS1 respectively), consistent with the strong induction of PaDXS2B by wounding and Ceratocystis polonica treatment in Norway

82 spruce (Phillips et al., 2007). Our data suggests that DXS WS00930_F08 is likely to be involved in providing substrate for fungal and wounding induced terpenoid biosynthesis.

Pathogenesis related proteins The most prominently induced group of transcripts in interior spruce and lodgepole pine treated with blue-stain fungi included transcripts representing pathogenesis related proteins (Table 2.2, 2.3, 2.7). Fourteen families of PR proteins have been previously described in angiosperm plants (Van Loon and Van Strien, 1999). In our study, highly induced transcripts in both species throughout the time course of fungal and wounding treatment included chitinases, thaumatin or thaumatin-like protein, osmotin or osmotin-like protein and a peroxidase (highly induced only in lodgepole pine) (Table 2.2, 2.3, 2.7). In interior spruce most of the PR transcripts were induced by wounding and, to a higher extent by fungus, and more fungus-specific induction of PR proteins annotated transcripts was observed in lodgepole pine. Induction of selected PR protein transcripts was previously demonstrated in conifer defense transcriptome studies, such as the induction of chitinases and peroxidase in slash pine induced by Fusarium circinatum (Morse et al., 2004), chitinase and thaumatin in Scots pine induced with Heterobasidion annosum (Adomas et al., 2007) and in Sitka spruce induced by wounding and weevil feeding (Ralph et al., 2006b). Similarly, transcript targeted studies demonstrated the induction of selected PR protein genes such as induction of chitinase transcripts in slash pine cell suspension culture induced by chitosan (Wu et al., 1997), in slash pine seedlings infected with the fungus Fusarium subglutinans (Davis et al., 2002) and in Norway spruce induced by Heterobasidion annosum (Fossdal et al., 2006). Thaumatin-like protein was also induced in white pine seedlings inoculated with the blister rust pathogen Cronartium ribicola and treated with wounding and methyl jasmonate (Piggott et al., 2004). Relevance of PR proteins in plant defense is supported by previously confirmed antifungal activity for selected PR proteins such as chitinases (Schlumbaum et al., 1986; Kirubakaran and Sakthivel, 2007), thaumatin-like proteins (Rajam et al., 2007) and osmotin (Abad et al., 1996). Among PR proteins, chitinases were most highly induced in interior spruce and lodgepole pine in response to fungal inoculation (Table 2.2, 2.3, 2.7). Chitinases catalyze the hydrolysis of chitin, a linear polymer of β-1,4-linked N-acetylglucosamine that is a common constituent of the fungal cell wall. Plant chitinases are known to be induced by pathogens in many plants (Kasprzewska, 2003), and were shown to contribute to fungal disease resistance when introduced as transgenes in other plants (Vellicce et al., 2006; Jayaraj and Punja, 2007; Xiao et al., 2007). Within plant species 83 chitinases are present as large multigene families with variation in biochemical and biological properties (Graham and Sticklen, 1994). Microarray analysis of the expression of 17 unique chitinase transcripts present on the 21K microarray revealed a strong induction of most of the members of chitinase family by fungal and, to a lesser extent, by wounding treatment in interior spruce and lodgepole pine (Table 2.7), which was confirmed by gene specific expression analysis for several chitinases (Chapter 4). This study, in addition to previous association of the conifer tree’s ability to induce chitinase expression with anti-fungal (Heterobasidion annosum) resistance in Norway spruce (Fossdal et al., 2006), indicates the potential importance of chitinases in the conifer defense response and merits further characterization of the biological role of identified target putative chitinase transcripts in interior spruce and lodgepole pine.

Conclusion Microarray analysis proved to be a reliable and powerful method to investigate the transcriptome response in interior spruce and lodgepole pine to the corresponding blue-stain fungal pathogens. It demonstrated similar complex defense strategies employed by both species, which involve the activation of defense-related genes and pathways including gene involved in the biosynthesis of secondary metabolites such as phenylpropanoids and terpenoid biosynthesis, as well as the involvement of PR genes, in particular chitinases, in conifer defense response. This study allowed us to analyze conifer defense responses to fungal inoculations in the context of the comprehensively represented conifer transcriptome and has provided multiple gene targets for further investigation of their roles in the conifer defense response.

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3 Comparative analysis of the interior spruce transcriptome response to white pine weevil feeding and fungal inoculation with blue-stain fungus Leptographium abietinum2

3.1 INTRODUCTION

Weevils and bark beetles that vector pathogenic blue-stain fungi are some of the most destructive pests of conifers. White pine weevil (Pissodes strobi) is a pest of spruce and pine forests in North America (Alfaro et al., 2000). White pine weevils can infest young saplings and mature trees where they feed on the bark of the leader, branches and stem. Weevils lay eggs on the tree leader and later emerging larvae feeds on the leader tissues and destroys it. Death of the leader results in the deformed and slower growth of the attacked conifer tree and reduction of wood quality. Given a choice weevils tend to select susceptible trees which insure brood survival and avoid resistant trees with strong anti-insect defense mechanisms which may cause mortality of weevil eggs and emerging larvae (Alfaro et al., 2002). Mass attack helps weevils to overcome conifer defenses (Alfaro, 1996). Destructive bark beetles such as spruce beetle (Dendroctonus rufipennis) and mountain pine beetle (Dendroctonus ponderosae) also employ mass attack on healthy or weakened conifer trees. Bark beetles usually attack large mature trees (Bjorklund and Lindgren, 2009). Beetle tunnelling and feeding in the phloem, cambium and outer sapwood caused tree death. Bark beetle vectored pathogenic blue-stain fungi contribute to the attack of bark beetles (Yamaoka et al., 1995; Paine et al., 1997; Ohsawa et al., 2000). The most pathogenic fungus associated with Dendroctonus rufipennis is the blue-stain fungus Leptographium abietinum that is capable of killing spruce trees if inoculated at a high enough density (Ohsawa et al., 2000). Conifer trees vary in resistance to weevil, bark beetles and beetle-associated fungal pathogens and multiple defense mechanisms contribute to successful defense (Alfaro et al., 2002; Paine et al., 1997). Similar constitutive and induced structural and chemical defenses are often effective against both insect attacks and fungal invasion. Among the most prominent defense related chemicals in conifers are terpenoids, which are produced and stored in constitutive or induced resin ducts (Keeling and Bohlmann,

2 A version of this chapter will be submitted for publication. Kolosova N, White R, Ralph S, Breuil C and Bohlmann J. Comparative analysis of interior spruce transcriptome response to white pine weevil feeding and fungal inoculation with blue-stain fungus Leptographium abietinum. 92

2006, Bohlmann, 2008) and phenolics, which are associated with polyphenolic parenchyma (Franceschi et al., 2005). Terpenoids released from damaged tissues flow into the wound sites and kill, or repel the invading insects and kill or inhibit growth of beetle-associated fungal pathogens (Bohlmann, 2008). High resin canal density was associated with spruce resistance to weevils (Alfaro et al., 1997). The ability of conifer trees to produce large quantities of resin upon fungal induction was shown to be associated with resistance to beetle attacks (Raffa and Berryman, 1982). Toxicity of terpenoids was demonstrated against bark beetles (Raffa et al., 1985) and beetle-associated blue-stain fungi (Paine and Hanlon, 1994; Kopper et al., 2005). Phenolic metabolites are present in high quantities in the bark of conifer trees (Franceschi et al., 2005). Phenolics, such as catechin, taxifolin, or resveratrol, were shown to have bark beetle antifeedant properties (Faccoli and Schlyter, 2007). In addition phenolics have antifungal properties and phenolic-storing cells were suggested to be important in defense against fungal pathogens (Woodward and Pearce, 1988; Beckman, 2000). Phenolics accumulation and increased expression of genes involved in their production was associated with resistance to fungi (Nagy et al., 2004b; Wallis et al., 2008). Higher density of sclereid cells, which have thickened highly lignified cell walls, was correlated with Sitka spruce resistance to white pine weevil (King and Alfaro, 2009) and involvement of these cells was demonstrated in spruce defense against bark beetle (Wainhouse et al., 1990; Wainhouse et al., 1998) and against bark beetle-associated fungus (Wainhouse et al., 1997). In addition to chemical defenses, several pathogenesis related (PR) proteins were shown to be involved in defense response in conifers (Kozlowski and Metraux, 1998; Davis et al., 2002; Hietala et al., 2004; Nagy et al., 2004a; Piggott et al., 2004; Fossdal et al., 2006). Antifungal activity of PR proteins and antiinsect chitinase activity was demonstrated in angiosperm biological systems (Van Loon and Van Strien, 1999; De Lucca et al., 2005; Sels et al., 2008; Lawrence and Novak, 2006). The recent development of genomics tools for conifers allowed transcriptome scale evaluation of the conifer defense response. Transcriptome profiling of Sitka spruce leader defense response to wounding, spruce budworm and white pine weevil feeding using 9.7K microarray (Ralph et al., 2006b), evaluation of Scots pine transcriptome response to root fungal pathogen Heterobasidion annosum using 2.1K microarray (Adomas et al., 2007) and evaluation of defense responses of interior spruce (Picea glauca x engelmannii) and lodgepole pine to inoculation with beetle vectored blue-stain fungi using 21.8K spruce microarray (Chapter 2) pointed to many similarities in conifer 93 response to insects and fungi on the transcriptome level. The comparisons of different responses have been hindered by different species used in the studies and different experimental designs. Understanding of the generic and specific roles of defense related pathways can be expanded with direct comparative studies of conifer defense responses to insect and fungal pathogens. Our goal was to compare molecular mechanisms of interior spruce response to white pine weevil and to necrotrophic blue-stain fungal pathogen L. abietinum. In our study we performed a microarray analysis of the effect of white pine weevil feeding on the transcriptome of interior spruce bark and compared this analysis with a previously performed microarray study of interior spruce bark response to inoculation with L. abietinum over the time course of 2 weeks (Chapter 2). Comparison of the transcriptome response of interior spruce to white pine weevil feeding and inoculation with L. abietinum allowed us to decipher generic conifer defense strategies and treatment specific strategies. Our results showed a large overlap of the spruce transcriptome response to white pine weevil and blue-stain fungal inoculation, with some treatment-specific variation in response. We observed some qualitative and some quantitative differences in the transcriptome response of interior spruce to different treatments. Overall this study contributed to our understanding of conifer defense to pathogens and insects, and characterized involvement of different transcripts in these defense responses on the transcriptome scale.

3.2 MATERIAL AND METHODS

3.2.1 Plant material and weevil treatment Interior spruce (Picea glauca x engelmannii, clone I1026) seedlings were propagated by somatic embryogenesis and generously provided by Dr. David Ellis (CellFor Inc., Victoria, Canada). Seedlings were grown to four years old outside at the University of British Columbia under natural light and environmental conditions. One week prior to treatment, saplings were transferred to the UBC greenhouse with greenhouse temperature fluctuating between 20 and 24oC and average humidity of 45% during summer months. Adult white pine weevils (P. strobi) were collected form infested leaders of Sitka Spruce growing on British Columbia Forest Service (BCFS) research plantations at Campbell River, Vancouver Island, BC. Weevils were kept without food on moist filter paper for 48 hr before they were placed on the trees. Five weevils were placed on each tree and caged on the lower stem section corresponding to the first year of growth (about 10-12 cm). Bark tissue from the area of weevil feeding was harvested 94 directly into liquid nitrogen from the treated and control (untreated) saplings 6 hours, 2 days, 2 weeks post-treatment. Weevil feeding during first 6 hours resulted in approximately one small puncture wound per five square centimeters, by 2 days feeding punctures were observed close to one per square centimeter and by 2 weeks there were 3-4 puncture wounds per square centimeter. Harvested bark tissue was stored at -80oC. Four trees were subjected to weevil feeding for each time point.

3.2.2 Microarray hybridization and gene expression data analysis Total RNA was extracted using previously published protocol (Appendix 3; Kolosova et al., 2004). Total RNA isolated was qualified and quantified by spectrophotometer. RNA quality was checked by a 2100 Bioanalyzer (Agilent Technologies). For microarray experiments, total RNA was isolated from the bark of interior spruce trees subjected to weevil feeding and untreated control for the three time points – 6 hours, 2 days and 2 weeks. RNA was isolated from four independent biological replicates for each treatment and each time point. All microarray experiments were designed to comply with MIAME guidelines (Brazma et al., 2001). The 21.8K cDNA microarray (containing 21,843 cDNA elements selected from cDNA libraries representing developmental stages of xylem, phloem, bark and roots, as well as elicitor- treated bark) and hybridization conditions are described in Chapter 2.2.4. A loop design with dye balance was chosen to allow comprehensive analysis of transcriptome changes between treatments at three time points (Figure 3.1).

I 4 C 6h 6h Figure 3.1: Microarray experimental 1 1 design. Each connection indicates two samples (specified by treatment and time point), which were hybridized on the I C same microarray. The number above the 1 4 1 connection indicates the number of 2d 2d replicates for each hybridization of two connected samples against each other. 1 1 Treatments: C-control, I-insect (weevil) treatment. Treatment time course included 6 hours (6h), 2 days (2d) and 2 I 4 C weeks (2w). 2w 2w

This microarray analysis was designed to have comparable statistical power to the previously performed microarray analysis of the effect of blue-stain fungus L. abietinum inoculation on interior spruce bark (Chapter 2). Four hybridizations were performed for treatment and control comparison within each time point and one hybridization was performed for each comparison between time points for both treatment and control. The experiment was designed to be Cy3 Cy5 dye balanced. Raw and processed data and Tiff images are available at the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/). EST sequence data for the microarray ESTs is available in the National Center for Biotechnology Information (NCBI) databases and is searchable by EST IDs. Data analysis was done as described previously (Holliday et al., 2008). To calculate changes in gene expression a linear mixed-effect model was used for 18 microarray slides (hybridizations) of the experiment. P values were calculated for each EST for treatment vs. control comparison and q values were calculated to correct for false discovery rate for each p value (Storey and Tibshirani, 2003). Microarray analysis of interior spruce bark inoculated with blue-stain fungus L. abietinum was performed as described in Chapter 2.

3.2.3 Real-time quantitative PCR and gene expression data analysis of selected transcripts in weevil treated interior spruce Total RNA (5µg per treatment) was pretreated with DNaseI (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions to remove genomic DNA. cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen) with an oligo dT18 primers. cDNA synthesis was evaluated using gel electrophoresis. Gene specific primers were designed using interior spruce full length and EST sequences available at the NCBI database and the lodgepole pine EST sequences produced as a result of the sequencing project reported in this paper. Methods for the primer design were previously described (Ralph et al., 2006b). Primer specificity was confirmed by analysing individual RT-PCR products on a 2% agarose gel, melting curve analysis and by sequencing. Primer sequences are provided in Supplemental Table 3.1. Real-time qPCR conditions and data analysis were as previously described (Ralph et al., 2007a). Three biological replicates were used for target quantification in each cDNA source. Transcript abundance was normalized to translation initiation factor 5A (TIF5A, IS0013_F24, GenBank: DR448953). Statistical analysis was performed as described in Chapter 2.2.5. Real-time qPCR data analysis of interior spruce subjected to weevil

96 feeding was compared with the real-time PCR data analysis of interior spruce inoculated with L. abietinum and treated with wounding that is reported in Chapter 2.

3.3 RESULTS

3.3.1 Weevil feeding induces a large transcriptome response in white spruce bark, similar to the transcriptome changes induced by blue-stain fungal inoculation and wounding We performed comparative transcriptome analysis using the 21.8K spruce cDNA microarray to study the effect of weevil feeding and blue-stain fungus (L. abietinum) inoculation on interior spruce. Bark was harvested from the lower stem section of interior spruce saplings exposed to weevil feeding at 6 hours, 2 days and 2 weeks after- treatment (corresponding to the similar area of interior spruce stem that was treated with wounding or inoculated with L.abietinum as described in Chapter 2.2.1). Transcripts were considered differentially expressed if transcript abundance exhibited fold change over 2x fold with p <0.05 and q <0.05. A complete list of expression data for interior spruce treated with weevil feeding or inoculated with Leptographium abietinum for all 21.8K microarray elements is available in Supplementary Table 3.2. Close to four thousand (3789) transcripts were differentially expressed in interior spruce subjected to weevil feeding throughout the course of the study (close to 20% of the transcriptome represented on the 21.8K microarray) (Supplemental Table 3.2) whereas 2052 transcripts were differentially expressed in response to inoculated with L. abietinum (Chapter 2, Supplemental Table 2.3). Weevil feeding induced 2197 transcripts throughout the time course as compared to 1440 transcripts induced by fungal inoculation (Supplemental Table 2.3, 3.2). The number of downregulated transcripts in interior spruce treated with weevil feeding over the time course was 1592 as compared to 612 transcripts downregulated by fungal treatment (Table 3.1, Supplemental Table 2.3, 3.2).

Table 3.1: Overall changes in gene expression induced by weevil feeding, wounding and L. abietinum inoculation in interior spruce bark. C-control, I-weevil, W-wounding, F-fungal treatment. Numbers in table indicate the number of genes with fold change >2, p<0.05, q<0.05 Common Common Common I/C W/C F/C F/W I/C and W/C I/C and F/C I/C and F/W Time point up down up down up down up down up down up down up down 6h 7 0 589 53 884 65 60 1 7 0 7 0 0 0 2d 1577 1293 343 77 858 566 222 100 265 67 559 377 177 48 2w 1427 803 0 0 61 10 1 0 0 0 56 4 1 0

The transcriptome response to weevil feeding seems to require a time lag as it only developed at 2 days post-treatment and is nearly absent at 6 h post-treatment. In contrast, response to fungal treatment and wounding is strong at 6 h post-treatment already involving close to a thousand transcripts in case of fungal treatment and six hundred transcripts in case of wounding. Two days after treatment, the number of upregulated (1577) and downregulated (1293) transcripts in interior spruce affected by weevil feeding is about twice as high as the number of upregulated (858) and downregulated (566) transcripts in interior spruce inoculated with L. abietinum. A number of transcripts induced by fungal treatment (559, 65% of all induced transcripts at 2 days) were also induced by weevil feeding in interior spruce bark and 377 transcripts were commonly downregulated in both responses. Two weeks after treatment, 2230 transcripts are differentially expressed in weevil treated interior spruce whereas only 71 transcripts are differentially expressed in response to L. abietinum inoculation. A comparison of the of transcripts either upregulated or downregulated by weevil or fungal treatment over the timecourse revealed overlap in the interior spruce response to weevil feeding and fungal inoculation. There was a large overlap of transcripts upregulated by both treatments (941, close to 50% of the weevil induced transcriptome) and downregulated by both treatments (428, close to 30% of the weevil downregulated transcriptome). The weevil induced transcriptome included 65% of the fungus induced transcriptome and weevil downregulated transcriptome included 75% of the fungus downregulated transcriptome in interior spruce (Supplemental Table 2.3, 3.2). In response to weevil feeding 1227 transcripts were uniquely induced and 1137 transcripts were uniquely downregulated, whereas 472 transcripts were uniquely induced and 155 transcripts were uniquely downregulated by fungal treatment over the time course (Supplemental Table 2.3, 3.2). Twenty nine transcripts were upregulated by weevil treatment and downregulated by fungal treatment; 27 transcripts were upregulated by fungal inoculation and downregulated by weevil treatment (Supplemental Table 2.3, 3.2). Most of the transcripts with opposing expression changes were only moderately up- or downregulated by weevil feeding or fungal inoculation. The transcripts induced by weevil treatment and downregulated by fungal inoculation included three peroxidase transcripts, two dirigent protein transcripts and a laccase transcript (Supplemental Table 2.3, 3.2). The set of transcripts downregulated by weevil feeding and induced by fungal inoculation included three transcripts from the shikimate pathway (two arogenate dehydratase transcripts and chorismate mutase), six transcripts from the phenylpropanoid pathway (phenylalanine ammonia-lyase (PAL), cinnamate-4- 100 hydroxylase (C4H), two chalcone/stilbene synthase (CHS/STS) transcripts, dihydroflavonol 4-reductase/leucoanthocyanidin reductase (DFR) and acetyl CoA: benzylalcohol acetyltransferase), and three transcripts involved in photosynthesis (three chlorophyll A-B binding family protein transcripts) (Supplemental Table 2.3, 3.2).

3.3.2 Cluster analysis of interior spruce transcriptome responses to weevil feeding and fungal inoculation Spruce microarray transcripts were clustered based on their expression profiles over time in weevil-treated interior spruce. Three possible expression modes: upregulated (U), downregulated (D) and no change (-) at three time points during the time course (6 h, 2 d, 2 w) can be grouped into 27 different possible clusters. Sorting of differentially expressed transcripts into these groups revealed that most of the transcripts populated only six major clusters with only four minor additional clusters (6 transcripts or less in a cluster) (Figure 3.2, Supplemental Table 3.3).

Figure 3.2: Cluster analysis of transcripts based on the pattern of expression through the time course of 6h, 2 days (2d) and 2 weeks (2w) for interior spruce treated with weevil feeding or fungal inoculation with Leptographium abietinum. A. Expression dynamics groups of weevil vs. control treatment comparison. B. Fungal treatment vs. control corresponding dynamics groups to weevil treatment. C. Additional major dynamic groups of fungal treatment vs. control comparison. The line passes through mean expression calculated on the logarithmic scale. Thickness of each line corresponds to the square root of the number of genes in each group shown (the numbers of transcripts are shown in the right bottom corner of each graph). Clusters are indicated by the expression change at three time points 6h, 2 days and 2 weeks, U – upregulated, (-) – no change, D – downregulated expression.

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Figure 3.2

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Although a similar number of transcripts were induced by weevil at 2 days (1577) and 2 weeks (1427) only about 50% (796) of the transcripts were induced at both time points (Table 3.1, Figure 3.2) The transcriptome changes induced by weevil were compared to changes induced by L. abietinum (Figure 3.2, Supplemental table 3.3, 2.4). Both weevil and fungal treatment have large clusters of genes induced (-U-) and downregulated (-D-) at 2 days post-treatment. Other major clusters are different between the two treatments, reflecting the slow response to weevil feeding compared to faster response to fungal inoculation, which is declining at 2 weeks post-treatment, while the response to weevil feeding is ongoing (Table 1). The overlap of transcripts in cluster (-U-) between weevil and fungal treatment includes 105 transcripts, whereas 125 transcripts overlap and in cluster (-D-), highlighting the different dynamics of transcriptome response to the different treatments throughout the time course.

3.3.3 Comparison of transcripts most strongly induced in interior spruce by weevil feeding or fungal inoculation

Microarray analysis revealed strong induction of a substantial number of transcripts in interior spruce exposed to weevil feeding (Table 3.2). The number of highly induced transcripts (fold change > 5) in weevil treated spruce was comparable to the number of transcripts strongly induced by fungal inoculation compared to control treatment (Table 3.2, Supplemental Table 2.3).

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Table 3.2: Transcripts most strongly induced in interior spruce by weevil feeding. Expression of the transcripts strongly induced by weevil feeding is compared to the expression in fungus or wounding treated interior spruce. I – weevil feeding, C – control, W – wounding, F – fungal treatment. Dynamic of expression is shown as three sign sequences indicating change in expression at 6h, 2 days and 2 weeks, U – upregulated, (-) –no change, D – downregulated transcript expression.

Gene ID E- value AGI BLASTX vs. Arabidopsis I/C F/C W/C F/W I/C W/C F/C F/W 6 h WS01012_J14 2.00E-06 At1g02820 LEA3 family protein 4.40 25.33 16.60 1.52* UUU U-- UU- -U- WS01033_E03 2.00E-38 At5g52390 photoassimilate-responsive protein 2.75 16.73 8.70 1.92* UUU UU- UU- --- WS0054_A06 n.a. No significant hit 2.71 9.41 6.48 1.45* UU- U-- U-- --- WS0078_I22 n.a No significant hit 2.18 15.25 7.85 1.96* UUU U-- UU- -U- WS00927_G05 1.00E-11 At2g46600 calcium-binding protein 2.16 4.42 2.42 1.82* UU- U-- U-- --- WS00930_G17 7.00E-46 At3g02650 (PPR) repeat-containing protein 2.02 5.30 3.12 1.69* UUU U-- U-- --- WS0039_H23 n.a No significant hit 2.02 3.20 2.61 1.22* UUU U-- U-- ---

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Table 3.2 (cont) Gene ID E- value AGI BLASTX vs. Arabidopsis I/C F/C W/C F/W I/C W/C F/C F/W 2 days WS01010_C04 n.a No significant hit 88.69 14.65 4.73 3.13 -UU UU- UU- -U- WS00926_A12 2.00E-30 At4g10300 expressed protein 79.74 13.39 4.05 3.33 -UU UU- UU- -U- WS00111_I09 3.00E-22 At1g77330 ACC oxidase 77.84 6.62 2.37 2.78 -UU UU- UU- -U- WS00917_K11 n.a No significant hit 76.92 16.23 2.80 5.88 -UU UU- UU- -U- WS00730_B15 4.00E-24 At5g03260 laccase 69.34 3.08 2.49 1.23* -UU UU- UU- --- WS01021_E17 5.00E-46 At2g30210 laccase 69.33 1.79* 1.64* 1.09* -UU U-- U-- --- WS00110_A15 6.00E-39 At1g08080 carbonic anhydrase 67.75 23.37 11.34 2.04 -UU UU- UUU -U- WS00928_K21 4.00E-75 At1g08080 carbonic anhydrase 64.12 23.56 13.63 1.72* -UU UU- UUU --- WS00923_N05 6.00E-75 At5g05390 laccase 59.48 5.01 3.25 1.54* -UU UU- UU- --- WS0014_N09 7.00E-24 At1g08080 carbonic anhydrase 53.13 7.70 3.80 2.04 -UU -U- UU- -U- WS0064_G03 2.00E-18 At3g14100 oligouridylate-binding protein 52.36 5.83 3.97 1.47* -UU UU- UU- --- IS0012_P10 n.a No significant hit 48.60 7.70 4.85 1.59* -UU UU- UU- --- WS01012_J14 2.00E-06 At1g02820 LEA3 family protein 46.64 3.88 1.61 2.38 UUU U-- UU- -U- WS0039_D14 7.00E-47 At4g35160 COMT like 46.20 0.89* 1.02 0.87* -UU U-- U-- --- WS00928_J21 1.00E-08 At1g19180 expressed protein 45.26 1.76* 1.12 1.59* -UU U-- U-- --- WS01010_B20 n.a No significant hit 44.39 1.23* 0.96 1.27* -UU U-- U-- --- WS0016_N04 8.00E-64 At5g05390 laccase 43.69 2.50 2.06 1.22* -UU UU- UU- --- WS00918_B02 n.a No significant hit 37.05 1.63* 1.28 1.28* -UU U-- U-- --- WS0063_C22 n.a No significant hit 36.78 7.41 5.12 1.45* -UU UU- UU- --- IS0012_L15 4.00E-24 At4g22710 cytochrome P450 36.33 35.29 8.42 4.17 -UU -U- -U- -U- WS00931_I06 2.00E-53 At4g10490 oxidoreductase 34.64 9.40 2.75 3.45 -UU -U- -U- -U- IS0013_E03 6.00E-40 At1g08080 carbonic anhydrase 34.20 9.09 5.62 1.61* -UU UU- UU- --- WS00916_N11 3.00E-12 At4g18910 aquaglyceroporin 33.57 7.50 4.31 1.75* -UU UU- UU- --- WS0091_H07 2.00E-18 At3g22400 lipoxygenase 30.86 6.99 4.82 1.45* -UU UU- UU- --- WS0102_J09 9.00E-52 At1g08080 carbonic anhydrase 29.53 5.11 3.18 1.61* -UU -U- UU- --- WS0105_K14 2.00E-07 At1g74950 expressed protein 28.47 6.20 3.38 1.85* -UU UU- UUU --- WS0082_A04 n.a No significant hit 27.91 2.91 1.57 1.85* -UU U-- UU- --- WS00723_D18 4.00E-45 At5g54160 quercetin 3-O-methyltransferase 1 26.63 0.75* 1.01 0.75* -UU U-- U-- --- WS01026_P09 n.a No significant hit 25.93 5.51 2.38 2.33 -UU -U- -UU -U- WS0105_M20 1.00E-67 At5g05390 laccase 25.52 0.96* 1.24 0.77* -UU U-- U-- --- WS00940_M12 n.a No significant hit 25.50 4.53 2.41 1.89* -UU UU- UU- --- WS0262_F02 9.00E-17 At4g22710 cytochrome P450 25.46 10.70 2.60 4.17 -UU -U- -U- -U- WS00914_H24 2.00E-09 At4g23690 dirigent 25.07 20.99 9.52 2.22 -UU UU- UUU -U- WS00915_P20 n.a No significant hit 24.84 6.91 2.91 2.38 -UU -U- UU- -U- WS0063_G08 n.a No significant hit 24.15 3.44 1.74 1.96* -UU U-- UU- --- WS00916_P13 3.00E-06 At5g13220 expressed protein 23.61 0.92* 0.92 1.00* -UU U-- U-- --- WS01032_M02 1.00E-48 At1g64160 dirigent 22.90 16.90 6.87 2.44 -UU UU- UUU -U- WS01024_C19 4.00E-55 At2g41380 embryo-abundant protein 22.07 4.90 2.42 2.04 -UU UU- UU- -U- WS0263_E22 2.00E-23 At1g12570 GMC oxidoreductase 22.01 2.27 1.70 1.33* -UU U-- UU- --- WS0262_K03 3.00E-44 At1g80160 lactoylglutathione lyase 21.96 3.02 2.72 1.11* -UU UU- UU- --- WS01033_B20 1.00E-07 At5g13220 expressed protein 21.92 1.98* 1.50 1.32* -UU U-- U-- --- WS00912_L05 1.00E-13 At1g74950 expressed protein 21.85 21.14 4.95 4.35 -UU UU- UUU -U- WS0044_J05 5.00E-20 At1g75390 bZIP transcription factor 20.38 9.94 6.15 1.61* -UU UU- UUU --- WS00931_J05 n.a No significant hit 20.12 5.93 2.70 2.17 -UU -U- UU- -U- WS0105_L13 n.a No significant hit 20.09 2.04 1.05 1.92* -UU U-- UU- ---

