Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.     

受体介导的鳞翅目昆虫对Bt毒素抗性机制进展

苏云金芽孢杆菌作为一种对人畜安全、环境友好型绿色杀虫剂在全球被广泛使用。Bt毒素与昆虫中肠上特定毒素受体结合并发挥作用,形成毒素穿孔导致昆虫死亡是其重要的杀虫机制之一,靶标害虫对Bt毒素产生抗性是制约转Bt作物长期有效种植和Bt毒素持续使用的重要因素。文中从鳞翅目昆虫中肠细胞Bt毒素重要受体的研究阐述昆虫对Bt的抗性机制,为Bt抗性机制的深入研究和对害虫的防控与治理提供了一定的理论参考。     

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae)

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.     

An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt-expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.     

Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae

Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F(2) screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.00045-0.0080) with a detection probability of 90%. These results demonstrate that (i) the F(2) screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates.     

害虫Bt抗性机制研究新方向：昆虫体液免疫系统

作为最成功的生物农药,苏云金芽孢杆菌Bacillus thuringiensis (Bt)杀虫剂已在农业生产中应用了约80年。Bt由于其特异性强、安全高效的特点而得到广泛、成功的应用,极大减少了化学农药的用量,为环境保护作出了巨大贡献。然而,由于长期使用,一些靶标害虫逐渐对Bt产生抗性。本文对昆虫体液免疫及昆虫Bt抗性机制的研究成果进行了总结,已有研究认为害虫对Bt产生抗性的主要原因是毒素激活受阻及(或)毒素受体突变或减少。然而近年越来越多的研究表明,昆虫的Bt抗性还与其免疫系统,特别是与Toll, IMD和proPO-AS等体液免疫通路有关。由此,本文对昆虫体液免疫系统参与昆虫Bt抗性形成的主要通路进行了归纳和推论。IMD免疫通路可能通过MAPK信号通路参与调节昆虫Bt抗性,或可能通过多种免疫反应对抗因中肠组织被Bt破坏而引起的败血症,并通过JNK信号通路促使中肠组织愈合,进而提高其对Bt的抗性。从体液免疫系统切入研究,可能成为深入探索昆虫Bt抗性机制的新方向。     

The compatibility of a nucleopolyhedrosis virus control with resistance management for Bacillus thuringiensis: co-infection and cross-resistance studies with the diamondback moth, Plutella xylostella

The use of genetically modified crops expressing Bacillus thuringiensis (Bt) toxins can lead to the reduction in application of broad-spectrum pesticides and an increased opportunity for supplementary biological control. Bt microbial sprays are also used by organic growers or as part of integrated pest management programs that rely on the use of natural enemies. In both applications the evolution of resistance to Bt toxins is a potential problem. Natural enemies (pathogens or insects) acting in combination with toxins can accelerate or decelerate the evolution of resistance to Bt. In the present study we investigated whether the use of a nucleopolyhedrovirus (AcMNPV) could potentially affect the evolution of resistance to the Bt toxin Cry1Ac in Plutella xylostella. At low toxin doses there was evidence for antagonistic interactions between AcMNPV and Cry1Ac resistant and susceptible insects. However, this antagonism was much stronger and more widespread for susceptible larvae; interactions were generally not distinguishable from additive for resistant larvae. Selection for resistance to Cry1Ac in two populations of P. xylostella with differing resistance mechanisms did not produce any correlated changes in resistance to AcMNPV. Stronger antagonistic interactions between Bt and AcMNPV on susceptible rather than resistant larvae can decrease the relative fitness between Bt-resistant and susceptible larvae. These interactions and the lack of cross-resistance between virus and toxin suggest that the use of NPV is compatible with resistance management to Bt products.     

