Poster Annual Conference of the Genetics Society of Australasia with the NZ Society for Biochemistry & Molecular Biology

A bimolecular luminescence complementation assay to identify a bee-friendly insecticide (544)

Charlotte Pushparajan 1 , Andrew G Cridge 1 , Peter K Dearden 1
  1. University of Otago, Dunedin, OTAGO, New Zealand

A critical event in insect development is specification of embryonic termini by the gap gene tailless in a process called terminal patterning.In the majority of insects,including several pest species,nuclear expression of tailless is triggered through a MAP kinase signalling cascade initiated by activation of the receptor tyrosine kinase torso by its ligand trunk.The honeybee is a beneficial insect whose genome lacks orthologues for torso and trunk and tailless expression is not activated by their interaction.Our aim is to identify a selective inhibitor of torso-trunk signalling which can be used as an insecticide to disrupt development in pests but spare bees.We have developed a bimolecular luminescence complementation reporter system in Sf9 cells,which allows us to quantify the strength of the interaction and its modulation in response to antagonists.Plasmid constructs encoding Torso and its downstream effector corkscrew fused to the respective amino- and carboxyl-terminal fragments of Renilla luciferase were transiently co-expressed in Sf9 cells,together with or without Trunk.Reconstitution of luciferase activity in the presence of substrate was significantly higher when trunk was present at 48h post-transfection,indicating ligand-mediated dimerization of torso and binding of phosphorylated corkscrew to activated torso.On the other hand,luciferase activity decreased when cells co-expressing trunk, torso and corkscrew were treated with tyrosine kinase inhibitors.A stable cell line is being generated for antibiotic-inducible expression of the interaction partners.Sustained gene expression at consistent levels in a clonal cell-based assay will improve reproducibility of hits obtained in high-throughput screening of compound libraries in our search for a bee-friendly insecticide.