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Table 3.2 (cont) Gene ID E- value AGI BLASTX vs. Arabidopsis I/C F/C W/C F/W I/C W/C F/C F/W 2 weeks WS0262_F02 9.00E-17 At4g22710 cytochrome P450 63.76 1.46* 1.12* 1.30* -UU -U- -U- -U- WS00924_I17 3.00E-36 At3g19390 cysteine proteinase 53.33 1.12* 1.00* 1.12* -UU -U- -U- -U- WS00110_A15 6.00E-39 At1g08080 carbonic anhydrase 51.37 2.64 1.40* 1.89* -UU UU- UUU -U- WS00928_K21 4.00E-75 At1g08080 carbonic anhydrase 38.12 2.86 1.51* 1.89* -UU UU- UUU --- WS00931_I06 2.00E-53 At4g10490 oxidoreductase 36.52 1.14* 1.14* 1.00* -UU -U- -U- -U- WS0014_N09 7.00E-24 At1g08080 carbonic anhydrase 33.94 1.48* 1.10* 1.35* -UU -U- UU- -U- WS01010_C04 n.a No significant hit 33.80 1.83* 1.58* 1.16* -UU UU- UU- -U- WS0102_C21 3.00E-46 At5g23300 dihydroorotate dehydrogenase 32.16 1.61* 0.73* 2.22* -UU UU- UU- --- IS0012_L15 4.00E-24 At4g22710 cytochrome P450 31.60 1.82* 1.08* 1.69* -UU -U- -U- -U- WS0091_H07 2.00E-18 At3g22400 lipoxygenase 31.26 1.90* 1.59* 1.19* -UU UU- UU- --- WS00926_A12 2.00E-30 At4g10300 expressed protein 30.88 1.56* 1.22* 1.28* -UU UU- UU- -U- WS00912_L05 1.00E-13 At1g74950 expressed protein 28.51 2.85 1.34* 2.13* -UU UU- UUU -U- WS0105_K14 2.00E-07 At1g74950 expressed protein 27.92 2.89 1.57* 1.85* -UU UU- UUU --- WS00916_N11 3.00E-12 At4g18910 aquaglyceroporin 25.87 1.57* 0.87* 1.82* -UU UU- UU- --- WS00113_G22 1.00E-34 At1g73050 (R)-mandelonitrile lyase 25.00 4.46 2.55* 1.75* -UU UU- UUU -U- IS0013_E03 6.00E-40 At1g08080 carbonic anhydrase 24.40 1.84* 1.21* 1.52* -UU UU- UU- --- IS0012_P10 n.a No significant hit 24.08 1.63* 1.51* 1.08* -UU UU- UU- --- WS0102_J09 9.00E-52 At1g08080 carbonic anhydrase 21.57 1.30* 1.07* 1.20* -UU -U- UU- --- WS00111_I09 3.00E-22 At1g77330 ACC oxidase 21.39 1.21* 1.27* 0.95* -UU UU- UU- -U- WS0064_G03 2.00E-18 At3g14100 oligouridylate-binding protein 20.63 1.39* 1.31* 1.05* -UU UU- UU- --- WS01021_E17 5.00E-46 At2g30210 laccase 20.41 1.48* 1.61* 0.92* -UU U-- U-- --- WS00923_N05 6.00E-75 At5g05390 laccase 20.16 1.33* 1.58* 0.84* -UU UU- UU- --- IS0011_F24 2.00E-46 At5g05340 peroxidase 18.91 2.91 2.02* 1.45* -UU UU- UUU --- WS00940_M12 n.a No significant hit 18.90 1.26* 1.19* 1.05* -UU UU- UU- --- WS00730_B15 4.00E-24 At5g03260 laccase 18.36 1.24* 1.23* 1.01* -UU UU- UU- --- WS0062_I06 n.a No significant hit 17.78 2.45* 0.84* 2.94* -UU -U- -U- --- WS00928_J21 1.00E-08 At1g19180 expressed protein 17.75 1.23* 1.00* 1.23* -UU U-- U-- --- WS00930_M06 2.00E-26 At1g73050 (R)-mandelonitrile lyase 17.73 2.91 2.12* 1.37* -UU UU- UUU -U- WS0091_M08 3.00E-38 At1g42970 GAPB 17.27 0.77* 0.95* 0.81* --U ------WS00929_G06 2.00E-56 At4g29270 acid phosphatase class B 16.54 1.39* 1.16* 1.20* -UU ------WS0016_N04 8.00E-64 At5g05390 laccase 16.42 1.39* 1.41* 0.98* -UU UU- UU- --- IS0011_B10 4.00E-28 At4g29260 acid phosphatase class B 16.21 1.86* 1.21* 1.54* -UU U------WS01039_L13 2.00E-14 At1g73260 trypsin and protease inhibitor 16.05 4.85 2.77* 1.75* --U -U- -UU --- WS0075_I06 2.00E-22 At1g62500 protease inhibitor 15.94 3.05* 1.78* 1.69* --U ------WS00937_E06 2.00E-27 At3g48140 senescence-associated protein 15.82 2.13 1.48* 1.43* -UU UU- UUU --- WS0063_G08 n.a No significant hit 15.22 1.03* 0.80* 1.28* -UU U-- UU- --- WS0093_G14 n.a No significant hit 15.21 5.53 2.03* 2.70* -UU -U- -UU -U- WS01010_B20 n.a No significant hit 14.36 1.02* 0.97* 1.05* -UU U-- U-- --- WS0024_B06 4.00E-17 At3g22400 lipoxygenase 14.18 1.70* 1.54* 1.10* -UU UU- UU- --- WS01021_C04 2.00E-13 At3g23380 P21-Rho-binding domain 14.08 2.65 1.61* 1.64* -UU UU- UUU --- WS0023_N05 5.00E-53 At5g42650 allene oxide synthase 14.07 1.42* 1.10* 1.28* -UU -U- -U- --- WS0063_E19 n.a No significant hit 13.09 1.72* 0.94* 1.82* -UU UU- -U- -U- WS0264_O06 8.00E-11 At3g50340 expressed protein 12.37 1.00* 0.82* 1.22* --U ------*- values marked with asterisk indicate transcript fold changes that did not meet the criteria of differentially expressed genes. All not marked fold change numbers in the table indicate fold change of differentially expressed transcripts with fold change > 2 and p,q values < 0.05

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Among the 45 most highly induced transcripts by weevil treatment in spruce at 2 days post-treatment were transcripts for ACC oxidase, lipoxygenase, laccases, COMT- like, dirigent protein and carbonic anhydrases. At two weeks post-treatment, carbonic anhydrase transcripts, ACC oxidase, lipoxygenase and laccases continued to be highly induced with the addition of allene oxide synthase, peroxidase and protease inhibitor transcripts. All of the highly induced transcripts at 2 days continued to be induced at 2 weeks (Table 3.2). Transcripts most strongly induced by fungal treatment compared to wounding and by fungal treatment compared to control included pathogenesis related proteins such as chitinases, which were more represented than following weevil treatment (Table 3.2, Table 2.2, Supplemental Table 2.3). Many of the highly induced transcripts (especially at 2 day post-treatment) were also highly induced by fungal treatment compared to control and most of the 97 highly induced transcripts in interior spruce induced by weevil feeding at different time points were also induced by fungal treatment compared to control throughout the time course of treatment and by wounding treatment (Table 3.2).

3.3.4 Comparative functional characterization of induced transcriptomes in interior spruce treated with weevil or fungal inoculation The 21.8K microarray transcripts were annotated using the available annotation and functional characterization from the Arabidopsis TAIR database (TAIR; http://www.arabidopsis.org) and the annotation available from conifer specific functional analysis (Ralph and Bohlmann, unpublished results). To gain an overview of the functional distribution of differentially expressed transcripts in interior spruce induced by weevil feeding, differentially expressed transcripts were segregated into gene ontology (GO) categories based on Arabidopsis GO categories (TAIR) (Chapter 2, Supplemental Table 2.5). In the GO category of the response to biotic and abiotic stimulus over a hundred transcripts were upregulated (130) and similar number of transcripts was downregulated (110) in interior spruce induced by weevil feeding (Table 3.3A, B).

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Table 3.3: Functional annotation of differentially expressed transcripts A. Upregulated transcripts Functional category1 weevil W/C F/C F/W GO:Response to abiotic and biotic stimulus (875)2 130 63 108 17 GO:Defense response (336) 59 34 50 13 *Phenylpropanoid pathway (340) 107 62 96 20 *Terpenoid pathway (82) 16 18 17 3 GO:Jasmonic acid biosynthesis and response (78) 25 15 26 1 GO:Ethylene biosynthesis and response (36) 13 9 14 4 GO:Salicylic acid biosynthesis and response (34) 7 3 5 4 GO:Primary metabolism (3948) 433 172 332 48 GO:Electron transport and energy pathways (417) 53 26 51 8 GO:Photosynthesis (58) 4 - 1 -

B. Down-regulated transcripts Functional category1 weevil W/C F/C F/W GO:Response to abiotic and biotic stimulus (875)2 110 19 40 2 GO:Defense response (336) 17 1 4 1 *Phenylpropanoid pathway (340) 39 1 21 4 *Terpenoid pathway (82) 3 - 1 - GO:Jasmonic acid biosynthesis and response (78) 7 - - - GO:Ethylene biosynthesis and response (36) 3 1 - - GO:Salicylic acid biosynthesis and response (34) 4 - - - GO:Primary metabolism (3948) 274 21 91 11 GO:Electron transport and energy pathways (417) 55 7 18 1 GO:Photosynthesis (58) 38 4 17 1 C-control, W-wounding, F-fungal treatment. 1- Functional categories marked with GO included spruce microarray transcripts annotated to functionally characterized Arabidopsis genes to respective GO category in TAIR database (TAIR; http://www.arabidopsis.org), *- functional categories marked with an asterisk were assembled based on characterization of conifer secondary metabolism by Ralph and Bohlmann (unpublished data). 2 Numbers in parentheses indicate the number of transcripts from a given functional category on the spruce microarray.

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The number of upregulated transcripts by weevil from this category is similar to the number of transcripts induced by fungal treatment (108) (Table 3.3A). Most of the highly induced transcripts in this category in interior spruce induced by weevil herbivory were PR protein genes (hevein-like protein, osmotin-like protein, thaumatin-like protein), transcripts involved in octadecanoid signaling and transcripts involved in the phenylpropanoid pathway (Supplemental Table 3.2). These transcripts were also induced by fungal treatment during the time course of the study with PR genes and genes involved in the phenylpropanoid pathway being the most upregulated (Supplemental Table 2.3). The majority of these transcripts were also induced by wounding treatment, to a lesser degree. Among the genes, which are the most downregulated by weevil feeding in this GO category, were genes involved in photosynthesis. Among the genes in the GO category of defense response that were strongly induced by weevil feeding in interior spruce were dirigent protein transcripts, phenylalanine ammonia-lyase transcripts and transcripts from octadecanoid signalling pathway (e.g. allene oxide synthase, lipoxygenase, osmotin-like protein transcripts, (R)- mandelonitrile lyase) (Supplemental Table 3.2). These transcripts were induced by weevil at 2 days and 2 weeks post-treatment and most of them were also highly induced by fungal treatment (compared to control) (Supplemental Table 3.2, 2.3). More transcripts in this category were downregulated by weevil treatment (17) compared to fungal treatment (4). Transcripts in this category downregulated by weevil feeding included accelerated cell death 2 (ACD2) transcript, glutathione S-transferase, basic endochitinase transcripts and several transcripts of LRR class disease resistance proteins and none of these transcripts were affected by fungal treatment. Evaluation of the transcript expression profile from the GO categories of biosynthesis and response to jasmonic acid, salicylic acid and ethylene in interior spruce treated with weevil feeding revealed induction of about a third of the transcripts from GO categories of jasmonic acid biosynthesis and response (24) and ethylene biosynthesis and response (13) with lower induction of transcripts from the GO category of salicylic acid biosynthesis and response (7) (Table 3.3A). Very few transcripts were downregulated from these GO signalling categories (Table 3.3B). A similar number of transcripts were induced in these signalling pathways by fungal treatment in interior spruce (Table 3.3A, B). Fifteen of the 25 transcripts induced by weevil from the jasmonic acid biosynthesis GO category were also induced by fungal treatment and 10 of these were induced by wounding throughout the study. Among the 13 induced transcripts 109 throughout the time course involved in ethylene biosynthesis and response nine transcripts were also induced by fungal treatment and four transcripts were induced by wounding (Supplemental Table 2.3, 3.2). To estimate the effect of weevil feeding on primary metabolic processes in interior spruce, we examined the effect of weevil feeding on transcripts from the primary metabolism GO category. Among 3948 transcripts from this GO category, 433 transcripts were induced and 274 downregulated by weevil feeding in interior spruce (Table 3.3A,B). Among the 433 primary metabolism transcripts induced by weevil in interior spruce, 204 were also induced by fungal treatment throughout the time course (Supplemental Table 2.3, 3.2). Among the 286 downregulated transcripts by weevil, 62 transcripts were downregulated by fungal treatment. Only 11 transcripts had different direction in expressional change between weevil and fungus treatment. This data indicates a large rearrangement of primary metabolism, with over 700 transcripts being differentially expressed as a result of weevil feeding. There is also a large overlap and coherence in expression changes of transcripts involved in primary metabolism in interior spruce treated with weevil feeding and fungal inoculation. Ten percent of transcripts from the GO category of electron transport and energy were induced, and similar number of transcripts were downregulated by weevil feeding (Table 3.3A, B). Of the 53 transcripts induced by weevil feeding during the time course, 31 transcripts were also induced during fungal treatment (Supplemental Table 2.3, 3.2). Of the 55 transcripts downregulated by weevil feeding, 13 were also downregulated by fungal treatment. Most of the transcripts downregulated in this category belong to GO category of photosynthesis (Table 3.3B). The number of photosynthesis-related downregulated transcripts was twice smaller in fungus treated interior spruce (Table 3.3B).

3.3.5 Comparative analysis of the phenylpropanoid pathway response in interior spruce induced by weevil feeding and fungal inoculation Based on the previous analysis of conifer phenylpropanoid pathway transcripts (Ralph and Bohlmann, unpublished data), 3124 spruce transcripts were annotated to this pathway and 340 of these transcripts were present on the 21.8K microarray (Chapter 2, Supplemental Table 2.5). 146 transcripts were differentially expressed in interior spruce induced by weevil feeding throughout the time course (Table 3.3A, B). Of the 107 transcripts induced by weevil feeding throughout the study, 71 were also induced by fungal treatment (among 96 induced transcripts by fungal treatment compared to control over the time course), and 49 transcripts induced by both weevil 110 feeding and fungal inoculation were also induced by wounding (among 62 total) (Table 3.3A, Supplemental Table 2.3, 3.2). Among the 39 transcripts downregulated by weevil treatment, 10 were also downregulated by fungal treatment (among 21 total) and only one transcript was downregulated by wounding treatment (Table 3.3B, Supplemental Table 2.3, 3.2). Twelve transcripts had an opposing pattern of expression in interior spruce treated with weevil and inoculated with fungus. This data indicates a strong induction of phenylpropanoid pathway in interior spruce induced by weevil feeding and a large overlap of the phenylpropanoid response in interior spruce induced by weevil feeding, fungal inoculation and wounding treatment. The expression data was visualized using heat maps, prepared using TIGR MeV software version 3.1 (Saeed et al., 2003), which were organised based on the dynamic of phenylpropanoid gene expression in interior spruce induced by weevil feeding. The data is shown in comparison to the expression of the same transcripts in interior spruce treated with fungal inoculation (Figure 3.3).

Figure 3.3: Expression of transcripts annotated to the phenylpropanoid pathway in interior spruce treated with weevil feeding compared to fungal inoculation. Transcripts with significant fold change (fold change >2, p,q<0.05) at least one time point in interior spruce treated with weevil feeding were grouped based on the dynamic of expression. Expression changes for the same transcripts in interior spruce inoculated with fungus are shown on the left side. I – weevil (insect) treatment, C – control, F – fungal treatment. A. Phenylpropanoid pathway transcripts with significantly upregulated expression at 2 days and 2 weeks post-treatment in interior spruce treated with weevil feeding compared to the expression of the same transcripts in interior spruce inoculated with fungus. B. Phenylpropanoid pathway transcripts with significantly upregulated expression at only 2 days (until WS00935_G13) and at only 2 weeks (remaining) post- treatment in interior spruce treated with weevil feeding as compared to the expression of the same transcripts in interior spruce inoculated with fungus. C. Phenylpropanoid pathway transcripts with significantly downregulated expression at 2 days and 2 weeks (until WS0261_E15) and at only 2 days (until WS0099_J20) and at only 2 weeks (remaining) post-treatment in interior spruce treated with weevil feeding compared to the expression of the same transcripts in interior spruce inoculated with fungus.

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A B C

I vs.C6h vs.C6h I I vs.C2d vs.C2d I I vs.C2w vs.C2w I I vs.C6h vs.C6h F F vs.C2d vs.C2d F F vs.C2w vs.C2w F F

I vs.C6h vs.C6h I I vs.C2d vs.C2d I I vs.C2w vs.C2w I I vs.C6h vs.C6h F F vs.C2d vs.C2d F F vs.C2w vs.C2w F F vs.C6h vs.C6h I I vs.C2d vs.C2d I I vs.C2w vs.C2w I I vs.C6h vs.C6h F F vs.C2d vs.C2d F F vs.C2w vs.C2w F F

Fold change (log2)

112 -3.0 0.0 3.0

Figure 3.3

The heat map suggests a generic response in phenylpropanoid metabolism in spruce response to weevil feeding and fungal inoculation. Many of the transcripts (especially highly induced) that are induced at 2 days and 2 weeks of treatment in interior spruce are also induced by fungal treatment in interior spruce (Figure 3.3A). Many similarities in the expression heat map are also observed for the phenylpropanoid pathway transcripts induced by weevil after 2 days of feeding in comparison to the expression map of the same transcripts in interior spruce (Figure 3.3B). The heat map of downregulated phenylpropanoid transcripts at both 2 days and 2 weeks and only at 2 days of weevil feeding also looks remarkably similar to the heat map of the expression of the same transcripts in fungus treated interior spruce (Figure 3.3C). The phenylpropanoid pathway transcripts in interior spruce that were strongly induced by weevil feeding include several laccase and dirigent proteins (DIR), flavonoid biosynthetic pathway transcripts such as flavanone 3-hydroxylase (F3H), anthocyanidin synthase/leucoanthocyanidin dioxygenase (ANS), flavonol 3-O-glucosyltransferase (3GT), and also phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) transcripts (Table 3.4). Among the 42 most induced phenylpropanoid transcripts, 38 transcripts were also induced by fungal treatment compared to control and 30 transcripts were also induced by wounding treatment. The most highly induced transcripts in these treatments included laccases and DIR transcripts (Table 3.4). Among 25 laccase transcripts present on the microarray, 16 were induced by weevil treatment, 7 of which were induced by fungal treatment, and one of which was downregulated in fungal treated spruce. Six of the 7 transcripts induced by fungal treatment were also induced by wounding treatment.

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Table 3.4: Expression of phenylpropanoid pathway transcripts in interior spruce treated with weevil feeding, fungal inoculation and wounding with >5 fold change in at least one time point in weevil treated sample and q<0.05 Numbers in the table indicate expression levels fold change in specified treatment comparisons (I – weevil (insect); C, control; W, wounding; F, fungal treatments). LMCO, laccase multicopper oxidase; COMT-like, caffeic acid O-methyltransferase like; C3H, p- coumarate-3-hydroxylase; F3H, flavanone 3-hydroxylase; DIR, dirigent protein; CCR, cinnamoyl-CoA reductase; ANS, anthocyanidin synthase/leucoanthocyanidin dioxygenase; F5H-like, ferulate-5-hydroxylase like; PAL, phenylalanine ammonia-lyase; 3GT, flavonol 3-O-glucosyltransferase, AD, arogenate dehydratase; C4H, cinnamate-4- hydroxylase; F3'H, flavonoid 3'-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; DHQD-SD, 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase. Colour scale from yellow to red correlates with fold-change expression and was applied only to fold change >2 values with q<0.05. Fold-change:

≥ 5.00 > 1.50 ≥ 10.00 < 0.2 < 5.00 < 10.00

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Table 3.4 I/C I/C I/C F/C F/C F/C W/C W/C W/C Clone ID Annotation E value 6h 2d 2w 6h 2d 2w 6h 2d 2w WS00730_B15 LMCO 2.00E-79 1.13 69.34 18.36 6.22 3.08 1.24 5.47 2.49 1.23 WS01021_E17 LMCO 1.00E-58 1.09 69.33 20.41 3.18 1.79 1.48 3.52 1.64 1.61 WS00923_N05 LMCO 0 1.96 59.48 20.16 9.67 5.01 1.33 9.65 3.25 1.58 WS0039_D14 COMT-like 1.00E-106 1.34 46.20 5.23 4.84 0.89 1.02 2.86 1.02 0.98 WS0016_N04 LMCO 7.00E-67 1.24 43.69 16.42 4.51 2.50 1.39 4.83 2.06 1.41 IS0012_L15 C3H 5.00E-32 0.90 36.33 31.60 1.49 35.29 1.82 1.68 8.42 1.08 WS00931_I06 F3H 8.00E-53 1.29 34.64 36.52 0.88 9.40 1.14 1.13 2.75 1.14 WS00723_D18 COMT-like 3.00E-89 1.46 26.63 2.45 3.14 0.75 0.94 2.55 1.01 0.95 WS0105_M20 LMCO 7.00E-71 0.78 25.52 10.53 2.16 0.96 1.62 2.91 1.24 1.79 WS00914_H24 DIRa 8.00E-22 2.02 25.07 4.41 21.61 20.99 3.32 17.87 9.52 1.74 WS01032_M02 DIRa 1.00E-117 1.94 22.90 3.70 18.86 16.90 3.21 17.53 6.87 1.79 WS0094_C01 CCR 8.00E-58 1.43 16.19 5.43 5.91 2.06 1.00 3.73 1.69 0.95 WS00912_H11 ANS 5.00E-37 1.22 15.06 3.37 5.19 1.43 0.96 3.37 1.47 1.02 WS00945_E03 F5H-like 3.00E-28 1.64 13.55 5.89 4.17 1.96 1.06 4.78 1.52 1.06 WS01011_J07 DIRa 4.00E-48 1.57 12.02 2.61 6.32 6.28 1.91 4.70 2.64 1.10 WS0044_O08 PAL 2.00E-22 1.73 11.91 3.35 6.93 8.46 1.24 5.14 4.03 1.00 WS00825_K05 DIRa 1.00E-132 1.23 11.64 2.37 9.80 7.47 1.63 8.50 3.57 1.13 WS00821_C05 PAL 0 1.29 10.22 2.83 6.54 7.67 1.11 3.57 3.39 0.98 WS01030_B11 PAL 1.00E-57 1.50 9.48 4.97 5.63 1.85 1.19 4.78 1.63 0.99 WS00928_N23 3GT 9.00E-38 1.35 9.45 2.78 10.15 3.42 1.28 4.05 1.38 1.03 WS00818_A11 DHQD-SD 1.00E-38 1.17 9.26 3.56 2.27 4.81 1.31 1.84 1.82 1.08 WS0071_D22 PAL 0 1.22 8.68 2.53 7.21 7.55 1.28 4.52 3.78 1.12 WS01014_N23 3GT 4.00E-43 1.24 8.63 4.25 2.08 1.39 1.33 1.85 1.33 1.19 WS00934_G23 F5H-like 1.00E-45 1.52 8.40 4.52 5.53 2.16 1.21 4.89 1.72 1.13 WS0097_O04 3GT 6.00E-48 1.29 8.10 2.77 4.64 3.95 1.36 2.97 1.40 1.14 WS0071_A18 AD 1.00E-83 1.19 7.40 3.96 2.72 2.47 1.29 2.50 1.69 1.09 WS00931_C11 C4H 4.00E-79 1.40 7.28 4.89 5.51 4.77 1.54 4.46 2.25 1.15 WS00916_A12 F3'H 4.00E-44 1.31 6.68 3.65 9.13 1.76 0.88 8.66 1.63 0.88 WS00720_P10 3GT 1.00E-32 1.17 6.30 3.21 4.64 3.79 1.18 2.51 1.53 1.06 WS00915_B09 COMT-like 4.00E-45 1.21 4.76 8.70 1.67 0.86 2.13 1.09 1.12 1.33 WS00923_A19 LMCO 3.00E-97 0.82 3.81 8.71 0.78 2.87 2.31 1.08 2.21 1.48 WS00815_F23 LMCO 4.00E-90 0.88 3.17 5.06 0.77 0.92 1.64 0.93 1.01 1.31 WS0038_B22 LMCO 2.00E-77 0.95 3.03 7.27 0.83 0.89 1.75 1.02 1.30 1.24 WS0039_C04 LMCO 1.00E-180 0.97 2.59 7.26 0.65 0.93 1.81 0.71 1.25 1.34 WS00821_H07 CAD 4.00E-39 1.02 6.61 1.54 2.66 1.09 0.91 1.89 0.80 1.02 WS00911_I09 DIRa 2.00E-73 0.99 6.57 1.90 2.18 1.89 0.92 1.82 1.12 0.92 WS01010_I07 LMCO 7.00E-82 1.08 6.00 1.82 1.00 3.29 1.64 1.01 1.54 1.32 WS00938_N21 CAD 1.00E-124 0.99 5.61 1.47 3.41 1.21 0.77 2.32 0.94 0.93 WS00920_P12 CCR 4.00E-25 1.18 5.24 0.49 1.66 2.29 1.08 1.88 1.55 1.30 WS00931_G15 F3H 2.00E-38 1.93 5.24 1.12 14.01 4.18 1.58 15.23 1.79 1.12 WS0262_I24 DIRb/d 3.00E-24 0.91 1.17 8.21 0.48 0.20 0.54 0.81 0.93 0.65 WS0078_K09 COMT-like 1.00E-34 1.23 0.89 6.82 1.41 8.65 1.67 1.17 2.15 1.76 WS0044_J08 CCR 4.00E-69 1.39 0.08 0.05 0.38 0.17 0.37 0.43 0.27 0.93 WS00912_M21 3GT 2.00E-28 0.91 0.15 0.32 0.58 0.30 1.41 0.50 0.65 1.91 WS0078_N05 CCR 6.00E-74 1.44 0.16 0.17 0.70 0.34 0.55 0.54 0.50 1.00 WS0076_C19 3GT 5.00E-42 0.86 0.17 0.18 0.76 0.52 0.93 0.73 0.59 1.13 WS00935_E02 3GT 1.00E-24 0.81 0.19 0.27 0.77 0.53 0.80 0.66 0.58 1.05

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Among the 39 phenylpropanoid transcripts downregulated by weevil feeding in interior spruce, only 5 transcripts were downregulated by more than 5 fold (Table 3.4). Three of these transcripts were also downregulated by fungal treatment compared to control and these included cinnamoyl-CoA reductase (CCR) and 3GT (flavonol 3-O- glucosyltransferase) transcripts. Twice as many transcripts were downregulated by weevil feeding compared to fungal treatment in interior spruce. The heat map revealed few phenylpropanoid pathway transcripts with the different patterns of expression in weevil and fungus treated interior spruce (Figure 3.3). These phenylpropanoid transcripts are among the transcripts that are induced by weevil treatment only at 2 weeks (14 transcripts at the lower half of the heat map Figure 3.3B), as well as among the transcripts that were downregulated by weevil treatment only at 2 weeks of weevil feeding (18 transcripts on the lower half of Figure 3.3C). Among transcripts induced by weevil and downregulated or uninduced by fungal treatment are select DIR and laccase transcripts. Among the transcripts downregulated by weevil feeding and induced or not changed by fungal treatment are PAL, cinnamate-4- hydroxylase (C4H) and several transcripts from flavonoid biosynthesis such as chalcone/stilbene synthase (CHS/STS), F3H, dihydroflavonol 4- reductase/leucoanthocyanidin reductase (DFR), leucoanthocyanidin reductase (LAR). These results indicate some differences in phenylpropanoid pathway involvement in defense response of interior spruce to weevil feeding and fungal inoculation.

Transcript specific analysis reveals induction of selected DIR protein transcripts in interior spruce by weevil feeding Microarray analysis revealed the strong induction of select DIR transcripts induced with weevil feeding throughout the time course of the study. High sequence similarity among different DIR genes (Ralph et al., 2007a) requires transcript specific analysis to differentiate the involvement of different DIR genes in spruce defense response. We used real-time qPCR for transcript specific expression analysis of selected DIR transcripts representing three sub-families of conifer DIR proteins family. Primers used in this study were previously designed based on the full-length sequences of DIR protein genes (Supplemental Table 3.1, Ralph et al., 2007a). Real-time qPCR analysis, using 12 primer pairs representing 18 DIR and DIR-like transcripts (6 pairs of the transcripts have too much sequence similarity to be distinguished by real-time PCR), is shown as a relative expression normalized to the transcript levels of the reference gene eukaryotic translation initiation factor TIF5A (Figure 3.4), and as a fold induction relative to control treatment (Supplemental Table 3.4). The real-time PCR data was 116 considered significant with a fold change >2 and p<0.05. This real-time PCR analysis demonstrated strong induction by weevil feeding of the four tested representatives of DIRa sub-family (DIR2/32, DIR5/15, DIR6, DIR16) at 2 days of the treatment (Figure 3.4). DIR16 was also strongly induced at 2 weeks of weevil feeding together with DIR2/32 and DIR6 that were induced at 2 weeks of treatment but to a lesser extent than at 2 days of treatment. One of the DIRb/d subfamily, DIR12/21 was also induced by weevil feeding at 2 days of the treatment. Among 6 tested DIRf sub-family transcripts DIR22 and DIR23/24 were induced by weevil feeding at 2 weeks of the treatment and DIR25 was induced at 2 days of weevil feeding (DIR27-DIRf control levels were variable during the time course compared to changes in treatment levels) (Figure 3.4). The DIRa subfamily representatives DIR2/32, DIR5/15 and DIR6 were also highly induced by fungal treatment in interior spruce throughout the same time course (Chapter 2, Supplemental Table 2.6). DIRa-DIR16 was not induced by fungal treatment in interior spruce. Two DIRb/d had similar patterns of expression in interior spruce treated with weevil feeding and fungal inoculation. DIR12/21 was moderately induced by both treatments and DIR1/20 had more evident pattern of downregulation in spruce inoculated with fungus compared to treatment with weevil feeding. Transcript specific evaluation of the expression of the DIRf subfamily revealed differences between weevil and fungus treated interior spruce. Most of the DIRf sub-family transcripts were not affected by fungal treatment and one of them (DIR23/24) was downregulated, whereas three DIRf sub-family transcripts (DIR22, DIR23/24 and DIR25) were induced by weevil feeding (Supplemental Table 2.6; 3.4).

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0.24 0.012 Dir 2/32 - DIRa * Dir 22-DIRf 0.18 0.009 *

0.12 0.006

0.06 * 0.003 * 0.00 0.000 0.24 0.04 Dir 5/15 - DIRa * DIR 23/24-DIRf *

0.18 0.03 Relative Relative expression level Relative Relative expression level 0.12 0.02

0.06 0.01

0.00 * 0.00 0.16 0.24 Dir 6-DIRa Dir 25-DIRf * * 0.12 0.18

0.08 0.12

0.04 0.06 * * 0.00 * 0.00 0.0012 1.2 Dir 16 - DIRa DIR 26-DIRf 0.0009 0.9

* *

Relative Relative Relative expression expression level level 0.0006 0.6

0.0003 0.3

0.0000 0.0 0.032 0.012 Dir 1/20-DIRb/d Dir 27-DIRf * 0.009 0.024

0.006 0.016 * 0.003 0.008

0.000 0.000 0.4 1.00 DIR 12/21-DIRb/d Dir 28/29-DIRf

0.3 * 0.75

Relative Relative Relative expression expression level level 0.2 0.50

0.1 * 0.25

0 0.00 C6h C2d C2w I6h I2d I2w C6h C2d C2w I6h I2d I2w Figure 3.4: Quantitative real-time PCR analysis of DIR transcripts levels in interior spruce. Relative expression levels of 12 representative transcripts of the DIR family in interior spruce bark untreated, treated with weevil feeding during the time course of 6h, 2 days (2d) and 2 weeks (2w). Values represent mean + standard error. Transcript abundance is shown relative to TIF5A transcript in the same sample. C- control, I – weevil (insect) treatment. * - indicates statistically significant difference as compared to control.

