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Mountain Pine Beetle (MPB; Dendroctonus Ponderosae) Pheromone Component Biosynthesis, Cytochromes P450 and Monoterpene Metabolism in Bark Beetles

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Mountain Pine Beetle (MPB; Dendroctonus Ponderosae) Pheromone Component Biosynthesis, Cytochromes P450 and Monoterpene Metabolism in Bark Beetles University of Nevada, Reno Mountain Pine Beetle (MPB; Dendroctonus ponderosae) Pheromone Component Biosynthesis, Cytochromes P450 and Monoterpene Metabolism in Bark Beetles A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Molecular Biology By Minmin Song Dr. Claus Tittiger/Dissertation Advisors May, 2012 THE GRADUATE SCHOOL We recommend that the dissertation prepared under our supervision by MINMIN SONG entitled Mountain Pine Beetle (MPB; Dendroctonus Ponderosae) Pheromone Component Biosynthesis, Cytochromes P450 and Monoterpene Metabolism in Bark Beetles be accepted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Claus Tittiger, Ph.D., Advisor Gary Blomquist, Ph.D., Committee Member Patricia Ellison, Ph.D., Committee Member Grant Mastick, Ph.D., Committee Member David Shintani, Ph.D., Committee Member Grant Mastick, Ph.D., Graduate School Representative Marsha H. Read, Ph. D., Associate Dean, Graduate School May, 2011 i Abstract Bark beetles successfully attack host trees by metabolizing host monterpenes efficiently and producing aggregation pheromones. Investigating enzymes involved in pheromone biosynthesis and monoterpene metabolism may identify unique molecular targets and provide information for developing new means to deal with bark beetle infestations. The three major mountain pine beetle (MPB, Dendroctonus ponderosae) pheromone components are frontalin, exo-brevicomin and trans-verbenol. Although these components were identified several decades ago, none of their biosynthetic pathways are known in detail and none of the involved enzymes are identified or characterized. This dissertation explores biochemical details of two important MPB pheromone components: exo-brevicomin and trans-verbenol, and bark beetle monoterpene metabolism in the context of specific enzymes. In vivo assays revealed that exo-brevicomin was predominantly produced in male fat bodies, and not in other tissues or by females, and that fat bodies catalyzed the conversion of decanoic acid to nonen-2-one, confirming that (6Z)-non-6-en-2-one is likely derived from fatty acid. Fat bodies converted (6Z)-non-6-en-2-one to 6,7- epoxynonan-2-one, the direct precursor of exo-brevicomin, and (6Z)-non-6-en-2-ol. The epoxide was stable under physiological conditions. These results implicate a cytochrome P450 and cyclase in the terminal steps of exo-brevicomin biosynthesis and (6Z)-non-6-en-2-ol may be the precursor of (6Z)-non-6-en-2-one. Two novel enzymes are implicated in exo-brevicomin production: a cytochrome P450, CYP6CR1, and a novel short chain dehydrogenase, ZnoDH. Their mRNA profiles were consistent with exo-brevicomin production suggests their coordinate regulation. ii Both enzymes were expressed in Sf9 cells for enzyme assays. Recombinant ZnoDH oxidized (6Z)-non-6-en-2-ol to the corresponding methyl-ketone, (6Z)-non-6-en-2-one, which serves as the substrate for CYP6CR1. CYP6CR1 converted (6Z)-non-6-en-2-one to 6,7-epoxynonan-2-one. These results suggest both CYP6CR1 and ZnoDH are in the exo-brevicomin biosynthetic pathway. Furthermore, two alternative pathways yielding production of the C9 precursor from a 10:1-fatty acid are discussed in light of these new data. While a direct decarboxylation of a β-ketoacyl-CoA intermediate has been suggested, evidence presented here supports oxidative decarbonylation of an unsaturated C10 aldehyle to produce 3-nonene, followed by hydroxylation to (6Z)-non-6- en-2-ol and oxidation by ZnoDH to (6Z)-non-6-en-2-one. Other cytochromes P450 were investigated to explore the evolutionary link between pheromone production and resin detoxification. Enzyme assays showed CYP6DH1 and its paralog, CYP6DH2, likely have complementary roles. CYP6DH1 produced verbenol from α-pinene, but did not hydroxylate other monoterpenes and was not induced by its substrates. Therefore, it likely functions as a pheromone (trans- verbenol) biosynthetic enzyme. In contrast, CYP6DH2 had a broad substrate range, suggesting a role in resin detoxification. CYP6DH1 most likely evolved from the detoxification enzyme, CYP6DH2, by duplication followed by genetic drift. Similarly, I. pini CYP9T2 had essentially the same substrate profile as CYP6DH2 even though CYP9T2 has been confirmed as a pheromone biosynthetic enzyme. Both enzymes converted the same substrates into different products, suggesting that different substrate binding regions may be involved in orienting the substrates. These results support the evolutionary mechanism that CYP9T2 or its ancestor originally worked as a detoxification enzyme, but it