Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination.

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Novel Field-Based Protein Detection Method Using Light Transmission Spectroscopy and Antibody Functionalized Gold Nanoparticles.

Protein detection is a universal tool critical to many applications in medicine, agriculture, and biotechnology. We developed a novel protein detection method combining light transmission spectroscopy and particle-size analysis of gold nanospheres monovalently functionalized with polyclonal antibodies and applied it to an emerging challenge for such technologies─the monitoring of environmental proteins (eProteins) present in natural aquatic systems. These are an underreported source of pollution and include the pseudopersistent Cry toxins that enter aquatic ecosystems from surrounding genetically engineered crops. The assay is capable of detecting proteins in complex matrices, such as water samples collected in the field, making it a competitive assay for eProtein detection. It is sensitive, reaching 1.25 ng mL-1, and we demonstrate its application to the detection of Cry1Ab from subsurface tile-drain and streamwater samples from agricultural waterways. The assay can also be quickly adapted for other protein detection applications in the future.

Inheritance and expression of Bt cry1Ba3 gene in progeny from transformed cabbage plants.

Stable inheritance and expression of transgene are important parameters for successful use of a transgenic crop. We previously transformed a Bt cry1Ba3 gene into cabbage inbred line CA21-3. To evaluate the stability of our Bt cabbage lineages, transgene inheritance and expression were examined in four successive generations under greenhouse conditions. In our study, T1, T2 and T3 progenies of the three independent transgenic lineages (YA-1, YA-2 and YA-3) were generated and then the inheritance and expression of cry1Ba3 were analyzed in sexually derived progeny. Segregation ratio of 2.81:1, 3.27:1 and 3.07:1 was found in T1 progeny of lineages YA-1, YA-2 and YA-3, respectively. Chi-square analysis indicated that these segregation ratios of corresponding population fit the 3:1 ratio. Segregation ratios of the transgene in T2 progeny showed either 3:1 or all expression of cry1Ba3. These data suggest that cry1Ba3 in CA21-3 can be inherited in a Mendelian manner. ELISA analysis of transgenic plants from four generations demonstrated that cry1Ba3 had been stably transmitted to the T3 progeny. Additionally, under artificial infestation conditions, the homozygous T3-YA-1-2-1 line exhibited excellent resistance to Plutella xylostella as compared with un-transformed CA21-3. All these results imply that the three cabbage lineages are genetically stable and can be used to inhibit damage on cabbage caused by P. xylostella.

Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins.

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258-1.407 µg/mL, the medium inhibition concentration (IC50) were 0.727-0.892 µg/mL and detection limit (IC10) were 0.029-0.074 µg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21-5.94 × 10(7) M(-1). The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%-100.8% with a CV at 2.5%-9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.

Targeting Design of Human Anti-idiotypic Genetically Engineered Antibody for Simulating the Structure and Insecticidal Function of Bt Cry1C Toxin.

The ß-type anti-Id (Ab2ß) is considered to have potential for simulating the structure and function of the antigen. In this study, a ß-type anti-Id (3A7 anti-I-GEAb) of the Cry1C toxin was captured from a GEAb library. Subsequently, a higher activity of mutant (3A7 mutant 8) was obtained from the mutagenesis library based on 3A7 anti-I-GEAb. The LD50 values of 3A7 anti-I-GEAb and 3A7 mutant 8 reach up to 38.9% and 46.8% of Cry1C toxin for P. xylostella and reach up to 32.9% and 37.4% of Cry1C toxin for H. armigera. Additionally, an IC-ELISA was established based on 3A7 mutant 8 (as the coated "antigen"), with an LOD value of 0.35 ng/mL, exhibiting good accuracy and stability for detecting Cry1C toxin in spiked samples. The present ß-type anti-I-GEAb not only exhibits insecticidal activity similar to Cry1C toxin, offering potential for environmentally friendly pest management, but it can also replace the Cry1C toxin structure to establish a highly sensitive and specific IC-ELISA for monitoring Cry1C toxin.

[Targeted innovative design of Bt Cry toxin insecticidal mimics].

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2ß anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2ß anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Construction of a Novel Degradation Model of Bacillus thuringiensis Protein in Soil and Its Application in Estimation of the Degradation Dynamics of Bt-Cry1Ah Protein.

