Jeanne A. Robert

An integrated genomic, proteomic and biochemical analysis of (+)‐3‐carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil

Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.

Disentangling Detoxification: Gene Expression Analysis of Feeding Mountain Pine Beetle Illuminates Molecular-Level Host Chemical Defense Detoxification Mechanisms

The mountain pine beetle, Dendroctonus ponderosae, is a native species of bark beetle (Coleoptera: Curculionidae) that caused unprecedented damage to the pine forests of British Columbia and other parts of western North America and is currently expanding its range into the boreal forests of central and eastern Canada and the USA. We conducted a large-scale gene expression analysis (RNA-seq) of mountain pine beetle male and female adults either starved or fed in male-female pairs for 24 hours on lodgepole pine host tree tissues. Our aim was to uncover transcripts involved in coniferophagous mountain pine beetle detoxification systems during early host colonization. Transcripts of members from several gene families significantly increased in insects fed on host tissue including: cytochromes P450, glucosyl transferases and glutathione S-transferases, esterases, and one ABC transporter. Other significantly increasing transcripts with potential roles in detoxification of host defenses included alcohol dehydrogenases and a group of unexpected transcripts whose products may play an, as yet, undiscovered role in host colonization by mountain pine beetle.

Global and comparative proteomic profiling of overwintering and developing mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae

BACKGROUND Mountain pine beetles, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), are native to western North America, but have recently begun to expand their range across the Canadian Rocky Mountains. The requirement for larvae to withstand extremely cold winter temperatures and potentially toxic host secondary metabolites in the midst of their ongoing development makes this a critical period of their lives. RESULTS We have uncovered global protein profiles for overwintering mountain pine beetle larvae. We have also quantitatively compared the proteomes for overwintering larvae sampled during autumn cooling and spring warming using iTRAQ methods. We identified 1507 unique proteins across all samples. In total, 33 proteins exhibited differential expression (FDR < 0.05) when compared between larvae before and after a cold snap in the autumn; and 473 proteins exhibited differential expression in the spring when measured before and after a steady incline in mean daily temperature. Eighteen proteins showed significant changes in both autumn and spring samples. CONCLUSIONS These first proteomic data for mountain pine beetle larvae show evidence of the involvement of trehalose, 2-deoxyglucose, and antioxidant enzymes in overwintering physiology; confirm and expand upon previous work implicating glycerol in cold tolerance in this insect; and provide new, detailed information on developmental processes in beetles. These results and associated data will be an invaluable resource for future targeted research on cold tolerance mechanisms in the mountain pine beetle and developmental biology in coleopterans.

Behavioral and Reproductive Response of White Pine Weevil (Pissodes strobi) to Resistant and Susceptible Sitka Spruce (Picea sitchensis)

White pine weevil (Pissodes strobi, Peck.) is a native forest insect pest in the Pacific Northwest of North America that attacks species of spruce (Picea spp.) and pine (Pinus spp.). Young Sitka spruce [Picea sitchensis (Bong.) Carr.] trees are particularly susceptible to weevil attack. Pockets of naturally occurring Sitka spruce resistance have been identified in high weevil hazard areas in coastal British Columbia. In this study, we characterize behavioral, physiological and reproductive responses of weevils to an extremely resistant Sitka spruce genotype (H898) in comparison to a highly susceptible genotype (Q903). The experiments relied on a large number of three-year-old clonally propagated trees and were therefore restricted to two contrasting Sitka spruce genotypes. When exposed to resistant trees, both male and female weevils were deterred during host selection and mating, females showed delayed or reduced ovary development, and successful reproduction of weevils was prevented on resistant trees.

Root Deformation Reduces Tolerance of Lodgepole Pine to Attack by Warren Root Collar Weevil

ABSTRACT Surveys were conducted on regenerating stands of lodgepole pine to determine the relationship between root deformation and susceptibility to attack by the Warren root collar weevil, Hylobius warreni Wood. The total number of trees attacked by H. warreni did not differ between planted and natural trees. A matched case-control logistic regression suggested that root crosssectional area was more important in predicting weevil attack for naturally regenerated trees than for planted trees, but weevils were associated with a larger reduction in height-to-diameter ratios for trees with planted root characteristics than for trees with natural root form. Neither the stability of attacked versus unattacked trees differed significantly and there was no significant interaction of weevil attack and tree type, but weevil-killed trees had different root characteristics than alive, attacked trees. Lateral distribution and root cross-sectional area were significant predictors of alive attacked trees versus weevil-killed trees, suggesting that trees with poor lateral spread or poor root cross-sectional area are more likely to die from weevil attack. We conclude that root deformation does not necessarily increase susceptibility to attack but may increase the likelihood of mortality. Thus, measures to facilitate good root form are needed when planting pine in areas with high risk of Warren root collar weevil attack.

Biomarkers and gene copy number variation for terpenoid traits associated with insect resistance in Sitka spruce: An integrated genomic, proteomic, and biochemical analysis of (+)-3-carene biosynthesis

Conifers have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. The large diversity of terpenoid metabolites is determined by the size and composition of the terpene synthase (TPS) gene family, and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (82-95% amino acid sequence identity) (+)-3-carene synthase (PsTPS-3car) genes. Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) which is expressed in both genotypes, one gene (PsTPS-3car2) expressed only in resistant trees, and one gene (PsTPS-3car3) expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring substantiated this pattern of differential expression of members of the PsTPS-3car family on the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3as a factor for the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by PsTPS-3car gene copy number variation, variation of gene and protein expression, and variation of catalytic efficiencies.