is now dedicated to pheromone production. iii Co-Authorship Bessie Liu and Tracy Nguyen (Department of Chemistry, UNR) produced penta- deuterium labeled (6Z)-non-6-en-2-one, (6Z)-non-6-en-2-ol and epoxide (Chapter 2, 3). Dr. C. Jeffery’s directed the syntheses. Mory Aw performed CYP6CR1 qRT-PCR analysis for developmental groups (Chapter 3). Patrick Delaplain contributed to ZnoDH sequencing, qRT-PCR analysis and generated the P3 viral stock (Chapter 4). Leah Plaugher assisted performing enzyme assays (Chapter 4). Dr. Chris Keeling (University of British Columbia) provided the unpublished CYP6DH1 cDNA clone (Chapter 5). Leah Plaugher generated the CYP6DH1 P3 viral stock and helped conduct enzyme assays (Chapter 5). Andrew Gorzalski and Patricia Kennel determined full-length sequences, performed the qRT-PCR, and produced CYP6DH2 and CYP9T2 viral stocks (Chapter 5). Most of that work appears in A.Gorzalski’s M.Sc. Thesis (Gorzalski, 2010). iv Table of Contents Chapter 1. Introduction: Mountain pine beetle pheromone component biosynthesis, cytochromes P450 and monoterpene metabolism in bark beetles…………………………1 Chapter 2. exo-Brevicomin biosynthesis in the mountain pine beetle……………………34 Chapter 3. CYP6CR1: a mountain pine beetle cytochrome P450 involved in exo- brevicomin biosynthesis……………………………………………………………………….70 Chapter 4. ZnoDH is a novel dehydrogenase involved in exo-brevicomin biosynthesis in the mountain pine beetle…………………………………………………………………….104 Chapter 5. Monoterpene-metabolizing P450s in pine bark beetle pheromone biosynthesis and resin detoxification………………………………………………………..134 . Chapter 6. Discussion and future directions…………………………………………….....183 Appendix……………………………………………………………………………………….203 1 Chapter 1 Introduction: Mountain pine beetle pheromone component biosynthesis, cytochromes P450 and monoterpene metabolism in bark beetles I. Biology of bark beetle: Dendroctonus ponderosae II. Cytochromes P450 III. Insect cytochromes P450 IV. Bark beetle cytochromes P450 V. Monoterpene metabolism and evolution in D. ponderosae and Ips pini VI. Objectives and hypotheses VII. References VIII. Figure legends I. Biology of bark beetle: Dendroctonus ponderosae I.1. Life cycle The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is the most destructive pest of coniferous forests in Alaska, Canada, and the western US. They destroy millions of acres of forest annually and cause significant economic damage through the devaluation of timber, soil destabilization, and increased risk of forest fires (CBC, 2003; Drooz, 1985; Furniss & Carolin, 1977; Pitman et al., 1968; USDA, 2002; Waters, 1985). MPBs preferentially attack lodgepole pine (Pinus contorta) and ponderosa pine (P. ponderosae), but they can attack any pine tree during outbreak conditions (Amman et al., 1990). These beetles infest the trunks of large, mature trees. The current outbreak in North America has extended the MPB range beyond its traditional habitat boundary, and has damaged millions of acres of mature forests. Up to 80% of pine trees in western Canada are predicted to be infested and killed by 2013 (Stickney, 2007). 2 The mountain pine beetle is an obligate parasite of pine trees, spending most of its life cycle beneath the bark. Egg laying, hatching and development through larvae, pupae to teneral adults all happen within the phloem of the brood tree. They emerge only for a short, pheromone-mediated flight to new host trees. Pioneer beetles chew through the outer bark to get into the inner phloem. During this process, the tree responds by producing a toxic resin to pitch out the attacking beetles (Phillips & Croteau, 1999; Steele et al., 1995). Healthy trees can produce enough volume to stop a beetle infestation, but under stressful conditions (e.g. drought), the attacked tree cannot produce enough resin to stop the infestation (Kurz et al., 2008; Rudinsky, 1996). Resin components produced by pine trees, such as myrcene, α and β-pinene, Δ3-carene, and limonene, appear most toxic to beetles (Byers, 1995; Raffa et al., 1985). The beetles also vector an associated blue-stain fungus that grows in the xylem. The extensive damage from bark beetles and fungi reduces water and nutrient flow, and causes tree mortality (Graham, 1967; Seybold et al., 2000). Like many bark beetles, D. ponderosae relies on aggregation pheromones to coordinate the “mass attack” required for successful tree colonization. The MPB pheromone system has three main components: trans-verbenol (4,7,7- trimethylbicyclo[3.1.1]hept-3-en-2-ol), exo-brevicomin (exo-7-Ethyl-5-methyl-6,8- dioxabicyclo[3.2.1]octane) and frontalin ((1S,5R)-1,5-dimethyl-6,8- dioxabicyclo[3.2.1]octane). Pioneer MPB females initially attack the host tree, and hydroxylate α-pinene to produce trans-verbenol, which attracts both males and females to the attacked tree (Billings et al., 1976; Libbey et al., 1985; Pitman, 1969; Pitman et al., 1969;
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