Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for pest resistance management in China.     

棉铃虫幼虫取食Vip3Aa蛋白后的中肠组织病理变化

为了进一步明确Vip3Aa的作用机制, 利用透射电镜观察了棉铃虫4龄幼虫取食含Vip3Aa蛋白饲料后中肠杯状细胞的病理变化, 并比较了其病变与取食含Cry1Ac饲料后棉铃虫组织病变的差异。取食含Vip3Aa饲料后, 棉铃虫幼虫的中肠杯状细胞逐渐发生病变, 主要表现为： 微绒毛肿胀、 脱落; 细胞核核膜界限不清晰, 染色质分布不均匀; 线粒体变形、 数量减少, 内脊不清晰; 内质网杂乱不规则、 数量减少。与取食Cry1Ac的棉铃虫相比, 取食Vip3Aa的棉铃虫中肠杯状细胞发生病变较为缓慢, 在取食12 h后才发现明显病变, 随着取食时间的增加病变越来越明显; 而取食Cry1Ac的棉铃虫2 h后中肠杯状细胞就出现明显病变。本研究可为Vip3Aa作为新毒素策略的重要蛋白在棉铃虫Helicoverpa armigera综合防治中更好地发挥作用提供理论依据。     

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.     

Vip3Aa及Cry1Ac对棉铃虫幼虫多种酶活力的影响

为了明确Vip3Aa的作用机制,为其作为新毒素策略重要蛋白的应用提供理论依据,本文比较了Vip3Aa、Cry1Ac对棉铃虫Helicoverpa armigera(Hübner)主要蛋白酶、解毒酶、APN活性的影响,并研究了Vip3Aa和Cry1Ac共同使用对几种酶活力的作用。室内生测结果表明,Vip3Aa对棉铃虫的杀虫效果低于Cry1Ac,但Vip3Aa对棉铃虫幼虫生长有明显的抑制作用。取食含Cry1Ac、Vip3Aa或Cry1Ac+Vip3Aa饲料的棉铃虫,总蛋白酶和类胰凝乳蛋白酶活性很快升高;但经Cry1Ac处理12 h后这2种酶活性与对照差异不显著或低于对照,而取食含Vip3Aa饲料的棉铃虫酶活力显著高于对照的时间明显延长,而且类胰蛋白酶活性也显著高于对照;表明Cry1Ac降解速度比Vip3Aa快,可能是由于降解2种蛋白参与的酶系存在差异,同时Cry1Ac+Vip3Aa混用可以延长蛋白被酶解的时间。谷胱甘肽S-转移酶和α-乙酸萘酯酶活性在棉铃虫取食含Vip3Aa、Cry1Ac或Cry1Ac+Vip3Aa蛋白的饲料后活性升高,说明这2种酶可能参与了对Cry1Ac、Vip3Aa的解毒作用。但Cry1Ac、Vip3Aa对氨肽酶活性影响不大,可能在毒蛋白发挥毒性的过程中与氨肽酶活力变化无关。     

Frequency of Bt resistance alleles in Helicoverpa armigera in the Xinjiang cotton-planting region of China

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a key insect pest of cotton in Xinjiang cotton-planting region of northwest China. In this region, cotton is grown on average ≈ 1.65 million ha (1.53 ≈ 1.80 million ha) annually in largely monoculture agricultural landscapes, similarly to cropping systems in the United States or Australia. Under such cropping regimes, naturally occurring refuges (with non-Bt crops) may be insufficient to prevent H. armigera resistance development to Bt toxins. Therefore, we assessed frequency of alleles conferring resistance to Cry1Ac toxin of F(1) and F(2) offspring of H. armigera isofemale lines from two distinct localities in the region during 2005-2009. More specifically, a total of 224 isofemale lines was collected from Korla County (≈ 70% Bt cotton adoption) and 402 lines from Shache County (≈ 5% Bt cotton planting). Subsequent offspring was screened on Cry1Ac artificial diet. From 2005 to 2009, resistance gene frequency in Korla fluctuated between 0.0000 and 0.0040, while being 0.0000-0.0008 in individuals collected from Shache, and there were no significant increases in both counties from 2005 to 2009. Relative average development rates (RADRs) of larvae in F(1) tests showed significant increases from Korla, but not in Shache. RADR of F(1) larvae is significantly correlated with RADR of F(2) offspring, indicating genetic variation in response to toxin in field H. armigera population. Although the occurrence of Cry1Ac resistance alleles was low in Xinjiang cotton-planting region of China, particular attention should be given to H. armigera resistance development in Korla County.     

Monitoring and management strategy for Helicoverpa armigera resistance to Bt cotton in China

The cotton bollworm, Helicoverpa armigera, is one of the most important insect pests in cotton growing regions of China. Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis (Bt) has been deployed for combating cotton bollworm since 1997. Natural refugees derived from the mixed planting system consisting of cotton, corn, soybean, vegetables, peanut and others on single-family farms of a small scale were used for delaying the evolution of resistance to Bt cotton. Susceptibility of H. armigera field populations to the Bt insecticidal protein Cry1Ac was monitored from 1997 to 2006. The results indicate that the field populations are still susceptible to Cry1Ac, and monitoring indication no apparent shifts in susceptibility in field populations of this important pest.     

