Doctor of Philosophy in Botany (PhD) 
Genomics of Systemic Induced Defense Responses to Insect Herbivory in Hybrid Poplar
Director of R&D
The mountain pine beetle (Dendroctonus ponderosae; MPB) is an irruptive bark beetle species affecting pine forests of western North America. A recent outbreak has spread over more than 25 million hectares of pine forests now affecting pine species of sensitive boreal and mountain ecosystems. Pine hosts produce a viscous oleoresin comprised of terpenoids; monoterpene, sesquiterpenes and diterpene resin acids, as a defense against insects and other herbivores. The MPB is exposed to terpenoids for most of its life cycle and these compounds act as host defenses, kairomones, and as pheromone precursors. Cytochromes P450 (P450s) have been proposed to function in MPB detoxification of host defenses, olfaction, and pheromone biosynthesis. My research addressed the role of terpenoids as toxic host defenses and pheromone precursors, and the role of MPB P450s in the modification of terpenoids in detoxification, odorant degradation, and pheromone biosynthesis.In this thesis, I assessed the toxic effects of monoterpenes to MPB, and analyzed the terpenoid metabolic products formed by MPB and by MPB P450s from host monoterpenes and diterpene resin acids. I assessed the toxicity of ten monoterpenes to the MPB. This study helps to quantitatively define the effects of individual monoterpenes towards MPB mortality, which is critical when assessing the variable monoterpene chemical defense profiles of its host species. I identified a set of novel monoterpenyl esters in the MPB, including verbenyl ester and showed that these esters are accumulated by the female beetle early in the life cycle for future release of the MPB aggregation pheromone trans-verbenol. I investigated seven different MPB P450s, specifically CYP6DE1, CYP6DE2, CYP6DJ1, CYP6BW1, CYP6BW3, CYP9Z18 and CYP345E2, for their potential roles in detoxification, odorant degradation, and pheromone biosynthesis by quantifying the transcript abundance in the antennae and alimentary canal. I have characterized the biochemical functions of four of these MPB P450s. The results of my thesis provide new insights into MPB interactions with host terpenoids defenses and the roles of P450s in these interactions.
Plant-specialized metabolites have long been utilized as medicines, cosmetics, flavours, and industrial raw materials. To explore the biosynthesis of a specialized metabolite in a non-model system and utilize the biosynthetic genes for future application, genomics-informed research typically flows through three phases: i) development of genomic or transcriptomic resources, ii) discovery and characterization of biosynthetic genes, and iii) application of the genes and enzymes for improved production of the specialized metabolite. This thesis describes hypothesis-driven research along these three phases in two different plant species and two different metabolic systems. My research with Crocosmia x crocosmiiflora focused on resource development and discovery of biosynthetic genes of a specialized metabolite of interest, montbretin A (MbA). I developed new resources for this system including metabolite-profiles and transcriptome sequences and annotations. This work resulted in insight into the spatial and temporal patterns of MbA accumulation in C. x crocosmiiflora and a first reference transcriptome with annotation for this species. Using these resources, I functionally characterized four UDP-xylose synthases and five UDP-rhamnose synthases. I discuss the application of these genes for possible use in an improved MbA production system and provide a proof of concept for using these genes to enable characterization of downstream MbA biosynthetic genes. I also identified 14 UDP-glycosyltransferases as candidate MbA biosynthetic genes through a guilt-by-association analysis; however, their functional characterization did not support a role in MbA biosynthesis. In the second biological system, Sitka spruce (Picea sitchensis), I performed a detailed characterization of a set of monoterpene synthases involved in the biosynthesis of the (+)-3-carene. Using domain swapping and site-directed mutagenesis, I demonstrated the catalytic plasticity of monoterpene synthases across a family of (+)-3-carene synthase-like genes associated with P. sitchensis resistance against the white pine weevil (Pissodes strobi). This work identified a single amino acid as most critical in determining both product profile and enzyme kinetics. Furthermore, I described mechanisms by which this amino acid directs product profiles through differential stabilization of the reaction intermediate. The work presented highlights the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against pests.
