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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The effect of abscisic acid (ABA) on the tolerance to oxidative stress in a freshwater green alga, Chlamydomonas reinhardtii , was investigated. Exogenously added ABA enhanced the growth of this alga, which was observed under continuous illumination but not in the dark. The cells treated with ABA for 24 h showed tolerance to oxidative stress caused by exposure to paraquat or hydrogen peroxide. In the ABA-treated cells, the activities of two antioxidant enzymes, catalase (CAT) and ascorbate peroxidase (APX), were significantly higher than those in the untreated control. The result suggests that ABA plays a role in the enhancement of tolerance to oxidative stress by increasing the activity of antioxidant enzymes.

Dark anaerobic fermentation in the green algae Chiamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chiamydomonas MGA 161, was unusual in having high H2 but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCI or NHI-CI concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCI concentrations; starch degradation, and formation of ethanol, H2, and CO2 increased with the addition of NH4CI to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO2 as its main products, with H2, formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chiamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H2 evolution of the Escherichia coli type could be ruled out in Chiamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells.Green algae produce hydrogen under dark anaerobic conditions, as first observed by Gaffron and Rubin (8). The whole process of dark H2 evolution is mediated through the fermentative degradation of accumulated starch. Use of this property in a stable biophotolysis system makes possible temporal separation of H2 production from photosynthetic 02 production. Lightdependent H2 production without an O2 defense system soon comes to a stop because of inhibition by the O2 produced (10, 28). Studies with a freshwater alga, Chlamydomonas reinhardtii, showed an intermittent but stable 02/H2 production system in an alternating light/dark cycle (24,25). A halotolerant green alga, Chlamydomonas MGA 161, has been isolated, and both growth and dark H2 evolution in this alga are active (26 ucts of green algae (9,17,19). Klein and Betz (16) reported on the fermentation metabolism with no formate formation in Chlamydomonas moewusii. Gfeller and Gibbs (9) and Kreuzberg (19) reported that formate, acetate, and ethanol are produced in a ratio of 2:1:1 in the dark incubation of C. reinhardtii. The fermentation process is regulated by ATP and reducing equivalents generated during starch degradation. The ATP concentration within Chlorella cells increases slightly with active hydrogenase under dark anaerobic conditions, accompanied by an increase in NADH and a decrease in NADPH (22). Reducing equivalents are consumed by the formation of fermentation products for a constant metabolic flux; the reducing power is used for the formation of H2 (from H+), formate (from C02), glycerol (from acetonephosphate) or ethanol (from acetyl-CoA) (17).In this paper, we rep...