Early detection of field-evolved resistance to Bt cotton in China: cotton bollworm and pink bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some major insect pests, but pests can evolve resistance and thereby reduce the effectiveness of such Bt crops. The main approach for slowing pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to cotton producing Bt toxin Cry1Ac, several countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. This strategy is designed for cotton bollworm (Helicoverpa armigera), which attacks many crops and is the primary target of Bt cotton in China, but it does not apply to pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we review evidence of field-evolved resistance to Cry1Ac by cotton bollworm in northern China and by pink bollworm in the Yangtze River Valley of China. For both pests, results of laboratory diet bioassays reveal significantly decreased susceptibility of field populations to Cry1Ac, yet field control failures of Bt cotton have not been reported. The early detection of resistance summarized here may spur countermeasures such as planting Bt cotton that produces two or more distinct toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

Vip3A resistance alleles exist at high levels in Australian targets before release of cotton expressing this toxin

Crops engineered to produce insecticidal crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt) have revolutionised pest control in agriculture. However field-level resistance to Bt has developed in some targets. Utilising novel vegetative insecticidal proteins (Vips), also derived from Bt but genetically distinct from Cry toxins, is a possible solution that biotechnical companies intend to employ. Using data collected over two seasons we determined that, before deployment of Vip-expressing plants in Australia, resistance alleles exist in key targets as polymorphisms at frequencies of 0.027 (n = 273 lines, 95% CI = 0.019-0.038) in H. armigera and 0.008 (n = 248 lines, 0.004-0.015) in H. punctigera. These frequencies are above mutation rates normally encountered. Homozygous resistant neonates survived doses of Vip3A higher than those estimated in field-grown plants. Fortunately the resistance is largely, if not completely, recessive and does not confer resistance to the Bt toxins Cry1Ac or Cry2Ab already deployed in cotton crops. These later characteristics are favourable for resistance management; however the robustness of Vip3A inclusive varieties will depend on resistance frequencies to the Cry toxins when it is released (anticipated 2016) and the efficacy of Vip3A throughout the season. It is appropriate to pre-emptively screen key targets of Bt crops elsewhere, especially those such as H. zea in the USA, which is not only closely related to H. armigera but also will be exposed to Vip in several varieties of cotton and corn.     

Helicoverpa armigera baseline susceptibility to Bacillus thuringiensis Cry toxins and resistance management for Bt cotton in India

Transgenic cotton that produces insecticidal proteins from Bacillus thuringiensis (Bt), often referred to as Bt cotton, is widely grown in many countries. Bt cotton with a single cry1A gene and stacked also with cry2A gene has provided satisfactory protection against the damage by the lepidopteran bollworms, especially the cotton bollworm, Helicoverpa armigera (Hübner) which is considered as a key pest. The baseline susceptibility of the larvae of H. armigera to Cry1Ac and other toxins carried out in many countries has provided a basis for monitoring resistance. There is no evidence of development of field-level resistance in H. armigera leading to the failure of Bt cotton crop anywhere in the world, despite the fact that Bt cotton was grown on the largest ever area of 12.1 million hectares in 2006 and its cumulative cultivation over the last 11 years has surpassed the annual cotton area in the world. Nevertheless, the Bt resistance management has become a necessity to sustain Bt cotton and other transgenic crops in view of potential of the target insects to evolve Cry toxin resistance.     

Disruption of a cadherin gene associated with resistance to Cry1Ac {delta}-endotoxin of Bacillus thuringiensis in Helicoverpa armigera

A laboratory strain (GY) of Helicoverpa armigera (Hubner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to "mode 1," the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.     

Increasing tolerance to Cry1Ac cotton from cotton bollworm, Helicoverpa armigera, was confirmed in Bt cotton farming area of China

Abstract.  1. Changes in the frequency of Cry1Ac resistance genes and shifts in tolerance of cotton bollworm, Helicoverpa armigera , to the Cry1Ac toxin were assessed using bioassays of F₁ and F₂ offspring of isofemale lines from Anci County of Hebei Province (a multiple-crop system including corn, soybean, peanut, and Bt cotton) and Xiajin County of Shandong Province (an intensive Bt cotton planting area) in Northern China during 2002–2005.
2. A conservative analysis of the overall results indicated that there was a small increase in the frequency of major, non-recessive resistance genes over time.
3. The relative average development ratings [RADR – growth rate of a line on a Bt diet in proportion to the growth rate on a non-Bt (NBT) diet] of the bollworm larvae in F₁ tests increased significantly from year to year, indicating a gradual trend towards higher tolerance to Cry1Ac in the field populations.
4. There were also significant positive correlations between RADR of the lines in the F₁ generation and the RADR of their F₂ offspring, indicating that the tolerance was genetically based.
5. Quantitative genetic simulation analysis showed that resistance of H . armigera to Bt cotton in Xiajin could evolve to a high level in 11–15 years if no effective resistance management measures are carried out.     

