Toward developing pheromone emitting trap crops: Metabolic engineering of an aggregation pheromone for enhanced attraction of Phyllotreta cruciferae

Pheromone lures and trap crops are appealing pest management tools that use insect and/or plant volatiles to reduce pest populations on crops of interest. Generating pheromone-emitting trap plants may allow for a continuing and highly-specific attraction of insect pests without repeated and costly application of synthetic pheromones. These trap plants may also be used to develop area-wide pest management strategies. As a proof-of-principle study we tested the possibility of producing the pheromone of the crucifer flea beetle Phyllotreta cruciferae in transgenic plants. P. cruciferae is an important pest of Brassica crops. In the presence of a host plant, males emit an aggregation pheromone, which attracts both males and females. Himachaladiene, a sesquiterpene, has been identified as a key component of the aggregation pheromone of P. cruciferae. In a close relative, Phyllotreta striolata, the compound is synthesized by a two-step pathway with an isoprenyl diphosphate synthase (PsIDS3) making (Z,E)-farnesyl diphosphate (FPP), which is converted by a terpene synthase (PsTPS1) to himachaladiene. Transient transformation of N. benthamiana with PsIDS3-TPS1 co-localized to the plastid resulted in the emission of himachaladiene and other known PsTPS1 products. Daily emissions of himachaladiene were approximately 1 µg per plant, which is six-fold higher than emissions from individual male flea beetles. Stable transformation of Arabidopsis thaliana with the same vector construct resulted in transgenic plants that expressed PsTPS1 and PsIDS3 transcripts, but no himachaladiene or other PsTPS1 products were present in volatile collections or leaf extracts of these plants. Moreover, no PsTPS1 enzyme activity was observed, indicating that post-transcriptional/translational effects prevent proper expression or targeting of functional PsIDS3 and/or PsTPS1 proteins in A. thaliana. Overall, this study demonstrates that the key component of the P. cruciferae aggregation pheromone, himachaladiene, can be transiently produced and emitted in a plant system at rates that are biologically relevant for insect attraction. However, further work is required for the stable production of the pheromone in plants. In addition, preliminary results are presented for the development of simple two-choice arenas that may allow for assessment of the movement of beetles toward host plant leaf tissue. This work can inform future efforts in developing methods for the economic production of himachaladiene in a plant system or the establishment of transgenic plants for the production and deployment of himachaladiene in a field setting.