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3.3.6 Comparative analysis of the effect of weevil feeding and fungal inoculation on terpenoid pathway transcripts in interior spruce To further explore the expressional changes in transcripts involved in secondary metabolism, we examined the effect of weevil feeding on terpenoid pathway expression in interior spruce. Eighty two transcripts on the spruce microarray were annotated to terpenoid pathway (Chapter 2, Supplemental Table 2.5). Weevil feeding induced 16 transcripts and downregulated 3 transcripts involved in the terpenoid pathway in interior spruce (Table 3.3). Thirteen of the upregulated terpenoid pathway transcripts were also upregulated by fungal treatment and 11 were upregulated by wounding treatment (Table 3.5). Only one transcript was downregulated by fungal treatment and it was also downregulated by weevil feeding. This data indicates strong induction of terpenoid pathway by weevil feeding and a large overlap of the induced terpenoid pathway in response to weevil feeding and fungal inoculation. The induced transcripts by weevil feeding included representatives from most of the major steps of terpenoid biosynthesis (Table 3.5). Two 1-deoxy-D-xylulose 5- phosphate synthase (DXS) transcripts from the methylerythritol phosphate (MEP) pathway were induced by weevil feeding and one of these (WS00930_F08) was also strongly induced by fungal treatment (Table 3.5). Two transcripts, mevalonate kinase (MK) and mevalonate diphosphate decarboxylase (MPDC), were moderately induced by weevil feeding in the mevalonic acid (MEV) pathway, another pathway involved in synthesis of terpenoid precursor isopentenyl pyrophosphate (IPP). MPDC was also induced by fungal inoculation. Three prenyltransferases were induced by weevil feeding and fungal inoculation (Table 3.5). Weevil feeding induced a subset of 9 terpene synthases out of 39 known and putative terpene synthases on the 21.8K microarray (Table 3.5). Six of these terpene synthases were among the most highly induced transcripts by weevil from the terpenoid pathway. Eight of the 9 terpene synthases were also induced by fungal treatment compared to control and by wounding treatment (Table 3.5). Only three putative terpene biosynthetic transcripts were downregulated by weevil feeding in interior spruce (Table 3.5). These transcripts included two 3-hydroxy-3- methylglutaryl-CoA reductase (HMGR) transcripts and one GGPPS transcript. One of the HMGR transcripts was also downregulated by fungal treatment (Table 3.5).

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Table 3.5: Expression of terpenoid biosynthesis transcripts in interior spruce treated with weevil feeding, fungal inoculation and wounding with >2 fold change in at least one time point in weevil treated sample and q<0.05. I/C I/C I/C F/C F/C F/C W/C W/C W/C Clone ID Annotation E value 6h 2d 2w 6h 2d 2w 6h 2d 2w MEP pathway WS00930_F08 DXS 1.00E-86 2.22 6.14 2.75 13.10 2.26 1.62 19.27 1.33 1.29 WS0097_H02 DXS 5.00E-45 0.79 4.84 2.62 0.97 1.05 0.86 1.15 1.12 0.94 MEV pathway WS01016_E19 HMGR 4.00E-23 1.04 0.49 0.67 0.84 0.65 1.11 0.89 0.73 1.10 WS01025_B12 HMGR 4.00E-23 0.92 0.47 0.65 0.92 0.46 1.12 1.07 0.63 1.17 WS00925_M22 MK 8.00E-72 1.06 2.05 1.57 1.28 1.73 1.08 1.19 1.29 1.02 WS0104_E21 MPDC 7.00E-60 1.10 2.77 1.55 1.71 2.94 1.33 1.53 1.67 1.07 Prenyl transferases WS01029_I02 IPPI 1.00E-112 1.10 4.44 2.12 2.32 3.02 1.14 2.27 1.66 1.02 WS0074_I12 FPPS 2.00E-74 1.00 2.38 1.96 2.15 2.07 1.02 1.60 1.30 1.00 WS00911_G14 GGPPS 1.00E-116 1.53 6.02 2.36 3.42 1.44 1.25 4.63 1.12 1.53 WS01030_E02 GGPPS 9.00E-77 0.82 0.60 0.48 1.01 0.84 1.17 1.11 0.83 1.11 Terpene synthases WS0078_K20 TPS 8.00E-52 1.26 14.72 3.50 7.23 5.89 1.44 9.73 3.06 1.35 WS0019_A03 TPS 4.00E-89 1.11 13.04 5.94 3.97 8.67 2.24 5.29 3.35 1.24 WS00724_C19 TPS 2.00E-97 1.43 11.83 3.98 5.20 4.43 1.98 5.94 2.33 1.46 WS0063_F08 TPS 2.00E-78 1.12 11.53 4.66 4.93 8.21 2.65 7.30 4.28 1.44 WS0094_F18 TPS 1.00E-100 1.11 8.50 2.69 6.88 4.93 1.94 6.25 2.72 1.36 WS00723_E14 TPS 3.00E-90 1.32 6.24 2.00 6.27 5.92 2.13 7.27 4.09 1.46 WS0092_L05 TPS 6.00E-94 1.48 2.75 1.82 6.90 3.93 1.64 6.73 2.62 1.45 WS00819_E12 TPS 2.00E-97 1.06 2.67 2.05 1.67 1.28 1.34 1.83 1.14 1.08 WS0092_I21 TPS 3.00E-86 1.32 2.44 1.55 2.85 3.45 1.59 3.37 2.53 1.56

Numbers in the table indicate expression levels fold change in specified treatment comparisons (I – weevil (insect); C, control; W, wounding; F, fungal treatments). DXS, 1-deoxy-D-xylulose 5-phosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; MK, mevalonate kinase; MPDC, mevalonate diphosphate decarboxylase; IPPI, isopentenyl diphosphate: dimethylallyl diphosphate isomerase; FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; TPS, terpene synthase. Colour scale from yellow to red correlates with fold-change expression and was applied only to fold change values >2.00 with q<0.05. Fold-change: ≥ 5.00 <0.50 > 1.50 ≥ 10.00 < 10.00 < 5.00

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Transcript specific expression analysis reveals differential DXS family expression in weevil and fungal-treated spruce We used real-time PCR to perform transcript specific expression profiling of three DXS transcripts present on the 21.8K microarray representing known conifer DXS family (Phillips et al., 2007). Real-time PCR data is presented as relative expression normalized to reference gene levels of the eukaryotic translation initiation factor TIF5A (Figure 3.5), and as fold induction relative to control treatment (Supplemental Table 3.4). The real time PCR data was considered significant with >2 fold change and p< 0.05. Real time PCR confirmed the strong induction of DXS (WS00930_F08) at 2 days of weevil feeding (FC 91.1) and at 2 weeks of feeding (FC 6.6) and DXS (WS0097_H02) at 2 days of weevil feeding (FC 19.3) with remaining, but reduced induction at 2 weeks (FC 4.5) (Figure 3.5, Supplemental Table 3.4). The third DXS (WS01028_M14) was not induced by weevil feeding, consistent with the microarray data (Table 3.5). DXS (WS00930_F08) was also strongly induced by fungal treatment in interior spruce already at 6h post inoculation (FC 73.7) and by wounding treatment (FC 94.4). DXS (WS0097_H02) was not induced by fungal or wounding treatment in interior spruce (Chapter 2, Supplemental Table 2.6).

0.8 DXS WS00930_F08 * 0.6 Figure 3.5: Quantitative real-time PCR 0.4 analysis of DXS transcripts levels in 0.2 interior spruce. Relative expression levels of three transcripts of DXS family in 0.0 * interior spruce bark untreated, or treated 0.16 DXS WS0097_H02 with weevil feeding during the time course * 0.12 of 6h, 2 days (2d) and 2 weeks (2w). Values represent mean + standard error. 0.08 Transcript abundance is shown relative to 0.04 * the TIF5A transcript in the same sample. C- control, I – weevil (insect) treatment. *

Relative Relative expression level 0.00 - indicated statistically significant 0.28 DXS WS01028_M14 difference compared to control. 0.21

0.14

0.07

0.00 C6h C2d C2w I6h I2d I2w

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3.3.7 Weevil feeding and fungal-treatment induces chitinases in interior spruce We evaluated chitinase transcript expression in spruce treated with weevil feeding to further investigate the involvement of chitinases in the interior spruce defense response. Among 17 putative chitinase transcripts and 4 chitinase-like transcripts present on the microarray 9 chitinase transcripts and 1 chitinase-like transcript were induced by weevil feeding in interior spruce. The 9 chitinase transcripts were also induced by fungal and wounding treatment (Table 3.6). More chitinase transcripts were induced at 2 weeks post-treatment compared to earlier time points in interior spruce treated with weevil feeding and all these transcripts were induced at 2 days in fungal- treated spruce (Table 3.6). Two chitinase transcripts were downregulated by weevil feeding at 2 weeks post-treatment. Transcript specific expression analysis confirmed high level of chitinase genes expression in interior spruce exposed to weevil feeding (Chapter 4).

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Table 3.6: Expression of chitinase and chitinase-like transcripts present on 21.8K spruce microarray in interior spruce treated with weevil feeding, fungal inoculation and wounding with >2 fold change in at least one time point in weevil treated sample and q<0.05. Numbers in the table indicate expression levels fold change in specified treatment comparisons (I – weevil (insect); C, control; W, wounding; F, fungal treatments. NSBI I/C I/C I/C F/C F/C F/C W/C W/C W/C Clone ID BlastX E value 6h 2d 2w 6h 2d 2w 6h 2d 2w WS00712_K20 BAD779321 1.00E-70 1.09 7.91 7.11 2.29 8.63 1.83 1.87 2.64 1.49 WS00924_F07 AAT094262 7.00E-128 0.98 5.56 6.09 1.02 35.97 1.80 1.04 5.58 1.46 WS00930_G18 AAQ100933 7.00E-66 1.17 4.22 5.83 1.92 14.09 1.89 1.15 4.66 1.59 WS00716_E14 AAV317434 8.00E-11 0.93 2.70 2.40 1.41 5.46 1.12 1.19 1.97 1.05 WS0092_L20 AAQ170505 7.00E-93 1.10 1.83 4.77 1.39 7.96 2.01 1.17 4.04 0.92 WS0018_O07 AY6395876 2.00E-24 1.42 1.93 4.66 1.03 10.11 1.99 1.11 4.93 0.77 WS01032_J21 AAV317427 2.00E-11 1.33 1.46 3.30 0.79 4.26 1.87 0.92 2.83 0.74 IS0014_F01 AAV317428 4.00E-88 0.95 1.36 2.85 1.07 3.55 1.36 1.06 2.42 0.80 WS01018_O22 BAD779329 8.00E-51 0.89 1.64 2.85 1.12 2.66 1.38 1.17 1.59 1.15 WS0061_N18 BAF4601210 2.00E-18 1.00 0.90 1.07 0.75 0.89 1.11 1.14 1.01 0.96 WS0046_M24 BAF4601211 1.00E-49 1.29 1.95 0.96 2.71 9.39 0.94 2.07 5.44 0.76 WS00922_B21 CAC8181212 8.00E-100 1.29 1.60 0.94 2.29 3.65 1.03 1.80 3.52 0.87 WS00927_N15 BAD0282413 7.00E-100 1.32 1.56 0.94 2.22 8.41 0.89 1.66 5.24 0.87 WS00924_E01 AAT0942714 3.00E-104 0.96 0.70 0.82 0.86 0.84 0.93 0.85 0.95 1.01 WS00929_K15 CAA1018915 2.00E-87 1.33 0.96 0.60 2.14 6.56 0.97 1.59 3.26 0.77 WS0106_P21 AAT0942716 7.00E-76 1.10 0.66 0.25 1.18 1.25 1.16 0.77 1.12 0.96 WS0045_M18 ABD9281917 1.00E-75 0.90 0.51 0.12 1.43 1.32 0.84 0.83 1.57 0.83 chitinase like WS0261_D24 NP_17207618 8.00E-08 0.94 0.85 1.07 0.75 1.19 0.93 1.19 0.94 0.98 WS0031_C03 NP_18831718 3.00E-53 0.84 1.64 2.35 0.84 1.12 1.01 1.12 1.26 0.89 WS00712_A06 NP_17207618 7.00E-27 0.88 0.55 0.81 0.66 0.96 0.91 0.96 0.79 0.98 WS02610_C03 NP_17207618 6.00E-04 1.04 0.65 1.08 1.06 0.95 0.76 0.95 0.98 1.13 *Chitinases were annotated based on the best annotated sequences match using blastX and NCBI database. Chitinase-like genes were annotated based on annotation of Arabidopsis thaliana. Annotation IDs represent the following species: 1- Cryptomeria japonica, 2- Picea abies, 3- Vitis vinifera, 4- Picea abies, 5- Picea abies, 6,7,8- Picea abies, 9- Cryptomeria japonica, 10- Chamaecyparis formosensis, 11- Chamaecyparis formosensis, 11- Musa acuminate, 13- Taxodium distichum, 14- Picea abies, 15- Cicer arietinum, 16-Picea abies, 17- Limonium bicolor, 18- Arabidopsis thaliana Color scale from yellow to red correlates with fold-change expression and was applied only to fold change values with q<0.05. Fold-change: ≥ 5.00 <0.50 > 1.50 ≥ 10.00 < 10.00 < 5.00

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3.4 DISCUSSION

We used the 21.8K cDNA microarray to compare the transcriptional response of interior spruce after exposure to weevil feeding with the response to blue-stain fungus L. abietinum inoculation (Chapter 2) over 2 weeks timecourse. Our study represents the first direct comparison of the effect of insect feeding and fungal inoculation on conifers species.

Weevil feeding induces a large transcriptome response in interior spruce similar to the response to fungal inoculation The microarray analysis revealed that weevil feeding induced a two-fold larger transcriptome response in interior spruce compared to fungal inoculations. This response included the induction of approximately 10% of all transcripts and downregulation of 7% of all transcripts represented on the 21.8K microarray, comparable to the large transcriptome rearrangement detected in Sitka spruce exposed to insect herbivory (Ralph et al., 2006b). The transcriptional response of interior spruce to weevil feeding included the majority (65% of induced and 75% of downregulated transcripts) of the transcripts involved in the response to fungal treatment (Chapter 2). The transcriptome responses correlated well to each other, with most of the common differentially expressed transcripts having a similar direction of expression change (upregulated or downregulated). The high correlation between the spruce transcriptome responses to weevil feeding and to fungal inoculation is consistent with previous studies describing the similar anatomical and chemical responses of spruce species to insect herbivory (Miller et al., 2005), and inoculations with necrotrophic fungi (Franceschi et al., 2000; Viiri et al., 2001). This comparison of the weevil-induced transcriptome with the fungal-induced transcriptome indicated that more transcripts specifically respond to weevil feeding as compared to fungal inoculation. Comparison of the spruce transcriptome responses to weevil feeding and fungal treatment revealed different dynamics of these responses. The response to weevil feeding was very low at 6 hr of feeding (0.03% of the transcriptome), but strongly escalated at 2 days of feeding (13.3% of the transcriptome) and stayed high at 2 weeks of feeding (10.3% of the transcriptome) (Table 3.1, Figure 3.2). A similar escalation of the transcriptome response over time was observed for Sitka spruce leaders induced by budworm feeding (Ralph et al., 2006b). In contrast, fungal inoculation induced a considerable transcriptome response (4.3% of all studied transcriptome) at 6 hr post- 124 treatment and the response at 2 days (6.5%) included about the same number of upregulated transcripts (3.9% of the transcriptome). The response to fungal inoculation decreased dramatically at 2 weeks post-treatment. The differences in dynamics is due to the time needed for insect feeding to mount, as compared to the fungal inoculation, which impacts spruce trees much sooner due to the immediate response to wounding and fungal inoculation which is applied at the beginning of the time course. The decrease response in spruce at 2 weeks (0.33% of the transcriptome) coincided with development of traumatic resin duct ring and the successful localization and arrest of fungal growth (Chapter 2). Comparison of the expression dynamics between weevil and fungal treatment revealed a small overlap of transcripts with the same dynamic (e.g. only 14% overlap of transcripts in a cluster of transcripts induced only at 2 days after treatment, Fig 3.2). These differences in response dynamics and the large overlap of differentially expressed transcripts throughout the time course indicate unique timing of the involvement of many transcripts in the defense responses to these treatments. Evaluation of the response over the time course allowed for a better appreciation of the similarities in response of spruce to weevil feeding and fungal inoculation, taking into account the differential timing of similar defense responses. Overall this data indicates that there is a large common defense response in interior spruce to different threats, with additional treatment-specific defense mechanisms. A large overlap of the transcriptome expression was also shown for weevil, budworm feeding and wounding treated Sitka spruce (Ralph et al., 2006b) as well as in Arabidopsis induced by the feeding of different herbivores (Ehlting et al., 2008). In addition, the effectiveness of systemically induced resistance by necrotrophic fungus Sphaeropsis sapine against herbivory by Neodiprion sertifer, and reciprocally, (Eyles et al., 2007) supports the involvement of similar defense mechanisms involved in conifer defense against necrotrophic fungi and insects.

Common and different trends in interior spruce response to weevil feeding and fungal inoculation The most highly induced transcripts by weevil feeding included transcripts involved in phenylpropanoid pathway such as C3H, F3H and transcripts putatively involved in phenylpropanoid biosynthesis (annotation limitations are discussed in Chapter 2.4) such as laccases and dirigent protein transcripts (Table 3.2; 3.4). Many other transcripts annotated to phenylpropanoid pathway were among very highly induced transcripts as well (Table 3.4). High levels of induction of selected 125 phenylpropanoid transcripts by insect or pathogenic fungal treatment was previously demonstrated in conifers (Ralph et al., 2006b; Adomas et al., 2007) and in poplar (Ralph et al., 2006a; Miranda et al., 2007). The most highly induced transcripts observed after fungal treatment included PR transcripts such as chitinase, thaumatin-like protein, hevein-like protein, osmotin-like protein, some phenylpropanoid pathway transcripts and transcripts involved in ethylene signalling (Chapter 2). Induction of selected PR proteins was shown to be a prominent feature in conifer response to fungal inoculation (Morse et al., 2004; Adomas et al., 2007). While both phenylpropanoid pathway and PR proteins were strongly induced by weevil and fungal treatment, the direct comparison of most highly induced transcripts may suggest a more prominent role of the phenylpropanoid pathway in spruce defense against weevil and a more prominent role of pathogenesis related proteins in spruce defense against the necrotrophic fungus L. abietinum. Transcripts putatively involved in jasmonate and ethylene signaling pathways were strongly induced by white pine weevil feeding (Table 3.2, Table 3.3A, B). Induced transcripts include allene oxide synthase (AOS) and allene oxide cyclase (AOS) involved in jasmonate biosynthesis and ACC synthase and ACC oxidase involved in ethylene biosynthesis. Previous transcript specific studies have demonstrated the induction of AOS and ACC transcripts in Sitka spruce by weevil feeding (Miller et al., 2005; Ralph et al., 2007b). In addition the involvement of ethylene in jasmonate signalling response events was demonstrated in Douglas-fir and in giant redwood (Hudgins and Franceschi, 2004). A large overlap of transcripts involved in jasmonate and ethylene signalling induced in interior spruce subjected to weevil feeding and fungal inoculation suggests similar signalling mechanisms in response to both treatments. Involvement of these signalling pathways in the interior spruce response to chewing insect weevil and to necrotrophic fungal pathogen in this study is consistent with the involvement of jasmonate and ethylene signaling in plant response to tissue damaging insects (Walling, 2000) and in plant defense against necrotrophic pathogens (McDowell and Dangl, 2000). The signalling response of interior spruce to weevil feeding and fungal treatment was more intense than the response to wounding treatment (Table 3.3). This is consistent with previous studies that demonstrated that herbivore feeding causes a larger jasmonic acid response than wounding treatment, due to insect oral secretions acting as additional stimulants (Walling, 2000). Overall weevil feeding did not cause any substantial downregulation of the expression of defense related transcripts similar to the effect of L. abietinum (Table 3.3). This pattern is consistent with plant defense against insects and necrotrophic pathogens, in which defense systems are mostly activated but not suppressed (Kessler 126 and Baldwin, 2002; Morse et al., 2004; Ralph et al., 2006b; Adomas et al., 2007) with suppression of defense response often required for biotrophic pathogens establishment (Schulze-Lefert and Panstruga, 2003; Miranda et al., 2007). Analysis of transcripts involved in primary metabolism revealed a large rearrangement of the primary metabolism transcriptome in interior spruce exposed to weevil feeding, with over 700 transcripts annotated to primary metabolism differentially expressed during the time course (Table 3.3). More than half of the transcripts of primary metabolism which were induced by weevil feeding were also induced by fungal inoculation. The connection between the rearrangement of primary metabolism and defense pathways was comprehensively reviewed by Bolton (Bolton, 2009) and demonstrated in defense-related plant systems using genomics approaches (Zulak et al., 2007). Plant defense was shown to require resource and energy allocation in several plant/pathogen, plant/insect biological systems for the biosynthesis of defense proteins and chemicals and requires reorganization of primary metabolic processes (Bolton, 2009). Expression of many transcripts involved in photosynthesis was downregulated by weevil feeding (Table 3.3b). A similar pattern is observed in fungus-treated interior spruce with two fold fewer transcripts involved in photosynthesis being downregulated (Table 3.3b). Presence of chlorophyll in bark and photosynthetic activity of young stems was previously demonstrated for spruce and pine (Berveiller et al., 2007). A similar downregulation of photosynthesis involved transcripts was observed in Sitka spruce shoot tips exposed to herbivory (Ralph et al., 2006b) and in Scots pine roots infected with a pathogenic fungus (Adomas et al., 2007). These studies suggest that defense related downregulation of photosynthesis related processes is a common phenomenon in conifer species. Although photosynthesis provides energy, shutting it down may prioritize the synthesis of defense compounds and may protect the photosynthetic apparatus against stress related damage (Bolton, 2009).

Phenylpropanoid pathway induction in interior spruce exposed to weevil feeding and fungal inoculation The analysis of the transcriptome of interior spruce bark induced by weevil feeding revealed a strong induction of large number of transcripts from the phenylpropanoid pathway (Table 3.4, Figure 3.3). Many of the 107 induced transcripts (out of 340 phenylpropanoid pathway transcripts present on the 21.8K microarray) were among the most highly induced in this response. Close to 70% of the weevil-induced phenylpropanoid pathway transcripts were also induced by fungal treatment, and close 127 to 50% of the weevil-induced phenylpropanoid transcripts were induced by both fungal and wounding treatment, indicating the general nature of the defense-related phenylpropanoid response in spruce. Since the phenylpropanoid response in lodgepole pine induced by fungal and wounding treatment was very similar to the interior spruce response to the same treatments (Chapter 2), we can conclude that the phenylpropanoid pathway response is common in the family Pinaceae. This is similar to an extensive involvement of the phenylpropanoid pathway in angiosperm plant species defense (Dixon et al., 2002). This conclusion is supported by the microarray study (using 9.7K array) of the Sitka spruce response to wounding, weevil and budworm, that showed the induction of several steps of the phenylpropanoid pathway by herbivory and wounding treatments (Ralph et al., 2006b). In angiosperms, phenolic-storing cells release their content as a result of wounding, fungal infection or herbivory, which causes lignification and suberization of cells, cell death and localization of infection, as well as deterrence of insect feeding (Beckman, 2000). The diverse nature of protective phenylpropanoid functions makes them promising candidates for further evaluation of their role in the conifer defense response. Sets of the most highly induced phenylpropanoid pathway transcripts largely overlapped in interior spruce treated with weevil feeding and fungal inoculation (Table 3.4, Figure 3.3), including several transcripts involved in the early steps of phenylpropanoid biosynthesis (PAL, C3H, C4H), CCR, transcripts involved in flavonoid biosynthesis (F3H, F3’H, 3GT and ANS), and transcripts with a potential role in phenylpropanoid metabolism such as laccases and dirigent protein (DIR) transcripts (Table 3.4). Induction of laccases and DIR transcripts was previously associated with fungal and insect treatment in conifers and is proposed to be associated with lignan and possibly lignin biosynthesis and strengthening of cell walls (Davin et al., 1997; Davin and Lewis, 2000; Kim et al., 2002; Boerjan et al., 2003; Ralph et al., 2006c; Sato and Whetten, 2006; Adomas et al., 2007). Flavonoids, such as condensed tannins, are known to be important in angiosperm defense against herbivores (Philippe and Bohlmann, 2007), and transcripts involved in condensed tannin synthesis were induced by wounding and herbivory in poplar (Peters and Constabel, 2002). In addition, the conifer flavonoid taxifolin was shown to have an antifeedant effect on bark beetles (Faccoli and Schlyter, 2007) and tannins amount was correlated with Norway spruce resistance to bark beetles (Brignolas et al., 1998). Strong induction of transcripts involved in flavonoid biosynthesis by weevil

128 feeding in interior spruce and the potential ability of selected flavonoids to serve as antiinsect compounds suggests possible role of flavonoids in conifer insect defense. A direct comparison of the effect of fungal inoculation and weevil feeding on the expression of phenylpropanoid pathway transcripts in interior spruce revealed few transcripts that were differentially expressed in opposing directions as a result of the treatments (Figure 3.3). Transcripts that were induced by weevil feeding and were downregulated by fungal inoculation in interior spruce included laccase and several DIR transcripts (Figure 3.3). Transcripts downregulated by weevil feeding and induced by fungal treatment included PAL, C4H, CHS/STS, DFR with LAR transcript being downregulated by weevil and unchanged fungus treated interior spruce (Figure 3.3). A possible role of flavonoids in antifungal defense is also supported by the accumulation of flavonoids in response to pathogen attack in conifers (Bonello and Blodgett, 2003) and poplar (Miranda et al., 2007). Although many transcripts potentially involved in lignin and flavonoid biosynthesis were highly upregulated by weevil feeding and fungal inoculation differential expression of few transcripts involved in these processes indicate possible differences in the involvement of lignification and flavonoid biosynthesis in interior spruce defense against weevil and fungus.

Strengthening of cell walls Analysis of the interior spruce defense response to weevil feeding and fungal inoculation revealed a large number of highly induced transcripts that are potentially involved in lignin biosynthesis and cell wall strengthening. In particular, laccases were among the most highly induced transcripts from the phenylpropanoid pathway in interior spruce induced by weevil feeding and fungal inoculation (Figure 3.3, Table 3.4). More than twice as many laccase transcripts (16 out of 25) were induced by weevil feeding compared to fungal inoculation (7 out of 25) and wounding treatment (6 out of 25), and induction of many laccase transcripts was much higher in response to weevil feeding compared to fungal inoculation or wounding. Some of the laccase enzymes are proposed to be involved in lignan and lignin biosynthesis due to their polyphenol oxidase activity, as demonstrated in loblolly pine (Bao et al., 1993; Sato and Whetten, 2006). The involvement of laccases in lignin biosynthesis is suggested due to their increased expression in lignifying cells of angiosperms (Ehlting et al., 2005) and gymnosperms (Bao et al., 1993; Sato et al., 2001; Friedmann et al., 2007). In particular, comparative analysis of the transcriptomes of the vegetative tip and woody base of the apical shoot of Sitka spruce revealed four laccases that were highly induced in the wood-forming apical shoot base with no laccase transcripts preferentially expressed in vegetative tip 129

(Friedmann et al., 2007). Three of these laccases were also present on the 21.8K array, two of which were moderately induced by weevil feeding (WS00813_M20 FC 3.5, 2 weeks post-treatment, WS0038_I15 FC 2.2, 2.4, 2 days and 2 weeks post-treatment) as compared to other highly induced laccases with up to 69 fold change (Table 3.4). None of the laccases induced in wood-forming Sitka spruce were induced by fungal and wounding treatment, and one of these laccases (WS00813_M20) was downregulated by fungal treatment (Figure 3.3). Among the set of 15 overlapping laccases on the 16.7K and 21.8K microarray, 11 laccases were induced by weevil feeding, 4 were induced by fungal treatment and 3 were induced by wounding treatment. Comparative analysis of the laccase transcript expression in weevil, and fungus-challenged tissues and in wood- forming tissues in spruce reveals possible differential involvement of different laccases in the defense response as opposed to developmental processes. Strong induction of laccases in response to fungal inoculation and weevil feeding together with the association of high density of lignified cells with spruce resistance to weevil, bark-beetle and beetle-associated fungus (Wainhouse et al., 1990; Wainhouse et al., 1997; Wainhouse et al., 1998; King and Alfaro, 2009) makes laccases an important target for further elucidation of their role in conifer defense against insects and fungi. In particular, evaluation of the association of induced and constitutive expression of laccases with conifer resistance to insect attacks and fungal pathogens may reveal useful targets for selection and breeding of resistant conifer trees. Strong induction of the cinnamyl-alcohol dehydrogenase (CAD) transcripts (Table 3.4) is also indicative of increased lignin synthesis as CAD catalyzes the final step of lignin monomer biosynthesis (Walter et al., 1988). Another transcript potentially involved in lignin biosynthesis, peroxidase (IS001_F24), was among the most strongly induced transcripts in interior spruce attacked by weevil (Table 3.2). Selected peroxidases are known to be expressed in lignifying cells and peroxidase expression has been correlated with lignin deposition (Boerjan et al., 2003). A further group of transcripts potentially involved in lignin biosynthesis that are highly upregulated by weevil feeding and fungal inoculation are the dirigent protein (DIR) transcripts, in particular the DIRa subfamily (Table 3.4). The spruce DIR family contains at least 35 unique genes (Ralph et al., 2007a). Induction of DIR transcripts expression, in particular the DIRa subfamily, was observed in Sitka spruce apical shoot exposed to weevil and budworm feeding (Ralph et al., 2007a). Some of the DIRa subfamily members were strongly expressed in apical shoot woody stem base compared to shoot tip (Friedmann et al., 2007) and particularly one of the DIRa transcripts (WS010011_J07) was also strongly induced by fungal and weevil treatment in interior 130 spruce (Table 3.4). Prominently preferentially expressed transcripts in apical shoot compared to woody base belonged to DIRb/d subfamily (Friedmann et al., 2007). This data may support the involvement of some of the DIRa subfamily members in lignin deposition. Dirigent proteins function in directing the stereoselective biomolecular coupling of phenoxy radicals resulting in the formation of lignans (Davin et al., 1997, Kim at al., 2002). The function of DIR genes in lignin biosynthesis was suggested based on specificity of linkages in lignin, which may require directed coupling of monolignols, and the localization of dirigent proteins in lignified cell walls (Davin and Lewis, 2000). However, requirement of DIR participation in lignin formation was challenged based on the possibility of the production of linkage specificity by random lignification controlled by metabolic regulation of monolignol synthesis and transport to cell wall, and by the lack of biochemical evidence of DIR protein involvement in lignin polymerization (Hatfield and Vermerris, 2001; Weng and Chapple, 2010). Further research is required for the establishment of DIR genes function in conifer defense.

