Plants as biofactories for insect pheromones: Production of aphid and bark beetle pheromones in plants

Crop pests, such as aphids and bark beetles, represent a big challenge for agriculture worldwide. Due to their high adaptability and more suitable conditions because of climate change, they can thrive on a large number of hosts. One of the strategies to control the population of these pests in agricultural areas is the use of pheromones, either to lure them, repel them or to attract predators as a way of biological control. In the case of aphids, nepetalactol and nepetalactone have proven to be powerful attractants to aphids and to their predators. For bark beetles, which encompass a broad number
of beetle species, some of the most relevant compounds are ipsdienol, cis-verbenol, trans-verbenol and 2-methyl-3-buten-2-ol. The aim of this project is to produce these compounds in plant species using genetic and metabolic engineering tools. The feasibility of this approach is due to the capacity of plants
to synthesize the precursors for all these compounds from their secondary metabolism. Considering this, plants could represent a good alternative to either act as natural dispensers or lures of insect pheromones, or to act as biofactories and avoid the chemical synthesis of these compounds.
Paper I describes the methodology to work with Camelina using antibiotic-selection methods to generate stable transformants. As this plant system was considered for this project, it was decided to establish the optimal dose for antibiotic selection corresponding to commonly used plasmids in Arabidopsis and grass transformation. mRNA expression from antibiotic selected plants is shown, as well as effective methods for seed sterilization and crossing.
Paper II describes the research done regarding the genetic engineering of a functional nepetalactol synthetic pathway into plants along with a major latex protein like (MLPL) enzyme. Results show transient expression of these enzymes in N. benthamiana caused production of nepetalactone, a ketone product of the oxidation of nepetalactol, without external supplementation with pathway substrates. In addition, nepetalactone production was achieved in stably transformed Camelina. These results provide the basis of a transferable nepetalactone producing pathway using a single plasmid vector into plants.
Paper III presents the research regarding the genetic engineering of metabolic pathways for the biosynthesis of bark beetle aggregation pheromones 2-methyl-3-buten-3-ol, ipsdienol, cis-verbenol and trans-verbenol. Results show Arabidopsis thaliana can produce intermediates β-myrcene (ipsdienol
pathway) and α-pinene (verbenol pathway), and the final products cis-verbenol and trans-verbenol. Verbenone, an anti-aggregation pheromone, was also produced, being the result of verbenol autooxidation.