Identification of an in vitro interaction between an insect immune suppressor protein (CrV2) and G alpha proteins.

The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gßγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gα(o) compared with rat Gα(i1). In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.

Mimicking nature's noses: from receptor deorphaning to olfactory biosensing.

The way in which organisms detect specific volatile compounds within their environment, and the associated neural processing which produces perception and subsequent behavioural responses, have been of interest to scientists for decades. Initially, most olfaction research was conducted using electrophysiological techniques on whole animals. However, the discovery of genes encoding the family of human olfactory receptors (ORs) paved the way for the development of a range of cellular assays, primarily used to deorphan ORs from mammals and insects. These assays have greatly advanced our knowledge of the molecular basis of olfaction, however, while there is currently good agreement on vertebrate and nematode olfactory signalling cascades, debate still surrounds the signalling mechanisms in insects. The inherent specificity and sensitivity of ORs makes them prime candidates as biological detectors of volatile ligands within biosensor devices, which have many potential applications. In the previous decade, researchers have investigated various technologies for transducing OR:ligand interactions into a readable format and thereby produce an olfactory biosensor (or bioelectronic nose) that maintains the discriminating power of the ORs in vivo. Here we review and compare the molecular mechanisms of olfaction in vertebrates and invertebrates, and also summarise the assay technologies utilising sub-tissue level sensing elements (cells and cell extracts), which have been applied to OR deorphanization and biosensor research. Although there are currently no commercial, "field-ready" olfactory biosensors of the kind discussed here, there have been several technological proof-of-concept studies suggesting that we will see their emergence within the next decade.