Transcript specific analysis confirms strong induction of DIRa transcripts by weevil feeding Transcript specific expression analysis is necessary to differentiate between members of DIR due to the high sequence similarity of different members of the spruce DIR family (Ralph et al., 2007a). Transcript specific real-time qPCR analysis revealed a high level of induction of the 4 DIRa transcripts, as well as induction of one DIRb/d transcript and 3 DIRf transcripts throughout the study (Figure 3.4). The induction pattern was similar to induction of DIRa and DIRb/d transcripts in interior spruce by fungal treatment (Chapter 2) and in Sitka spruce apical shoot (Ralph et al., 2007a). DIRf subfamily members DIR22, DIR23/24 and DIR25 were induced by weevil feeding in interior spruce and these transcripts were either not differentially expressed or were downregulated during the time course in interior spruce inoculated with fungus or in Sitka spruce exposed to weevil or budworm feeding (Ralph et al., 2007a). Based on constitutive expression of DIRf subfamily transcripts in Sitka spruce, it was suggested that they may play a role in a primary process, or in constitutive defense (Ralph et al., 2007a). Induction of some of the DIRf subfamily transcripts by weevil feeding in interior spruce may indicate their role in induced conifer defense. Induction of selected members of DIRf subfamily in more developed interior spruce bark, but not in apical shoot of Sitka Spruce by weevil feeding may reflect developmental differences in conifer defense-related responses. This analysis confirmed strong induction of DIRa transcripts

131 in interior spruce by weevil feeding and suggested potential importance of DIRa and selected DIRf transcripts in conifer defense against weevil.

Weevil feeding and fungal inoculation induce the terpenoid biosynthetic pathway in interior spruce A number of transcripts involved in different steps of the terpenoid pathway were induced by weevil feeding in interior spruce and many of these transcripts were also induced by fungal and wounding treatment (Table 3.5). Many terpene synthases were highly induced, as was the 1-deoxy-xylulose-phosphate synthase (DXS) transcript involved in methylerythritol phosphate (MEP) pathway and one prenyltransferase (geranygeranyl diphosphate synthase (GGPPS). Strong induction of DXS, which is known to play a major role in regulation of MEP pathway (Cordoba et al., 2009), is indicative of activation of the MEP pathway by weevil feeding, fungal- and wounding- treatment. Induction of DXS is particularly relevant to resin production, as the MEP pathway is localized in chloroplasts (Cordoba et al., 2009) and supplies terpenoid precursors for the biosynthesis of monoterpenoids and diterpenoids that are produced also in chloroplast (Huber et al., 2004), and constitute the majority of the terpenoids in conifer resin (Keeling and Bohlmann, 2006). Larger contribution of IPP from the MEP pathway in Taxus baccata diterpenoid taxane production and the MEP pathway importance in production of monoterpenoids (e.g. peppermint monoterpene essential oil) support the primary importance of the MEP pathway in diterpenoid and monoterpenoid biosynthesis with some possibility of contribution of IPP from the mevalonic acid (MEV) pathway through shuffling of the MEV-produced IPP through chloroplast membrane (Roberts, 2007). In addition, two HMGR transcripts are downregulated by weevil feeding in interior spruce (among three downregulated transcripts involved in terpenoid biosynthesis) and one HMGR transcript was the only transcript involved in terpenoid pathway downregulated by fungal inoculation (Table 3.5). HMGR is well regulated in plants (Roberts, 2007) and was shown to be a rate limiting step in the biosynthesis of sterols in tobacco (Chappell et al., 1995). Downregulation of HMGR by weevil feeding and fungal inoculation in interior spruce may indicate decreased biosynthesis of terpenoids involved in primary metabolism in favour of biosynthesis of defense related terpenoids in conifers. High levels of induction of one of the GGPPS transcript and downregulation of another (Table 3.5) can be explained by the differential regulation and function of different GGPPS genes in conifers as was previously demonstrated in Norway spruce (Schmidt and Gershenzon, 2007). One (PaIDS5) of two Norway spruce GGPPS genes 132 was highly induced by methyl jasmonate treatment, whereas methyl jasmonate had no effect on the expression of the other gene (PaIDS6) in Norway spruce bark. Based on phylogenetic analysis it was suggested that PaIDS5 is involved in resin production in conifers (Schmidt and Gershenzon, 2007). Sequence alignment of two differentially expressed GGPPS transcripts in interior spruce revealed higher sequence similarity of the highly induced transcript (WS00911_G14) to PaIDS5 as compared to a downregulated transcript (WS01030_E02) (Table 3.5, data for sequence alignment analysis is not shown). Thus, microarray analysis supports specificity in GGPPS gene involvement in the interior spruce defense response to weevil feeding. Induction of GGPPS transcripts can lead to higher diterpenoid accumulation as was observed in the bark of methyl jasmonate treated spruce as well as in spruce exposed to weevil feeding (Miller et al., 2005). Induction of many terpene synthases by weevil feeding in interior spruce indicates activation of resin production (Table 3.5). Previous analysis of the effect of weevil feeding on Sitka spruce revealed increased accumulation of monoterpenoids and diterpenoids as well as the strong induction of monoterpene synthases (Miller et al., 2005). It was demonstrated that constitutive and induced production of resin contributes to spruce defense against weevil (Alfaro et al., 2002). Monoterpenoids are important to decrease resin viscosity, to allowing the resin to flood and kill weevil eggs and larva (Alfaro et al., 2002). Spruce resin with high diterpene resin acids concentration was associated with resistance to weevil, possibly due to higher toxicity or stronger feeding deterrence (Tomlin et al., 1996). Similar induction of transcripts involved in terpenoid biosynthesis by weevil feeding, fungal inoculation and wounding treatment supports the broad function of terpenoids in conifer defense response.

Transcripts specific analysis confirms strong induction of two DXS transcripts by weevil feeding in interior spruce Strategic positioning of DXS at the beginning of MEP pathway, which most likely plays central role in providing precursors for resin biosynthesis (Roberts, 2007), and establishing of DXS enzyme major role in regulation of the MEP pathway, makes DXS enzyme a promising candidate for the regulation of induced terpenoid defense in conifers. To further investigate DXS transcript involvement in the conifer defense response to weevil feeding we performed transcript specific expression analysis using real-time PCR. The DXS gene family was characterized in Norway spruce and is currently represented by three DXS genes, divided into type I and type II (Phillips et al., 2007). Transcript specific expression of the three DXS transcripts present on the 21.8k 133 spruce microarray confirmed strong induction of two of the three analysed DXS transcripts by weevil feeding in interior spruce (Figure 3.5). The most highly induced DXS transcript (WS00930_F08) by weevil feeding was also highly induced by fungal inoculation and wounding treatment in interior spruce (Figure 3.5, Chapter 2). This transcript is an ortholog of the PaDXS2B gene, a type II DXS from Norway spruce which was induced by methyl jasmonate, wounding and by the fungal pathogen Ceratocystis polonica in Norway spruce (Phillips et al., 2007). Strong induction of this type II DXS in interior spruce and induction of its ortholog in Norway spruce by a variety of treatments supports the involvement of this DXS in defense related induced terpenoid biosynthesis in conifers. DXS (WS0097_H02) was also induced by weevil. This DXS is the ortholog of the type I PaDXS1 gene in Norway spruce and WS0097_H02 was not induced by wounding or fungal treatment in interior spruce (Table 3.5, Figure 3.5) and in Norway spruce (Phillips et al., 2007). Type I DXS genes are considered to be involved in primary terpene biosynthetic processes due to their constitutive expression (Cordoba et al., 2009). Induction of the type I DXS in interior spruce may indicate the activation of primary metabolism in response to weevil feeding or the potential involvement of this DXS in defense response through the biosynthesis of additional substrate for resin terpenoids.

Pathogenesis related proteins A number of PR protein transcripts, such as chitinases, thaumatin-like protein, hevein-like protein, osmotin-like protein, were induced in interior spruce exposed to weevil feeding. Induction of selected PR protein transcripts by weevil and budworm feeding was also demonstrated by a microarray study for apical part of Sitka spruce (Ralph et al., 2006b) The PR proteins were shown to have antifungal properties (Van Loon and Van Strien, 1999; Sels et al., 2008). The role of the PR proteins in plant insect defense is not well established. Chitinases can damage the insect peritrophic matrix, which contains chitin, as was demonstrated in vitro and/or in vivo for entomopathogenic fungi chitinases and nematode chitinase (Kramer and Muthukrishnan, 1997). Antiinsect activity of a poplar chitinase expressed in transgenic tomato against Colorado potato beetle has been demonstrated (Lawrence and Novak, 2006), and supports the involvement of plant chitinases in plant antiinsect defense. A direct comparison of chitinase expression in interior spruce induced by weevil feeding and fungal inoculation provided an opportunity to compare the involvement of chitinases in the spruce defense response. Microarray analysis of 17 chitinases present on the 21.8K spruce microarray revealed induction of 9 chitinases by weevil feeding in interior spruce (Table 3.6) and the 134 strong induction of several chitinases was confirmed using gene specific analysis (Chapter 4). Induction of the same chitinases by weevil feeding, fungal and wounding treatments in interior spruce observed in our study (Table 3.6, Chapter 4), as well as induction of chitinases by wounding, fungal and insect treatment observed in other conifers (Davis et al., 2002; Hietala et al., 2004; Ralph et al., 2006b) indicates the generic function of these chitinases and their potential importance in protecting plants from infection by pathogenic fungi that may occur through wounding and insect feeding. The specific roles of different chitinases in conifer defense against fungi and insects have yet to be elucidated.

Additional targets for conifer defense research suggested by the microarray analysis In addition to genes known to be involved in conifer defense, the microarray analysis suggested a role for transcripts annotated as protease inhibitors in spruce defense against weevil, as these transcripts were among the most induced transcripts in interior spruce exposed to weevil feeding and were either not induced or induced to a much lesser degree by fungal treatment (Table 3.2). A protease inhibitor transcript was also induced in Sitka spruce treated by wounding and exposed to weevil feeding (Ralph et al., 2006a), and the importance of protease inhibitors in antiinsect and antimicrobial defense was demonstrated in angiosperms (Dunaevsky et al., 2005). The microarray analysis also pointed to a possible defense involvement of several transcripts annotated as (R)-mandelonitrile lyase. These transcripts were highly upregulated by weevil feeding and to a smaller degree by fungal inoculation in interior spruce (Table 3.2). (R)-mandelonitrile lyase is a cyanogenic enzyme that releases cyanide from mandelonitrile and represent a broader enzyme group of hydroxynitrile lyases (Hickel et al., 1996). Cyanogenic compounds are widely distributed among thousands of different plant species (Bennett and Wallsgrove, 1994), including the tree species Eucalyptus (Goodger et al., 2004) and yew (Chattopadhyay et al., 2002). Upon insect feeding, cyanide is released from cyanogenic compounds in plants and often serves as a feeding deterrent due to its toxicity (Bennett and Wallsgrove, 1994). Although presence of cyanogenic glucosides was not previously reported for Pinaceae species, further investigation into genes with homology to hydroxynitrile lyases may uncover new secondary metabolite defense strategies for conifer species against insects. Similarly, the strong induction of several carbonic anhydrase transcripts by weevil feeding and to a lesser degree by fungal inoculation and wounding treatment in 135 interior spruce points to possible involvement of carbonic anhydrase transcripts in the conifer defense response (Table 3.2). Carbonic anhydrase catalyzes the reaction of conversion of carbon dioxide to bicarbonate, it also has been shown to have antioxidant activity and is capable of binding salicylic acid (Slaymaker et al., 2002). Carbonic anhydrase was upregulated in incompatible interaction of potato with Phytophtora infestans and its silencing resulted in faster Phytophtora infestans pathogen growth in tobacco (Restrepo et al., 2005). The exact role of carbonic anhydrase in defense is not clear, however, may be involved in development of hypersensitive response (Slaymaker et al., 2002), in photosynthetic processes in stressful conditions or in mediating jasmonic acid response by increasing rate of lipid biosynthesis (Restrepo et al., 2005).

Conclusion This microarray study allowed a thorough comparison of the interior spruce transcriptome response to weevil feeding and to blue-stain fungal inoculation, highlighting the importance of the phenylpropanoid pathway, the terpenoid pathway and chitinases in the spruce defense responses to both treatments. Analysis of highly induced transcripts provided candidate genes that may play generic roles in spruce defense, as well as candidate genes which may have specific functions in weevil defense. Further investigation of the biological function of proposed genes will develop a specific understanding of the role of these genes in conifer defense against insects and fungal pathogens.

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4 Cloning and characterization of chitinases from interior spruce and lodgepole pine3

4.1 INTRODUCTION Conifer trees are often exposed to insect pests and pathogenic fungi (Paine et al., 1997; Alfaro et al., 2000). Major defense responses in conifers include constitutive and induced formation of terpenoid resin, phenolics and protein based defenses, such as chitinases (Keeling and Bohlmann, 2006a, 2006b, Bohlmann, 2008; Franceschi et al., 2005; Schmidt et al., 2005). Chitinases catalyze the hydrolysis of chitin, a linear polymer of β-1,4-linked N acetylglucosamine that is a common constituent of fungal cell walls and the peritrophic matrix of insect digestive systems. Chitinolytic activity has been demonstrated for a number of angiosperm plant chitinases in vitro using chitin or chitin analogs as a substrate (Collinge et al., 1993; Kasprzewska, 2003). The effective hydrolysis of chitin resulted in growth inhibition of a variety of fungal pathogens in vitro for selected plant chitinases (Schlumbaum et al., 1986; Verburg and Huynh, 1991; Li et al., 2003; Ye and Ng, 2005; Kirubakaran and Sakthivel, 2007; Singh et al., 2007). The chitinolytic activity of chitinases was confirmed using purified chitin or chitin analogs and fungus exposure to chitinases resulted in observable lysis, fragmentation and swelling of fungal mycelia (Kirubakaran and Sakthivel, 2007; Singh et al., 2007). The induction of chitinases by pathogens was demonstrated in many angiosperm species (Kasprzewska, 2003) and overexpression of selected chitinases in transgenic plants resulted in increased resistance against fungal pathogens (Vellicce et al., 2006; Jayaraj and Punja, 2007; Xiao et al., 2007). The ability of chitinase to damage insect peritrophic matrix was demonstrated in vitro and in vivo for chitinases of entomopathogenic fungi and nematodes (Kramer and Muthukrishnan, 1997). Expression of a poplar chitinase in transgenic tomato led to slower development of Colorado potato beetle (Lawrence and Novak, 2006). In conifers, induction of chitinase expresison by pathogen attack, insect herbivory and wounding was demonstrated for spruce and pine species (Kozlowski and Metraux, 1998; Davis et al., 2002; Hietala et al., 2004; Nagy et al., 2004; Liu et al., 2005; Ralph et al., 2006), and increased expression of chitinases was associated with Norway spruce resistance to the pathogenic fungus Heterobasidion annosum (Fossdal et al., 2006). In addition to their involvement in defense, chitinases also function in plant development. For example, chitinases are

3 A version of this chapter will be submitted for publication. Kolosova N, Breuil C and Bohlmann J. Cloning and characterization of a set of novel chitinases from interior spruce and lodgepole pine. 144 involved in the control of early embryogenesis (Kragh et al., 1996), and in gymnosperms, a chitinase is involved in Norway spruce embryogenesis through stimulating programmed cell death (Wiweger et al., 2003). Chitinases are represented by large gene families in plants (Graham and Sticklen, 1994) and members vary in biochemical and biological properties. Rapid evolution of chitinases was suggested to be driven by an arms race in plant-pathogen interactions (Bishop et al., 2000). Plant chitinases are divided into seven classes (class I-VII) based on their sequence and domain structure (Meins et al., 1994; Neuhaus, 1999). Class I chitinases contain the catalytic domain present in all chitinases, and an additional cysteine-rich domain, which is suggested to have chitin binding capacity. These chitinases are usually localized to the vacuole, although some are apoplastic (Graham and Sticklen, 1994). Class I are known to have high chitinolytic activity compared to class II chitinases. Class II chitinases have been localized extracellularly and their catalytic domain is highly similar to that of class I chitinases. Class II chitinases lack the cysteine-rich domain (Graham and Sticklen, 1994). Class III chitinases have no obvious sequence similarity with class I and II chitinases but instead, they have some sequence similarity to bacterial chitinases (Graham and Sticklen, 1994). Class IV chitinases have a high level of sequence similarity with class I chitinases but have several deletions, a truncated C-terminus (Meins et al., 1994) and appear to be localized extracellularly (Graham and Sticklen, 1994). Class V chitinases have a duplicated cysteine-rich domain (Meins et al., 1994). Class VI chitinases have sequence similarity to bacterial chitinases, but no obvious sequence similarity to the class I-V chitinases (Meins et al., 1994). Class VII chitinases are highly similar to class IV chitinases but are lacking the chitin binding domain (Neuhaus, 1999). Previously cloned and characterised chitinases in conifers include class I, II and IV chitinases (Wu et al., 1997; Davis et al., 2002; Wiweger et al., 2003; Hietala et al., 2004; Liu et al., 2005). Involvement of many of these chitinases in conifer defense was proposed based on their induction by wounding, fungal inoculation, or defense related elicitor treatments (e.g. methyl jasmonate) (Kozlowski and Metraux, 1998; Davis et al., 2002; Hietala et al., 2004; Nagy et al., 2004; Liu et al., 2005; Ralph et al., 2006). To date, there are no reports on the functional characterization of conifer chitinases. In this study we present the cloning and functional characterization of cDNAs encoding six different chitinases from interior spruce (Picea engelmannii x glauca) representing classes I, II, IV and VII and four different chitinases from lodgepole pine (Pinus contorta) representing classes I, IV and VII. Functional analysis of recombinant proteins demonstrates chitinolytic activity for two interior spruce and one lodgepole pine 145 chitinases all belonging to class I. Gene specific expression analysis revealed induction of most of the tested chitinases by fungal and wounding treatments in interior spruce and lodgepole pine and by weevil feeding in interior spruce. These results suggest a biological role for chitinases in conifer defense against fungal pathogens and/or insects.

4.2 METHODS

4.2.1 Nucleotide sequence accession numbers The nucleotide sequences of the six interior spruce chitinases and four lodgepole pine chitinases have been deposited into GenBank database with the following accession numbers: HM219843 (PgeChia1-1), HM219844 (PgeChia1-2), HM219845 (PgeChia2-1), HM219846 (PgeChia4-1), HM219847 (PgeChia7-1), HM219848 (PgeChia7-2), HM219849 (PcChia1-1), HM219850 (PcChia4-1), HM219851 (PcChia7- 1), HM219852 (PcChia7-2).

4.2.2 Lodgepole pine cDNA library construction and DNA sequencing The cDNA library was prepared from bark of three year old lodgepole pine inoculated with the fungus G. clavigera (Chapter 2). Bark tissue, with phloem attached, was harvested at 6 hrs, 2, and 14 days after treatment initiation (four trees per time point). Total RNA was extracted using previously published protocol (Appendix 3; Kolosova et al., 2004). The same amounts of RNA from each sample were pooled for library construction. The cDNA library was constructed using pBluescript II XR cDNA Library Construction Kit (Stratagene). Sequencing was performed as previously described (Ralph et al., 2008) and 1222 lodgepole pine ESTs, which represent 1030 unique transcripts, were submitted to NCBI GenBank (and Treenomix.ca).

4.2.3 Subcloning and sequence analysis of chitinase cDNA Six full length FLcDNAs for interior spruce chitinases sequences were identified in the ESTs of the Treenomix project (Ralph et al., 2008) and completely sequenced. Physical cDNA clones were obtained from the Treenomix project (Treenomix.ca). The FLcDNA clones, representing four different classes of chitinases, were characterized: PgeChia1-1 was cloned based on EST WS00922_B21, PgeChia1-2 based on EST WS00929_K15, PgeChia2-1 based on EST WS00927_N15, PgeChia4-1 based on EST WS00924_F07, PgeChia7-1 based on EST WS00951_K15, PgeChia7-2 based in EST WS00949_I04 (NCBI, Treenomix.ca). Two full length lodgepole pine chitinases sequences were identified in the lodgepole pine EST sequences developed as part of 146 the present project. These chitinases represent class VII: PcChia7-1 was cloned based on EST WS0353_A23 (GB: GW725974.1) and PcChia7-2 based in EST WS0352_P07 (GB GW725947.1). Two additional lodgepole pine chitinases PcChia1-1 and PcChia4-1 were cloned using lodgepole pine partial EST sequences of WS0354_C23 (Treenomix.ca) and WS0354_D22 (Treenomix.ca), respectively. Full length cDNA for these chitinases was obtained by PCR amplification of the coding region. A 5' end degenerate primer for PcChia1-1 (5' GCTGAKATAGAAACGAGTGTCTGN) was designed just upstream of the coding region based on the alignment of orthologous full length EST sequences available from loblolly pine (TA991_3352, TA1024_3352). Similarly, a 5' primer for PcChia4-1 (5' GSGGARTCTTAAGRAATTATTSGN) was designed using Sitka spruce (WS02717_M22) and loblolly pine (TA795_3352, TA906_3352) ESTs. The 3' primers for these genes were designed based on available EST sequences. PCR products of 1kb (PcChia1-1) and 0.8kb (PcChia4-1) were amplified using fungus induced lodgepole pine cDNA (Chapter 2). These PCR products were initially subcloned into pJET1.2 vector using CloneJET PCR Cloning Kit (Fermentas) for sequence analysis. The six full length interior spruce and four full length lodgepole pine chitinase cDNAs were subcloned into HindIII and BamHI restriction cites of the pMal-p4X expression vector (New England Biolabs, Pickering, Ontario).

4.2.4 Heterologous expression and purification of chitinases E.coli BL21(DE3) competent cells were transformed with the spruce and lodgepole pine pMal-p4X chitinase constructs. Cells were grown at 37oC and 250 rpm to an OD600 of 0.5 in LB medium containing 100 µg/ml of ampicillin and 2 g/L glucose, after which 0.3 mM IPTG was added and the cultures were incubated for another 16-20 h. Cells were collected by centrifugation and chitinase protein was extracted with buffer containing 20 mM Tris-HCl pH7.4, 200 mM NaCl, 1 mM EDTA, 40 µg/ml DNAse (Sigma, Oakville, ON, Canada) and 40 µg/ml RNAse (Sigma) and protease inhibitor coctail [50M TPCK (chymotrypsin-like), 50M TLCK (trypsin-like), 2 M Leupeptin (serine, cysteine), 1 M E64 (cysteine), 1 M Pepstatin (aspartic), 10 mM 1,10- phenanthroline (mettaloproteases), 0.5 mM PMSF (serine), 0.5 mM AEBSF (serine)]. Recombinant chitinases were partially purified using amylose resin (NEB) following the pMal-p4X expression and purification manufacturer’s instructions. Briefly, cells were sonicated and cell debris spun down, then supernatant was applied to the amylose column. After washing with 12 column volumes of wash buffer containing 20 mM Tris-HCl pH7.4, 200 mM NaCl, 1 mM EDTA chitinase protein was eluted with elution 147 buffer containing 20 mM Tris-HCl pH7.4, 200 mM NaCl, 1 mM EDTA and 10 mM maltose. The size of the expressed chitinases was confirmed using protein gel.

4.2.5 Enzyme assay Chitinolytic activity was assayed using a previously described protocol with modification (Ramirez et al., 2004). Four to 25 µg of protein in 30 µl of 0.1 M Tris-HCl pH7 was combined with 90 µl of carboxymethyl Remazol Brilliant Violet-Chitin (2 mg/ml) (CM-Chitin-RBV, Loewe Biochemica, Munich, Germany). The resulting assay mixture was incubated at 50oC for 30 min, then reactions were stopped by adding 10 µl of 1 M HCl (to adjust to pH2), CM-Chitin-RBV that was not digested was precipitated on ice for 10 min and then samples were centrifuged for 5 min at 6000g. The supernatant absorbance was measured at 560 nm using a Spectra Fluor Plus plate reader (Tecan, San Jose, CA). Three independent measurements were performed per each of three sample replicates. Samples at 0 min of reaction time were used as controls and their absorption was subtracted from the absorption of the assay samples to evaluate the amount of released chitin. A change of absorbance (Δ560) of 0.1 corresponded to 13 µg of released RBV chitin. Protein isolated from IPTG induced cells containing empty plasmid did not show detectable chitinolytic activity.

4.2.6 Determination of optimum pH and temperature The effect of pH on the enzymatic activity of the purified chitinases was assessed within a pH range between 3.0 and 10.0 using 0.1 M sodium acetate buffer (pH 3.0, 4.0, 5.0), 0.1 M phosphate buffer (pH 6.0), 0.1M Tris-HCl buffer (pH 7.0, 8.0) and 0.1M Glycine-NaOH buffer (pH 9.0, 10.0). To assess the effect of temperature on chitinase activity enzymatic assay of purified chitinases was performed at a temperature range from 20 to 60oC.

4.2.7 Quantitative real-time PCR (QRT-PCR) Bark samples were collected from interior spruce inoculated with Leptographium abietinum and lodgepole pine inoculated with Grossmannia clavigera at 6h, 2 days and 2 weeks of treatment as described in Chapter 2.2.1 and from interior spruce exposed to weevil feeding following the same time course as described in Chapter 3.2.1. Total RNA was extracted following a previously reported protocol (Appendix 3; Kolosova et al., 2004). QRT-PCR was performed and analyzed as previously described (Ralph et al., 2007). Gene specific primers (Table 4.1) were designed using previously described 148 guidelines (Ralph et al., 2006). Transcript abundance was normalized to translation initiation factor 5A (TIF5A, IS0013_F24, GenBank: DR448953). Statistical analysis was performed as described in Chapter 2.2.5.

Table 4.1: Primer sequences used for real-time PCR (5' to 3' orientation) Gene ID Forward primer Reverse primer TIF5A GTGCCATCTTCACACAACTGC CAGATTCAGTCAGCAGGCTAAC PgeChia1-1 GACTCGAGGCAGGTGGACCGC GGAGATCCAACTGCTTTAACC PgeChia1-2 GCAGCAATGTTAAGCAGGATG AATGGACCGCGTTATTGCTTC PgeChia2-1 GACTCAAGGCAGGAGGACCG TGCAAGACTAACAACGCAAAG PgeChia4-1 TGTCGACCCTGGAGCCAATG TGCACGCCACGAGAGGTGCA WS0092_L20 GTGCCACAATCAAAGCCATC CCACATCAGTGACGTCTTATTG IS0014_F01 GCCAAGTCAATAGCAGAGTTAC TTGAGGACACCCGTTATCAGC PcChia1-1 CCGGTCCATAGTTGTAATTCC CAAAGAGGAGCAAGGCAATCC PcChia4-1 GATCCTGGAGCCAATGTTTCG TTGCACAGAGATGCGCAATG PcChia7-1 GCCATCGCCTTCATAACTATC TGTATCAACTTACATACGACC PcChia7-2 GCAACGATCAAAGCAATCAGC GAACTATTCTGGATCCTTTGAC

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4.3 RESULTS

4.3.1 Cloning, sequencing and identification of conifer chitinases Six different chitinases from interior spruce and four different chitinases from lodgepole pine representing four chitinase classes were cloned as full length cDNAs based on EST sequences. Chitinase sequences were assigned to classes based on the previously described classification and nomenclature system (Meins et al., 1994; Neuhaus, 1999). Sequence alignment of the conifer chitinases with previously characterized chitinases (Hamel et al., 1997; Liu et al., 2005) confirmed the presence of characteristic plant chitinase domains of different chitinase classes, including catalytic domain and hydrophobic N-terminal signal peptides present in all cloned interior spruce and lodgepole pine chitinases and the chitin binding domain and C-terminal extension present in selected chitinases (Figure 4.1 and 4.2).

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Signal peptide Chitin binding domain PgeChia1-1 ------MKSIRF-SAMAIALVTMGTMNLYFASAEQCGRQASGALCPGGLCCSKWGWCGN 52 PgeChia1-2 MAGNVGKMSSIFLGTTLAIFTAVAMIMSLPSVSSEDCGQQAGGALCPGGLCCSKWGWCGN 60 PcChia1-1 ------MKSMKF-SAMAIALLTMATMNLYFVSAEQCGQQAGGALCPGGLCCSKWGWCGN 52 PgeChia2-1 METSVRLMKSMRF-SVMVMALVTMATMNFYFVSAEQCGRQASGVLCTG------47 PgeChia4-1 ------MALVLVLLLVGVSVNAQNC------GCASGVCCSQYGYCGT 35 PcChia4-1 ------MGSSSGNWVMAVLVLLLVSVSVNAQNC------GCASGLCCSKYGYCGT 43 PgeChia7-1 ------MATHFRVNVIFLWLAFALSALSIC------24 PgeChia7-2 ------MATHFRVNVIFLWLAFALSALSIC------24 PcChia7-1 ------MATHFTVRAVILWFVFALSALYIC------24 PcChia7-2 ------MRNYLRVMTLSAIILWLVLAFDLVSMC------27 Hinge region Catalytic domain PgeChia1-1 TEAHCGQDCQSQCGG------STPTPPSPTPGGQGVSSVITESIFNDLLKHRNDAAC 103 PgeChia1-2 TQAHCGQDCQSQCGGGGSTPTPTTPTPTPTTPTPSGQGVASIMTEDLFNQLLKYKDDSRC 120 PcChia1-1 TDAHCGQDCQSQCGG------STPTPPSPTPGGQGVASIITESIFNELLKHRNDAGC 103 PgeChia2-1 ------GVQGVSSVITESIFNNLLKHRNDAGC 73 PgeChia4-1 TSAYCGKGCKSGPCY------SSGGGSPSAGGGSVGGIISQSFFNGLAGGAGSS-C 84 PcChia4-1 TSAYCGAGCKSGPCS------SSGGGSPSGGGGSVGTIISQSFFNGLAGGAASS-C 92 PgeChia7-1 ------RGAVSDIATQDFFNGILSAATDG-C 48 PgeChia7-2 ------RGAVSDIATQDFFNGILSAATDG-C 48 PcChia7-1 ------RGAVSDIATQDFFNGILSAATDG-C 48 PcChia7-2 ------RGDVGDFATQDFFNGILSGASDS-C 51

PgeChia1-1 KAKGFYSYAAFIAAANAFPAFGTTGDLSTRKRELAAFFGQTSHETTGGWPAAPDGPYAWG 163 PgeChia1-2 KANGFYSYAAFIAAASAFSGFGTAGDLTTNKRELAAFLAQTSHETTGGWQSAPDGPYAWG 180 PcChia1-1 KASGFYTYSAFIAAANAFPSFGTTGDVATRKRELAAFFGQTSHETTGGWATAPDGAYAWG 163 PgeChia2-1 KAKGFYSYDAFIAAANAFPGFGTTGDLTSQKRELAAFFGQTSHETTGGWPTAPDGPYAWG 133 PgeChia4-1 EGKGFYTYNAFIAAANAFSGFGTTGSNDVKKRELAAFFANVMHETGG------131 PcChia4-1 EGKGFYTYNAFIAAANAYSGFGTTGSADVRKRELAAFFANVMHETEG------139 PgeChia7-1 AGKTFYTYTDFINAANSFSSFGTTGTSDDNKREIAAFFANVAHETTN------95 PgeChia7-2 AGKTFYTYTDFINAAISFSSFGTTGTSDDNKREIAAFFANVAHETTN------95 PcChia7-1 AGKTFYTYSDFINAANSFSSFGTTGTSDDNKREIAAFFANVAHETTN------95 PcChia7-2 TGKTFYTYNNFMDAATAFSGFGTTGPDVDHKREIAAFFANVAHETSR------98

PgeChia1-1 YCFKEEQGNPPGEYCQASSQWPCASGKRYYGRGPVQISWNYNYGPAGRAIGFDGINNPDI 223 PgeChia1-2 YCFKEEQ-DPVSDFCQASSQWPCASGKRYYGRGPIQISWNYNYGQAGSALQFDGINNPDI 239 PcChia1-1 YCFKEEQGNPPAEYCQATSQWPCASGKRYYGRGPVQLSWNYNYGPAGKAIGFDGINNPDI 223 PgeChia2-1 YCFKEEQGNPPGEYCQASSQWPCASGKRYYGRGPVQISWNYNYGPAGKAVGFDGINNPDI 193 PgeChia4-1 LCYINEKNPPMKYCQSSST-WPCTSGKSYHGRGPLQLSWNYNYGAAGKSIGFDGLNNPEK 190 PcChia4-1 MCYINEINPQSNYCNSSAT-WPCASGKSYHGRGPLQLSWNYNYGAAGQTIGFDGVNNPEK 198 PgeChia7-1 LCYVEEID-KSDYCDSSNTQYPCASGQQYYGRGPLQLTGNANYGAAGTYLSADLLNNPGL 154 PgeChia7-2 LCYVEEIA-KSDYC-SSNTQYPCASGQQYYGRGPLQLTGNGNYGAAGDYLGVDLLNNPGL 153 PcChia7-1 LCYVEEIA-KSAYCDSTNTQYPCASGQQYYGRGPLQLTGNANYGAAGAYLAVDLRNNPGL 154 PcChia7-2 LCYVEQIE-KSDYCDSTNQKYQCVAGKQYYGRGPLQLTWNYNYGAAGDYLGFDGLNHPEI 157

PgeChia1-1 VANDATVSFKTAVWFWMTAQSPKPSCHDVMTGRWSPSGSDSAAGRAAGYGVVTNIINGGL 283 PgeChia1-2 VASDPTVSFKTAVWFWMTAQSPKPSCHDVMTGTWSPSGSDSAAGRAAGYGLVTNIINGGL 299 PcChia1-1 VASDATVSFKTAIWFWMTAQSPKPSCHDVMTGKWTPSGSDSAAGRAAGYGAVTNIINGGL 283 PgeChia2-1 VANDATVSFKTAVWFWMTEQSPKPSCHNVMAGGWGPSGSDTAAGRAAGYGVVTNIINGGL 253 PgeChia4-1 VGQDSTISFKTAVWFWMK----NSNCHSAIT-----SGQ------GFGGTIKAINSM- 232 PcChia4-1 VGQDPTISFKTAVWFWMK----NSNCHSAIT-----SGQ------GFGGTIKAINSQ- 240 PgeChia7-1 VAQDDLTSWKTALWFWNV----NSNCHTAIT-----SGQ------GFGATIQAINGAI 197 PgeChia7-2 VAQDDLTSWKTALWFWNV----NSNCHTAIT-----SGQ------GFGATIQAINGAI 196 PcChia7-1 VAQDDLTSWKTALWFWNV----NSNCHTAIT-----SGQ------GFGATIQAINGAV 197 PcChia7-2 VAQNGSISWKTAVWFWMK----HSNCHSAIT-----SGQ------GFRATIKAISGD- 199 CTE PgeChia1-1 ECGKGSDSRQVDRIGFYKRYCDILGVSYGSNLDCNSQKPFGFAAQSHPRLIKTVV 338 PgeChia1-2 ECGKGSNVKQDDRIGFYKRYCDILGVSYGSNIDCNSQTPYGG------341 PcChia1-1 ECGKGSDSRQQDRIGFYKRYSDILGVSYGSNLDCNNQRPFGAAVQSEARLIKTVV 338 PgeChia2-1 ECGKGSDSRQEDRIGFYKRYCDILGVSYGSNLDCNSQKPFGFAAQSQPRLIKTVV 308 PgeChia4-1 ECNGGNSGEVSSRVNYYKKICSQLGVDPGANVSC------266 PcChia4-1 ECNGGKTGEVNNRVNYYKNICSQLGVDPGANVSC------274 PgeChia7-1 ECNGGNPDEVNDRISHYTNYCSQFGVDPGSNLSC------231 PgeChia7-2 ECNGGNTDQVNDRISRYTNYCSQFGVDPGSNLSC------230 PcChia7-1 ECNGGNTAEVNDRVSRYTTYCSQLGVDPGSNLTC------231 PcChia7-2 ECNGGDSNAVDERVNYYTNYCNEFGVDPGNNLSC------233

Figure 4.1: Alignment of the amino acid sequences of six interior spruce (Pge) and four lodgepole pine (Pc) chitinases. Numbers correspond to the last amino acid position on each line. Hyphens represent gaps in sequences introduced for the best alignment.

