Cloning and Expression of a Receptor for an Insecticidal Toxin of Bacillus thuringiensis

Abstract

Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins.

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

“…31,50 BT-R 1 homologs are the principal determinant for Cry1A toxin action in lepidopteran insects. [30][31][32][33][34][35] No BT-R 1 homologs has been identified in vertebrates, suggesting that these particular cadherin receptors represent a unique family of proteins in invertebrates, particularly insects, and may explain why Cry toxins are not toxic to mammalian cells. Perhaps, Cry toxins once constituted virulence factors with the ability to destroy cells through oligomerization and incorporation of toxin molecules into cell membranes.…”

“…The cadherin receptor BT-R 1 , identified in the tobacco hornworm Manduca sexta, [30][31][32] represents a family of cadherins that are expressed in the midgut epithelium of various insects susceptible to Cry1A toxins. Cry toxins bind to their respective cadherin receptors with high affinity and specificity, the disruption or absence of which results in loss of susceptibility to Cry toxin.…”

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

“…In the case of Cry1A toxins, two receptors have been characterized in several lepidopteran species, cadherin-like proteins, (named Bt-R 1 in the case of M. sexta) [42], and the GPI-anchored proteins aminopeptidase-N (APN) and alkaline phosphatase [22,25].…”

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

“…Even more, the principal mechanism of resistance to Cry toxins are mutations that affect toxin-receptor interaction [8]. Different proteins such as cadherins, aminopeptidase-N (APN), and alkaline phosphatase (ALP) have been characterized as Cry-receptors in different insect species [17,18,21,33]. Thus, understanding the molecular basis of the interaction of Cry toxins with their receptor molecules would be useful not only for engineering Cry proteins with different specificities or with enhanced insecticidal activity but also for coping with the problem of insect resistance in the field.…”

“…At that time, two M. sexta proteins that bind Cry1A toxins had been cloned and characterized, a 210 kDa cadherin protein known as Bt-R 1 and a 120 kDa glycosylphosphatidy-linositol (GPI) anchored aminopeptidase N (APN) [21,33]. As a way to determine which was the functional receptor and the amino acid epitopes involved in the toxinreceptor interaction, we decided to select by phage display peptides that bind Cry1Ab toxin and identify those that competed toxin interaction with both receptor molecules, then we used these phages as tools to determine the role of both M. sexta proteins in toxicity.…”