Tobacco plants expressing the Cry1AbMod toxin suppress tolerance to Cry1Ab toxin of Manduca sexta cadherin-silenced larvae

Cry toxins produced by Bacillus thuringiensis bacteria are insecticidal proteins used worldwide in the control of different insect pests. Alterations in toxin-receptor interaction represent the most common mechanism to induce resistance to Cry toxins in lepidopteran insects. Cry toxins bind with high affinity to the cadherin protein present in the midgut cells and this interaction facilitates the proteolytic removal of helix ??-1 and pre-pore oligomer formation. Resistance to Cry toxins has been linked with mutations in the cadherin gene. One strategy effective to overcome larval resistance to Cry1A toxins is the production of Cry1AMod toxins that lack helix ??-1. Cry1AMod are able to form oligomeric structures without binding to cadherin receptor and were shown to be toxic to cadherin-silenced Manduca sexta larvae and Pectinophora gossypiella strain with resistance linked to mutations in a cadherin gene.We developed Cry1AbMod tobacco transgenic plants to analyze if Cry1AMod toxins can be expressed in transgenic crops, do not affect plant development and are able to control insect pests. Our results show that production of the Cry1AbMod toxin in transgenic plants does not affect plant development, since these plants exhibited healthy growth, produced abundant seeds, and were virtually undistinguishable from control plants. Most importantly, Cry1AbMod protein produced in tobacco plants retains its functional toxic activity against susceptible and tolerant M. sexta larvae due to the silencing of cadherin receptor by RNAi. These results suggest that CryMod toxins could potentially be expressed in other transgenic crops to protect them against both toxin-susceptible and resistant lepidopteran larvae affected in cadherin gene.     

Colorado potato beetle larvae on potato plants expressing a locust proteinase inhibitor

The natural defence system of plants often involves inhibitors of digestive enzymes of their pests. Modem and environmental-friendly methods try to increase this plant resistance by expressing heterologous protease inhibitors in crops. Here we report the effects of expressing a gene from desert locust (Schistocerca gregaria) encoding two serine protease inhibitors in potato on Colorado potato beetle (Leptinotarsa decemlineata) larvae. The gene encoding both peptides on a single chain was used for Agrobacterium-mediated transformation of potato plants. The presence of the active inhibitor protein in the leaves was verified. The feeding bioassays in the laboratory showed that despite the low level of the peptide in leaves, CPB larvae on transgenic plants have grown slightly but significantly more slowly than those on control potato plants. The results support the notion that expression of multifunctional proteinase inhibitors of insect origin in plants might be a good strategy to improve insect resistance.     

Resistance of potatoes transgenic for a cry1Ac9 gene, to Phthorimaea operculella (Lepidoptera: Gelechiidae) over field seasons and between plant organs

Stable performance of insect‐resistant transgenic plants across field seasons and between plant organs damaged by the insect pest is critical for management of this resistance in the field. To evaluate this, potato (Solanum tuberosum) lines transgenic for a cry1Ac9 gene with resistance to potato tuber moth (Phthorimaea operculella) were established in the field during the southern hemisphere summers of 1997/98, 1998/99 and 1999/00 as small field plots, each of 10 plants. Replicate plots of the non‐transgenic parent cultivars (at least one for every three independently derived transgenic lines) were planted randomly throughout the trials. Field‐grown foliage was challenged with larvae in the laboratory and a growth index (GI) was calculated for recovered larvae from each transgenic and non‐transgenic potato line. Larval growth on young and mature leaves, and on newly harvested or stored tubers was also measured in the laboratory. Foliage from the transgenic lines inhibited larval growth in all seasons tested. For both control and transgenic lines, larvae had slightly lower GIs when reared on mature leaves compared with young leaves, although the correlation between mean GI for young and mature transgenic leaves was high (r = 0.97). The correlation between the mean GIs of larvae on newly harvested tubers and on those stored for 5 months was also high (r = 1.0). However, the GIs of larvae on newly harvested transgenic tubers were larger than on transgenic tubers stored for 5 months. The relative growth indices (RGI = mean GI/number days before final weighing) of larvae reared on newly harvested tubers from transgenic lines were generally higher than those from young transgenic foliage, while the RGIs of larvae reared on non‐transgenic tubers were slightly lower than those fed non‐transgenic foliage. The correlation between mean RGIs of larvae fed tubers or foliage was 0.62. The transgenic potato lines exhibited stable resistance to larvae across field seasons, between affected plant organs, and between plant organs of different ages.     

Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests

A member of the potato proteinase inhibitor II (PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco (Nicotiana tabacum) usingAgrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R₀) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R₀ plants (designated R₁) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae ofChrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper),Spodoptera litura (F.) (Lepidoptera: Noctuidae) andThysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently,C. eriosoma larvae fed leaf tissue from transgenic plants expressing thePPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectablePPI II protein accumulation. However, larvae of bothS. litura andT. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured inC. eriosoma digestive tract extracts, but not in extracts fromS. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants.     

转抗虫基因植物生态安全性研究进展

转抗虫基因植物如Bt棉花等已在美国、中国和澳大利亚等国家大规模商业化种植 ,有关转抗虫基因植物潜在的生态风险已引起广泛的关注。该文综述了转抗虫基因植物研究应用现状与安全性研究进展。主要内容包括 :转抗虫基因植物的种类及其对靶标害虫的抗性 ,对非靶标害虫和天敌发生的影响 ,对农田生态系统生物多样性的影响 ,靶标昆虫的抗性治理及转抗虫基因植物的基因漂移等     

转基因抗虫作物对非靶标昆虫的影响

转基因抗虫作物自 1996年被批准商业化种植以来 ,它的抗虫性和经济效益已得到了普遍肯定 ,同时 ,转基因抗虫作物对非靶标生物的影响 ,如转基因抗虫作物的长期种植 ,是否会导致次要害虫上升为主要害虫 ,是否会影响有益昆虫 ,包括重要经济昆虫、捕食性和寄生性天敌以及重要蝶类的种类及种群数量 ,已成为转基因抗虫作物生态风险评估的重要内容。一些研究结果表明 ,转基因抗虫作物在对靶标害虫有效控制的同时 ,一些对杀虫蛋白不敏感的非靶标害虫有加重危害的趋势 ,由于种植转基因抗虫作物 ,减少了化学农药的使用 ,客观上也使非靶标害虫种群数量上升 ,这对转基因抗虫作物害虫综合治理提出了新的要求。靶标害虫数量的减少直接影响了害虫天敌种群数量 ,靶标害虫取食转基因抗虫作物后发育迟缓 ,也间接影响了天敌昆虫的生长发育 ,转基因抗虫作物的花粉或花蜜是一些重要经济昆虫如蜜蜂、熊蜂和一些寄生蜂 ,甚至捕食性天敌的食物来源 ,或花粉飘落到一些鳞翅目昆虫如家蚕或重要蝶类昆虫的寄主植物上 ,直接或间接对这些昆虫造成一定影响。目前大多数研究表明转基因抗虫作物对非靶标昆虫 ,特别是对有益昆虫没有明显的不利影响 ,也有研究报道认为对某些有益昆虫有一定的不良影响。这为深入开展转基因抗虫作物的生态安全     

Transgenic Plants for Insect Pest Control: A Forward Looking Scientific Perspective

One of the first successes of plant biotechnology has been the creation and commercialisation of transgenic crops exhibiting resistance to major insect pests. First generation products encompassed plants with single insecticidal Bt genes with resistance against major pests of corn and cotton. Modelling studies predicted that usefulness of these resistant plants would be short-lived, as a result of the ability of insects to develop resistance against single insecticidal gene products. However, despite such dire predictions no such collapse has taken place and the acreage of transgenic insect resistance crops has been increasing at a steady rate over the 9 years since the deployment of the first transgenic insect resistant plant. However, in order to assure durability and sustainability of resistance, novel strategies have been contemplated and are being developed. This perspective addresses a number of potentially useful strategies to assure the longevity of second and third generation insect resistant plants.     

Age-specific bioassays and fecundity of Phthorimaea operculella (Lepidoptera: Gelechiidae) reared on cry1Ac -transgenic potato plants

