Cloning, Sequencing, and Expression of theBombyx moriReceptor forBacillus thuringiensisInsecticidal CryIA(a) Toxin

Abstract

Bacillus thuringiensis strains produce insect-specific Bt toxins. Bt CryIA(a) toxin binds to a 175-kDa glycoprotein (BtR175) on the microvillus membranes of columnar cells in the Bombyx mori midgut and causes lysis of the cells. BtR175 was purified, and its cDNA was cloned. The cDNA encodes a newly identified 193.3-kDa preproprotein form of BtR175 that includes nine extracellular cadherin repeats, a 23.5-kDa membrane-proximal domain, a membrane-spanning region, and a 13.6-kDa cytoplasmic domain. Spodoptera frugiperda cells transfected with a recombinant baculovirus DNA carrying the cDNA produced a 175-kDa protein that reacted with anti-BtR antibodies and the Bt CryIA(a) toxin.

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.…”

“…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. [33][34][35] So far, the role of BT-R 1 in the cytotoxic action of Cry toxin has not been defined.…”

The Molecular Genetics of Insecticide Resistance

“…These new approaches are particularly well illustrated by recent advances in our understanding of Bt resistance in Lepidoptera. For example, although numerous potential Cry1Ac-binding proteins have been identified from the brush border membrane of the lepidopteran midgut (Vadlamudi et al 1995;Nagamatsu et al 1998;McNall and Adang 2003), only one potential receptor, a 12-cadherin domain protein, has been linked to various resistant strains of the different caterpillar pests Heliothis virescens (Gahan et al 2001), Pectinophora gossypiella (Morin et al 2003), and Helicoverpa armigera (Xu et al 2005). This situation has now changed dramatically following the mapping of a second Cry1Ac resistance locus in H. virescens encoding the ABC transporter C2 (Gahan et al 2010).…”

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

“…1; fractions [26][27]. Initially these fractions were predicted to contain the cadherin-like Cry1Aa-binding protein [13,14,16]. The candidate receptor fractions for APN and cadherin were separately loaded on a size-exclusion column for further purification ( Fig.…”

“…Antibodies produced to BtR175 blocked toxin binding to the receptor in BBMV. The antibody serum also reduced Cry1Aa activity against B. mori when it was fed to larvae prior to toxin addition to the diet [14]. The same group cloned and introduced the BtR175 gene with a baculovirus vector intoSpodptera frugiperda Sf9 cells.…”