Functional characterization of Bacillus thuringiensis Cry toxin receptors explains resistance in insects.
Bacillus thuringiensis produces Cry toxins, which are used as insecticides in sprays and in transgenic crops. However, little is known about the function of Cry toxin receptors and the mechanisms that determine their binding specificity and activity. In this study, the cRNAs of Bombyx mori ABC transporter C2 (BmABCC2), the toxin-binding region of cadherin-like receptor (BtR175-TBR), or aminopeptidase N1 (BmAPN1) were injected into Xenopus oocytes, and the Cry1Aa-dependent cation-selective pore formation activities of these receptors were analyzed using a two-electrode voltage clamp. Cation current passing through the pores was detected within 25 s, and increased in a linear fashion in BmABCC2-expressing oocytes treated with 88 nm Cry1Aa. This result suggested that Cry1Aa continuously made stable pores with the help of BmABCC2. In contrast, no cation current was observed until 60 min after incubation with 500 nm Cry1Aa in BtR175TBR-expressing oocytes even though oligomerization of Cry1Aa progressed. This result indicated that in the presence of BtR175-TBR most of the oligomerized toxin could not enter the cell membrane. However, oocytes that simultaneously expressed both receptors demonstrated that BtR175-TBR exerted a synergistic effect with BmABCC2 on pore formation in the presence of 22 nm Cry1Aa. These results confirm that the main reason for moderate-level resistance in insects lacking the cadherin-like receptor but expressing ABCC2 is the absence of a similar synergistic promotion of toxin oligomerization. Similar to results from our previous report evaluating ectopic expression in the Sf9/Baculovirus system, BmAPN1 could not by itself cause Cry1A-related pore formation, despite the fact that BmAPN1 gathered toxin on the oocytes as well as BmABCC2 did.
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
Shiho
S
Tanaka
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
Haruka
H
Endo
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
Satomi
S
Adegawa
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
Shingo
S
Kikuta
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
Ryoichi
R
Sato
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D012831:Bombyx / Q000235:genetics / Q000378:metabolism; D018826:CD13 Antigens / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D005260:Female; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007313:Insecta / Q000382:microbiology; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D018826:CD13 Antigens; D019476:Insect Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011956:Receptors, Cell Surface; C095416:Cry toxin receptors

Bacillus thuringiensis
; 
Bombyx mori
; ABC transporter C2; cadherin-like receptor; two-electrode voltage clamp