151

SP CBD HR Catalytic domain CTE Chia1 Class I Chia2 Class II Chia2 Class II Chia4 Class IV Chia7 Class VII

Figure 4.2: Schematic structure of chitinases characterized in conifer species so far. SP – signal peptide, CBD – chitin binding domain, HR – hinge region, CTE – C terminal extension. The schematic representation was modified from Hamel et al., 1997 and extended based on Neuhaus, 1999

The cloned chitinase genes were named based on the previously suggested nomenclature and included abbreviated species name followed by Chia, which indicates the chitinase family, followed by a number indicating chitinase class with the final number indicating individual class members (Neuhaus, 1999). Cloned interior spruce chitinases PgeChia1-1 (338 amino acids), PgeChia1-2 (341 amino acids) and lodgepole pine chitinase PcChia1-1 (341 amino acids) contained the cysteine-rich (chitin binding) domain and had over 50% amino acid sequence identity with tobacco class I chitinase (GenBank Accession No. X64519.1), therefore, they were assigned to class I (Figure 4.1 and 4.2). Interior spruce chitinase PgeChia2-1 (308 amino acids) had 79% amino acid sequence identity with class I chitinase PgeChia1-1, but was lacking cysteine-rich domain and was therefore assigned to class II (Figure 1 and 2). Chitinase PgeChia2-1 represents class II chitinases without an internal deletion in the catalytic domain, in addition to the previously cloned class II eastern white pine (Pinus strobus) chitinase Pschi4 (Wu et al., 1997) that represents conifer class II chitinases with an internal deletion in the catalytic domain (Figure 2). The cloned chitinases of class I and II contain C-terminal extensions of variable length. Interior spruce chitinase PgeChia4-1 (266 amino acids) and lodgepole pine chitinase PcChia4-1 (274 amino acids) were assigned to class IV based on a deletion in the chitin binding domain, several deletions in the catalytic domain and a lack of a C-terminal extension (Figure 1 and 2). In addition, PgeChia4-1 and PcChia4-1 have 93% and 85% amino acid sequence identity respectively with the previously characterized Norway spruce class IV chitinase Chia4- Pa1 (Wiweger et al., 2003). Interior spruce chitinases PgeChia7-1 (231 amino acids) and PgeChia7-2 (230 amino acids) and lodgepole pine chitinases PcChia7-1 (231 amino

152 acids), PcChia7-2 (233 amino acids) were assigned to class VII due to their highly homologous catalytic domain to class IV chitinases and the absence of the cysteine-rich domain and C-terminal extension (Figure 4.1 and 4.2).

4.3.2 Heterologous expression and functional characterization of conifer chitinases The ten chitinases cloned from interior spruce and lodgepole pine were expressed in E.coli using the pMAL-4X (NEB) expression vector that contains a maltose binding protein (MBP) tag. Initial attempts of expression of the chitinases using the His- Tag expression vector pET-28b(+) resulted in the production of insoluble chitinase protein that was unsuitable for biochemical analysis (data not shown). Soluble recombinant chitinase proteins were obtained using pMAL-4X expression vector. The maltose binding protein tag is known to support proper protein folding and consequently solubility of proteins (PerezMartin et al., 1997). All chitinase proteins were partially purified using amylose resin. The yield of purified chitinases varied from 2 mg/L to 30 mg/L depending on the chitinase construct. The most highly expressed chitinases were PgeChia1-1, PgeChia1-2 and all four of the class VII chitinases. The partially purified chitinase proteins were used to determine the presence of chitinolytic activity. Utilising CM-chitin-RBV as a substrate revealed the presence of chitinolytic activity for all of the tested class I chitinases: PgeChia1-1, PgeChia1-2 and PcChia1-1. Boiled chitinases and maltose binding protein tag isolated from E.coli culture carrying an empty vector did not exhibit chitinolytic activity. All other interior spruce and lodgepole pine tested chitinases of class II, IV and VII did not exhibit chitinolytic activity under the conditions tested. To further explore the chitinolytic activity of the cloned chitinases, we tested other methods for determination of chitinase activity, including measuring the release of the reducing end group N-acetamino-glucose from colloidal chitin using dinitrosalicylic acid (Kirubakaran and Sakthivel, 2007) and using 4-methylumbelliferyl (4MU) labelled chitin analogs such as 4-MU-(GlcNAc)1 and 4-MU-(GlcNAc)3 (Eilenberg et al., 2006). Both of these methods we were not able to detect chitinolytic activity with the tested chitinases.

Increased absorbance at A560 in CM-chitin-RBV assay results from the digestion and solubilization of the CM-chitin-RBV by chitinases. Initial absorbance at the start of the reactions was subtracted as a background. This assay revealed similar range of linearity (up to 5-10µg of protein per assay) for all three class I chitinases (Figure 3). Chitinolytic activity of the three chitinases was comparable to previously characterized Aeromonas schubertii chitinase that was assayed using a similar method (Guo et al., 2004). We used chitinase protein amounts from the linear range of the enzyme activity 153 plotted against protein amount (Figure 4.3) (10 µg for PgeChia1-1, 5 µg for PgeChia1-2 and 4 µg for PcChia1-1) for the determination of temperature and pH dependence of the class I chitinases. All three class I chitinases had optimal activity at 50oC and at pH 7 (Figure 4.3).

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1.0 0.5 0.8 0.8 0.4 0.6 A 0.6 0.3

0.4

OD 560560560 OD OD OD

OD 560560 OD OD 560560560 OD OD OD

0.4 560 OD 0.2

  

      0.2 0.1 0.2 0 0 0 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 10 protein amount, μg temperature, oC pH

0.8 0.8 0.8 0.6 0.6 0.6

B 0.4 0.4 0.4

OD 560560560 OD OD OD

OD 560560560 OD OD OD 560560560 OD OD OD

  

     0.2  0.2 0.2 0 0 0 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 10 protein amount, μg temperature, oC pH

1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6

C 0.4

OD 560560560 OD OD OD

OD 560560 OD OD 560560560 OD OD OD

0.4 560 OD 0.4

  

      0.2 0.2 0.2 0 0 0 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 10 protein amount, μg temperature, oC pH

Figure 4.3: Effect of the amount of protein, pH and temperature on the activity of conifer class I chitinases. A - PgeChia1-1; B - PgeChia1-2; C - PcChia1-2. Chitinase activity was determined by spectrophotometric assay as described in material and methods. All the results were averaged from three replicates and the standard deviation is shown.

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4.3.3 Gene specific QRT-PCR analysis of the expression of chitinases in interior spruce inoculated with Leptographium abietinum Expression of chitinases was analysed in interior spruce inoculated with blue- stain fungus Leptographium abietinum at 6h, 2 days and 2 weeks of inoculation. Gene- specific analysis of the expression of six interior spruce chitinases revealed a strong induction of five out of six tested chitinases (Figure 4.4A). QRT-PCR data are represented as relative expression normalized to housekeeping gene levels of eukaryotic translation initiation factor TIF5A (Figure 4.4A), and as fold induction relative to control treatment in the case of the wounding treatment and to control and wounding treatment in the case of fungal treatment (Supplemental Table 4.1). The QRT-PCR data was statistically analyzed and considered significant with >2 fold change and p< 0.5. The most strongly induced chitinases were PgeChia1-1 (wounding vs. control - 53.1 fold change (FC), fungal treatment vs. control - 189.4 FC at 2 days post treatment) and PgeChia4-1 (wounding vs. control - 36.6 FC, fungal treatment vs. control - 97.7 FC at 2 days post treatment). Among the other chitinases, PgeChia1-2, PgeChia2-1, and putative class IV chitinase represented by ESTs WS0092_L20 were also induced by fungal treatment and to a lesser degree by wounding. The putative class IV chitinase represented by EST IS0014_F01 demonstrated moderate induction by fungal treatment compared to control at 2 days and 2 weeks post-treatment (FC 3.2 and 4.5 respectively). The significance of this induction pattern is reduced due to similar fluctuation of the level of this chitinase in control samples. This QRT-PCR analysis indicated a strong induction of interior spruce chitinases in response to wounding treatment and increased induction by fungal inoculation at two days post-treatment.

156

3.2 16 A PgeChia1-1 PgeChia4-1 * * 2.4 12

1.6 8 * 0.8 * 4 * * 0.0 0

0.08 0.56 PgeChia1-2 * WS0092_L20 0.06 0.42 * * 0.04 * 0.28 * 0.02 0.14 *

0.00 0.00 Relative Relative expression level Relative Relative expression level 1.8 0.04 PgeChia2-1 IS0014_F01 * * 1.4 0.03

0.9 0.02 * * 0.5 0.01 * * 0.0 0.00 C C C W W W F F F C C C W W W F F F 6h 2d 2w 6hr 2d 2w 6hr 2d 2w 6h 2d 2w 6hr 2d 2w 6hr 2d 2w

6.4 1.6 B PcChia1-1 * PcChia7-1 4.8 1.2 *

3.2 0.8 * * 1.6 * * 0.4 * * * 0.0 0.0 * 0.44 PcChia4-1 * 0.4 PcChia7-2

0.33 0.3 Relative Relative expression level Relative Relative expression level 0.22 0.2

0.11 0.1

0.00 0.0 C C C W W W F F F C C C W W W F F F 6h 2d 2w 6hr 2d 2w 6hr 2d 2w 6h 2d 2w 6hr 2d 2w 6hr 2d 2w

Figure 4.4: Quantitative real-time PCR analysis of chitinase transcripts levels in interior spruce and lodgepole pine. A. Relative expression levels of six transcripts of chitinase family in interior spruce bark untreated, treated with wounding and inoculated with L. abietinum during the time course of 6hours (6h), 2 days (2d) and 2 weeks (2w). Values represent mean + standard error of the mean. Transcript abundance is shown relative to TIF5A transcript in the same sample. B. Relative expression levels of four transcripts of chitinase family in lodgepole pine bark untreated, treated with wounding and inoculated with G. clavigera during the time course of 6h, 2 days (2d) and 2 weeks (2w). Values represent mean + standard error of the mean. Transcript abundance is shown relative to TIF5A transcript in the same sample. * - indicated statistically significant difference compared to control, and a dot indicated statistically significant difference compared to wounding. 157

4.3.4 Gene specific QRT-PCR analysis of the expression of chitinases in lodgepole pine inoculated with Grosmannia clavigera Expression of chitinases was analysed in lodgepole pine inoculated with blue- stain fungus Grosmannia clavigera at 6h, 2 days and 2 weeks of inoculation. QRT-PCR analysis of the expression of four lodgepole pine chitinases revealed a strong induction of PcChia1-1 chitinase transcript in lodgepole pine treated with wounding (22 FC) and fungal inoculation (144.7 FC) at 2 days post-treatment. This transcript was induced to high levels compared to the housekeeping gene, translation initiation factor TIF5A. The chitinases transcripts PcChia4-1 and PcChia7-1 were also strongly induced by fungal inoculation at 2 days post-treatment (28.4 and 572.7 FC respectively) as compared to control. Induction of the PcChia7-1 transcript by fungal treatment remained high at 2 weeks post-treatment compared to control (366.0 FC) (Figure 8B, Supplemental Table 4.1). This transcript was also induced by wounding, but to lesser extent as compared to control at 6h (15.9 FC) and at 2 days (13.8 FC). The chitinase PcChia7-2 transcript WS0352_P07 was not differentially expressed in lodgepole pine treated with wounding and fungal inoculation throughout the time course of the study (Figure 4.4B, Supplemental Table 4.1).

4.3.5 Gene specific QRT-PCR analysis of the expression of chitinases in interior spruce inoculated exposed to white pine weevil (Pissodes strobi) feeding

The QRT-PCR analysis revealed high induction of chitinases PgeChia1-1 and PgeChia4-1 by weevil feeding in interior spruce. Induction of chitinase PgeChia4-1 started at 2 days of weevil feeding (FC 47.6) and reached the highest expression level at 2 weeks of weevil feeding (FC 88.4) (Figure 4.5, Supplemental Table 4.1). Chitinase PgeChia1-1 was highly induced at 2 days post treatment (FC 125.6) and continued to be induced but to a lesser extent at 2 weeks post-treatment (FC 18.0) (Figure 4.5, Supplemental Table 4.2). Three other chitinase transcripts PgeChia1-2, PgeChia2-1 and putative class IV chitinases transcript WS0092_L20 were induced by weevil treatment in interior spruce, mostly at 2 days of weevil feeding with decreased induction observed at 2 weeks post- treatment (Figure 4.5). Expression of one of the tested putative class IV chitinase transcript IS0014_F01 was not affected by weevil feeding (Figure 4.5).

158

2.0 16 PgeChia1-1 * PgeChia4-1 1.5 12 *

1.0 8

0.5 4 * * 0.0 0 0.08 0.20 PgeChia1-2 * WS0092_L20 * 0.06 0.15

0.04 * 0.10

0.02 0.05 * Relative Relative expression level Relative Relative expression level 0.00 0.00 1.6 0.020 PgeChia2-1 IS0014_F01 1.2 * 0.015

0.8 0.010 * 0.4 0.005

0.0 0.000 C6h C2d C2w I6h I2d I2w C6h C2d C2w I6h I2d I2w

Figure 4.5: Quantitative real-time PCR analysis of chitinase transcripts levels in interior spruce and lodgepole pine. A. Relative expression levels of six transcripts of the chitinase family in interior spruce bark untreated, or treated with weevil feeding during the time course of 6h, 2 days (2d) and 2 weeks (2w). Values represent mean + standard error. Transcript abundance is shown relative to the TIF5A transcript in the same sample. C- control, I – weevil (insect) treatment. * - indicated statistically significant difference compared to control.

159

4.3.6 Evaluation of antifungal activity The three class I chitinases (PgeChia1-1, PgeChia1-2, PcChia1-1) did not demonstrate antifungal activity against blue-stain fungi Grosmannia clavigera (strain SLKw1407) and Leptographium abietinum (strain 2PG6P-La) when tested using three different spore germination methods, including spore germination in liquid suspension in the presence of a chitinase protein (Chen et al., 2007), germination of the spores on agar plate in the presence of a chitinase protein (Mauch et al., 1988) and germination of preincubated (for 24-48h) spores with chitinase protein on agar plate. In all cases concentration of chitinases was 2 mg/ml. MBP tag protein (2 mg/ml) was used as a control. In all tested cases fungal growth and germination of the tested spores was not inhibited by the presence of the tested chitinases.

160

4.4 DISCUSSION

The involvement of the chitinase gene family in conifer defense has been suggested by a number of studies that correlated chitinase expression with the conifer defense response and disease resistance (Kozlowski and Metraux, 1998; Davis et al., 2002; Hietala et al., 2004; Nagy et al., 2004; Liu et al., 2005; Fossdal et al., 2006; Ralph et al., 2006). In this study, we cloned six interior spruce chitinases and four lodgepole pine chitinases and for the first time established the presence of chitinolytic activity in three conifer class I chitinases: PgeChia1-1, PgeChia1-2 and PcChia1-1. The six interior spruce chitinases and four lodgepole pine chitinases represented four different classes of chitinases class I, class II, class IV and class VII and had characteristic protein structures for these classes which was previously established for angiosperm and conifer chitinases (Hamel et al., 1997; Neuhaus, 1999; Liu et al., 2005). All ten cloned chitinases had a characteristic N-terminal signal peptide with a highly hydrophobic core that presumably targets proteins into the secretory pathway (Graham and Sticklen, 1994). The cloned interior spruce chitinases of class I and II (PgeChia1-1, PgeChia1-2 and PgeChia2-1) and lodgepole pine chitinase class I (PcChia1-1) also possess a C-terminal extension that may target these chitinases to vacuoles (Graham and Sticklen, 1994). It was previously demonstrated that a short (6aa) C-terminal amino acid sequence is necessary and sufficient for targeting of tobacco class I chitinase to the vacuole (Neuhaus et al., 1991). Only the class I and IV chitinases cloned from interior spruce and lodgepole pine had the cysteine rich domain that has been shown to have chitin binding properties in angiosperm chitinases (Iseli et al., 1993). Class I chitinases generally have higher chitinolytic activity than class II chitinases (that lack chitin binding domain) as has been demonstrated for barley and tobacco chitinases, perhaps supporting the importance of the chitin binding domain for catalytic activity (Graham and Sticklen, 1994). However, another study involving a direct comparison of the tobacco class I chitinases activity, with and without a chitin binding domain, demonstrated that this domain may not be necessary for catalytic activity (Iseli et al., 1993). Functional characterization revealed chitinolytic activity in class I interior spruce (PgeChia1-1, PgeChia1-2) and lodgepole pine (PcChia1-1) chitinases (Figure 4.3), consistent with previous observation that class I chitinases contribute much of the chitinolytic activity in plant tissues where different chitinases are present (Graham and Sticklen, 1994). The absence of catalytic activity in interior spruce class II chitinase PgeChia2-1 that lacks the chitin binding domain but share 93% sequence identity in the catalytic domain with class I chitinase PgeChia1-1 supports the relevance of the chitin 161 binding domain for chitinolytic activity of interior spruce chitinases. All of the cloned interior spruce and lodgepole pine class I chitinases had an optimal temperature of 50oC, consistent with previous reports of angiosperm chitinases (Li et al., 2003; Kirubakaran and Sakthivel, 2007). QRT-PCR gene specific expression analysis revealed strong induction of interior spruce chitinases PgeChia1-1 and PgeChia4-1 by blue-stain fungus Leptographium abietinum inoculation and by white pine weevil (Pissodes strobi) feeding (Figure 4.4, 4.5). Three other tested chitinases were also induced by both treatments in interior spruce. Similarly, lodgepole pine chitinases PcChia1-1 and PcChia4-1 were strongly induced by blue-stain fungus Grosmannia clavigera inoculation in lodgepole pine. These results are in agreement with the microarray data that showed hight induction of chitinase transcripts in interior spruce and lodgepole pine inoculated with corresponding blue-stain fungus (Chapter 2) and in interior spruce exposed to weevil feeding (Chapter 3). Similar induction levels of chitinases by both weevil and fungal treatments in interior spruce indicate a generic role for these chitinases in spruce defense. Induction of chitinases with antifungal properties and/or antiinsect properties by both fungal and insect treatment may be advantageous to conifers that are often exposed to insect and fungal attack simultaneously (Paine et al., 1997; Huber et al., 2004). The presence of chitinolytic activity and the high induction of class I chitinases by blue-stain fungal inoculation in interior spruce and lodgepole pine suggests a role for these chitinases in antifungal defense. The lack of the antifungal activity of the three interior spruce and lodgepole pine class I chitinases in spore germination tests could be attributed to the complexity of the fungal cell wall (Bowman and Free, 2006), the successful digestion of which may require additional enzymes such as β-1,3-glucanase (Mauch et al., 1988). Highly induced chitinases PgeChia4-1 and PcChia4-1 may have a role in antifungal defense as class IV representatives are considered to be extracellularly localized and possibly involved in releasing fungal elicitors for additional activation of plant defense (Collinge et al., 1993; Graham and Sticklen, 1994). Rapid accumulation of class IV chitinase was associated with resistance of Norway spruce to pathogenic fungus Heterobasidion annosum (Hietala et al., 2004). The antifungal activity of plant chitinases makes them attractive candidates for enhancing plant disease resistance. However, they present a challenge due to the presence of antifungal activity in only selected chitinases, which, in turn, are effective against selected and not all fungi (Graham and Sticklen, 1994). Conifer defense mechanisms include the interaction of chemical defenses such as terpenoids and phenolics with multiple pathogenesis related proteins such as chitinases, thaumatins 162 and others (Piggott et al., 2004; Franceschi et al., 2005). This interaction of different defense mechanism may contribute to effective antifungal and antiinsect conifer defense. Further testing of antifungal and antiinsect activity of the chitinases and association of the expression of the chitinases with conifer disease resistance is necessary to determine the role of these chitinases in the complex mechanism of conifer defense. Preliminary annotation of the chitinases available from full length Sitka spruce cDNA collection (Ralph et al., 2008) revealed at least 22 different chitinase transcripts, indicating the large size of conifer chitinase family. The large chitinase family in conifers is consistent with rapid evolution of plant chitinases in connection to evolving pathogen threats (Bishop et al, 2000) and is another indication of the importance of this family in conifer defense. In conclusion, our study resulted in the cloning of six interior spruce and four lodgepole pine chitinases and the establishment of chitinolytic activity of all three studied interior spruce and lodgepole pine class I chitinases. The establishment of chitinolytic activity in the conifer chitinases and induction of these chitinases in interior spruce and lodgepole pine by fungal treatment and in interior spruce by weevil feeding indicates a possibility for antifungal and/or antinsect biological function of the chitinases and supports further investigation of the role of chitinase family in conifer defense.

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4.5 REFERENCES

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Kasprzewska A (2003) Plant chitinases - regulation and function. Cellular & Molecular Biology Letters 8: 809-824 Keeling CI, Bohlmann J (2006a) Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defence of conifers against insects and pathogens. New Phytologist 170: 657-675 Keeling CI, Bohlmann J (2006b) Diterpene resin acids in conifers. Phytochemistry 67: 2415-2423 Kirubakaran SI, Sakthivel N (2007) Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli. Protein Expression and Purification 52: 159- 166 Kolosova N, Miller B, Ralph S, Ellis BE, Douglas C, Ritland K, Bohlmann J (2004) Isolation of high-quality RNA from gymnosperm and angiosperm trees. Biotechniques 36: 821-824 Kozlowski G, Metraux JP (1998) Infection of Norway spruce (Picea abies (L) Karst) seedlings with Pythium irregulare Buism and Pythium ultimum Trow: histological and biochemical responses. European Journal of Plant Pathology 104: 225-234 Kragh KM, Hendriks T, deJong AJ, LoSchiavo F, Bucherna N, Hojrup P, Mikkelsen JD, deVries SC (1996) Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant ts11. Plant Molecular Biology 31: 631-645 Kramer KJ, Muthukrishnan S (1997) Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochemistry and Molecular Biology 27: 887-900 Lawrence SD, Novak NG (2006) Expression of poplar chitinase in tomato leads to inhibition of development in colorado potato beetle. Biotechnology Letters 28: 593-599 Li YC, Chang CT, Hsiao ESL, Hsu JSF, Huang JW, Tzen JTC (2003) Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes. Plant and Cell Physiology 44: 1162-1167 Liu RJ, Ekramoddoullah AKM, Zamani A (2005) A class IV chitinase is up-regulated by fungal infection and abiotic stresses and associated with slow-canker-growth resistance to Cronartium ribicola in Western white pine (Pinus monticola). Phytopathology 95: 284-291 Mauch F, Mauchmani B, Boller T (1988) Antifungal Hydrolases in Pea Tissue .2. Inhibition of Fungal Growth by Combinations of Chitinase and Beta-1,3- Glucanase. Plant Physiology 88: 936-942 Meins F, Fritig B, Linthorst H, Mikkelsen J, Neuhaus J, Ryals J (1994) Plant chitinase genes Plant Molecular Biology Reporter 12: S22-S28 Nagy NE, Fossdal CG, Dalen LS, Lonneborg A, Heldal I, Johnsen O (2004) Effects of Rhizoctonia infection and drought on peroxidase and chitinase activity in Norway spruce (Picea abies). Physiologia Plantarum 120: 465-473 Neuhaus JM (1999) Plant chitinases (PR-3, PR-4, PR-8, PR-11). In SK Datta, S Muthukrishnan, eds, Pathogenesis-related Proteins in Plants. CRC Press, Boca Ranton, pp 77-105 Neuhaus JM, Sticher L, Meins F, Boller T (1991) A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proceedings of the National Academy of Sciences of the United States of America 88: 10362-10366 Paine TD, Raffa KF, Harrington TC (1997) Interactions among scolytid bark beetles, their associated fungi, and live host conifers. Annual Review of Entomology 42: 179-206 PerezMartin J, Cases I, deLorenzo V (1997) Design of a solubilization pathway for recombinant polypeptides in vivo through processing of a bi-protein with a viral protease. Protein Engineering 10: 725-730 165

Piggott N, Ekramoddoullah AKM, Liu JJ, Yu XS (2004) Gene cloning of a thaumatin- like (PR-5) protein of western white pine (Pinus monticola D. Don) and expression studies of members of the PR-5 group. Physiological and Molecular Plant Pathology 64: 1-8 Ralph SG, Chun HJE, Kolosova N, Cooper D, Oddy C, Ritland CE, Kirkpatrick R, Moore R, Barber S, Holt RA, Jones SJM, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2008) A conifer genomics resource of 200,000 spruce (Picea spp.) ESTs and 6,464 high-quality, sequence-finished full-length cDNAs for Sitka spruce (Picea sitchensis). Bmc Genomics 9: 484 Ralph SG, Jancsik S, Bohlmann J (2007a) Dirigent proteins in conifer defense II: Extended gene discovery, phylogeny, and constitutive and stress-induced gene expression in spruce (Picea spp.). Phytochemistry 68: 1975-1991 Ralph SG, Yueh H, Friedmann M, Aeschliman D, Zeznik JA, Nelson CC, Butterfield YSN, Kirkpatrick R, Liu J, Jones SJM, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2006) Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome. Plant Cell and Environment 29: 1545-1570 Ramirez MG, Avelizapa LIR, Avelizapa NGR, Camarillo RC (2004) Colloidal chitin stained with Remazol Brilliant Blue R-(R), a useful substrate to select chitinolytic microorganisms and to evaluate chitinases. Journal of Microbiological Methods 56: 213-219 Schlumbaum A, Mauch F, Vogeli U, Boller T (1986) Plant Chitinases Are Potent Inhibitors of Fungal Growth. Nature 324: 365-367 Schmidt A, Zeneli G, Hietala AM, Fossdal CG, Krokene P, Christiansen E, Gershenzon J (2005) Induced chemical defences in conifers: Biochemical and molecular approaches to studying their function. In JT Romeo, ed, Chemical Ecology and Phytochemistry in Forest Ecosystems. Elsevier, Amsterdam, pp 1- 28 Singh A, Kirubakaran SI, Sakthivel N (2007) Heterologous expression of new antifungal chitinase from wheat. Protein Expression and Purification 56: 100-109 Vellicce GR, Ricci JCD, Hernandez L, Castagnaro AP (2006) Enhanced resistance to Botrytis cinerea mediated by the transgenic expression of the chitinase gene ch5B in strawberry. Transgenic Research 15: 57-68 Verburg JG, Huynh QK (1991) Purification and Characterization of an Antifungal Chitinase from Arabidopsis-Thaliana. Plant Physiology 95: 450-455 Wiweger M, Farbos I, Ingouff M, Lagercrantz U, von Arnold S (2003) Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases. Journal of Experimental Botany 54: 2691- 2699 Wu HG, Echt CS, Popp MP, Davis JM (1997) Molecular cloning, structure and expression of an elicitor-inducible chitinase gene from pine trees. Plant Molecular Biology 33: 979-987 Xiao YH, Li XB, Yang XY, Luo M, Hou L, Guo SH, Luo XY, Pei Y (2007) Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants. Bioscience Biotechnology and Biochemistry 71: 1211-1219 Ye XY, Ng TB (2005) A chitinase with antifungal activity from the mung bean. Protein Expression and Purification 40: 230-236

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5 Concluding discussion

5.1 OVERVIEW OF THE THESIS WORK

Conifer trees have a variety of defense mechanisms which allow them to survive and thrive during their long generation time throughout which they are subjected to a variety of abiotic and biotic stresses (Alfaro et al., 2002; Franceschi et al., 2005; Raffa et al., 2005; Bohlmann, 2008). Development of successful forest management, including breeding of resistant trees and prevention of epidemic outbreaks requires a thorough understanding of tree defense mechanisms. The recent development of conifer genomics resources has allowed profiling of conifer defense responses using EST and microarray and proteomics resources (Ralph et al., 2006b; Adomas et al., 2007; Lippert et al., 2007; Ralph et al., 2008). In this thesis work, I performed two comparative transcriptome profiling studies of conifer defense. In the first study, I evaluated the transcriptome response of interior spruce (Picea glauca x engelmannii) inoculated with spruce beetle (Dendroctonus rufipennis)-associated blue-stain fungus Leptographium abietinum and lodgepole pine (Pinus contorta) inoculated with mountain pine beetle (Dendroctonus ponderosae)-associated blue-stain fungus Grosmannia clavigera (Chapter 2). In the second study, I performed a comparative transcriptome analysis of the interior spruce response to white pine weevil (Pissodes strobi) feeding with the response to blue-stain fungus Leptographium abietinum inoculation (Chapter 3). Building on the previous research in our group (Ralph et al., 2006b; 2008; Holliday et al., 2008), I used the newly developed 21.8K spruce microarray for transcriptome profiling of conifer defense responses. Testing of the spruce microarray application with other conifer species revealed the effective performance of this microarray platform with other species of the Pinaceae family (Appendix 2) including lodgepole pine, which was used for the experiments described in Chapter 2. In addition, the development of a reliable method for RNA isolation from gymnosperms (Appendix 3; Kolosova et al., 2004) contributed to the quality of the microarray analyses. The comparative study of spruce and pine responses to bark beetle pathogenic fungal associates and of the spruce response to weevil herbivory on the transcriptome level revealed substantial changes in the transcriptomes of conifer hosts in response to fungal inoculation or insect feeding. The fungus-induced and weevil-induced transcriptomes of hybrid spruce and the fungus-induced transcriptome of lodgepole pine shared a large number of similarly responding transcripts with some unique dynamics of the response in each species and treatment. Among the strongest up-regulated 167 transcripts in all interactions were phenylpropanoid pathway genes, dirigents, laccases, chitinases and transcripts of the terpenoid pathway. Gene specific expression analysis confirmed the strong induction of selected chitinases in interior spruce and lodgepole pine inoculated with fungal pathogens and in spruce exposed to weevil feeding. It also showed differential expression of DXS genes of the terpenoid pathway and dirigent protein genes in spruce response to fungal inoculation as compared to weevil feeding. Following the results of the microarray profiling, which indicated a substantial involvement of chitinases in conifer defense, I performed functional characterization of six interior spruce and four lodgepole pine chitinases belonging to four different classes of Chia chitinase family. This functional analysis revealed chitinolytic activity in all three chitinases belonging to class I and tested in this study (Chapter 4). Chitinolytic activity has not previously been shown for any conifer chitinase. The presence of chitinolytic activity, in addition to strong induction of these chitinases by fungal treatment in interior spruce and lodgepole pine and by weevil treatment in interior spruce (Chapter 2 and 3), strongly supports a role for these chitinases in conifer defense.

