Bt Cry毒素抗虫模拟物靶向创新设计

Bt Cry毒素是当前研究最深入、应用最广的生物抗虫蛋白，对农业害虫的绿色防治发挥了重大作用。然而，随着其制剂和转基因抗虫作物的广泛应用，由此驱动诱发的靶标害虫抗药性及潜在生态安全风险等问题日益凸显。探寻具备模拟Bt Cry毒素杀虫功能的新型抗虫蛋白材料，不仅可为农作物持续健康生产保驾护航，也能在一定程度上缓解靶标害虫对Bt Cry毒素的抗药性压力。近年来，笔者团队以抗体“免疫网络学说(immune network theory)”中Ab2β类型抗独特型抗体(anti-idiotype antibody, Anti-Id)具备模拟抗原结构和功能的特性为理论依据，借助噬菌体展示抗体库及特异性抗体高通量筛选与鉴定技术，设计Bt Cry毒素抗体为包被靶点抗原，从噬菌体抗体库中靶向筛选到了一系列具备模拟Bt Cry毒素抗虫功能的Ab2β类型抗独特型抗体(即Bt Cry毒素抗虫模拟物)，其中活性最强的Bt Cry毒素抗虫模拟物对靶标害虫的致死率接近相应原Bt Cry毒素的80%，初步实现了Bt Cry毒素抗虫模拟物的靶向设计。本文从理论依据、技术条件、研究现状等方面进行系统概述，并就相关技术发展...     

Cry毒素杀虫活性分子改造方法研究进展

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)产生的Cry毒素为防治害虫提供了一种宝贵的资源。但昆虫中肠蛋白酶的活化作用在不同的昆虫中对Cry毒素的杀虫活性有限制性。分析了几种不同的策略来增加Cry毒素对靶标昆虫的毒性,包括结构域Ⅲ交换、结构域Ⅱ和结构域Ⅲ的突变以及其他突变。此外,还详细阐述了噬菌体展示技术进行毒素优化的方法,毒素优化能够增加Cry毒素对亲本Cry毒素敏感度较低的害虫的杀虫活性,以期为Cry毒素的分子改造和应用提供参考。     

棉铃虫中肠氨肽酶APN4与Cry1Ac、Cry2Aa结合能力的比较

【目的】Bt杀虫蛋白(Bacillus thuringiensis)具有高度的靶标特异性,已经被广泛用于农业害虫防治。Bt杀虫蛋白要发挥杀虫活性,必须首先与其受体蛋白结合,氨肽酶N(Aminopeptidase N)是一类重要的Bt受体蛋白。因此,分析该受体与Bt杀虫蛋白的结合能力,可为进一步明确不同Bt的分子作用机制、Bt的抗性治理以及新Bt的开发应用等提供借鉴。【方法】本文利用Ligand blot和Elisa方法比较了棉铃虫Helicoverpa armigera中肠APN4(Aminopeptidase N4,APN4)与Cry1Ac、Cry2Aa的结合能力。【结果】原核表达的APN4片段与活化的Cry1Ac、Cry2Aa都可以结合,解离常数(Kd)分别是48.59 nmol/L和21.73 nmol/L。【结论】APN4片段与Cry1Ac、Cry2Aa的结合能力在数量级上不存在显著性差异。     

