Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93 , which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

Reactive oxygen species (ROS) such as superoxide (O 2 ÀC ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (HOC) are important mediators of pathological processes in various diseases.[1] Detection by fluorescent probes is one of the most useful methods for evaluating the roles of ROS in pathological processes. 2',7'-Dichlorofluorescin (DCFH) and its diacetyl derivative (DCFH-DA) [2] have been widely used as fluorescent probes for measuring cell-derived H 2 O 2 , [3] but these compounds suffer from the major drawback that they are poorly selective toward H 2 O 2 . Researchers have demonstrated that oxidation of DCFH to dichlorofluorescein is also induced by peroxidase [4] and other hemoproteins [5] as well as by hydroperoxides in the presence of peroxidase, [6] nitric oxide, [7] and peroxynitrite. [8] Therefore, the fluorescent response based on the oxidation of DCFH provides an index, not for cell-derived H 2 O 2 , but for the total oxidants present in biological systems. This limitation stems from its mechanism of fluorescence, which is based on oxidation. Dihydro derivatives of fluorescent compounds such as dihydrorhodamine 123 [3c,g] and N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) [9] have been shown to function as probes for detecting H 2 O 2 . However, their mechanism of action is similar to that of DCFH, which implies that low selectivity toward H 2 O 2 is a shortcoming that must be accepted when utilizing these probes. In fact, dihydrorhodamine 123 was shown to react with various ROS, [3c, 7b] and although Amplex Red seems to have high selectivity toward H 2 O 2 , peroxidase is essential for its fluorescence, similar to the case of DCFH. Thus, developing probes for H 2 O 2 based on a non-oxidative fluorescence mechanism, which would allow the highly specific and peroxidase-independent detection of H 2 O 2 under the complicated oxidative circumstances found in biological systems, is a worthwhile goal.Recently, we found that perhydrolysis of acyl resorufins is a useful reaction that acts as a fluorescent indicator for H 2 O 2 assays.[10] The method is based on simple deprotection, not on oxidation, thus allowing acyl derivatives of fluorescent compounds such as resorufin and fluorescein to work as probes for detecting cell-derived H 2 O 2 with higher selectively than that provided by DCFH and its analogues. Unfortunately, the competition between perhydrolysis and hydrolysis of acyl resorufins and fluoresceins in biological systems was not altered in a manner favorable towards H 2 O 2 -based deacylation.We thus designed pentafluorobenzenesulfonyl fluoresceins (1 a-c, Scheme 1) as selective fluorescent probes for H 2 O 2 but would eliminate, or at least significantly reduce, competition from hydrolysis reactions of the acetyl derivatives. These compounds were chosen for the following reasons: sulfonates are more stable to hydrolysis than are esters; fluoresceins have high fluorescence quantum yields in aqueous solution; and the pentafluorobenzene ring enhances the reactivity of the sulfonates tow...

Bacillus thuringiensis is the most widely used biopesticide, and its Cry toxin genes are essential transgenes for the generation of insect‐resistant transgenic crops. Recent reports have suggested that ATP‐binding cassette transporter subfamily C2 (ABCC2) proteins are implicated in Cry intoxication, and that a single amino acid insertion results in high levels of resistance to Cry1 toxins. However, there is currently no available direct evidence of functional interactions between ABCC2 and Cry toxins. To address this important knowledge gap, we investigated the role of Bombyx mori ABCC2 (BmABCC2) or its mutant from a Cry1Ab‐resistant B. mori strain on Cry1A toxin action. When we expressed BmABCC2 ectopically on Sf9 cells, it served as a functional receptor, and the single amino acid insertion found in BmABCC2 from Cry1Ab‐resistant larvae resulted in lack of susceptibility to Cry1Ab and Cry1Ac. Using the same expression system, we found that Bo. mori cadherin‐like receptor (BtR175) conferred susceptibility to Cry1A toxins, albeit to a lower degree than BmABCC2. Coexpression of BtR175 and BmABCC2 resulted in the highest cell susceptibility to Cry1A, Cry1F, and even the phylogenetically distant Cry8Ca toxin, when compared with expression of either receptor alone. The susceptibility observed in the coexpressing cells and that in Bo. mori larvae are likely to be correlated, suggesting that BtR175 and BmABCC2 are important factors determining larval susceptibility. Our study demonstrates, for the first time, Cry toxin receptor functionality for ABCC2, and highlights the crucial role of this protein and cadherin in the mechanism of action of Cry toxin.

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