5.2 TRANSCRIPTOME PROFILING OF CONIFER DEFENSE AGAINST FUNGAL PATHOGENS AND INSECTS

The recent development of conifer genomics resources provided an opportunity to evaluate conifer defense responses at the transcriptome, proteome and biochemical levels (Ralph et al., 2006b; Adomas et al., 2007; Lippert et al., 2007, Zulak et al., 2009). Several different microarrays were applied to conifer defense studies: 2.1K pine microarray, which was applied in transcriptome profiling of Scots pine inoculated with Heterobasidion annosum (Adomas et al., 2007), the 9.7K spruce microarray, which was applied in transcriptome profiling of Sitka spruce induced by mechanical wounding, spruce budworm and white pine weevil herbivory (Ralph et al., 2006b), and the 21.8K spruce microarray, which was applied in the comparative transcriptome analysis of interior spruce and lodgepole pine inoculated with blue-stain fungi Leptographium abietinum and Grosmannia clavigera respectively (Chapter 2). In addition, the effect of Leptographium abietinum on the interior spruce transcriptome was compared with the effect of weevil feeding (Chapter 3). Evaluation of the interspecies application of the 16.7K spruce microarray revealed a highly reliable (based on comparative analysis with spruce) and efficient (close to 80% based on the microarray transcripts with significant signal) performance of the microarray with other species of the Pinaceae family, including lodgepole pine, Douglas-fir and Noble fir (Appendix 2). This study, in addition 168 to previous smaller studies of pine microarray application with different pine and spruce species (van Zyl et al., 2002), supports the application of the spruce microarrays with other species of the Pinaceae family, allowing genomics research on species for which development of genomics resources is not yet established.

5.2.1 Response of the conifer transcriptome to fungal pathogens The conifer defense response to fungal inoculations has been primarily studied on the anatomical and chemical levels, with targeted gene expression characterization available only for a small number of genes (Franceschi et al., 2005; Schmidt et al., 2005; Bonello et al., 2006). The first large scale microarray study of conifer fungal interaction investigated a transcriptome response of the roots of Scots pine to infection with Heterobasidion annosum and used a 2.1K-pine microarray platform (Adomas et al., 2007). The comparative microarray analysis of interior spruce inoculated with the spruce beetle fungal associate Leptographium abietinum and lodgepole pine inoculated with the mountain pine beetle fungal associate Grosmannia clavigera using the spruce 21.8K microarray substantially expanded the transcriptome analyses of conifer defense against fungal pathogens (Chapter 2). Compared to the 2.1K pine microarray, which was constructed primarily from xylem with some representation from shoot tissue, the 21.8K spruce microarray was constructed using a wide variety of spruce tissues, including tissues from wounded and methyl jasmonate treated trees, which makes it particularly suitable for studies of the conifer defense response. A direct comparison of the transcriptome response of spruce and pine to corresponding fungal inoculations provided a broader context for the evaluation of conifer defense mechanisms, which was strengthened by the parallel transcriptome analyses, allowed the identification of common and species specific trends. A substantial transcriptome change was detected following blue-stain fungal inoculation in both spruce and pine. This corresponded to the differential expression of genes represented with almost 10% of the 21.8K EST array platform during the two weeks time course of the study and correlated well with the scale of expression changes of the Scots pine transcriptome following inoculation with Heterobasidion annosum (Adomas et al., 2007) and of the Sitka spruce transcriptome following wounding and herbivory (Ralph et al., 2006b). The transcriptome analysis revealed that conifers can detect and respond to wounding and fungal inoculation with substantial transcriptome expression changes within just six hours after treatment, with a stronger response to fungal treatment compared to wounding. Such rapid defense related change in the expression of conifer 169 transcriptome was previously demonstrated only for Sitka spruce induced by budworm herbivory (Ralph et al., 2006b). Rapid induction of the high number of spruce transcripts in response to fungal inoculation, as compared to the lower level of the lodgepole pine response, including the absence of a fungal-specific lodgepole pine response in the first 6 hours after treatment, may suggest that interior spruce can induce defense responses faster than lodgepole pine, and is consistent with the rapid traumatic duct formation in interior spruce as compared to lodgepole pine (Chapter 2) and previously hypothesized differences in the spruce and pine defense response strategies (Christiansen et al., 1987). My microarray study (described in Chapter 2) showed an overlap of about 50% of interior spruce and lodgepole pine transcriptome responses to fungal inoculations, indicating large common transcriptome component in defense of the two species. Upregulation of most of the defense related transcripts by fungal treatment in both species and lack of noticeable downregulation is consistent with the previously observed similar pattern in plant response to necrotrophic pathogens as contrasted with downregulation of defense responses in case of biotrophic pathogens (Adomas et al., 2007; Mendgen and Hahn, 2002; Voegele and Mendgen, 2003; Miranda et al., 2007). Among the transcripts involved in primary metabolism, expression of which was considerably affected by fungal treatment in interior spruce and pine, the transcripts involved in photosynthesis were downregulated. Common occurrence of this phenomenon in fungus-treated conifers and angiosperms may indicate the importance of these rearrangements in defense strategy (Adomas et al., 2007; Ralph et al., 2006a, 2006b; Miranda et al., 2007; Bolton, 2009). Induction of transcripts involved in methyl jasmonate and ethylene signalling by fungal treatment in interior spruce and lodgepole pine provided additional support for the importance of these signalling mechanisms in conifer defense against fungi which were previously suggested based on elicitation of defense by external application of these compounds (Liu et al., 2003; Hudgins and Franceschi, 2004; Schmidt et al., 2005). Microarray analysis of the interior spruce and lodgepole pine response to wounding and fungal inoculation highlighted the involvement of the phenylpropanoid pathway in the defenses response of both species (Chapter 2). Substantial induction of phenylpropanoid pathway by wounding, fungal and insect treatment suggests common role of this pathway in conifer defense (Adomas et al., 2007; Ralph et al., 2006b) Many genes potentially involved in lignans and lignin biosynthesis (such as laccases and dirigent proteins), the flavonoid pathway and early steps of phenylpropanoid biosynthesis were highly induced in response to fungal inoculation and 170 wounding in interior spruce and lodgepole pine (Chapter 2). A similar induction of laccases and of flavonoid biosynthesis was observed in Scots pine inoculated with Heterobasidion annosum (Adomas et al., 2007). A gene specific expression study of dirigent proteins, previously characterized in spruce (Ralph et al., 2006c; 2007), confirmed the high induction of several DIR-a protein subfamily members by wounding and fungal treatment in interior spruce (Chapter 2). Induction of these pathways is consistent with the previously demonstrated involvement of lignin and flavonoid accumulation in conifer defense against fungal pathogens (Brignolas et al., 1998; Bonello and Blodgett, 2003; Wallis et al., 2008). In addition, to pissible lignin biosynthesis and production of lignans, which may have antifungal properties (Vargas- Arispuro et al., 2005; Cho et al., 2007), laccases are also known to facilitate wound sealing and possible iron uptake that may be indirectly important for lignin biosynthesis (McCaig et al., 2005). The microarray analysis also demonstrated strong induction of the terpenoid pathway in both interior spruce and lodgepole pine, including transcripts in the MEP and MEV pathways, prenyltransferases, and terpene synthases (Chapter 2). The gene specific analysis of the DXS family of interior spruce confirmed the strong induction of one of the type II DXS transcripts in response to wounding and fungal treatment, consistent with the previously reported induction of an orthologous transcript by wounding and the blue-stain fungus in Norway spruce (Phillips et al., 2007). These results are also consistent with the previously demonstrated induced resin accumulation in lodgepole pine (Chapter 2; Miller et al., 1986; Croteau et al., 1987) and Norway spruce (Viiri et al., 2001; Baier et al., 2002). The involvement of the terpenoid biosynthesis pathway in the interior spruce and lodgepole pine defense response was also supported by the formation of traumatic resin ducts in the xylem of both species following inoculation with fungi (Chapter 2). The induction of terpenoid transcripts and the development of resin ducts occurred more rapidly in interior spruce as compared to lodgepole pine. In addition, the transcriptome response of the terpenoid pathway to wounding and fungal treatment was similar in spruce whereas in lodgepole pine, the terpenoid pathway was predominantly induced by fungal treatment, again possibly indicating the involvement of unique defense strategies for interior spruce and lodgepole pine. The transcriptome analysis also demonstrated a strong induction of antimicrobial proteins such as chitinases, thaumatins and osmotins in interior spruce and lodgepole pine in response to fungal treatment (Chapter 2), consistent with the previously reported induction of antimicrobial proteins in slash pine and Scots pine following fungal 171 treatment (Morse et al., 2004; Adomas et al., 2007). The microarray study highlighted the involvement of chitinases in the defense responses of spruce and pine, with many of these transcripts being the most strongly induced transcripts following inoculation with fungus. The induction of chitinases by wounding and the much stronger induction of chitinases by fungal treatment suggest a common defense function of chitinases in conifer defenses with additional activation of the chitinases by fungal threat as compared to wounding. Gene specific analysis of chitinase transcripts confirmed the strong induction of chitinase transcripts in spruce and pine by wounding and fungal treatment (Chapter 4), and the functional characterization of a subset of these genes confirmed their chitinolytic function (Chapter 4). This research supports the involvement of chitinases in conifer antifungal defense and corresponds with previous studies that showed the induction of chitinases in fungus- or fungal-elicitor treated conifer species (Wu et al., 1997; Davis et al., 2002), and the association of chitinase expression with spruce resistance to fungal pathogen (Fossdal et al., 2006).

5.2.2 A direct comparison of spruce response to insect feeding and fungal pathogen The first direct comparison of a conifer defense response to insect attack and fungal inoculation [i.e. interior spruce inoculated with blue-stain fungus (Chapter 2) or exposed to white pine weevil (Chapter 3)] provided an opportunity to evaluate common and treatment specific conifer defense responses (Chapter 3). Weevil feeding induced a larger transcriptome change compared to fungal treatment, and included about 20% of the studied transcriptome with more transcripts induced than downregulated. Substantial rearrangement of the spruce transcriptome was consistent with previous studies that revealed similar effects of insect feeding on spruce and poplar (Ralph et al., 2006b; Miranda et al., 2007) Comparison of weevil and fungus effect on spruce transcriptome revealed a large overlap of induced and downregulated transcripts by two treatments. This overlap included the majority of fungus-affected transcriptome and is consistent with previous microarray studies of insect effect on spruce transcriptome (Ralph et al., 2006b) and fungal effect on pine transcriptome, which showed similar trends in conifer response to insects and fungus on a smaller transcriptome scale (Adomas et al., 2007). In addition, similar anatomical and chemical responses were observed in conifers exposed to insects or fungal pathogens (Chapter 2; Franceschi et al., 2000; Viiri et al., 2001; Miller et al., 2005).

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The activation of the jasmonate and ethylene signalling pathways in interior spruce exposed to weevil herbivory and necrotrophic fungal inoculation (Chapter 3) was consistent with previous studies in angiosperms that showed the involvement of these signalling mechanisms in insect and necrotrophic fungi induced plants (McDowell and Dangl, 2000; Walling, 2000). A detailed analysis of the spruce transcriptome response to weevil feeding and fungal inoculation revealed predominantly similar defense responses with some treatment specific trends. Phenylpropanoid pathway transcripts were among the most abundant (30% of the transcripts represented on the microarray) and were also among the transcripts most highly induced by weevil treatment. In the case of the fungal treatment, antimicrobial proteins, specifically chitinases, were among the most induced transcripts (Chapter 3), with similar high number phenylpropanoid transcripts induced as well. Similar to the response of interior spruce to fungal inoculation, the most induced phenylpropanoid transcripts by weevil were laccases and dirigent proteins. Weevil feeding induced more laccase transcripts and many of them to a higher level than did fungal treatment. Gene specific analysis of the expression of dirigent protein transcripts in interior spruce revealed similar expression patterns of DIR-a and DIR-b subfamilies, with DIR-a subfamily members strongly induced by both treatments, whereas selected members of the DIR-f subfamily were induced by weevil feeding in interior spruce and were either unaffected or downregulated by fungal treatment. DIR-a subfamily members were also strongly induced in Sitka spruce exposed to weevil feeding (Ralph et al., 2007a). The potential involvement of laccases and dirigent proteins in lignin biosynthesis suggests that cell wall strengthening is one of the key defense mechanisms in spruce against weevil, which is also important in spruce protection against fungi (Chapter 2). This is consistent with reports which positively correlate lignin content in bark (mostly concentrated in stone cells) with spruce resistance to weevil, bark beetle and beetle- associated fungus (Wainhouse et al., 1990; Wainhouse et al., 1998; King and Alfaro, 2009). Genes of the terpenoid pathway were strongly induced in interior spruce by weevil feeding, fungal inoculation and wounding treatment, which indicated a common role of terpenoid defenses conifers. Gene specific analysis of DXS transcripts revealed the increased expression of DXS type II transcripts by weevil (Chapter 3) and fungal (Chapter 2) treatments, consistent with the homologous DXS transcript being induced in Norway spruce by wounding and fungal treatments (Phillips et al., 2007). In addition, DXS type I was induced in weevil treated spruce but not in fungus treated spruce (Chapter 2, 3). DXS type I is considered to be involved predominantly in primary 173 metabolism (Cordoba et al., 2009) and differential involvement of this DXS in the spruce response to fungus and weevil suggests specific regulation of terpenoid defense in response to different threats. Among the antimicrobial protein transcripts, chitinases were highly induced by weevil feeding, fungal inoculation and wounding in interior spruce as was demonstrated by the microarray and particularly by gene specific analysis of the expression of selected chitinases (Chapter 2, 3, 4). The high level of chitinase induction in response to different treatments indicates the common involvement of these chitinases in the spruce defense response, consistent with the ability of chitinases to damage insect peritrophic matrix (Kramer and Muthukrishnan, 1997; Lawrence and Novak, 2006), slow fungal growth and convey increased antiinsect and antifungal resistance when introduced as transgenes (Graham and Sticklen, 1994; Kasprzewska, 2003). The transcriptome analysis in interior spruce also suggested the downregulation of selected chitinases by weevil treatment (Chapter 3), consistent with the previously observed downregulation of a chitinase protein in Sitka spruce exposed to weevil feeding (Lippert et al., 2007) and may be an example of weevil-specific downregulation of transcripts in spruce response to weevil. The microarray analysis suggested new candidate genes with a possible role in spruce defense against weevil including protease inhibitors, transcripts annotated to (R)- mandelonitrile lyase and carbonic anhydrase transcripts all of these candidates were previously implicated as potentially involved in plant defense (Hickel et al., 1996; Dunaevsky et al., 2005; Restrepo et al., 2005; Ralph et al., 2006b). Overall this comparative microarray analysis of spruce defense response to weevil and fungus is currently the largest study of the transcriptome of conifer defense. The comparative evaluation of the defense response of two different conifer species lodgepole pine and interior spruce and the direct comparison of insect and fungal treatment provided an additional level of complexity to our study. The spruce microarray proved to be a reliable tool for conifer transcriptome analysis. The strengths of this genomics research included the comparative analysis of the conifer transcriptome response at three time points using either three treatments (control, wounding and fungal treatment) or two treatments (control and weevil herbivory) with both experiments having a similar statistical power. The large number of treatments and samples allowed for a good statistical representation of the microarray data. In addition, the inclusion of multiple time points post-treatment allowed for a more thorough analysis of the conifer defense transcriptome response due to the coverage of early, medium and late induced responses. The microarray analysis and complementary gene specific expression study provided a variety of candidate genes and pathways for further study into their role in 174 conifer defense. In addition, the transcriptome analyses can be further mined as additional annotation of conifer genes becomes available.

5.2.3 Biological relevance of the research methodology used to study conifer defense responses

Treatments used in the conifer defense studies In order to study defense mechanisms that are used by conifer trees against herbivory and pathogens in natural conditions a variety of treatment techniques have been developed that can be used for controlled experimental conditions. In the process of the attack by bark beetles, conifer trees are wounded and are also inoculated with a variety of pathogenic blue stain fungi (Paine et al., 1997). To simulate this type of attack different wounding treatments and fungal inoculation procedures have been used. These treatments include application of a variety wounding and fungal inoculation techniques, such as wounding with razor blade (Ralph et al., 2006b; Phillips et al., 2007), needle (Chapter 2; Croteau et al., 1987) or cork borer (Christiansen et al., 1999) and fungus is introduced by spraying spores on the wounded trees (Phillips et al., 2007) or inoculation into the wounds (Chapter 2; Croteau et al., 1987; Christiansen et al., 1999). Application of these treatments resulted in the activation of conifer defense responses, which were investigated on anatomical and molecular levels. However, certain limitations are associated with the simulation techniques. Damage by bark beetles or other insects occurs over time whereas, in experimental conditions, wounding is applied at the time zero, which limits the comparison of the conifer trees response to wounding and insect treatment. In addition, bark beetles transfer a variety of microorganisms (Paine et al., 1997; Cardoza et al., 2006; Alamouti et al., 2007), which may affect conifers, and inoculation with just one strain of blue stain fungus may not entirely reflect the effects of conifer tree exposure to the combined presence of all bark beetle associated microorganisms. Elicitors of conifer defense such as MeJa and ethylene are also used to study conifer defense responses (Martin et al., 2002; Hudgins et al., 2003; Hudgins and Franceschi, 2004; Miller et al., 2005; Zulak et al., 2009). In particular, MeJa is easy to apply, does not require wounding, and it produces remarkably similar defense responses in spruce compared to weevil feeding and fungal inoculations (Franceschi et al., 2000; Franceschi et al., 2002; Huber et al., 2004; Miller et al., 2005). MeJa application can help to standardise defense response studies between different species, which may be complicated in case of using herbivory or fungal inoculations with different 175 pathogens due to additional variability in biological interaction of the insects or fungi with different hosts (Chapter 2). However, the application of methyl jasmonate may reflect only part of the conifer defense response to herbivory or fungal inoculation and may not precisely reflect the intensity of the responses. Performance of the experiments in green house conditions (e.g. Chapter 2, 3; Miller et al., 2005; Ralph et al., 2006; Phillips et al., 2007) allows to control the environmental conditions (temperature, humidity, soil quality), and ease of access to the tree material, including availability of clonal or previously characterised tree seedlings, as compared to the experiments done in natural forest conditions. However, experiments conducted in greenhouse impose restriction on the age of tree material and are most suitable for seedlings and very young trees. Young trees can be easily used to evaluate conifer defense responses to weevil feeding because weevils infest young saplings as well as older trees in natural conditions (Alfaro et al., 2002). Bark beetles attack older trees (at least approaching 10 cm in diameter and preferably larger) (Bjorklund and Lindgren, 2009) and the effect of bark beetle attack in greenhouse conditions can only be studied using simulation techniques such as separate evaluation of the effect of wounding and beetle transferred microorganisms on conifer trees. In general, similar anatomical and biochemical defense responses were observed in young saplings and older trees, such as formation of traumatic resins ducts and induction of terpenoid biosynthesis in spruce saplings and older spruce trees in response to methyl jasmonate treatment, weevil feeding and blue stain fungal inoculations (Chapter 2, 3; Alfaro et al., 1995; Franceschi et al., 2000, 2002; Viiri et al., 2001; Martin et al., 2002; Faldt et al., 2003; Miller et al., 2005, Erbilgin et al., 2006) induction of terpenoid biosynthesis in young (Chapter 2, Croteau et al., 1987) and mature lodgepole pines (Miller et al., 1986), or induction of antimicrobial proteins such as chitinases by pathogenic fungi in spruce saplings (Chapter 2) and older spruce trees (Fossdal et al., 2006). Experiment performed in the controlled greenhouse conditions provide initial level of evaluation of conifer defense responses and need to be followed up with further studies of the effects of herbivory and pathogens on conifer trees in natural forest environment. In particular, the microarray studies of the effect of fungal pathogens on spruce and pine (Chapter 2) and the effect of weevil on spruce (Chapter 3) performed with spruce and pine saplings provided initial assessment of the level of the defense responses and a number of candidate genes with potential importance in conifer defense. This kind of controlled evaluation of conifer defenses would be challenging in the forest environment due to high variability of environmental conditions and tree 176 material. However follow up on few selected candidate genes can be done in forest conditions using mass sampling (to allow for better statistical power) and such methods as QRT-PCR, proteomics or metabolite analysis for further characterisation of the role of these genes in conifer defense.

Biological relevance of the research approach used in the conifer defense studies The microarray studies of spruce and pine defense response to blue stain fugal pathogens and spruce response to weevil herbivory revealed rearrangement of the studied transcriptomes and highlighted biosynthetic pathways and gene families potentially important in conifer defense (Chapter 2,3). The effect of the induction of transcripts of genes on the protein levels and corresponding accumulation of metabolites (if applied) need to be further confirmed using proteomics (Lippert et al., 2007) and metabolite analysis. In this study, all transcriptome responses induced by wounding, herbivory or pathogens in conifers were discussed as “defense responses”. It is important to point out the limitation in this notion of the nature of the responses. If the induction of transcripts generally known to be associated with resistance, such as secondary metabolism and antimicrobial protein transcripts, is likely aimed at defense (Franceschi et al., 2005; Schmidt et al., 2005; Keeling and Bohlmann, 2006 a,b; Bohlmann, 2008), as well as some rearrangement of the primary metabolism, which may be necessary for effective mobilization of resources (Bolton, 2009), there is also a possibility of the presence of the transcriptome response component that is not aimed at defense but rather is a result of stress and sickness caused by destructive agent or treatment (Bonello, personal communication). In addition, the effectiveness of different defense responses against a variety of threats needs to be evaluated for their contribution to the resistance of conifers to the threat. This requires detailed understanding of gene function, expression and effect of the expression on the resistance of conifers and may be evaluated using comparative studies of defense responses in resistant and susceptible trees or using transgenic gene silencing approach.

5.3 FUNCTIONAL CHARACTERIZATION OF CONIFER DEFENSE RELATED GENES

Transcriptome analyses of the conifer defense responses (Chapter 2, 3; Ralph et al., 2006b; Adomas et al., 2007) identified a large number of genes, with putative roles in conifer defense. A number of previous studies resulted in the cloning and gene 177 specific expression analysis of a variety of conifer defense related genes involved in terpenoid (Keeling and Bohlmann, 2006a, 2006b, Phillips et al., 2007; Zulak et al., 2009) and phenylpropanoid pathways (Chiron et al., 2000; Richard et al., 2000; Nagy et al., 2004; Ralph et al., 2006c; Ralph et al., 2007a; Gronberg et al., 2009), ethylene signaling (Hudgins et al., 2006; Ralph et al., 2007b), and antimicrobial proteins (AMP) such as chitinases (Wu et al., 1997; Davis et al., 2002; Wiweger et al., 2003; Hietala et al., 2004; Liu et al., 2005; Fossdal et al., 2006), thaumatin-like protein (Piggott et al., 2004), defensins (Sharma and Lonneborg, 1996; Kovalyova and Gout, 2008), other AMPs (Asiegbu et al., 2003) and additional pathogenesis related proteins such as peroxidase (Fossdal et al., 2001) and PR-10 protein (Ekramoddoullah et al., 2000; Liu et al., 2003; Mattheus et al., 2003). Functional characterization is mostly available for the genes involved in terpenoid biosynthesis, such as prenyltransferases (Keeling and Bohlmann 2006a, Schmidt and Gershenzon, 2007, 2008; Schmidt et al., 2009), terpene synthases (Keeling and Bohlmann, 2006a, 2006b) and cytochrome P450 enzymes (Keeling and Bohlmann, 2006a, 2006b; Hamberger and Bohlmann, 2006). Several genes involved in phenylpropanoid pathway in conifers have also been functionally characterized, including PAL (Whetten and Sederoff, 1992), CAD (O’Malley et al., 1992), chalcone synthase and stilbene synthase (Fliegmann et al., 1992), laccases (Sato and Whetten, 2006) and dirigent protein (Kim et al., 2002). Despite strong evidence of the involvement of antimicrobial proteins in conifer defense, biological function has only been demonstrated for selected defensins (Elfstrand et al., 2001; Pervieux et al., 2004). The involvement of chitinases in the conifer defense response has been demonstrated based on strong induction in response to insect herbivory, fungal inoculation, wounding and defense elicitor treatments (Kozlowski and Metraux, 1998; Davis et al., 2002; Hietala et al., 2004; Nagy et al., 2004; Liu et al., 2005; Ralph et al., 2006b). The functional characterization of chitinases in this study is the first report of chitinolytic function of any conifer class I chitinases (Chapter 4). This study resulted in the cloning of six interior spruce and four lodgepole pine chitinases representing chitinase class I, II, IV and VII. Functional characterization established chitinolytic activity of two interior spruce and one lodgepole pine classes I chitinases. The ability of these chitinases to digest chitin (Chapter 4), an important constituent of the fungal cell wall and insect peritrophic matrix, and the high induction of these chitinases in interior spruce and lodgepole pine by fungal inoculation, weevil herbivory and wounding treatment suggests the importance of these chitinases in the conifer defense response.

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5.4 FUTURE RESEARCH

5.4.1 Suggested research directions based on the thesis work The research of this thesis contributed to our knowledge of conifer defense genomics and conifer functional gene identification and characterization and has general implications in the study of conifer defense against fungal pathogens and insects. As such this thesis work will serve as a foundation for new research projects in the field of conifer defense.

1. The availability of the 21.8K spruce microarray and the evaluation of spruce microarray performance (Appendix 2), which supports the application of this array with other species of family Pinaceae, provide an opportunity to apply this microarray in transcriptome studies of conifer defense in other species of the Pinaceae family, including lodgepole pine (Chapter 2), Douglas-fir and Noble fir. 2. Analysis of the defense response of spruce and lodgepole pine against the highly pathogenic blue-stain fungi revealed a large transcriptome rearrangement and the strong activation of defense-related pathway transcripts in both species (Chapter 2). A comparative transcriptome study of the spruce and pine responses to highly pathogenic fungi (e.g. Grosmannia clavigera) with weakly pathogenic (e.g. Ophiostoma montium (Yamaoka et al., 1995)) or non pathogenic fungi will further clarify the unique defense strategies employed by conifers against different fungi (Adomas et al., 2008). 3. This study (Chapter 2) and previous studies of the transcriptome response in conifers used necrotrophic fungal pathogens (Morse et al., 2004; Adomas et al., 2008). A comparative study of the conifer response to necrotrophic and biotrophic pathogens will reveal specificity of conifer defense response against different pathogens, including possible different signaling mechanisms and possible inhibition of conifer defenses by biotrophic pathogens as has been demonstrated in angiosperms (McDowell and Dangl, 2000; Miranda et al., 2007). 4. This study identified a variety of genes in spruce and pine that are involved in defense response against fungi (Chapter 2) and weevil (Chapter 3). They include phenylpropanoid and terpenoid biosynthetic pathway genes and transcripts representing antimicrobial proteins. The involvement in conifer defense of dirigent proteins, laccases, DXS transcripts and antimicrobial proteins (Chapter 2, 3) needs to be further elucidated by cloning, functional characterization and

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gene specific transcript expression profiling of these genes in resistant and susceptible trees to uncover possible association with resistance. 5. Transcriptome analysis revealed the involvement of the chitinase family in the spruce and pine response to fungus and the spruce response (Chapter 2) to weevil feeding (Chapter 3). In addition, functional characterization confirmed chitinolytic activity of spruce and pine selected class I chitinases (Chapter 4). This research, in addition to previously shown antifungal and antiinsect activity of chitinases, suggests the importance of chitinases in conifer defense against fungi and insects. Analysis of the Sitka spruce full length cDNA collection (Ralph et al., 2008) identified 22 unique chitinase transcripts in spruce. Characterization of the chitinase family in different conifer species including the cloning and biochemical analysis of the recombinant protein and verification of biological function in vivo (using transgenic approaches) and the analysis of the expression association of these chitinases with resistance has a high potential in discovering effective conifer defense mechanisms. 6. In addition to previously identified conifer defense related genes, this study suggests the involvement of a variety of genes which have not been previously studied in relation to conifer defense including genes annotated as protease inhibitors, carbonic anhydrase, (R)-mandelonitrile lyase which were highly induced by fungus in spruce and pine and by weevil in spruce (Chapter 2, 3). Further study of these genes will be aided by the development of improved conifer gene annotation and may result in the discovery of new conifer defense mechanisms.