Variation in the susceptibility of lepidopterous pest larvae of different ages to transgenic crops and the potential for survivors to reproduce could have important consequences for the development of resistance in such pests. Experiments were undertaken in the laboratory to determine if larvae of the potato tuber moth, Phthorimaea operculella, of different ages (0 (< 1 day old), 3, 5, 7 days) varied in their susceptibility to cry1Ac9–transgenic potato (Solanum tuberosum) foliage grown in the glasshouse or field. The survival and fecundity of larvae reared on transgenic tubers was also determined in the laboratory. There were no apparent differences in susceptibility of larvae of different ages to transgenic foliage. Larvae fed glasshouse or field‐grown non‐transgenic foliage had significantly larger relative growth indices and more larvae pupated, than those fed transgenic foliage, regardless of larval age. Eggs from a laboratory colony were placed on transgenic or non‐transgenic tubers to measure survival and fecundity. Between 6% and 15% of eggs placed on transgenic tubers developed into pupae for three of the four transgenic potato lines tested. On one transgenic line, only six adults emerged from 1300 eggs. In contrast, between 71% and 97% of the eggs placed on non‐transgenic tubers developed into pupae. Male and female pupae from transgenic lines weighed less than those from non‐transgenic lines. The fecundity of females from two of four transgenic lines was lower than from the non‐transgenic parent cultivar. Although larvae of different ages did not exhibit any overall age‐dependent pattern of increasing or decreasing susceptibility to transgenic foliage of glasshouse or field‐grown plants, the ability of larvae to survive and reproduce on transgenic tubers suggests this pest has the ability to evolve resistance to the transgenic plants used in the present study.     

Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene

AIMS: To search for novel Vip3A proteins for controlling insect pests. METHODS AND RESULTS: A pair of universal primers was designed based on the conserved regions of five vip3A genes. Amplified products were digested with the HindIII and EcoR enzymes so as to confirm different restriction fragment length polymorphism (RFLP) patterns used to identify vip3A-type genes. The vip3A gene types of 606 Bacillus thuringiensis strains were screened and three patterns of RFLP were successfully identified. Two novel vip3A genes were found and one of these, vip3Aa19, was further characterized and its product was confirmed toxic to Spodoptera exigua, Helicoverpa armigera and Plutella xylostella larvae. Partial sequences of another novel vip3A-type gene were obtained that shared 83% homology with that of the vip3Af1 gene. CONCLUSIONS: A polymerase chain reaction (PCR)-RFLP system we developed could be used for identifying novel vip3A-genes from B. thuringiensis strains. A novel Vip3A protein was found to have a broader insecticidal spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The reported method is a powerful tool to find novel Vip3A proteins from large-scale B. thuringiensis strains. The novel Vip3A protein may be used to control insect pests or resistant insect pests by constructing genetically engineered strains or transgenic plants.     

Tri-trophic interactions involving pest aphids,predatory 2-spot ladybirds and transgenic potatoes expressing snowdrop lectin for aphid resistance

Transgenic crops genetically engineered for enhanced insect resistance should be compatible with other components of IPM for the pest resistance to be durable and effective. An experimental potato line was genetically engineered to express an anti-aphid plant protein (snowdrop lectin, GNA), and assessed for possible interactions of the insect resistance gene with a beneficial pest predator. These extended laboratory studies are the first to demonstrate adverse tri-trophic interactions involving a lectin- expressing transgenic crop, a target pest aphid and a beneficial aphidophagous predator. When adult 2-spot ladybirds (Adalia bipunctata[L.]) were fed for 12 days on peach-potato aphids (Myzus persicae Sulzer) colonising transgenic potatoes expressing GNA in leaves, ladybird fecundity, egg viability and longevity significantly decreased over the following 2–3 weeks. No acute toxicity due to the transgenic plants was observed, although female ladybird longevity was reduced by up to 51%. Adverse effects on ladybird reproduction, caused by eating peach-potato aphids from transgenic potatoes, were reversed after switching ladybirds to feeding on pea aphids from non-transgenic bean plants. These results demonstrate that expression of a lectin gene for insect resistance in a transgenic potato line can cause adverse effects to a predatory ladybird via aphids in its food chain. The significance of these potential ecological risks under field conditions need to be further evaluated.     

Managing Insect Resistance to Plants Producing Bacillus thuringiensis Toxins

ABSTRACT:?

Insect-resistant transgenic plants have become an important tool for the protection of crops against insect pests. The acreage of insecticidal transgenic plants is expected to increase significantly in the near future. The bacterium Bacillus thuringiensis is currently the source of insecticidal proteins in commercial insect-resistant transgenic plants and will remain the most important source during the next decade. Insect resistance to B. thuringiensis Cry toxins is the main problem. Only one species, the diamondback moth, has evolved a resistance to B. thuringiensis-based formulations under field conditions. However, many other insect species were selected for resistance under laboratory conditions, indicating that there is a potential for evolution of resistance in most major pests. Many studies were conducted to elucidate the mode of action of the Cry toxins, the mechanisms and genetics of resistance, and the various factors influencing its development. This article reviews insect resistance to B. thuringiensis insecticidal proteins and related aspects, including the development of insect-resistant transgenic plants, B. thuringiensis toxins, their mode of action, mechanisms, stability, and genetics of resistance and management strategies for delaying resistance.     

