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。     

Geographic variation in susceptibility of Chilo suppressalis (Lepidoptera: Pyralidae) to Bacillus thuringiensis toxins in China

Geographic variation in the susceptibility of the striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), in China to Bacillus thuringiensis (Bt) insecticidal crystal proteins Cry1Ac and Cry1Ab was studied to establish baseline information for comparing the future response of populations with increased exposure to Bt products. Rice is the major host of C. suppressalis, and Bt rice ma) be released in China in the near future. Twelve populations of the pest were collected from the major rice-growing regions of China. LC50 estimates were determined for all populations for Cry1Ac and for eight populations for Cry1Ab. The bioassay results indicated that the range of LC50 in neonate larvae to Cry1Ac and Cry1Ab was from approximately 15 to approximately 157 mg (AI)/L and approximately 2 to approximately 34 mg (AI)/L, respectively. LC50 values were lower for Cry1Ab than for Cry1Ac, and there was a significant positive correlation between the two toxins tested.     

Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae)

Ten transgenic Bacillus thuringiensis Bt rice, Oryza sativa L., lines with different Bt genes (two Cry1Ac lines, three Cry2A lines, and five Cry9C lines) derived from the same variety Minghui 63 were evaluated in both the laboratory and the field. Bioassays were conducted by using the first instars of two main rice lepidopteran insect species: yellow stem borer, Scirpophaga incertulas (Walker) and Asiatic rice borer, Chilo suppressalis (Walker). All transgenic lines exhibited high toxicity to these two rice borers. Field evaluation results also showed that all transgenic lines were highly insect resistant with both natural infestation and manual infestation of the neonate larvae of S. incertulas compared with the nontransformed Minghui63. Bt protein concentrations in leaves of 10 transgenic rice lines were estimated by the sandwich enzyme-linked immunosorbent assay. The cry9C gene had the highest expression level, next was cry2A gene, and the cry1Ac gene expressed at the lowest level. The feeding behavior of 7-d-old Asiatic rice borer to three classes of Bt transgenic rice lines also was detected by using rice culm cuttings. The results showed that 7-d-old larvae of Asiatic rice borer have the capacity to distinguish Bt and non-Bt culm cuttings and preferentially fed on non-Bt cuttings. When only Bt culm cuttings with three classes of different Bt proteins (CrylAc, Cry2A, and Cry9C) were fed, significant distribution difference of 7-d-old Asiatic rice borer in culm cuttings of different Bt proteins also was found. In the current study, we evaluate different Bt genes in the same rice variety in both the laboratory and the field, and also tested feeding behavior of rice insect to these Bt rice. These data are valuable for the further development of two-toxin Bt rice and establishment of appropriate insect resistance management in the future.     

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.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker)

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.     

Determination of Binding of Bacillus thuringiensis (delta)-Endotoxin Receptors to Rice Stem Borer Midguts

Insecticidal activity and receptor binding properties of Bacillus thuringiensis toxins to yellow and striped rice stem borers (Sciropophaga incertulas and Chilo suppresalis, respectively) were investigated. Yellow stem borer (YSB) was susceptible to Cry1Aa, Cry1Ac, Cry2A, and Cry1C toxins with similar toxicities. To striped stem borer (SSB), Cry1Ac, Cry2A, and Cry1C were more toxic than Cry1Aa toxin. Binding assays were performed with (sup125)I-labeled toxins (Cry1Aa, Cry1Ac, Cry2A, and Cry1C) and brush border membrane vesicles (BBMV) prepared from YSB and SSB midguts. Both Cry1Aa and Cry1Ac toxins showed saturable, high-affinity binding to YSB BBMV. Cry2A and Cry1C toxins bound to YSB BBMV with relatively low binding affinity but with high binding site concentration. To SSB, both Cry1Aa and Cry1Ac exhibited high binding affinity, although these toxins are less toxic than Cry1C and Cry2A. Cry1C and Cry2A toxins bound to SSB BBMV with relatively low binding affinity but with high binding site concentration. Heterologous competition binding assays were performed to investigate the binding site cross-reactivity. The results showed that Cry1Aa and Cry1Ac recognize the same binding site, which is different from the Cry2A or Cry1C binding site in YSB and SSB. These data suggest that development of multitoxin systems in transgenic rice with toxin combinations which recognize different binding sites may be useful in implementing deployment strategies that decrease the rate of pest adaptation to B. thuringiensis toxin-expressing rice varieties.     