利用噬菌体展示技术筛选Cry2Ab毒素受体表位的方法

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)产生的内毒素具有杀虫活性,Cry2Ab毒素作为Bt棉花的杀虫活性蛋白,其在靶标昆虫体内的结合受体及作用位点尚不清楚,本研究采用噬菌体展示(phage display)的方法,经4轮的"吸附—洗脱—扩繁"筛选,并对阳性克隆所携带的外源DNA片段进行序列测定后,得到2段能够与活化Cry2Ab毒素相互作用的多肽序列,通过酶联免疫结合试验(ELISA)进一步证明,这2段多肽序列与活化Cry2Ab毒素具有较高的亲和力和特异性,结果表明,利用该方法能够由噬菌体随机肽库中高效捕获亲和序列,筛选到与活化Cry2Ab毒素具有高亲和力的多肽,该序列可以模拟Cry2Ab毒素的受体表位,为进一步研究Cry2Ab毒素作用机制奠定了基础,并为今后田间抗性基因频率检测,以及毒素—受体作用机制研究工作提供更有力的技术支持。     

家蚕氨肽酶和类钙粘蛋白与苏云金芽孢杆菌Cry1Ac毒素相互作用

苏云金芽孢杆菌Bacillus thuringiensis生产的晶体毒素被广泛用作农林害虫的杀虫剂。鳞翅目昆虫受体蛋白是阐明其与晶体毒素相互作用的重要模式。文中纯化了苏云金芽孢杆菌的晶体毒素蛋白,质谱鉴定为Cry1Ac毒素,然后重组表达家蚕氨肽酶N (BmAPN6) 和类钙粘蛋白 (CaLP) 结合结构域。利用免疫共沉淀、Far-Western印迹和酶联免疫吸附试验,证明Cry1Ac毒素蛋白和BmAPN6之间的相互作用。在Sf9细胞中,对Cry1Ac毒素的细胞毒活性分析,表明BmAPN6参与Cry1Ac毒素诱导的细胞形态异常和裂解死亡。文中也利用相同的方法,对钙粘蛋白的3个结合位点CR7、CR11和CR12进行相互作用分析,结果表明3个重复结构域是CaLP的Cry1Ac结合位点。上述结果表明,BmAPN6和CaLP可作为Cry1Ac毒素致病的功能性受体,为进一步揭示晶体毒素的致病机制和基因编辑增强家蚕抗病性提供了研究靶标。     

Sodium Solute Symporter and Cadherin Proteins Act as Bacillus thuringiensis Cry3Ba Toxin Functional Receptors in Tribolium castaneum

Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that this protein is not relevant in the Cry3Ba toxin mode of action in Tc. Remarkable features of TcSSS protein were the presence of cadherin repeats in its amino acid sequence and that a TcSSS peptide fragment containing a sequence homologous to a binding epitope found in Manduca sexta and Tenebrio molitor Bt cadherin functional receptors enhanced Cry3Ba toxicity. This is the first time that the involvement of a sodium solute symporter protein as a Bt functional receptor has been demonstrated. The role of this novel receptor in Bt toxicity against coleopteran insects together with the lack of receptor functionality of aminopeptidase N proteins might account for some of the differences in toxin specificity between Lepidoptera and Coleoptera insect orders.     

Structure of Cry2Aa suggests an unexpected receptor binding epitope

BACKGROUND: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. RESULTS: We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. CONCLUSIONS: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.     

Insect resistance to Bacillus thuringiensis: uniform or diverse

Resistance to the insecticidal proteins produced by the soil bacterium Bacillus thuringiensis (Bt) has been documented in more than a dozen species of insect. Nearly all of these cases have been produced primarily by selection in the laboratory, but one pest, the diamondback moth (Plutella xylostella), has evolved resistance in open-field populations. Insect resistance to Bt has immediate and widespread significance because of increasing reliance on Bt toxins in genetically engineered crops and conventional sprays. Furthermore, intense interest in Bt provides an opportunity to examine the extent to which evolutionary pathways to resistance vary among and within species of insect. One mode of resistance to Bt is characterized by more than 500-fold resistance to at least one Cry1A toxin, recessive inheritance, little or no cross-resistance to Cry1C, and reduced binding of at least one Cry1A toxin. Analysis of resistance to Bt in the diamondback moth and two other species of moth suggests that although this particular mode of resistance may be the most common, it is not the only means by which insects can attain resistance to Bt.     