5.4.2 General future directions Research into conifer defense revealed a number of defense mechanisms and suggests that the cooperative nature of these mechanisms allows conifer trees to survive for a long time in the environment in which they are exposed to a variety of potentially faster evolving insect pests and pathogens. Conifer defense studies need to be expanded on molecular, metabolic and structural levels and interactions of different defense mechanisms need to be evaluated. The development of conifer EST resources, full-length cDNA collections (Ralph et al., 2008) and conifer genome sequencing (e.g. Spruce Genome Project, Umea Plant Science Centre and new initiatives in the Treenomix project) will allow for further gene discovery, functional characterization and improvement in conifer gene annotation. The available transcriptome profiling of conifer defense can be further extended by including 180 new conifer/insect, conifer/pathogen systems, by comparing defense responses of resistant and susceptible trees and by evaluating systemic defense mechanisms. The systematic application of metabolic analysis, targeted proteomics and transcript analysis of target defense related genes, was recently used in the study of genes involved in conifer terpenoid biosynthesis (Zulak et al., 2009), and can now be applied in future work to studies of the involvement of phenylpropanoid pathway and pathogenesis related proteins in conifer defense. Further analysis of the localization of metabolites, proteins and transcripts in different cell types using advanced methods such as laser-microdissection (Li et al., 2007; Abbott et al., 2010) may clarify defense related roles of different pathways and genes, in particular, it might be important for genes with uncertain biochemical function, among which are different laccases and dirigent proteins. A large variety of candidate genes involved in the conifer defense response, which was suggested by this and other genomics studies (Ralph et al., 2006b; Adomas et al., 2007), requires functional characterization to clarify the role of these genes in conifer defense. Biological functions of these genes need to be assessed by correlating metabolite accumulation and gene expression with the resistance level of conifer trees, and by evaluation of the effect of these metabolites and proteins on pathogens and insects. Furthermore, biological function of conifer defense related genes can be evaluated in vivo in transgenic angiosperms or conifer systems, which have been successfully applied in conifer research (Davis et al., 2002; Chatthai et al., 2004; Wadenback et al., 2008; Bedon et al., 2009; Wagner et al., 2009). A better understanding of conifer defense mechanisms can be applied to the development of pretreatment strategies, which can be used to effectively increase conifer tree resistance (Franceschi et al., 2002; Zeneli et al., 2006; Krokene et al., 2008; Moreira et al., 2009). Further improvement of pretreatment strategies may include an optimization of the application of the elicitor methyl jasmonate for large scale applications or an identification of other potent elicitors suitable for the induction of acquired resistance in conifer trees. Finally, analysis of the integration of conifer defense mechanisms with the ecology and biology of complex interactions of conifer species, insect herbivores and fungal pathogens will allow a better understanding of forest epidemics and will contribute to the development of forest management strategies (Raffa et al., 2008).

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Appendix 1. Supplementary data

Chapter 2

Supplemental Table 2.1: Primer sequences used for real-time PCR (5' to 3' orientation) Gene ID Forward primer Reverse primer TIF5A GTGCCATCTTCACACAACTGC CAGATTCAGTCAGCAGGCTAAC DIR1/20 TTCGATCAAATGGCAGTGTACTC ACCATGACAAATATGCACAGAAG DIR2/32 AGTCTACTGATCTCTCCTGCTG ATTATTTCACTCTACGCTAGCTG DIR5/15 TGCATGAAGCTCCGAAATGCTG TATATTGGATATGGCCTTCCAAC DIR6 CTACTGATCTTTCATGCATGTTC TTCACTCTACCAATGCAGATTTG DIR12/21 TGCCGTTCCATCCATCTATCTC GGTCATGATTAGACTGCCCTTG DIR16 GCTCTCCATTTCTATGTTCATTG TTTGGGAGATTCATGCATATCTC DIR22 TTATTCAGTGCTCAGAGTATGAC CATATAATACTTGCCTGTCTGTG DIR23/24 ACAAAGCAGAGGGAGATATCAG TTTGCTCCCATTGACAACTGTG DIR25 TGGCTATGCGGTTGTTACTGTG ACAAGGTAACCTCTCCACAACG DIR26 CAAGAATGCTGTTCCCAGATTC TAAGCCATACAAACGGAAACAAG DIR27 TTCACATCCCGTGCCTTATAAAG TCAACGACACCACTAGGCAAAC DIR28/29 CTAGCACAGAAGCTCTTTCTAG AAACTAACCGTCTGATAGGAACC DXS WS00930_F08 TCCAGTGATTGAATATTCTTCC ATTCACTGCACAAGGCGACAC DXS WS0097_H02 CATTTCTGAGCTGCTCTTCATAC TTGAATTGCCTGTCACGTAGTC DXS WS01028_M14 AAGCCTCACGATGCTCTTCTC TGCGGCGTAGCCTTATTGATAG All DIR primers were used from previously reported work by S Ralph (Ralph et al., 2007).

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Supplemental Table 2.2: Terpenoid composition of constitutive and induced resin in outer stem tissues of interior spruce and lodgepole pine.* A. Interior spruce Time point 6h 6h 6h 2d 2d 2d 2w 2w 2w 6w 6w 6w Treatment C W F C W F C W F C W F mg/g dry weight Monoterpenoids α-pinene 8.96 10.97 11.84 15.63 7.79 10.05 8.11 6.34 10.24 10.22 10.73 12.86 camphene 0.12 0.15 0.16 0.22 0.09 0.13 0.12 0.08 0.14 0.14 0.15 0.18 β-pinene 7.71 9.71 10.67 14.00 6.58 8.22 6.23 5.29 7.91 8.69 8.25 9.07 Sabinene 0.06 0.11 0.10 0.13 0.05 0.07 0.07 0.05 0.10 0.09 0.08 0.13 3-carene 0.35 0.78 0.74 0.74 0.26 0.24 0.49 0.34 0.58 0.65 0.49 0.74 myrcene 1.25 1.63 1.38 1.85 0.80 1.06 1.19 0.75 1.10 1.47 1.04 1.45 limonene 7.06 8.53 7.33 10.47 4.65 5.70 6.66 4.37 6.34 8.73 6.20 9.06 β-phelandrene 4.83 6.05 6.19 8.33 3.63 4.61 4.91 2.91 3.65 5.29 4.02 4.56 terpinolene 0.12 0.22 0.22 0.26 0.09 0.12 0.14 0.09 0.17 0.20 0.14 0.21 bornyl acetate 0.16 0.20 0.21 0.29 0.12 0.15 0.11 0.10 0.12 0.16 0.15 0.18 citronellyl acetate 0.58 0.55 0.66 0.76 0.40 0.44 0.76 0.20 0.26 0.38 0.25 0.20 Diterpenoids sandarocopimarate 1.01 1.27 1.32 1.52 0.96 1.03 0.25 0.38 0.47 0.31 0.47 1.13 levopimarate 6.05 7.98 8.17 9.63 3.94 6.50 2.73 1.67 5.65 2.64 5.32 9.22 palustrate 7.49 10.02 10.52 10.78 3.87 6.27 2.31 1.34 3.81 2.24 4.17 5.48 isopimarate 11.44 13.99 13.91 16.65 9.44 11.24 5.71 4.75 6.83 8.17 9.91 13.57 abietate 7.18 8.17 7.40 5.45 2.51 7.04 3.43 3.11 4.68 5.49 6.71 7.17 dehydroabietate 0.99 1.35 1.21 6.53 5.96 2.27 1.34 2.28 3.42 1.40 4.05 9.79 neoabietate 6.00 6.50 6.47 8.04 3.46 5.07 2.50 1.90 3.61 3.33 4.49 7.41 *Listed terpenoids were identified by GC/FID or GC/MS. Terpenoids that could not be identified are not shown. Data are the mean of nine samples. C-control, W-wounding, F- fungus.

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Supplemental Table 2.2 (cont.): Terpenoid composition of constitutive and induced resin in outer stem tissues of lodgepole pine*

B. Lodgepole pine 6h 6h 6h 2d 2d 2d 2w 2w 2w 6w 6w 6w C W F C W F C W F C W F mg/g dry weight Monoterpenoids α-pinene 1.12 1.54 1.18 2.03 1.48 1.48 1.98 1.96 3.51 1.45 1.16 1.70 camphene 0.09 0.10 0.12 0.11 0.10 0.11 0.19 0.10 0.25 0.08 0.09 0.11 β-pinene 2.43 3.99 1.32 2.89 4.19 5.66 5.14 10.82 14.52 6.85 4.53 7.37 sabinene 0.23 0.23 0.36 0.22 0.29 0.82 0.31 0.26 0.89 0.13 0.23 0.33 3-carene 1.10 2.06 3.24 1.28 2.50 1.19 0.79 2.59 10.96 0.34 2.38 4.15 myrcene 0.43 0.51 0.64 0.55 0.51 0.51 0.40 0.41 1.22 0.37 0.37 0.53 limonene 0.73 0.45 0.56 0.38 0.40 0.42 0.36 0.36 0.85 0.53 0.53 0.47 β-phelandrene 7.76 9.08 12.45 6.37 10.89 10.71 6.48 5.59 21.13 5.45 6.57 8.65 terpinolene 0.16 0.19 0.25 0.12 0.22 0.49 0.12 0.23 1.04 0.05 0.14 0.36 α-terpineol 0.16 0.16 0.21 0.36 0.21 0.19 0.14 0.18 0.26 0.15 0.14 0.15 geranyl acetate 1.13 1.36 1.48 1.87 0.63 1.06 2.12 0.63 0.73 1.70 0.62 0.50 Diterpenoids pimarate 0.82 0.72 1.28 0.73 0.82 1.53 0.82 1.48 1.30 0.34 1.44 0.87 sandarocopimarate 0.53 0.71 0.79 0.52 0.74 0.62 0.55 0.91 2.09 0.57 0.80 1.03 levopimarate 0.54 4.70 0.61 0.21 7.36 6.21 2.11 5.66 32.82 1.72 2.16 7.92 palustrate 0.20 0.78 0.25 0.15 2.08 0.80 0.33 1.22 13.69 0.31 0.36 3.13 isopimarate 5.56 6.57 7.02 6.70 8.99 6.57 6.10 10.33 16.72 5.96 6.92 7.99 abietate 5.21 6.51 11.41 5.08 6.25 7.21 4.81 7.08 23.50 3.92 5.88 15.45 dehydroabietate 6.43 9.24 11.04 6.61 8.37 7.82 6.99 12.55 14.23 8.59 13.34 13.19 neoabietate 1.19 2.18 2.51 1.66 3.23 2.78 1.35 3.15 8.99 1.69 1.62 4.48 *Listed terpenoids were identified by GC/FID or GC/MS. Terpenoids that could not be identified are not shown. Data are the mean of nine samples. C-control, W-wounding, F- fungus.

Supplemental Tables 2.3, 2.4, 2.5 are too large for including in the thesis document and are provided on a compact disc:

Supplemental Table 2.3: Transcript expression in response to fungal inoculation in lodgepole pine and interior spruce trees determined using a 21.8K cDNA microarray (CD).

Supplemental Table 2.4: Distribution of transcripts in clusters (CD).

Supplemental Table 2.5: Functional annotation of the spruce microarray ESTs (CD).

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Wounding treatment vs. Control Fungal treatment vs. Control Fungal treatment vs. Wounding 6hours 2days 2weeks 6hours 2days 2weeks 6hours 2days 2weeks Interior spruce FC P FC P FC P FC P FC P FC P FC P FC P FC P Dir 1/20 0.46 0.206 0.50 0.279 0.61 0.11 0.48 0.208 0.22 0.014 0.66 0.511 1.05 0.997 0.44 0.126 1.07 0.326 Dir 2/32 111.56 <0.001 23.29 <0.001 1.22 0.913 131.83 <0.001 83.85 <0.001 3.23 0.094 1.18 0.636 3.60 0.037 2.66 0.076 Dir 5/15 9.52 0.006 13.24 0.005 0.76 0.734 8.73 0.003 18.79 0.001 0.55 0.234 0.92 0.977 1.42 0.339 0.72 0.386 Dir 6 24.88 <0.001 8.80 0.001 1.05 0.597 50.12 <0.001 43.47 <0.001 6.34 0.001 2.01 0.083 4.94 0.006 6.03 0.003 Dir 12/21 3.67 0.008 1.57 0.348 1.10 0.581 4.64 0.003 1.53 0.349 0.82 0.39 1.27 0.707 0.98 0.999 0.75 0.166 Dir 16 1.06 0.852 7.99 0.187 0.63 0.599 2.32 0.262 7.15 0.24 1.28 0.294 2.18 0.345 0.89 0.877 2.04 0.124 Dir 22 0.58 0.604 0.98 0.915 1.28 0.603 0.46 0.498 0.23 0.106 0.50 0.485 0.79 0.928 0.23 0.087 0.39 0.856 Dir 23/24 1.18 0.952 0.68 0.909 1.02 0.915 0.57 0.604 0.20 0.015 0.40 0.129 0.48 0.564 0.29 0.019 0.39 0.106 Dir 25 3.26 0.082 1.17 0.907 0.97 0.634 2.61 0.108 1.19 0.864 0.81 0.849 0.80 0.882 1.01 0.957 0.84 0.507 Dir 26 4.56 0.114 2.69 0.321 1.67 0.359 1.99 0.382 1.81 0.55 0.67 0.275 0.44 0.399 0.67 0.685 0.40 0.054 Dir 27 3.21 0.15 2.40 0.34 0.76 0.201 1.13 0.929 1.17 0.963 1.04 0.941 0.35 0.174 0.49 0.318 1.37 0.178 Dir 28/29 3.01 0.02 1.49 0.429 1.45 0.218 1.89 0.174 1.84 0.155 0.93 0.647 0.63 0.274 1.23 0.508 0.64 0.428 DXS WS00930_F08 94.37 <0.001 1.29 0.533 1.63 0.198 73.74 <0.001 2.01 0.148 3.30 0.009 0.78 0.898 1.56 0.392 2.02 0.134 DXS WS0097_H02 1.12 0.339 1.40 0.391 1.15 0.738 1.01 0.664 1.10 0.803 0.74 0.458 0.90 0.596 0.78 0.539 0.64 0.287 DXS WS01028_M14 1.40 0.882 0.70 0.886 1.88 0.802 0.59 0.691 0.88 0.718 1.55 0.749 0.42 0.586 1.25 0.827 0.82 0.945

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Chapter 3

Supplemental Table 3.1: Primer sequences used for real-time PCR (5' to 3' orientation) Gene ID Forward primer Reverse primer TIF5A GTGCCATCTTCACACAACTGC CAGATTCAGTCAGCAGGCTAAC DIR1/20 TTCGATCAAATGGCAGTGTACTC ACCATGACAAATATGCACAGAAG DIR2/32 AGTCTACTGATCTCTCCTGCTG ATTATTTCACTCTACGCTAGCTG DIR5/15 TGCATGAAGCTCCGAAATGCTG TATATTGGATATGGCCTTCCAAC DIR6 CTACTGATCTTTCATGCATGTTC TTCACTCTACCAATGCAGATTTG DIR12/21 TGCCGTTCCATCCATCTATCTC GGTCATGATTAGACTGCCCTTG DIR16 GCTCTCCATTTCTATGTTCATTG TTTGGGAGATTCATGCATATCTC DIR22 TTATTCAGTGCTCAGAGTATGAC CATATAATACTTGCCTGTCTGTG DIR23/24 ACAAAGCAGAGGGAGATATCAG TTTGCTCCCATTGACAACTGTG DIR25 TGGCTATGCGGTTGTTACTGTG ACAAGGTAACCTCTCCACAACG DIR26 CAAGAATGCTGTTCCCAGATTC TAAGCCATACAAACGGAAACAAG DIR27 TTCACATCCCGTGCCTTATAAAG TCAACGACACCACTAGGCAAAC DIR28/29 CTAGCACAGAAGCTCTTTCTAG AAACTAACCGTCTGATAGGAACC DXS WS00930_F08 TCCAGTGATTGAATATTCTTCC ATTCACTGCACAAGGCGACAC DXS WS0097_H02 CATTTCTGAGCTGCTCTTCATAC TTGAATTGCCTGTCACGTAGTC DXS WS01028_M14 AAGCCTCACGATGCTCTTCTC TGCGGCGTAGCCTTATTGATAG All DIR primers were used from previously reported work by S Ralph (Ralph et al., 2007).

Supplemental Tables 3.2, 3.3 are too large for including in the thesis document and are provided on a compact disc:

Supplemental Table 3.2: Transcript expression in response to weevil feeding in interior spruce trees determined using a 21.8K cDNA microarray (CD).

Supplemental Table 3.3: Distribution of transcripts in clusters (CD).

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Supplemental Table 3.4: Fold-change differences of selected gene expression in interior spruce exposed to weevil feeding measured using qRT- PCR.

Weevil treatment vs. Control 6hours 2days 2weeks Interior spruce FC P FC P FC P DIR 1/20 0.41 0.046 0.53 0.195 2.15 0.073 DIR 32/2 34.35 0.001 248.17 <0.001 61.06 <0.001 DIR 15/5 3.02 0.177 11.74 <0.001 0.09 <0.001 DIR 6 23.43 <0.001 1624.02 <0.001 41.50 <0.001 DIR 12/21 1.11 0.673 5.31 <0.001 2.94 0.002 DIR 16 2.72 0.861 37.79 0.001 41.89 0.009 DIR 22 2.22 0.332 2.27 0.101 4.86 0.003 DIR 23/24 0.17 0.104 0.98 0.333 15.26 0.001 DIR 25 1.45 0.515 2.03 0.012 0.22 <0.001 DIR 26 2.04 0.341 2.77 0.154 0.45 0.089 DIR 27 1.92 0.964 10.79 0.005 1.21 0.316 DIR 28/29 1.47 0.388 1.34 0.224 0.63 0.133 DXS WS00930_F08 36.10 0.005 91.12 <0.001 6.63 0.062 DXS WS0097_H02 0.86 0.444 19.34 <0.001 4.54 <0.001 DXS WS01028_M14 1.09 0.715 0.82 0.499 1.10 0.916

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Chapter 4

Wounding treatment vs. Control Fungal treatment vs. Control Fungal treatment vs. Wounding 6hours 2days 2weeks 6hours 2days 2weeks 6hours 2days 2weeks Interior spruce FC P FC P FC P FC P FC P FC P FC P FC P FC P PgeChia1-1 17.52 <0.001 53.09 <0.001 0.98 0.958 23.72 <0.001 189.43 <0.001 1.26 0.507 1.35 0.286 3.57 0.009 1.29 0.54 PgeChia1-2 4.47 <0.001 2.41 0.008 0.78 0.396 4.83 <0.001 8.37 <0.001 0.99 0.967 1.08 0.882 3.48 <0.001 1.27 0.374 PgeChia2-1 2.63 0.007 6.21 <0.001 0.89 0.972 2.06 0.026 14.70 <0.001 0.61 0.331 0.78 0.55 2.37 0.017 0.69 0.348 PgeChia4-1 1.89 0.294 36.56 <0.001 0.82 0.376 1.50 0.516 97.74 <0.001 4.50 0.09 0.79 0.681 2.67 0.14 5.50 0.015 WS0092_L20 0.68 0.911 18.10 <0.001 0.36 0.131 2.09 0.05 61.82 <0.001 0.88 0.834 3.08 0.062 3.42 0.04 2.45 0.09 IS0014_F01 1.11 0.721 2.99 0.091 0.97 0.83 0.87 0.92 3.18 0.032 4.52 0.018 0.78 0.797 1.06 0.6 4.67 0.011 Lodgepole pine PcChia4-1 3.05 0.168 9.84 0.143 1.70 0.138 0.45 0.909 28.39 0.035 4.05 0.092 0.15 0.234 2.88 0.482 2.38 0.829 PcChia1-1 12.27 <0.001 22.03 <0.001 1.20 0.778 11.13 <0.001 144.74 <0.001 51.17 <0.001 0.91 0.809 6.57 0.001 42.66 <0.001 PcChia7-1 15.90 0.042 13.83 0.018 1.51 0.05 34.94 0.003 572.71 <0.001 365.97 <0.001 2.20 0.296 41.42 0.004 243.08 <0.001 PcChia7-2 2.19 0.718 1.99 0.612 0.76 0.975 0.30 0.394 0.71 0.397 0.27 0.113 0.14 0.26 0.35 0.181 0.36 0.107

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Weevil treatment vs. Control 6hours 2days 2weeks Interior spruce FC P FC P FC P PgeChia1-1 1.85 0.183 125.64 <0.001 17.97 <0.001 PgeChia1-2 1.12 0.619 4.95 <0.001 2.72 0.002 PgeChia2-1 0.85 0.762 5.58 0.001 3.12 0.013 PgeChia4-1 1.76 0.388 47.62 <0.001 88.41 <0.001 WS0092_L20 2.43 0.068 12.27 <0.001 3.14 0.017 IS0014_F01 1.76 0.15 0.96 0.271 1.05 0.866

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Appendix 2. Evaluation of spruce microarray performance in heterologous hybridizations

Introduction Genomics tools for conifer species include the development of EST databases and microarrays, which contribute to the understanding of conifer biology and provides new resources for the development of tree breeding programs. These genomic advancements are particularly important for conifer species where studies are complicated by long generation time and large genome size (100 times the size of Arabidopsis genome). Transcriptional analysis using conifer microarray resources provides transcriptome scale characterization of biological processes and candidate gene lists for further characterization. The largest resources available for conifer studies are EST collections and microarrays for spruce and loblolly pine (Stasolla et al., 2003; Ritland et al., 2006; Ralph et al., 2006a; Ralph et al., 2008). Loblolly pine EST resource and microarrays (first microarray: 384 clones and second microarray: 2.2 thousand transcripts (2.2K)) were developed with an emphasis on wood formation (van Zyl et al., 2002; Stasolla et al., 2003). Spruce microarrays (9.7K, 16.7K and 22K) represent a variety of tissues (xylem, bark, roots), under constitutive, wounding-induced and methyl jasmonate-induced conditions (Ritland et al., 2006). Comprehensive representation of genes from different and induced tissues make these microarrays suitable to study the transcriptional response of a wide variety of biological processes including conifer defense systems (Ralph et al., 2006a; Ralph et al., 2006b). The implementation of large, genome-level projects is not practical for many conifer species because of the high cost and time commitment. Characterization of cross-species compatibility of existing microarrays to other conifer species is necessary to extend the application of the previously developed microarray resources. Currently available microarrays are built using cDNA not oligos, which facilitates interspecies usage due to anticipated higher signal and increased probability of homologous gene hybridization. In this paper we present the comprehensive characterization of spruce microarray performance with different conifer species. This study was done to support the application of the newly developed Spruce 9.7K, 16.7K and 21.8K microarrays (Ritland et al., 2006) to cross species hybridizations. Previously, loblolly pine microarrays were evaluated for use with other species of pine and spruce using RNA isolated from small seedlings with a xylem-based loblolly pine array that included 384 clones (van Zyl et al., 2002). Most of the genes on this microarray were chosen based on their homology to known angiosperm genes thus

198 introducing bias in the evaluation of the microarray usage in heterologous hybridizations. The 16.7K spruce microarray was constructed using the set of unique transcripts from the spruce EST collection, independent of their homology to angiosperm genes necessitating the evaluation of the performance of this array with other species. In addition, the previously evaluated loblolly pine and the current spruce microarrays were built using different technologies (loblolly pine microarray was printed on nylon membrane (van Zyl et al., 2002), spruce microarrays were printed on glass slides (Ralph et al., 2006a) and involve different hybridization process. We extended our analysis of heterologous hybridization to include the closely related genuses Picea (Picea glauca), and Pinus (Pinus contorta) and the more distant genuses of Pseudotsuga (Pseudotsuga menziesii) and Abies (Abies procera) in family Pinacea, as well as conifer family Cupressaceae (Chamaecyparis nootkatensis) and the angiosperm species Populus trichocarpa as an outgroup. In addition to the basic characterization of spruce array performance with other species we also included analysis of the relationship between sequence similarity and microarray signal intensity for the species used in this comparison. This correlation provides a better understanding of heterologous array applications in conifers, allowing correlation of signal intensity differences (using the spruce array with other conifer species) to observed differences in transcript expression and sequence divergence. Our results show that the spruce microarray may be efficiently used with species in family Pinacea and produces meaningful microarray results throughout wide range of sequence similarity.

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Methods

Plant material Individual mature trees representing different species were used for the experiments: white spruce (Picea glauca: clone PG-29) mature tree grown in Vernon, lodgepole pine (Pinus contorta) (about 15 year old) grown at UBC (University of British Columbia) South Campus farm, Douglas-fir (Pseudotsuga menziesii) mature tree grown at UBC South Campus farm, noble fir (Abies procera) mature tree (about 30 year old) grown at UBC South Campus farm, yellow cedar (Chamaecyparis nootkatensis) mature tree grown at UBC South Campus farm, Populus trichocarpa: clone 383-2499 mature tree grown at Biose cascade. All trees were grown outside in natural conditions. Developing xylem tissue was collected in late spring-early summer by scraping the trunk from which bark was removed. Tissue was stored at –80oC.

Microarray hybridization and gene expression data analysis RNA was isolated following the protocol of Kolosova et al. (2004). mRNA was isolated from total RNA using the Poly(A) Pure kit (Ambion, Austin, USA). The quality of RNA and mRNA was evaluated using reverse transcription with incorporation of radioactive nucleotide and subsequent gel autoradiography previously described (Kolosova et al., 2004). The construction of the spruce 16.7K array is similar to the previously described 9.7K spruce microarray (Ralph et al., 2006a) and will be reported elsewhere (Ralph S. and Bohlmann J., in preparation). A summary of the distribution of ESTs on the 16.7K spruce microarray is given in Table 1. Microarray hybridization was performed as described in Ralph et al., 2006a with small modifications. Hybridizations were performed using the Genisphere Array350 kit (Genisphere, Hatfield, USA) following the manufacturer’s instructions. Forty µg of total RNA (or 1µg mRNA) was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) (400U for total

RNA and 600U for mRNA) and oligo d(T18) primers with a 5’ unique sequence overhang specific to either the Cy3 or Cy5 labeling reactions provided with the Array 350 kit. The reverse transcription reaction was performed for 2 hrs at 42oC. The RNA strand of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65°C for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0). Following pooling of the appropriate cDNAs, samples were precipitated with linear acrylamide and resuspended in a 45 µL hybridization solution consisting of 0.25 M NaPO4, 1 SSC, 0.5% SDS, 2 Denhardt’s solution, 1 mM EDTA, 4.0 µL LDNA d(T) blocker, 2 µg sheared salmon testes DNA (Invitrogen) and 0.3 µL of Cy5-labeled GFP cDNA (Cy5- 200 dUTP and Ready-To-Go labeling beads, Amersham Pharmacia Biotech). Immediately prior to use, arrays were pre-washed 3 in 0.1% SDS at room temperature for 5 min each, followed by two washes in MilliQ-H2O for 2 min each, 3 min at 95°C in MilliQ-H2O, and dried by centrifugation (3 minutes at 2000 rpm in an IEC Centra CL2 centrifuge with rotor IEC 2367-00 in 50 mL conical tube). The cDNA probe was heat denatured at 80°C for 10 min, then maintained at 65°C prior to adding to a microarray slide heated to 55°C, covered with a 22  60  1.5 mm glass coverslip (Fisher Scientific), and incubated for 16 h at 60°C. Arrays were washed in 2 SSC, 0.2% SDS at room temperature for 5 min to remove the coverslip, followed by 15 min at 65°C in the same solution, then three washes of 5 min in 2 SSC at room temperature, and three washes of 5 min in 0.2 SSC at room temperature, and dried by centrifugation. The Cy3 and Cy5 3DNA capture reagent (Genisphere) were then hybridized to the bound cDNA on the microarray in a 45

µL volume consisting of 0.25 M NaPO4, 1 SSC, 0.5% SDS, 2 Denhardt’s solution, 1 mM EDTA, 2.5 µL Cy3 capture reagent and 2.5 µL Cy5 capture reagent. The second hybridization was performed for 3 h at 60°C, then washed and dried as before. Fluorescent images of hybridized arrays were acquired by using ScanArray Express (Perkin Elmer, Foster City, USA). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm, respectively. All scans were performed at the same laser power (90%), but with the photomultiplier tube settings for the two channels adjusted to maintain background levels below 300 fluorescent units and the number of saturated spots below 1% of total number of array elements. Fluorescent intensity data were extracted by using the ImaGene 5.5 software (Biodiscovery, El Segundo, USA). Tiff images, raw data outputs were archived in a BioArray Software Environment (BASE).

Bioinformatics analysis of EST sequences The bioinformatics analysis was performed by Nima Farzaneh (Treenomix, UBC). For the Spruce 16.7K microarray, data for 16,740 EST sequences were obtained from Treenomix, UBC and Genome Sciences Center, Vancouver. EST sequences for Pinus taeda (loblolly pine), Pseudotsuga menziesii (Douglas- fir), Cryptomeria japonica (Japanese cedar), Populus trichocarpa (Poplar) were retrieved from NCBI Gen Bank using a PERL script on July 4, 2007. The total number of EST sequences retrieved was 329,469 for Pinus taeda, 18,142 for Pseudotsuga menziesii, 19,605 for Cryptomeria japonica, and 89,943 Populus trichocarpa. The 16.7K spruce microarray ESTs database was first filtered using the program DUST (Hancock, J. M. and J. S. Armstrong, 1994) available as an option with Wu-

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BLASTn (Gish, W. (1996-2006) http://blast.wustl.edu) to remove low complexity sequence regions, including polyA tails. The edited spruce 16.7K microarray EST database was used for four independent BLASTn analyses separately for each species using Wu-BLASTn with the extracted sequences specified above. Wu-BLASTn was run with the following parameters: matrix set as default, e-value cut off 1e-05. The output was then parsed using a script in PERL to retrieve hit ID, hit size, E-value, matched size, matched percent.

202

Results

Evaluation of spruce array performance in heterologous hybridization with different plant species A spruce microarray containing 16,756 EST that representing 16,032 unique transcripts from different spruce tissues (Table A2.1) was used for heterologous hybridizations with three species from the family Pinaceae: lodgepole pine (Pinus contorta), Douglas-fir (Pseudotsuga menziesii), noble fir (Abies procera), one species from the family Cupressaceae - yellow cedar (Chamaecyparis nootkatensis) and the angiosperm species, poplar (Populus trichocarpa) as a distant reference. To characterize of spruce array performance across phylogenetic distance we chose to minimize biological variability by using developing xylem tissue, that is more uniform in cell structure than other tissues, that was collected from all tested species at the same developmental stage. Equal amounts of RNA from each species was reverse transcribed and an autoradiogram of the labelled cDNA revealed that unequal amounts of cDNA was produced from the same amount of total RNA from each species used (data not shown). The amount of cDNA obtained was relatively similar in white spruce and lodgepole pine and it increased in Douglas-fir, noble fir, and was highest in yellow cedar and poplar. Using mRNA from each species resulted in equal amounts of cDNA produced as was revealed by autoradiogram (data not shown). This data indicated that different species have different amounts of mRNA in total RNA. To compare microarray performance, we used mRNA for producing cDNA in order to remove the mRNA content bias in total RNA of different species. Xylem mRNA of each species was reverse transcribed and the resulting cDNA was hybridized against white spruce cDNA as a reference. Four technical replicates of microarray hybridization were performed for each of the six species including a dye flip (two replicates per each dye). In addition to these experiments we included one comparison of spruce microarray performance with the total RNA of white spruce and lodgepole pine. As most of the heterologous microarray experiments were performed with mRNA, the source of cDNA will be specified only for total RNA hybridization of lodgepole pine and white spruce.