"Active" refuges can inhibit the evolution of resistance in insects towards transgenic insect-resistant plants

Negative cross-resistance (NCR) toxins that hitherto have not been thought to have practical uses may indeed be useful in the management of resistance alleles. Practical applications of NCR for pest management have been limited (i) by the scarcity of high toxicity NCR toxins among pesticides, (ii) by the lack of systematic methodologies to discover and develop such toxins, as well as (iii) by the lack of deployment tactics that would make NCR attractive. Here we present the concept that NCR toxins can improve the effectiveness of refuges in delaying the evolution of resistance by herbivorous insect pests to transgenic host plants containing insecticidal toxins. In our concept, NCR toxins are deployed in the refuge, and thus are physically separated from the transgenic plants containing the primary plant-protectant gene (PPPG) encoding an insecticidal toxin. Our models show: (i) that use of NCR toxins in the refuge dramatically delays the increase in the frequency of resistance alleles in the insect population; and (ii) that NCR toxins that are only moderately effective in killing insects resistant to the PPPG can greatly improve the durability of transgenic insecticidal toxins. Moderately toxic NCR toxins are more effective in minimizing resistance development in the field when they are deployed in the refuge than when they are pyramided with the PPPG. We explore the potential strengths and weaknesses of deploying NCR toxins in refuges.     

Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis

Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.     

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K_i values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.     

Capturing the interaction types of two Bt toxins Cry1Ac and Cry2Ab on suppressing the cotton bollworm by using multi‐exponential equations

Transgenic crops are increasingly promoted for their practical effects on suppressing certain insect pests, but all transgenic crops are not equally successful. The insect pests can easily develop resistance against single Bacillus thuringiensis (Bt) toxin transgenic crops. Therefore, transgenic crops including two or more mixed Bt‐toxins can solve this problem by delaying the resistance development and killing the majority of targeted pests before the evolution of resistance. It is important to test the controlling effects of transgenic crops including multiple mixed toxins on a particular insect pest. Previous research has checked the cross‐resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against one susceptible and four resistant strains of cotton bollworm. The results showed that independence was the main interaction type between two toxins for the susceptible strain, whereas synergism was the main interaction type for any one resistant strain. However, the optimal combinations of two toxins were not obtained. In the present study, we developed two multi‐exponential equations (namely bi‐ and tri‐exponential equations) to describe the combination effects of two Bt toxins. Importantly, the equations can provide predictions of combination effects of different continuous concentrations of two toxins. We compared these two multi‐exponential equations with the generalized linear model (GLM) in describing the combination effects, and found that the bi‐ and tri‐exponential equations are better than GLM. Moreover, the bi‐exponential equation can also provide the optimal dose combinations for two toxins.     

基于微生物源的抗虫基因发掘

植物转基因抗虫技术在害虫控制方面取得了巨大成功。商业化运用的抗虫基因目前全部来源于苏云金杆菌（Bacillus thuringiensis,Bt）的杀虫晶体蛋白基因,存在抗虫谱较窄及害虫逐渐产生抗性等问题,表明新型抗虫基因的筛选尤为重要。已有的文献研究表明,除了继续发掘Bt来源的新型杀虫蛋白基因以外,非Bt杀虫细菌及杀虫真菌也具有重要的发掘价值。     

抗草甘膦抗虫植物表达载体的构建及其转基因烟草的分析

构建了含草甘膦抗性突变基因（aroAM12）和人工合成重组Bt抗虫基因（Bts1m）的植物表达载体pCM12_s1m。aroAM12基因的表达由CaMV35S启动子控制,Bts1m基因的表达由2E_CaMV35S启动子和Ω因子控制。通过农杆菌介导,将aroAM12和Bts1m基因转化到烟草中,转基因烟草通过在含草甘膦的MS培养基上筛选而获得。Southern blot分析表明所有经过草甘膦筛选出的转化植株都整合有aroAM12基因,约70%的转化植株同时整合有aroAM12和Bts1m基因。Northern blot、Immunodot blot分析进一步证明整合的两个基因在转录、翻译水平上均进行了表达,不同植株之间表达存在着差异。草甘膦抗性和虫试实验证明,获得的转基因烟草对草甘膦和烟青虫具有很强的抗性。