Conifers produce large quantities of diterpene resin acids (DRAs) as major components of the constitutive and induced oleoresin defense system. Like all vascular plants, conifers also produce gibberellin (GA) diterpene phytohormones, which influence growth and development. Conifers thus provide an interesting biological system for comparing the GA and DRA diterpene biosynthetic pathways. Despite serving different functions in growth and defense, respectively, the GA and DRA biosynthetic pathways are biochemically similar, utilizing the same isoprenoid precursors, evolutionarily related diterpene synthases (diTPSs), and functionally similar cytochrome P450 monooxygenases (CYP450s) to produce structurally similar diterpene intermediates and diterpene acid products. Functional characterization of central diTPS genes (ent-copalyl diphosphate synthase [CPS], ent-kaurene synthase [KS], levopimaradiene/abietadiene synthase [LAS]) and CYP450 genes (ent-kaurene oxidase [CYP701] and CYP720B4) in white spruce (Picea glauca), described in this thesis, allowed for comparative analysis of GA and DRA pathway genes. This thesis characterized the DXS (1-deoxy-D-xylulose 5-phosphate synthase) gene family in white spruce as additional analysis of the isoprenoid biosynthetic pathway producing the common precursor to both GAs and DRAs. Transcript expression of genes was analyzed to understand their seasonal (year-long time course of apical bud and shoot development), sample-specific (e.g. needle, stem, bud, bark/phloem, wood/xylem), and stress-specific (methyl jasmonate [MeJA] exposure) spatial and temporal patterns. Functionality of the DRA pathway was also assessed via quantification of DRA products. Expression of DRA and GA pathway genes was generally spatially separated. Expression of DRA genes was low in photosynthetic tissues but up-regulated during the time of year when trees are most likely to encounter seasonal attack from insect pests; expression declined sharply well before dormancy showing a strong seasonality to DRA production. GA related genes had broader expression across sample types and throughout the year, but spatially were mainly allocated to photosynthetic tissues. GA and DRA pathway genes all showed differential responses to MeJA treatment, and within corresponding sample types, age also played a role in expression. These studies improve our understanding of the organization of conifer chemical defenses, showing distinct differences compared with GA gene expression, and providing information on the spatial, seasonal and stress-responsive expression of DRA pathway genes.
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.
White pine weevil, Pissodes strobi, is an insect that occurs throughout Canada that attacks a number of conifers including Sitka spruce, Picea sitchensis, a commercially and ecologically important tree for coastal B.C.. Because of attack by weevils, Sitka spruce is no longer replanted as a commercial species. The re-introduction of this species would be a valuable asset for sustainable coastal forestry. My research addresses the terpene composition and the molecular-genetic underpinning of Sitka spruce resin defenses against attack by white pine weevil. In this thesis, I report that terpene profiles can be used to classify resistant tree genotypes. I analysed 111 different genotypes in order to determine the relationship of mono- and diterpenoid oleoresin compounds with the resistance rating. Dehydroabietic acid, a diterpene, was identified as a strong indicator of resistance. Two monoterpenes, (+)-3-carene and terpinolene were also associated with resistance in genotypes originating from the Haney region, an area which may have been subject to higher weevil pressure. In addition, I characterized weevil behavior and physiology (feeding patterns, host choice, ovary development, egg laying behavior, and larval development) in response to an extremely resistant Sitka spruce genotype (H898) in comparison to a highly susceptible genotype (Q903). My results suggest that the highly resistant genotype H898 has defense mechanisms that deter both male and female weevils during host selection and mating, that cause delayed ovary development in females, and prevent successful reproduction of weevils on H898 trees. Finally, I have identified the first (+)-3-carene and (+)-sabinene synthase genes in Sitka spruce. These terpene synthase (TPS) genes have very similar sequences, yet the encoded enzymes have different product profiles; this shows a new level of genetic diversity in the spruce TPS gene family. In addition, different (+)-3-carene synthase genes are expressed in the resistant H898 tree genotype producing large amounts of (+)-3-carene, versus the susceptible Q903 tree genotype that produces trace amounts of (+)-3-carene. This information will support the identification and breeding of resistant Sitka spruce in order to re-introduce it as a viable, native commercial species.