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Table A2.1: Distribution of ESTs on Spruce 16.7 K microarray by cDNA libraries* Library Library name Species (genotype) library type Tissue Treatment/Conditions number number of ESTs WS001 WS-ES-A-1 Picea glauca standard young none 1,976 (PG-29) EST shoots WS002 WS-PS-A-2 Picea glauca standard shoots developmental stages 809 (PG-29) EST WS003 WS-X-A-3 Picea glauca standard xylem developmental stages 1,469 (PG-29) EST WS004 SS-R-A-5 Picea sitchensis standard roots young, mature 1,477 (Gb2-229) EST WS005 WS-PP-A-6 Picea glauca standard phloem developmental stages 714 (PG-29) EST WS006 IS-B-A-7 Picea engelmannii x standard bark 0.01% MeJa and wounding 445 Picea glauca EST treatment (tissue harvested 1, 2, 4, (Fa1 – 1028) 8 days post treatment) WS007 WS-PS-N-A-8 Picea glauca normalized shoots developmental stages 2,020 (PG-29) WS008 WS-X-N-A-9 Picea glauca normalized xylem developmental stages 1,974 (PG-29) WS009 IS-B-N-A-10 Picea engelmannii x normalized bark 0.01% MeJa and wounding 2,689 Picea glauca treatment (tissue harvested 3 h, 6 h, (Fa1 – 1028) 12 h, 1 day, 2 days, 4 d ays, 8 days post treatment and untreated control) WS010 SS-R-N-A-11 Picea sitchensis normalized roots young, mature 2,370 (Gb2-229) IS001 IS-B-A-4 Picea engelmannii x standard EST bark 0.01%MeJa and wounding treatment 797 Picea glauca (tissue harvested 3h, 6h, 12 h post (Fa1 – 1028) treatment and untreated control) * Information about libraries and ESTs sequences is available in Gene Bank at the NCBI web page. 204

The obtained microarray data was not normalized or modified to preserve signal differences resulting from using different species (e.g. most common normalization protocol LOESS makes an assumption that signal intensity is the same for most of the genes of treatment and control (Edwards, 2003)). Hybridization performance of the microarray with different species was evaluated using the number of genes that have signal of 2 and 5 fold above background and by the comparison of the mean total raw signal intensity of each species to the white spruce control (Table A2.2A). Raw signal and background intensity for the following analysis were calculated as the average of the median signal and background for four replicates per microarray transcript. White spruce cDNA was hybridized against itself to provide a reference of optimal spruce microarray performance. Hybridization performance of the spruce microarray was similar for all white spruce cDNA samples hybridized against itself and against cDNA all studied species (Table A2.2B). The number of genes with a signal intensity of 2 fold above background in lodgepole pine (mRNA and total RNA), Douglas-fir and noble fir was relatively high, varying between 63 and 70% of all transcripts on the array compared to 81% for white spruce, while for yellow cedar and poplar it decreased to 46% and 22% respectively. The performance of the microarray drops with species outside the family of Pinaceae. It is especially evident from the low number of genes in yellow cedar (19%) and poplar (6%) with signal that is 5 times above background, as compared to 66% in white spruce and 39%, 43 and 50% in lodgepole pine, noble fir and Douglas-fir respectively. The average signal intensity relative to the white spruce control was 39% for lodgepole pine mRNA, 55% for lodgepole pine total RNA, 60% for Douglas-fir mRNA, 43% for noble fir mRNA, 21% for yellow cedar mRNA and 10% with poplar mRNA. Visualization of the signal intensity data as a boxplot demonstrates that signal intensity is similarly distributed in interspecies hybridization of the three Pinaceae species lodgepole pine, Douglas-fir and noble fir (Figure A2.1). Correlation of signal intensity with the number of transcripts (Figure A2.2) clearly groups lodgepole pine, Douglas-fir and noble fir together in terms of spruce array performance. Even though overall performance of spruce microarray drops significantly outside the family Pinaceae there is a number of genes in yellow cedar and poplar that hybridise with high signal intensity.

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Table A2.2: Analysis of interspecies 16.7K spruce microarray performance. A. Comparison of the microarray performance of different conifer species and white spruce. WS - white spruce, LP – lodgepole pine, DF – Douglas-fir, NF – noble fir, YC – yellow cedar, P – poplar, bckg. – background. WS LP DF NF YC P WS LP Microarray performance mRNA mRNA mRNA mRNA mRNA mRNA total RNA total RNA average background (16756 genes) 195 182 190 178 178 170 266 284 number of genes 2 fold above bckg. 13636 10540 11795 11277 7714 3733 13243 11080 % of genes 2 fold above bckg. 81 63 70 67 46 22 79 66 number of genes 5 fold above bckg. 11108 6534 8301 7133 3221 1070 9522 6448 % of genes 5 fold above bckg. 66 39 50 43 19 6 57 38 average signal (16756 genes) 3909 1514 2353 1696 810 397 3812 2135 average signal to background ratio 20.04 8.34 12.37 9.51 4.56 2.34 14.3 7.5

B. Microarray performance with white spruce reference for all species used in interspecies microarray study. Species for which WS was a reference is indicated in parentheses, LP – lodgepole pine, DF – Douglas-fir, NF – noble fir, YC – yellow cedar, P – poplar, bckg. – background. WS (WS) WS (LP) WS (DF) WS (NF) WS (YC) WS (P) Microarray performance mRNA mRNA mRNA mRNA mRNA mRNA average background (16756 genes) 208 209 189 187 193 194 number of genes 2 fold above bckg. 14003 12807 13252 13344 13383 14161 % of genes 2 fold above bckg. 84 76 79 80 80 85 number of genes 5 fold above bckg. 11571 10785 10206 10526 10509 11351 % of genes 5 fold above bckg. 69 64 61 63 63 68 average signal (16756 genes) 4720 3872 3343 3471 3704 4307 average signal to background ratio 22.70 18.54 17.71 18.60 19.17 22.20

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WS LP DF NF YC P Pinaceae Cupressaceae Angiosperm

Figure A2.1: Spruce microarray performance in interspecies hybridizations. Boxplot of signal intensity (mean of four replicates). The upper edge of the box indicates the 75th percentile of the data set, and the lower edge of the box indicates the 25th percentile. The line in the box indicates the median value of the data. WS – white spruce, LP – lodgepole pine, DF – Douglas-fir, NF – noble fir, YC – yellow cedar, P – poplar.

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1000 YC signal intensity signal intensity P 500

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0 0 2500 5000 7500 10000 12500 15000 number of genes

Figure A2.2: Comparison of the microarray signal intensity distribution among different species. Each point on the graph represents the number of microarray elements that have microarray signal intensity equal to or lower than the value on the y axis. WS – white spruce, LP – lodgepole pine, DF – Douglas-fir, NF – noble fir, YC – yellow cedar, P – poplar.

To test the ability of the spruce microarray to adequately detect a gene expression pattern in other species we performed pairwise Spearman and Pearson correlations of the log-transformed signal intensity (mean of the medians for four replicates) of cDNA from different species (Table A2.3A).

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Table A2.3 Correlation of log transformed signal intensity between species used in interspecies hybridizations. A. Comparison of different species. Each species, including white spruce, was hybridized against the white spruce. WS – white spruce, LP – lodgepole pine, DF – Douglas-fir, NF – noble fir, YC – yellow cedar, P – poplar.

Spearman correlation Pearson correlation species WS LP DF NF YC species WS LP DF NF YC WS 0.98 WS 0.97 LP 0.81 LP 0.80 DF 0.81 0.86 DF 0.80 0.87 NF 0.79 0.84 0.87 NF 0.78 0.85 0.88 YC 0.69 0.70 0.70 0.71 YC 0.67 0.75 0.74 0.76 P 0.57 0.53 0.51 0.52 0.57 P 0.58 0.65 0.61 0.63 0.76

B. Correlation of log transformed signal intensity of white spruce samples hybridized against different species. Species for which WS was a reference is indicated in parentheses, WS – white spruce, LP – lodgepole pine, DF – Douglas-fir, NF – noble fir, YC – yellow cedar, P – poplar. Spearman correlation Pearson correlation species WS WS WS WS WS species WS WS WS WS WS (WS) (LP) (DF) (NF) (YC) (WS) (LP) (DF) (NF) (YC) WS(LP) 0.96 WS(LP) 0.96 WS(DF) 0.96 0.97 WS(DF) 0.95 0.96 WS(NF) 0.97 0.97 WS(NF) 0.96 0.96 0.97 WS(YC) 0.96 0.96 0.97 0.97 WS(YC) 0.95 0.96 0.96 0.96 WS(P) 0.96 0.97 0.97 0.97 0.97 WS(P) 0.95 0.96 0.97 0.97 0.96

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The correlations were performed for log transformed signal intensity from each species (including white spruce that was hybridized against white spruce). In the case of white spruce, one set of data from four hybridizations (with balanced dye flip pattern) was considered the reference and the other set was used for comparison in the same pattern as was done for other tested species. Correlation between white spruce data and white spruce reference data from the same experiment was very high (Table A2.3A, Spearman correlation - 0.98, Pearson correlation - 0.97). A high correlation was observed for all white spruce references for all other species: spearman correlation ranged from 0.96 to 0.97 and Pearson correlation ranged from 0.95 to 0.97 (Table A2.3B). This high correlation indicates a high similarity of microarray data for white spruce controls used for hybridization against all other studied species. Thus we compared data sets for all of the studied species to the white spruce data set. For each interspecies comparison of the other Picea species to white spruce, the Spearman correlation coefficients were high ranged from 0.79 to 0.81 and Pearson correlation coefficients ranged from 0.78 to 0.8. Similar correlation was observed for lodgepole pine and white spruce signal intensity obtained from total RNA hybridizations: Spearman correlation coefficient of 0.85, Pearson correlation coefficient of 0.71. The Spearman and Pearson correlation coefficients were also high for yellow cedar (0.69 and 0.67 respectively) and for poplar (0.57 and 0.58 respectively).

Correlation of spruce microarray performance in heterologous hybridizations with sequence similarity of microarray ESTs between species In order to understand if the observed loss of signal intensity in species with different phylogenetic distance is correlated with the loss of gene homology we looked at the expression of homologous genes in different species. From public databases we extracted EST data available for poplar (Populus trichocarpa), loblolly pine (Pinus taeda), Douglas-fir and Japanese cedar (Cryptomeria japonica). We used BLASTn to select ESTs from each of the species that had sequence similarity with the spruce 16.7K microarray ESTs (Table A2.4A). ESTs from loblolly pine were used to identify a group of genes on the spruce microarray that are likely to have sequence similarity to lodgepole pine. ESTs from Japanese cedar were used for identifying genes on the spruce microarray ESTs that likely have sequence similarity to yellow cedar as a representative of the same family.

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Table A2.4: Correlation of sequence similarity and average signal intensity for genes with an E value equal or less than E-05 in sequence alignments with the spruce microarray ESTs. (A) Summary of EST data retrieved from NCBI Gen Bank. (B) Average microarray signal intensity from interspecies hybridizations for groups of spruce 16.7K array ESTs that have sequence similarity to tested species based on Blastn analysis with E value cut off of E-05. WS – white spruce, LP – lodgepole pine, DF – Douglas-fir, NF – noble fir, YC – yellow cedar, P – poplar.

A. Sequence similarity to Number of ESTs in the Number of ESTs from BLAST Spruce 16.7K array NCBI database (on search July 4, 07) E value cut off E-05 Loblolly pine 329469 13325 Douglas-fir 18142 8147 Japanese cedar 19605 5300 Poplar (Populus trichocarpa) 89943 5223

B. Average microarray signal intensity Subset of genes WS LP DF NF YC P all genes 3909 1514 2352 1695 809 396 Loblolly pine 4493 1772 2766 1984 923 430 Douglas-fir 5511 2218 3522 2474 1170 522 Japanese cedar 5720 2599 3982 2860 1424 606 Poplar 5406 2506 3786 2807 1457 608

We chose an E value cut off of E-05 because it resulted in sequence matches of near 50 base pairs or higher, that are relevant for hybridization analysis. To evaluate the overall performance of the spruce array with the subsets of genes in each of the four species that have sequence similarity to the spruce array genes we looked at the average microarray signal intensity of the subsets of genes with sequence similarity to spruce array ESTs in interspecies hybridizations (Table A2.4B). For each group of genes with sequence similarity to the spruce array ESTs the average signal intensity was higher than that of all the genes on the microarray. In addition, these four sets of ESTs with sequence similarity to the spruce microarray ESTs had higher average signal intensity in all interspecies hybridizations (Table A2.4B).

Correlation of E value and microarray signal intensity To better illustrate the correlation of microarray signal intensity with sequence similarity we plotted the microarray signal intensity (mean of four replicates) of lodgepole pine, Douglas-fir, yellow cedar and poplar against the E value of loblolly pine, Douglas- fir, yellow cedar and poplar ESTs with sequence similarity to spruce microarray ESTs using a hexbin graph (Figure A2.3).

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A B 0 0 Figure A2.3: Correlation of the distribution of E values and 16.7K spruce microarray signal intensity (mean of four replicates) for genes -40 -50 with E value

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Hexbin plots showed some correlation of increased microarray signal with decreased E value for lodgepole pine and Douglas-fir. In order to quantify this trend we calculated a spearman correlation for microarray signal intensities and E values for each of the four species. Spearman correlation coefficient was also calculated for the E values of the same subsets of genes (for each of the four species) with hybridization signal intensity of these subsets of genes from white spruce reference hybridization in order to estimate the contribution of differences in expression of different genes in addition to difference of array performance with these genes to the correlations (Table A2.5).

Table A2.5: Spearman correlation of E value and microarray signal intensity (mean of four replicates) for genes that have E value equal or less than E-05.

Species Spearman cor. Spearman cor. WS reference Lodgepole pine -0.358 -0.191 Douglas-fir -0.323 -0.212 Yellow cedar -0.343 -0.144 Poplar -0.238 -0.157

Spearman correlation revealed a negative correlation of E value with signal intensity (ranging from -0.24 to -0.36) for all subsets of genes from the four species. Correlation of the E values of the same sets of genes with white spruce hybridization signal intensity of these genes also revealed a negative correlation (from -0.14 to -0.21).

Correlation of sequence identity and microarray signal intensity To further investigate the interaction between sequence identity, gene expression and microarray signal intensity in heterologous hybridizations we compared sequence identity obtained through BLASTn analysis with microarray signal. For each of the four species with available EST sequences (poplar, Douglas-fir, loblolly pine (for lodgepole pine analysis) and Japanese cedar (for yellow cedar analysis) we sorted sequences (with E value -05 or lower) into groups based on sequence identity equal to or over 90%, equal or more than 80%, equal or more than 70% and the rest of the sequences (most of the sequences had a sequence overlap of 50 base pairs or higher, except for 2 loblolly pine ESTs and 1 Japanese cedar EST (data not shown). For each of the four species the distribution of signal intensity (mean of four replicates) in each of these groups was plotted as a boxplot with the corresponding boxplot of the signal intensity of the same genes from white spruce hybridization with white spruce (Figure A2.4). The boxplots for lodgepole pine, Douglas-fir and poplar demonstrate a clear 213 decrease in signal intensity with a decrease in sequence identity. In the case of yellow cedar, there is a decrease in signal intensity with decrease in sequence identity except for the 90% sequence identity group compared to 80% group. This inconsistency could be the result of a low number of genes in the 90% group in case of yellow cedar (Figure A2.4) that resulted in a skewed statistical representation. A comparison of the changes in signal intensity with sequence identity to the changes in signal intensity of the same genes in white spruce revealed a similar decrease in signal intensity with a decrease in sequence identity. The average signal intensity was calculated for sequences with a matched length of 150 bases or less and for sequences with a matched length of over 150 bases for sequences sorted into groups based on sequence identity values (Table A2.6). Our data showed that there is an increase in signal intensity with an increase in the sequence length with a given sequence identity (except for groups with few genes). The average signal intensity was also calculated for white spruce cDNA for the same groups of genes. In most cases longer matched sequences length resulted in higher hybridization signal in interspecies hybridizations. The ratio of average signal intensity for the same groups of genes from the hybridization of other species to white spruce is increases with a longer matched length with the exceptions of groups where the gene number is small.

Figure A2.4: Microarray performance correlated with the sequence identity of BLAST aligned sequences (E value < E-05) to spruce sequences present on the 16.7K spruce microarray. X axis values represent groups of genes with sequence similarity equal or above the group value. Following each species data there is white spruce reference (R) data for the hybridization of the same genes with white spruce. Y axis represents the mean signal intensity of four replicates. The upper edge of the box indicates the 75th percentile of the data set, and the lower edge of the box indicates the 25th percentile. The line in the box indicates the median value of the data. Number in each box specifies the number of genes in the given boxplot. (A) Lodgepole pine (LP) data boxplot; (B) Douglas-fir (DF) data boxplot (C) Yellow cedar (YC) data boxplot. (D) Poplar (P) data boxplot. WS – white spruce.

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Lodgepole pine Douglas-fir sequence identity sequence identity at at at at at at at at matched least least least least least least least least length 90 80 70 50 90 80 70 50 (bases) <90 <80 <70 <90 <80 <70 Number of genes all genes 3840 6126 2389 970 1536 3558 1959 1094 Average signal all genes 2145 1864 1383 677 4253 3832 3275 1927 Average signal WS ref. all genes 4671 4805 4152 2655 5700 6073 5452 3525 Average signal ratio to WS ref. (%) all genes 46 39 33 25 75 63 60 55

Number of genes 150 or less 149 340 153 15 140 285 219 29 Average signal 150 or less 1208 949 1088 893 2626 2575 2162 2120 Average signal WS ref. 150 or less 3145 3549 3500 2708 3824 4838 4939 6193 Average signal ratio to WS ref. (%) 150 or less 38 27 31 33 69 53 44 34

Number of genes >150 3691 5786 2236 955 1395 3273 1740 1065 Average signal >150 2183 1918 1403 674 4416 3942 3415 1922 Average signal WS ref. >150 4732 4879 4197 2654 5882 6180 5516 3453 Average signal ratio to WS ref. (%) >150 46 39 33 25 75 64 62 56

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Table A2.6 (cont): Correlation of microarray signal intensity with matched length and sequence identity of EST sequences from tested species to spruce microarray EST sequences. WS ref. – white spruce reference (signal intensity was taken for white spruce samples that were hybridized against reported species).

Yellow cedar Poplar sequence identity sequence identity at at at at at at at at matched least least least least least least least least length 90 80 70 50 90 80 70 50 (bases) <90 <80 <70 <90 <80 <70 Number of genes all genes 49 727 2348 2176 69 187 1861 3106 Average signal all genes 1690 2583 1558 886 1889 2329 698 421 Average signal WS ref. all genes 5124 7022 6141 4845 10014 8982 5828 4835 Average signal ratio to WS ref. (%) all genes 33 37 25 18 19 26 12 9

Number of genes 150 or less 14 80 188 41 6 32 171 37 Average signal 150 or less 1294 1149 949 886 279 698 480 297 Average signal WS ref. 150 or less 5465 5308 5215 4975 1393 7845 5077 3412 Average signal ratio to WS ref. (%) 150 or less 24 22 18 18 20 9 9 9

Number of genes >150 35 647 2160 2135 63 155 1690 3069 Average signal >150 1849 2761 1611 886 2042 2665 720 423 Average signal WS ref. >150 4988 7235 6222 4843 10835 9217 5904 4853 Average signal ratio to WS ref. (%) >150 37 38 26 18 19 29 12 9

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Discussion

Spruce microarray performance in heterologous hybridizations Our results demonstrated that the spruce microarray performs efficiently with cDNA from three species of the Pinaceae family in heterologous hybridizations and this performance is comparable to the performance of the spruce microarray with white spruce (Table A2.2a). Phylogenetically, lodgepole pine, Douglas-fir and noble fir are grouped together in the Pinaceae family with approximately 140, 150 and 170 million years of divergence from white spruce respectively (Wang et al., 2000). The performance of the spruce microarray with these species is similar and produces adequate signal intensity to allow allows analysis of 80-85 % of genes (compared to spruce as 100%) with a signal intensity of at least two fold above background with majority of these genes having a signal intensity of five fold or more above background (Table 2). Performance of the spruce array with yellow cedar (a representative of the family Cupressaceae) is poorer than performance with the Pinaceae species as expected by the greater divergence of yellow cedar from spruce species (Bowe et al., 2000). Performance of the spruce microarray with poplar is lower than yellow cedar and is consistent with the larger phylogenetic distance between poplar and spruce (Bowe et al., 2000). When using the spruce microarray with different species from the Pinaceae family it can be expected to have about 50% of the average signal to that of spruce, with 60% (compared to 80% in spruce) of the genes with a signal that is 2 fold above background and 40% (compared to over 60% in spruce) of genes with a high signal (5 fold above background). The spruce microarray may be considered for use with yellow cedar and possibly other conifer species outside the Pinaceae family an expected 20% average signal intensity compared to white spruce with 45% of genes having a signal that is two fold above background and 20% of genes with a high signal. Phylogenetic distance of family Cupressaceae from Pinaceae is similar to that of gymnosperms and angiosperms (Bowe et al., 2000). The better performance of the spruce microarray with cedar (than poplar) may be explained by the slower evolutionary rates in gymnosperms as compared to angiosperms (Ritland et al., 2006). This feature of gymnosperm evolution is advantageous for transferring genomic tools from model species to species with no available genomics resources within conifers and possibly within the whole group of gymnosperms.

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Spearman and Pearson correlations revealed the ability of the spruce microarray to reliably detect a gene expression pattern in interspecies hybridizations with species from the Pinaceae family and to some extent with yellow cedar and poplar (Table A2.3a). Similar values of Spearman and Person correlation coefficients indicate that signal intensity in examined interspecies hybridizations correlates well with white spruce using the ranking values and absolute expression values. The observed high correlation of gene expression between different species not only in family Pinaceae but also between gymnosperms and angiosperms indicates a similarity in gene expression in the xylem of different species, as well as the ability of the spruce array to detect this expression pattern in distant species from spruce. These results are consistent with heterologous hybridizations performed with loblolly pine microarray (van Zyl et al., 2002) that revealed a high correlation in gene expression within the needles of pine and spruce (Spearman correlation 0.88 to 0.93, Pearson correlation 0.90 to 0.93) as well as a correlation of signal intensity of pine and spruce with the angiosperm species tobacco (Spearman correlation 0.73-0.79, Pearson correlation 0.76 to 0.82). The higher correlation reported in van Zyl et al., 2002 may result from the inclusion of conserved genes on the microarray that were functionally identified (van Zyl et al., 2002). These results indicate the efficient performance of the spruce array with species from the Pinaceae family.

Correlation of spruce microarray performance in interspecies hybridizations with sequence similarity The availability of EST databases for some of the tested species (or closely related species) allowed the correlation of microarray performance with sequence similarity. Based on BLASTn sequence similarity to the spruce microarray ESTs, the loblolly pine EST data set was used to correlate spruce microarray performance with lodgepole pine cDNA, the Douglas-fir EST data set was used to correlate spruce microarray performance with Douglas-fir, the Japanese cedar data set was used for the analysis of spruce microarray performance with yellow cedar and poplar (Populus trichocarpa) ESTs were used for the evaluation of spruce microarray performance with poplar (Table A2.4a). Each subset of sequences of the four species with similarity to the spruce microarray ESTs exhibited a higher average signal for each hybridized species as compared to the average signal intensity of all genes on the microarray (Table A2.4b). Since the expression of these homologous genes is also higher in white spruce we may conclude that the increase in signal intensity is partly due to an increase in expression and not only due to an increase in sequence similarity. 219

Correlation of E values with signal intensity for the hybridization of the four species revealed a small negative correlation. A negative correlation (to a lesser degree) was also observed in the hybridization of white spruce for the same subsets of genes as was analyzed in other species (Table A2.5), indicating that the increase in signal intensity with the increase in sequence similarity (decrease in E) is likely due to both an increase in hybridization efficiency, as well as a general increased in expression of more conserved genes between species. E value gives a general characterization of sequence similarity that is based on the score, which is determined using scoring matrix that takes into to account different sequence parameters (e.g. sequence identity and gaps) (Baxevanis, 2005). It was shown that sequence identity is the best predictor of the efficiency of cross hybridization (Evertsz et al., 2001) with 80% sequence identity resulting in lower (26%-57%) but still significant cross reactivity. It was also demonstrated that sequences with at least 75% of identity over 50mer (50bases) will cross hybridize (Kane et al., 2000) and the length of similar sequence plays a significant role in determining cross hybridization efficiency up to 150mer (Chou et al., 2004). Analysis of the correlation of signal intensity with sequence similarity revealed decrease in signal intensity with decrease in sequence identity in all four analyzed species (Figure A2.4). Interestingly a similar decrease in signal intensity was observed for the same subsets of genes in the hybridization of white spruce cDNA. If we would assume that gene expression is independent of the degree of conservation or homology with other species we would expect signal intensity to stay the same in all sequence similarity groups for white spruce hybridization and decrease in signal intensity as sequence identity decreases for other species. Assuming that white spruce and the other studied species have a similar gene expression pattern in xylem, a correlation of the overall microarray signal of genes in these species sorted by sequence identity with the signal intensity of the same groups of genes in white spruce hybridization will suggest the contribution of the expression and sequence identity to changes in average microarray signal. This analysis revealed that there is a strong correlation in the changes of overall signal intensity in white spruce and tested species through different gene groups assembled based on sequence identity to spruce EST present on microarray. In the in case of spruce array performance with lodgepole pine cDNA there is a 50% drop in the median of signal intensity as compared to white spruce starting with the group of genes with highest sequence identity. The median of signal intensity in lodgepole pine is decreasing and the box including middle 50% of the data is shifting to lower numbers in groups of genes with decrease in sequence identity. The 220 same sets of genes exhibit a decrease in the median of signal intensity and a downward shift in the position of the box in the white spruce hybridization (Figure A2.4), and a similar pattern is observed for other species (Figure A2.4). This analysis suggests that more conserved genes have higher levels of expression and a drop in sequence similarity contributes only partially to the decrease of the overall signal intensity with decreasing sequence similarity. The drop in signal intensity correlates with previously characterized cross hybridization within homologous gene families, where 80% sequence identity resulted in 26 to 57% signal intensity of hybridization of the same sequences to each other (Evertsz et al., 2001). A similar pattern of lower signal intensity compared to white spruce and correlation of the decrease of the average signal intensity with white spruce signal intensity over different sequence similarities is observed for other species as well (Figure A2.4). On average the microarray signal intensity drops with the increase of phylogenetic distance from spruce although the performance of the spruce microarray is reliable in detecting expression pattern across a large variation in sequence identity. Evaluation of spruce microarray performance with matched sequence length for genes with the same sequence similarity revealed that the spruce microarray produces a higher average signal intensity with the groups of genes that have a longer matched length, in our case longer than 150 bases (Table A2.6). Previous research demonstrated that 150mer probes are similar in hybridization properties to long cDNA probes (Chou et al., 2004). Our data showed that sequences with longer matches are more likely to produce higher signal in interspecies gene hybridizations. The white spruce average microarray signal also increases for the gene groups that have longer matched length in the four investigated species with spruce sequences, indicating that an increase in average signal intensity with increased matched length contributes because of a higher expression level of the more conserved genes (assuming that overall gene expression pattern is similar in all studied species to white spruce). An increase in the ratio of the average signal for all of the four species to the white spruce signal for the groups of genes with longer sequence matched length (that is observed in most cases) indicates that the spruce microarray on average performs better in interspecies hybridizations with sequences that have a longer matched length to the spruce sequences. Overall, the spruce microarray can be efficiently used with species of Pinaceae family and can be applied to other conifer families for the study of the expression of more conserved genes.

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References Baxevanis A (2005) Assesing pairwise sequence similarity: BLAST and FASTA. In A Boxevanis, F Ouellette, eds, Bioinformaitcs: a practical guide to the analysis of genes and proteins, Ed Third. John Wiley and Sons, Inc, Hoboken, New Jersey, pp 295-324 Bowe LM, Coat G, dePamphilis CW (2000) Phylogeny of seed plants based on all three genomic compartments: Extant gymnosperms are monophyletic and Gnetales' closest relatives are conifers. Proceedings of the National Academy of Sciences of the United States of America 97: 4092-4097 Chou CC, Chen CH, Lee TT, Peck K (2004) Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression. Nucleic Acids Research 32 Evertsz EM, Au-Young J, Ruvolo MV, Lim AC, Reynolds MA (2001) Hybridization cross-reactivity within homologous gene families on glass cDNA microarrays. Biotechniques 31: 1182-1192 Kane MD, Jatkoe TA, Stumpf CR, Lu J, Thomas JD, Madore SJ (2000) Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. Nucleic Acids Research 28: 4552-4557 Kolosova N, Miller B, Ralph S, Ellis BE, Douglas C, Ritland K, Bohlmann J (2004) Isolation of high-quality RNA from gymnosperm and angiosperm trees. Biotechniques 36: 821-824 Ralph SG, Chun HJE, Kolosova N, Cooper D, Oddy C, Ritland CE, Kirkpatrick R, Moore R, Barber S, Holt RA, Jones SJM, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2008) A conifer genomics resource of 200,000 spruce (Picea spp.) ESTs and 6,464 high-quality, sequence-finished full-length cDNAs for Sitka spruce (Picea sitchensis). Bmc Genomics 9: 484 Ralph SG, Yueh H, Friedmann M, Aeschliman D, Zeznik JA, Nelson CC, Butterfield YSN, Kirkpatrick R, Liu J, Jones SJM, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2006a) Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome. Plant Cell and Environment 29: 1545-1570 Ralph S, Park JY, Bohlmann J, Mansfield SD (2006b) Dirigent proteins in conifer defense: gene discovery, phylogeny, and differential wound- and insect-induced expression of a family of DIR and DIR-like genes in spruce (Picea spp.). Plant Molecular Biology 60: 21-40 Ritland K, Ralph S, Lippert D, Rungis D, Bohlmann J (2006) New directions for conifer genomics. In Landscapes, genomics and transgenic conifer forests. Springer Netherland, Dordrecht Netherland, pp 75-84 Stasolla C, van Zyl L, Egertsdotter U, Craig D, Liu WB, Sederoff RR (2003) The effects of polyethylene glycol on gene expression of developing white spruce somatic embryos. Plant Physiology 131: 49-60 Van Zyl L, von Arnold S, Bozhkov P, Chen YZ, Egertsdotter U, MacKay J, Sederoff RR, Shen J, Zelena L, Clapham DH (2002) Heterologous array analysis in Pinaceae: hybridization of Pinus taeda cDNA arrays with cDNA from needles and embryogenic cultures of P-taeda, P-sylvestris or Picea abies. Comparative and Functional Genomics 3: 306-318 Wang XQ, Tank DC, Sang T (2000) Phylogeny and divergence times in Pinaceae: Evidence from three genomes. Molecular Biology and Evolution 17: 773-781

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Appendix 3. Isolation of high-quality RNA from gymnosperm and angiosperm trees (Published paper)

Natalia Kolosova, Barbara Miller, Steven Ralph, Brian E. Ellis, Carl Douglas, Kermit Ritland, and Jörg Bohlmann (2004) Isolation of high-quality RNA from gymnosperm and angiosperm trees. BioTechniques 35:821-824.

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