二化螟APN1的原核表达及其与Cry2Aa蛋白的结合特性研究

【目的】Bt杀虫蛋白发挥杀虫活性的重要前提是Cry蛋白能够与昆虫中肠上皮细胞刷状缘膜囊(BBMVs)上的受体蛋白结合。在前期获得二化螟氨肽酶N1(Aminopeptidase N,APN1)基因全长序列的基础上,明确二化螟APN1多肽片段与Cry2Aa的结合能力。【方法】将二化螟APN1序列片段在大肠杆菌BL21(DE3)中表达,利用蛋白质单向电泳和ligand blotting技术分析二化螟APN1多肽片段与Cry2Aa的结合能力。【结果】重组载体可在表达菌株BL21(DE3)中表达一个约70 ku的蛋白,纯化后的多肽条带单一,纯度较好。Ligand blot分析结果显示,表达的二化螟APN1多肽片段可以与活化的Cry2Aa杀虫蛋白结合,且结合条带随着重组蛋白上样量的降低而减弱。【结论】APN1多肽片段可以与Cry2Aa结合,为阐明APN1基因的功能奠定基础,也为其他Bt蛋白的受体蛋白相关研究提供新的借鉴。     

Cry1A毒素与二化螟中肠刷状缘膜囊泡受体蛋白配基结合分析

分离和鉴定二化螟Chilo suppresalis幼虫中肠刷状缘膜囊泡（BBMV）中Cry1A毒素的受体蛋白,对于阐明Cry1A毒素作用机理和二化螟抗性机理具有十分重要的意义。为此,本文就Cry1A毒素对二化螟杀虫活性及Cry1Ac与二化螟中肠受体的配基结合进行了研究。结果表明: Cry1Ab对二化螟室内品系(CN)的毒力高于Cry1Ac,而Cry1Ac高于Cry1Aa。配基结合分析表明二化螟CN品系幼虫中肠BBMV中有6个Cry1Ac结合蛋白(分子量分别为50,70,90,120,160和180 kDa), 其中180,160和90 kDa结合蛋白的条带颜色明显深于其他结合蛋白的条带,表明这3个受体蛋白具有较高的结合浓度。同源竞争结合研究表明,180和90 kDa结合蛋白为Cry1Ac的低亲合性结合蛋白,其他4个为高亲合性结合蛋白。为了研究Cry1Ac和Cry1Ab受体结合部位的相互作用,进行了异源竞争结合研究。Cry1Ab可以与Cry1Ac所有的6个结合蛋白进行竞争性结合,与180,120,70和50 kDa结合蛋白具有高亲合性,而与160和90 kDa结合蛋白具有低亲合性。结果显示,Cry1Ac与Cry1Ab在二化螟幼虫中肠BBMV上拥有多个共享的结合位点,但对每个结合位点的亲合性有差异。基于毒素结合部位的相似性,Cry1Ac和Cry1Ab不宜同时用于转基因Bt水稻来控制二化螟。     

Cry毒素自然进化与作用机制研究进展

苏云金芽胞杆菌(简称Bt)产生的杀虫Cry毒素被广泛的运用于转基因作物害虫的有效防治。在Cry毒素家族中,Cry毒素的三维结构域引导它们自然进化,并产生大量结构和行为方式相似但在不同的昆虫中表现出不同特异性的Cry蛋白,同时Cry毒素家族也是对昆虫特异性分子研究的基础。简述了Bt本身及其发展历程,还详细阐述了Cry毒素在自然中完成昆虫特异性的进化及其作用机制。最后,对于Cry毒素杀虫活性的进化提出了一般性的策略。     

