Applied Forest Health Genomics: Diversification Of Gene Families Associated With Conifer Resistance Against Insect Pest

The Genome Canada-funded projects SMarTForests (www.smartforests.ca) and Tria (www.thetriaproject.ca) are exploring a pathway of genomics research from fundamental science to practical applications in forest health. A focus of these projects is the applied genomics of conifer resistance against environmental disturbance in the form of outbreaks of insect pests and pathogens. For example, the pine weevil is one of the most devastating pests of spruce and pine. Variation in the level sof the monoterpene(+)-3-carene is associated with resistance to weevil attack in Sitka spruce. Variation in (+)-3-carene is due to variations in gene copy number, transcript and protein levels, and catalytic efficiency in a family of carene synthase genes. This family includes three carene synthases: PsTPS-3car1, PsTPS-3car2, PsTPS-3car3 and sabinene synthase PsTPS-sab. PsTPS-3car2is responsible for the high levels of (+)-3-carene in weevil-resistant trees. Structural comparisons of PsTPS-sab and PsTPS-3car guided a series of experiments to explore the amino acids responsible for the evolution of these specific defence gene functions in Sitka spruce. The product profiles of all of PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, relative to PsTPS-sab, were found to be predominantly controlled by three amino acids located in the helix J region: L596, which is vital for (+)-3-carene biosynthesis, and G595 and F599, which provide synergistic effects. In PsTPS-sab, the amino acids in the corresponding positions were shown to have the same control on product profile with F596 being essential for sabinene biosynthesis and A595 and L599 providing synergistic effects. Kinetic analysis revealed the amino acid in position 596 plays a strong role in the enzymes’ catalytic efficiencies. Homology modeling of PsTPS-3car2 and PsTPS-sab predicts this 596 amino acid to affect carbocation position in the reaction intermediate, directly affecting the enzymes specific product profile. Further mutation of the 596 amino acid to those with different side chain properties created a series of enzymes with new product profiles, highlighting the plasticity of terpene synthases in conifers. Investigation of the amino acids responsible for enzyme activity provides insights into the evolution of this family of (+)-3-carene synthase-like genes associated with insect resistance in Sitka spruce and their potential use in marker-assisted breeding.