DIVERSITY IN GUT MICROFLORA OF Helicoverpa armigera POPULATIONS FROM DIFFERENT REGIONS IN RELATION TO BIOLOGICAL ACTIVITY OF Bacillus thuringiensis δ‐ENDOTOXIN Cry1Ac

Transgenic crops expressing toxin proteins from Bacillus thuringiensis (Bt) have been deployed on a large scale for management of Helicoverpa armigera. Resistance to Bt toxins has been documented in several papers, and therefore, we examined the role of midgut microflora of H. armigera in its susceptibility to Bt toxins. The susceptibility of H. armigera to Bt toxin Cry1Ac was assessed using Log‐dose‐Probit analysis, and the microbial communities were identified by 16S rRNA sequencing. The H. armigera populations from nine locations harbored diverse microbial communities, and had some unique bacteria, suggesting a wide geographical variation in microbial community in the midgut of the pod borer larvae. Phylotypes belonging to 32 genera were identified in the H. armigera midgut in field populations from nine locations. Bacteria belonging to Enterobacteriaceae (Order Bacillales) were present in all the populations, and these may be the common members of the H. armigera larval midgut microflora. Presence and/or absence of certain species were linked to H. armigera susceptibility to Bt toxins, but there were no clear trends across locations. Variation in susceptibility of F₁ neonates of H. armigera from different locations to the Bt toxin Cry1Ac was found to be 3.4‐fold. These findings support the idea that insect migut microflora may influence the biological activity of Bt toxins.     

Binding of Bacillus thuringiensis Cry1 Toxins to the Midgut Brush Border Membrane Vesicles of Chilo suppressalis (Lepidoptera: Pyralidae): Evidence of Shared Binding Sites

Binding and competition among Cry1Aa, Cry1Ac, and Cry1Ba toxins were analyzed quantitatively in vitro by using (sup125)I-labeled activated toxins and brush border membrane vesicles isolated from Chilo suppressalis larval midguts. The three toxins bound specifically to the midgut brush border membrane vesicles. Direct binding experiments showed that Cry1Aa and Cry1Ba recognized a single class of binding sites with different affinities, whereas Cry1Aa recognized two classes of binding sites, one with a high affinity and a low concentration and the other with a lower affinity but higher concentration. Competition experiments showed that toxins Cry1Ac and Cry1Ba shared a binding site in the C. suppressalis midgut membranes and that this site was also the low-affinity binding site for Cry1Aa.     

水稻螟虫神经肽PBAN及其受体序列的生物信息学分析

【目的】性信息素合成激活肽(PBAN)是控制昆虫产生性信息素的激素,本文旨在分析水稻螟虫神经肽PBAN及其受体的序列。【方法】通过t Blastn同源检索从水稻螟虫基因组和转录组数据库中鉴定水稻螟虫PBAN神经肽及其受体序列,在此基础上进行序列比对及系统发生分析。【结果】发现二化螟Chilo suppressalis、三化螟Tryporyza incertulas和大螟Sesamia inferens的PBAN成熟肽序列均含有33个氨基酸残基,其C端五肽序列完全相同,3种水稻螟虫PBAN多肽相似度为54.55%~63.64%;发现二化螟PBAN受体3个异构体全长氨基酸序列(PBANR-A、PBANR-B和PBANR-C),均含有7个跨膜区域。【结论】进化树分析发现不同昆虫PBAN神经肽及其受体存在一定的保守性和多样性,并且在进化树上的位置几乎与昆虫系统发育分类一致,推测PBAN神经肽和PBAN受体在昆虫系统进化过程中可能存在协同进化现象。本研究为水稻螟虫PBAN神经肽及其受体的结构和功能分析提供基础。     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins’ mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD₅₀ concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.     

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: identification of proteins binding the delta-endotoxin, Cry1Ac of Bacillus thuringiensis

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) delta-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.     

Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) for detecting novel Bt toxins

Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) was used to study crystal (Cry) toxins from different Bacillus thuringiensis (Bt) strains. Known Cry toxins such as Cry1Ac and Cry2A, as well as novel toxins for which the protein sequences were predicted by their gene sequences, were used as controls in this study. The peptide masses, obtained after in-gel trypsin digestion for all these proteins, matched correctly to the corresponding proteins. Also, MALDI-TOF MS was able to resolve and identify multiple Cry toxins of very similar molecular weights and highly similar isoelectric points, from a single protein band. Furthermore, in novel Bt strains for which PCR techniques were unable to detect the cognate genes, this method was able to detect novel Cry toxins. Hence, present data clearly suggest that MALDI-TOF MS could be used as a tool for identifying Cry toxins from novel Bt strains.     

Effectiveness in the field of Bt rice lines against target pests under various cultural regimes

China has a long history of rice cultivation, incorporating several cultural practices known to influence damage by insect pests. Transgenic Bt rice expresses lepidopteran‐specific insecticidal proteins that primarily target lepidopteran insect pests. However, the effectiveness of Bt rice against target insect pests under different cultural regimes has not been evaluated. In this study, the effectiveness of Bt rice lines against rice leaffolder, Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), and striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), was evaluated under various transplanting densities, crop establishment methods, and planting times. The results showed that Bt rice lines (T2A‐1 and T1C‐19, containing Cry2A and Cry1C, respectively) could prevent damage by these target pests under a range of cultural practices. Injury by C. medinalis or C. suppressalis on rice did not differ with the rice lines under various transplanting densities. Direct‐seeded non‐Bt rice MH63 suffered heavier injury by C. medinalis and C. suppressalis than it did with transplanting, whereas injury to the two Bt rice lines did not differ with planting methods. Planting time significantly affected injury by C. medinalis or C. suppressalis on non‐Bt rice, whereas injury to Bt rice lines did not differ with planting time. These results suggest that transplanting density, planting method, and planting time did not significantly affect the resistance of two Bt rice lines, due to their high insecticidal activity against target insects.     

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.     

Toxicity and Receptor Binding Properties of Bacillus thuringiensisδ-Endotoxins to the Midgut Brush Border Membrane Vesicles of the Rice Leaf Folders, Cnaphalocrocis medinalis and Marasmia patnalis

Pesticidal activity and receptor-binding properties of Bacillus thuringiensis toxins to rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis, were investigated. Saturation and competition binding experiments were done with iodine (¹²⁵1)-labeled Bt proteins and brush border membrane vesicles prepared from the midgut of C. medinalis and M. patnalis. The results show saturable, specific, and high-affinity binding of all toxins except Cry2A toxin. Cry1Aa and Cry2A toxins were bound with low affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab, and Cry1Ac recognized or shared the same binding site that is different from the binding site for Cry2A toxin. Iodine (¹²⁵I)-labeled Cry1Ac and Cry1Ab toxins were used in ligand blot experiments to detect specific binding proteins in brush border membrane vesicles of C. medinalis and M. patnalis. Cry1Ab toxin protein binds to 205-kDa and 200-kDa proteins respectively in case of C. medinalis and M. patnalis. The apparent molecular mass of the protein bound to labeled Cry1Ac toxins was identified as a 120-kDa protein in both C. medinalis and M. patnalis. Received: 10 April 2000 / Accepted: 23 May 2000     

Synergistic activity between Bacillus thuringiensis Cry1Ab and Cry1Ac toxins against maize stem borer (Chilo partellus Swinhoe)

Aim: To select a toxin combination for the management of maize stem borer (Chilo partellus) and to understand possible mechanism of synergism among Bacillus thuringiensis Cry1A toxins tested. Methods and Results: Three Cry1A toxins were over expressed in Escherichia coli strain JM105 and used for diet overlay insect bioassay against C. partellus neonate larvae, both alone and in combinations. Probit analysis revealed that the three Cry1A toxins tested have synergistic effect against C. partellus larvae. In vitro binding analysis of fluorescein isothiocyanate (FITC)‐labelled Cry1A toxins to midgut brush border membrane vesicle (BBMV) shows that increase in toxicity is directly correlated to an increase in binding of toxin mix. Conclusions: A high Cry1Ac to Cry1Ab ratio leads to an increase in efficacy of these toxins towards C. partellus larvae and this increase in toxicity comes from an increase in toxin binding. Significance and Impact of the Study: Use of Cry1Ab and Cry1Ac combination could be an effective approach to control C. partellus. Furthermore, we show it first time that possible reason behind increase in toxicity of synergistic Cry1A proteins is an increase in toxin binding.