Multidrug transporters and organic anion transporting polypeptides protect insects against the toxic effects of cardenolides

In the struggle against dietary toxins, insects are known to employ target site insensitivity, metabolic detoxification, and transporters that shunt away toxins. Specialized insects across six taxonomic orders feeding on cardenolide-containing plants have convergently evolved target site insensitivity via specific amino acid substitutions in the Na/K-ATPase. Nonetheless, in vitro pharmacological experiments have suggested a role for multidrug transporters (Mdrs) and organic anion transporting polypeptides (Oatps), which may provide a basal level of protection in both specialized and non-adapted insects. Because the genes coding for these proteins are evolutionarily conserved and in vivo genetic evidence in support of this hypothesis is lacking, here we used wildtype and mutant Drosophila melanogaster (Drosophila) in capillary feeder (CAFE) assays to quantify toxicity of three chemically diverse, medically relevant cardenolides.We examined multiple components of fitness, including mortality, longevity, and LD50, and found that, while the three cardenolides each stimulated feeding (i.e., no deterrence to the toxin), all decreased lifespan, with the most apolar cardenolide having the lowest LD50 value. Flies showed a clear non-monotonic dose response and experienced high levels of toxicity at the cardenolide concentration found in plants. At this concentration, both Mdr and Oatp knockout mutant flies died more rapidly than wildtype flies, and the mutants also experienced more adverse neurological effects on high-cardenolide-level diets. Our study further establishes Drosophila as a model for the study of cardenolide pharmacology and solidifies support for the hypothesis that multidrug and organic anion transporters are key players in insect protection against dietary cardenolides.     

昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展

鳞翅目昆虫中肠上皮细胞刷状缘膜(BBM)上的Bt杀虫晶体蛋白毒素受体氨肽酶N(APN)的结构和位点密度的改变是昆虫对Bt毒素的主要抗性机制之一,该文简要综述了APN受体的研究进展。每种昆虫中肠上皮细胞中有数种APNs,彼此间同源性较高,其中部分APNs为crylA家族毒素的功能性受体。不同种类昆虫的APNs受体,甚至同一种昆虫的不同类型APNs,其所结合的毒素种类可能不同。APNs决定该昆虫对crylA类毒素的敏感程度差异。有些抗性昆虫的APNs基因编码区发生了多个点突变。     

Peritrophic membrane contribution to Bt Cry δ-endotoxin susceptibility in Lepidoptera and the effect of Calcofluor

In an effort to determine a reason for differing susceptibilities to Bacillus thuringiensis (Bt) Cry δ-endotoxins amongst lepidopteran species, the peritrophic membrane (PM), and a number of agents that target the PM, were investigated to determine their effect on the efficacy of Bt toxins. In particular Calcofluor is able to disrupt the PM that acts as a barrier to microorganisms. Although Bt toxins have been shown to traverse the PM in some lepidopteran species, new data shows that toxins can bind the PM. Lepidopteran larval PMs also vary in thickness and composition that may determine the passage of Bt toxins. In non-susceptible insects the toxin associates with PM proteins and frass and is thought to be retained by the PM and then excreted before binding to the exposed target midgut membrane. However, the addition of Calcofluor to Bt toxins at an LC₅₀ for the recipient species did not result in an increase in the efficacy of the toxin. It is evident that Calcofluor does disrupt the PM but the toxin preferentially binds PM fragments and is excreted instead of binding the exposed target midgut membrane, therefore having little toxic effect. This study therefore concludes that Calcofluor is not as suitable as other toxin enhancing agents such as chitinase.     

Fitness of Bt‐resistant cabbage loopers on Bt cotton plants

Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt‐resistant insects on Bt and non‐Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt‐resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt‐resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non‐Bt cotton and on transgenic Bt cotton leaves expressing a single‐toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R₀) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4‐day asynchrony of adult emergence between the susceptible T. ni grown on non‐Bt cotton leaves and the dual‐toxin‐resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.     

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.     

Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.     

转Bt基因玉米的生态安全性研究进展

随着转基因作物的应用和推广 ,转 Bt基因作物释放后对生态环境及其它方面产生的潜在影响越来越受到重视。分别从生物活性杀虫晶体蛋白在土壤中的残留特性、杀虫晶体蛋白对土壤中非目标生物的影响、转 Bt基因玉米植株体成分的变化、转Bt基因玉米花粉中杀虫晶体蛋白的表达特性及其在田间和马力筋叶片上的散积状况、花粉中表达的杀虫晶体蛋白对君主斑蝶的毒性、君主斑蝶幼虫暴露在 Bt花粉中的概率及综合风险评价估算等方面对转 Bt基因玉米产生的杀虫晶体蛋白与土壤生态环境的相互作用、花粉对非目标生物影响的研究现状进行了综述。通过对转 Bt基因作物生态安全性的科学评价和广泛宣传 ,以确保生物技术的健康发展。