鳞翅目昆虫中肠Bt杀虫蛋白受体研究进展

杀虫晶体蛋白(insecticidal crystal proteins,ICPs;含有Cry和Cyt 2大家族)和营养期杀虫蛋白(vegetative insecticidal proteins,Vips)等Bt杀虫蛋白可有效防治鳞翅目害虫,其中Cry应用最广泛。然而,一些地区的鳞翅目害虫已对Bt杀虫蛋白产生了抗性。目前,普遍认为鳞翅目昆虫中肠受体与Bt杀虫蛋白结合能力的改变是导致其对Bt杀虫蛋白产生抗性的最主要因素。在鳞翅目昆虫中,Cry受体是研究得最为透彻的Bt受体,已经被证实的有氨肽酶N、钙黏蛋白、碱性磷酸酶和ABC转运蛋白等。Vips杀虫蛋白类与鳞翅目昆虫中肠受体的结合方式与Cry杀虫蛋白相似,但结合位点与Cry杀虫蛋白不同。本文从结构特点、作用机制及不同鳞翅目昆虫间的表达差异等角度对以上4种鳞翅目昆虫中肠Bt受体进行了综述,并提出如下展望:(1)以棉铃虫或小菜蛾等鳞翅目昆虫为农业害虫模式生物进行深入研究,阐明其对Bt杀虫蛋白产生抗性的机制,为研究其他鳞翅目农业害虫对Bt杀虫蛋白产生抗性的机制提供理论借鉴;(2)鉴于在不同鳞翅目昆虫间,中肠Bt受体与Bt杀虫蛋白结合存在差异,且同一Bt杀虫蛋白与鳞翅目昆虫Bt受体并不专一性结合,Bt杀虫蛋白多基因组合策略是较为有效的田间鳞翅目昆虫防治策略,是今后一段时间内Bt杀虫蛋白应用的发展方向。     

棉铃虫对不同Bt蛋白的抗性及交互抗性研究

【目的】棉铃虫Helicoverpa armigera(Hübner)对Bt(Bacillus thuringiensis)的抗性问题是阻碍Bt抗虫棉持续发展的关键,探究棉铃虫对不同Bt蛋白的抗性演化趋势及交互抗性,可为选择合适的抗虫棉花备选基因提供参考。【方法】在室内通过连续多代筛选,获得了5个对Bt具有不同抗性的品系,利用饲料混合法测定了这些品系棉铃虫的交互抗性。【结果】经筛选后棉铃虫对Cry1Ac产生了超高水平的抗性、对Cry2Ab、Vip3Aa产生了超低抗性(耐性);Cry1Ac抗性棉铃虫停止筛选而换成Cry2Ab或Vip3Aa继续筛选后,棉铃虫对Cry1Ac抗性水平明显降低,对Cry2Ab或Vip3Aa产生低抗或超低抗性(耐性)。Cry1Ac抗性棉铃虫对Cry1Ab敏感性显著降低,而对Cry2Ab、Cry2Ah及Vip3Aa敏感性变化不显著;Cry2Ab抗性品系棉铃虫对Cry1Ac敏感性显著降低;Vip3Aa抗性品系棉铃虫对Cry1Ac敏感性变化不显著。说明Cry1Ac与Cry1Ab间存在明显交互抗性,与Cry2Ah、Vip3Aa没有交互抗性;而Cry2Ab与Cry1Ac间存在不对称的交互抗性。【结论】在筛选新杀虫基因或叠加基因时,应充分考虑抗性发展及交互抗性问题,Cry2Ah、Vip3Aa是治理Cry1Ac抗性棉铃虫或与Cry1Ac叠加最优的选择。     

Cry1Ac和Cry2Ab杀虫蛋白对粘虫幼虫生长发育的影响

【目的】为探究Bt杀虫蛋白对次要靶标害虫粘虫Mythimna separata (Walker)(鳞翅目:夜蛾科)的杀虫活性及对其生长发育的影响。【方法】本文通过浸叶法饲喂初孵及2龄末粘虫不同剂量的Cry1Ac及Cry2Ab杀虫蛋白后,观察其死亡率,称量幼虫重,并统计了幼虫历期、化蛹率、蛹重、蛹期、蛹的羽化率、畸形率等指标。【结果】初孵幼虫取食浸泡含16、64、128μg/mLCry1Ac及Cry2Ab的玉米叶片后,随着时间的延长及浓度的增加,死亡率逐渐增加,且Cry1Ac杀虫蛋白对粘虫的生物活性高于Cry2Ab蛋白,在128μg/mL浓度下,取食Cry1Ac和Cry2Ab蛋白13d时的死亡率分别达到了65%及60%。取食两种蛋白后,初孵幼虫和2龄末幼虫重量均受到显著抑制,短期取食两种蛋白对幼虫历期、化蛹率、蛹重、蛹期、蛹的羽化率、畸形率没有影响。【结论】取食Cry1Ac和Cry2Ab杀虫蛋白后,对初孵幼虫有很好的杀虫活性,且Cry1Ac杀虫活性高于Cry2Ab杀虫蛋白;短期饲喂两种杀虫蛋白时,对2龄粘虫后期生长影响不大。本文结果为转Bt基因作物更好的应用于粘虫的防治提供了理论基础。     

