The use of specific insecticides is indispensable for the control of a wide range of pests. To date, the most widely applied biologically produced insecticides are derived from Bacillus thuringiensis, a ubiquitous Gram-positive, spore-forming bacterium initially described as an insect pathogen. Bacillus thuringiensis-derived insecticides are environmentally benign and mainly used to combat gypsy moths in forestry and against mosquitoes and blackflies, which transmit malaria and river blindness,respectively.
The insecticidal strains of Bacillus thuringiensis produce parasporic crystals that embed a variety of toxic proteins; a significant portion of them belongs to the Cry δ-endotoxins. The proteins are released into the midgut of infected insects, where the Cry prototoxin is processed by proteases. The toxin then binds to receptors in the insect's gut,inserts into the apical membrane of the midgut epithelial cells and creates lesions, which disable vital ion regulation, by pore or channel formation.
In vitro experiments, performed with proteins prepared from lepidopteran larvae, revealed several putative Cry protein receptors,including aminopeptidase N (APN). Preliminary support that this protein may be the receptor for the Cry1Ac1 toxin was provided by indirect evidence from reconstitution of the tobacco hornworm APN into phospholipid vesicles, which resulted in an increase of Cry1Ac1 binding and ion permeability. However,unequivocal evidence that APN is the toxin's receptor was missing until Michael Gill and David Ellar decided to express the lepidopteran APN in Drosophila, a fly that is not usually susceptible to Cry toxins.
Expressing a gene in a cell in which it is not active under normal circumstances is a powerful way to determine a protein's function. The GAL4 gene expression system allows selective expression of any cloned gene in a wide variety of cells and tissues of Drosophila. So Gill and Ellar decided to use this nifty technique to generate transgenic flies, which produced the tobacco hornworm APN in the midgut and salivary gland of developing Drosophila larvae. When the team fed active Cry1Ac1 toxin to the transgenic larvae, the insects stopped feeding and died within several days, in contrast to the control animals, which were insensitive to the Cry1Ac1 in their diet. Gill and Ellar also generated transgenic flies that expressed APN in the mesodermal tissue of developing larvae, in order to demonstrate whether the receptor functions in an anomalous cellular environment. When they treated these transgenic larvae with the active Cry1Ac1 toxin, the larval muscle wall disintegrated and extensive internal damage was observable.
These results provide unequivocal evidence for the function of APN as an in vivo receptor of Cry1Ac1. They also agree with Rajagopal and colleagues findings, who demonstrated in a recent paper that APN is the functional in vivo receptor for another Cry δ-endotoxin, Cry1C from Spodoptera litura, by dsRNA mediated gene silencing. Knowing the in vivo receptors for both Cry1Ac1 and Cry1C endotoxins may help to develop new strategies to counteract insect's increasing resistance to Bacillus thuringiensis toxins, which has emerged over the past two decades.