The availability of a poplar (Populus trichocarpa Torr & A. Gray, black cottonwood) genomesequence is enabling new research approaches in angiosperm tree biology. Much of the recentgenomics research in poplars has been on wood formation, growth and development, and abiotic stresstolerance, motivated, at least in part, by the fact that poplars provide an important system for largescale, short-rotation plantation forestry in the Northern Hemisphere. Given their widespreaddistribution and long lifespan, poplar trees are threatened by a large variety of insect herbivore pests,and must deal with their attacks with a successful defense response. To sustain productivity andecosystem health of natural and planted poplar forests, it is of critical importance to develop a betterunderstanding of the molecular mechanisms of defense and resistance of poplars against insect pests.Previous research has established a solid foundation of the chemical ecology of poplar defense againstinsects. In this study, I buiLd on this base with large-scale profiling of transcriptome responses ofpoplar trees to insect herbivory. A 15,496-clone cDNA microarray was developed and used to analysetranscriptome responses through time to a variety of insect, mechanical, and chemical elicitortreatments in treated source leaves, as well as in undamaged systemic source and sink leaves of hybridpoplar (Populus trichocarpa x deltoides).Comparing mechanical wounding with insect feeding and chemical eLicitor treatment withmethyl jasmonate demonstrated that qualitatively similar profiles of transcriptome response wereeLicited with differences in the timing of induction. Transcriptome analysis in undamaged systemicleaves of treated trees uncovered distinct early changes in primary metabolism (e.g. sugar metabolism)and general stress responses (e.g. heat shock proteins) prior to the activation of insect herbivoryresponse genes (e.g. Kunitz-type protease inhibitors). Source-sink relationships are maintained andstrengthened by insect damage on source leaves, emphasizing changes in resource allocation patternsas being important for poplar defense. Overall, a model of poplar defense begins to emerge where acascade of transcriptome profiles through space and time lead to reorganization of metabolism fortolerance and induction of defense.
Laser-assisted microdissection has been established for isolation of individual tissue types from herbaceous plants. However, there are few reports of cell- and tissue-specific analysis in woody perennials. While microdissected tissues are commonly analyzed for gene expression, reports of protein, enzyme activity and metabolite analysis are limited due in part to an inability to amplify these molecules. Conifer stem tissues are organized in a regular pattern with xylem, phloem and cortex development controlled by the activity of the cambial zone (CZ). Defense responses of conifer stems against insects and pathogens involve increased accumulation of terpenoids in cortical resin ducts (CRDs) and de novo formation of traumatic resin ducts from CZ initials. Woody plants are difficult to study at the level of individual tissues or cell-types and are thus good candidates for application of LMD. This thesis describes robust methods for isolation of individual tissue-types from white spruce (Picea glauca) stems for analysis of RNA, enzyme activity and metabolites. A tangential cryosectioning approach was important for obtaining large quantities of CRD and CZ tissues using LMD. Differential expression is reported for genes involved in terpenoid metabolism between CRD and CZ tissues and in response to treatment with methyl jasmonate (MeJA). Transcript levels of β-pinene synthase and levopimaradiene/abietadiene synthase were constitutively higher in CRDs, but induction was stronger in CZ in response to MeJA. 3-Carene synthase was more strongly induced in CRDs compared to CZ. A differential induction pattern was observed for 1-deoxyxyulose-5-phosphate synthase, which was up-regulated in CRDs and down-regulated in CZ. We identified terpene synthase enzyme activity in CZ protein extracts and terpenoid metabolites in both CRD and CZ tissues. Combined analysis of transcripts, proteins and metabolites of individual tissues will facilitate future characterization of complex processes of woody plant development, including periodic stem growth and dormancy, cell specialization, and defense and may be applied widely to other plant species.