Characteristics of resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa punctigera (Lepidoptera: Noctuidae) isolated from a field population

In 1996, the Australian cotton industry adopted Ingard that expresses the Bacillus thuringiensis (Bt) toxin gene cry1Ac and was planted at a cap of 30%. In 2004-2005, Bollgard II, which expresses cry1Ac and cry2Ab, replaced Ingard in Australia, and subsequently has made up >80% of the area planted to cotton, Gossypium hirsutum L. The Australian target species Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are innately moderately tolerant to Bt toxins, but the absence of a history of insecticide resistance indicates that the latter species is less likely to develop resistance to Bt cotton. From 2002-2003 to 2006-2007, F2 screens were deployed to detect resistance to CrylAc or Cry2Ab in natural populations of H. punctigera. Alleles that conferred an advantage against CrylAc were not detected, but those that conferred resistance to Cry2Ab were present at a frequency of 0.0018 (n = 2,192 alleles). Importantly, the first isolation of Cry2Ab resistance in H. punctigera occurred before significant opportunities to develop resistance in response to Bollgard II. We established a colony (designated Hp4-13) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony. Through specific crosses and bioassays, we established that the resistance present in Hp4-13 is due to a single autosomal gene. The resistance is fully recessive. Homozygotes are able to survive a dose of Cry2Ab toxin that is 15 times the reported concentration in field grown Bollgard II in Australia (500 microg/ml) and are fully susceptible to Cry1Ac and to the Bt product DiPel. These characteristics are the same as those described for the first Cry2Ab resistant strain of H. armigera isolated from a field population in Australia.     

Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the "stacked" gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of (125)I-labeled Cry1Ab protein ((125)I-Cry1Ab) and (125)I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either (125)I-Cry1Ab or (125)I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with (125)I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. (125)I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, (125)I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to promote survival of susceptible pests. To delay pest resistance to transgenic cotton producing Bt toxin Cry1Ac, farmers in the United States and Australia planted refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. Here we report data from a 2010 survey showing field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera), in northern China. Laboratory bioassay results show that susceptibility to Cry1Ac was significantly lower in 13 field populations from northern China, where Bt cotton has been planted intensively, than in two populations from sites in northwestern China where exposure to Bt cotton has been limited. Susceptibility to Bt toxin Cry2Ab did not differ between northern and northwestern China, demonstrating that resistance to Cry1Ac did not cause cross-resistance to Cry2Ab, and implying that resistance to Cry1Ac in northern China is a specific adaptation caused by exposure to this toxin in Bt cotton. Despite the resistance detected in laboratory bioassays, control failures of Bt cotton have not been reported in China. This early warning may spur proactive countermeasures, including a switch to transgenic cotton producing two or more toxins distinct from Cry1A toxins.     

Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. The "natural" refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005-2007 to 56% in 2008-2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

一株对苹果树鳞翅目害虫高毒力的苏云金芽胞杆菌YX-1

苏云金芽胞杆菌Bacillus thuringiensis(Bt)YX-1是从土壤中分离的对多种鳞翅目害虫具有杀虫活性的新菌株。为了探索该菌株在果树上应用的可行性,本研究测定了Bt YX-1菌株对苹果树上6种鳞翅目害虫的杀虫毒力,同时对该菌株的晶体形态特征、蛋白型、生长特性、基因型等进行了分析。结果表明,Bt YX-1菌株产生菱形伴胞晶体,SDS-PAGE分析表明该菌株表达的主要蛋白条带分子量约为130ku和60ku;基因型鉴定表明,Bt YX-1菌株含有cry1Ac、cry2Ac、cry1I、vip3Aa和cry34-35基因;生物活性测定表明,Bt YX-1菌株的孢晶混合物对美国白蛾Hlyphantria cunea、棉铃虫Helicoverpa armigera、斜纹夜蛾Prodenia litura、梨小食心虫Grapholitha molesta、苹小卷叶蛾Adoxophyes orana以及苹掌舟蛾Phalera flavescens的LC50分别为14.48、2.72×103、6.24×104、1.01×102、3.52×104、4.73×103mg/L,均低于标准菌株Bt HD-1的LC50。发酵上清液的杀虫活性很低,2龄棉铃虫幼虫的死亡率仅为8.33%,但是该上清液能显著提高孢晶混合物的毒力,说明上清液中含有增效物质。研究结果表明该菌株具有进一步开发为商品制剂的潜力。     

Mutated cadherin alleles from a field population of Helicoverpa armigera confer resistance to Bacillus thuringiensis toxin Cry1Ac

The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r(1)) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r(2) and r(3)) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r(2) and r(3) alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera.     

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.     

Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.