苏云金芽孢杆菌B-Pr-88菌株中cry2Ab4基因的表达和杀虫活性研究

以我室自行分离的对鳞翅目夜蛾科害虫具有高毒力的Bt菌株B-Pr-88为材料,用PCR-RFLP方法从其质粒DNA文库中筛选到含cry2Ab基因的一个阳性克隆pZF858,序列测定发现,该片段含有cry2Ab全长基因,开放读码框为1902bps,编码由633个氨基酸组成的70.7kD蛋白,氨基酸同源性与已公布的cry2Ab基因同源性均为99.8%,经Bt基因国际命名委员会正式命名为cry2Ab4。根据cry2Ab4基因开放阅读框架(ORF)两端序列,设计合成一对特异引物L2ab5和L2ab3,PCR扩增获得cry2Ab4完整ORF,与大肠杆菌表达载体pET-21b连接,构建了重组表达质粒pET-2Ab4,质粒导入大肠杆菌BL21(DE3),IPTG诱导后,SDS-PAGE电泳证实该基因表达了60kD的蛋白,生物测定表明,Cry2Ab4对棉铃虫和大豆食心虫具有高毒力,同时对小菜蛾和二化螟有一定的杀虫活性,而对亚洲玉米螟和甜菜夜蛾没有杀虫活性。     

Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with ¹²⁵I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with ¹²⁵I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K_d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R_t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

Molecular basis for Bacillus thuringiensis Cry1Ab toxin specificity: two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops alpha-8 and 2 in domain II of Cy1Ab toxin

The identification of epitopes involved in Cry toxin-receptor interaction could provide insights into the molecular basis of insect specificity and for designing new toxins to overcome the potential problem of insect resistance. In previous works, we determined that the Manduca sexta Cry1A cadherin-like receptor (Bt-R(1)) interacts with Cry1A toxins through epitope (865)NITIHITDTNN(875) and by loop 2 of domain II in the toxin (Gomez, I., Miranda-Rios, J., Rudi?o-Pi?era, E., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2002) J. Biol. Chem. 277, 30137-30143.). In this work, we narrowed to 12 amino acids a previously identified Bt-R(1) 66 amino acids epitope (Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S. A., Midbo, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Insect Biochem. Mol. Biol. 32, 1025-1036) and identified loop alpha-8 of Cry1Ab domain II as its cognate binding epitope. Two amino acid Bt-R(1) toxin binding regions of 70 residues, one comprised of residues 831-900 containing the (865)NITIHITDTNN(875) epitope (TBR1) and the other comprised of residues 1291-1360 (TBR2) were cloned by RT-PCR and produced in Escherichia coli. Cry1A toxins bind with the two TBR regions in contrast with the nontoxic Cry3A toxin. The loop 2 synthetic peptide competed with the binding of Cry1Ab toxin to both TBR regions in contrast to the alpha-8 synthetic peptide that only competed with Cry1Ab binding to TBR2. Western blots and competition ELISA analysis showed that the Cry1Ab loop 2 RR368-9EE mutant did not show observable binding to TBR1 but still bound the TBR2 peptide. This result suggests that loop alpha-8 interacts with the TBR2 region. Competition ELISA analysis of Cry1Ab binding to the two TBR peptides revealed that the toxin binds the TBR1 region with 6-fold higher affinity than the TBR2 region. The amino acid sequence of TBR2 involved on Cry1Ab interaction was narrowed to 12 amino acids, (1331)IPLPASILTVTV(1342), by using synthetic peptides as competitors for Cry1Ab binding to Bt-R(1). Our results show that the specificity of Cry1A involves at least two structural determinants on both molecules.     

Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with (125)I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K(com)] = 1.1 nM) for (125)I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for (125)I-Cry1Ab binding sites, though the K(com) values ranged from 179 to 304 nM. Cry1Ab competed for (125)I-Cry1Ac binding sites (K(com) = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the (125)I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.     

Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R(1) in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis

Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.     

Hydropathic complementarity determines interaction of epitope (869)HITDTNNK(876) in Manduca sexta Bt-R(1) receptor with loop 2 of domain II of Bacillus thuringiensis Cry1A toxins

In susceptible insects, Cry toxin specificity correlates with receptor recognition. In previous work, we characterized an scFv antibody (scFv73) that inhibits binding of Cry1A toxins to cadherin-like receptor. The CDR3 region of scFv73 shared homology with an 8-amino acid epitope ((869)HITDTNNK(876)) of the Manduca sexta cadherin-like receptor Bt-R(1) (Gomez, I., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2001) J. Biol. Chem. 276, 28906-28912). In this work, we show that the previous sequence of scFv73 CDR3 region was obtained from the noncoding DNA strand. However, most importantly, both scFv73 CDR3 amino acid sequences of the coding and noncoding DNA strands have similar binding capabilities to Cry1Ab toxin as Bt-R(1) (869)HITDTNNK(876) epitope, as demonstrated by the competition of scFv73 with binding to Cry1Ab with synthetic peptides with amino acid sequences corresponding to these regions. Using synthetic peptides corresponding to three exposed loop regions of domain II of Cry1Aa and Cry1Ab toxins, we found that loop 2 synthetic peptide competed with binding of scFv73 to Cry1A toxins in Western blot experiments. Also, loop 2 mutations that affect toxicity of Cry1Ab toxin are affected in scFv73 binding. Toxin overlay assays of Cry1A toxins to M. sexta brush border membrane proteins showed that loop 2 synthetic peptides competed with binding of Cry1A toxins to cadherin-like Bt-R(1) receptor. These experiments identified loop 2 in domain II of as the cognate binding partner of Bt-R(1) (869)HITDTNNK(876). Finally, 10 amino acids from beta-6-loop 2 region of Cry1Ab toxin ((363)SSTLYRRPFNI(373)) showed hydropathic pattern complementarity to a 10-amino acid region of Bt-R(1) ((865)NITIHITDTNN(875)), suggesting that binding of Cry1A toxins to Bt-R(1) is determined by hydropathic complementarity and that the binding epitope of Bt-R(1) may be larger than the one identified by amino acid sequence similarity to scFv73.     

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins.     

Interaction of Bacillus thuringiensis δ-endotoxins with Midgut Brush Border Membrane Vesicles of Helicoverpa armigera

Pesticidal activity of different Bacillus thuringiensis (Bt) δ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac and Cry2A, were investigated against Helicoverpa armigera infesting cotton crop worldwide. Cry1Ac toxin was found to be the most potent toxin towards H. armigera. All selected Bt toxins were found stable in vitro processing by midgut juice of H. armigera. Saturation and competition binding experiments were performed with iodine-125 labeled proteins and brush border membrane vesicles prepared from the midgut of H. armigera. The results show saturable, specific and high affinity of all toxins except for Cry2A. Both the toxins were bound with low binding affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab and Cry1Ac recognized or share the same binding site which is different from that of Cry2A. The data suggest that development of multiple toxin system in transgenic plants with toxin pyramiding, which recognize different binding sites, may be useful in the deployment strategies to decrease the rate of pest adaptation to Bt toxins in transgenic plants.     

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.     

Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre)

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with (125)I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC(3)(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.