Bacillus thuringiensis (Bt) protein expressed by genetically modified (GM) crops is released into the soil ecosystem, where it accumulates for a long time; therefore, degradation of Bt protein has gained increased attention for environmental risk assessments. A first-order kinetic model (Y = ae-b*X) is usually used to evaluate the degradation of Bt proteins, including Bt-Cry1Ab and Bt-Cry1Ac; this has some limitations regarding the precise fitting and explanation of the influence of various factors on Bt protein degradation in the later stage. Therefore, to amend these limitations, we report a new degradation model Y = Y0 + ae-b*X. The effects of soil temperature, water content, soil types, and soil sterilization on the degradation of Bt-Cry1Ah protein in soil were estimated in a 96d long laboratory study using a GM maize leaf-soil mixture. The results showed that the Bt-Cry1Ah protein degraded rapidly in the early stage and then slowly in the middle and late stages. Temperature was identified as the key factor affecting the degradation of Cry1Ah protein-a relatively higher temperature favored the degradation. The degradation rate of Cry1Ah protein was the fastest when the water content was 33 and 20% in the early and later stages, respectively. The soil types had a significant effect on the degradation of Cry1Ah protein. Moreover, soil sterilization slowed down the rate of protein degradation in both the early and later stages. In conclusion, the model Y = Y0 + ae-b*X established in this study provided a more robust model for exploring and simulating the degradation of Bt protein in soil growing GM crops and overcame the shortcomings of the Y = ae-b*X model. The findings of this study enriched the understanding of Bt protein degradation in soil ecosystems. They would be helpful for evaluating the environmental safety of GM crops.

Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops.

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.

Effects of temperature on baseline susceptibility and stability of insecticide resistance against Plutella xylostella (Lepidoptera: Plutellidae) in the absence of selection pressure.

Plutella xylostella L. (Lepidoptera: Plutellidae) is an important pest causing significant losses to vegetables worldwide. Insecticides resistance in P. xylostella is a serious issue for scientists since last 30 years. However, deltamethrin and Bt Cry1Ac are commonly used insecticides against P. xylostella but studies involving development of resistance in P. xylostella against these two insecticides at different temperatures are lacking. The current study was aimed to find out the toxicity of deltamethrin and Bt Cry1Ac, and resistance development in P. xylostella. Results showed that the positive correlation between the temperature and toxicities of deltamethrin and Bt Cry1Ac. The results indicated -0.051, -0.049, -0.047, and -0.046 folds of deltamethrin resistance at 15 °C, 20 °C, 25 °C, and 30 °C temperatures, respectively from 1st to 12th generations. The toxicity of Bt Cry1Ac after 24 h was 2.2 and 4.8 folds on 1st generation at 20 °C and 25 °C temperatures, respectively compared to the toxicity recorded at 15 °C (non-overlapping of 95% confidence limits). Based on the results of this study, it is concluded that the temperature has a positive correlation with the toxicity of deltamethrin and Bt Cry1Ac against the larvae of P. xylostella. This study suggests that deltamethrin and Bt Cry1Ac can be included in the management program of P. xylostella on many vegetable crops. The baseline susceptibility data might be helpful to understand the resistance mechanisms in P. xylostella.

Cry1 Bt Susceptibilities of Fall Armyworm (Lepidoptera: Noctuidae) Host Strains.

The fall armyworm, Spodoptera frugiperda (Smith; Lepidoptera: Noctuidae), is a highly polyphagous, multivoltine pest of commercial crops including corn (Zea mays L.), cotton (Gossypium spp. L.), rice (Oryza sativa L.), and pasture grasses. Fall armyworm has become a growing concern in agricultural communities across the Americas as field populations in many locales have evolved resistance to several Cry1 toxins derived from the bacterium Bacillus thuringiensis Berliner (Bt). An often overlooked aspect of fall armyworm biology is the existence of two host strains, the 'rice' and 'corn' strains. There has been little research devoted to the characterization of fall armyworm host strains, although there is evidence that the rice and corn-strains may differ in their tolerances to Bt toxins expressed by transgenic plants. In this study, diet-based bioassays were conducted to compare the susceptibilities of one rice-strain, two corn-strains, and one rice-corn hybrid population to Cry1Ab, Cry1Ac, and Cry1F protein. Results indicate that the corn-strains and hybrid populations are more tolerant to the Bt toxins, especially to Cry1F, than the rice-strain population. Results from this study, when combined with existing techniques for host strain identification, may aid in the development of regional insect resistance management programs for fall armyworm.

Targeted innovative design of Bt Cry toxin insecticidal mimics / 生物工程学报

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Stable integration and expression of a cry1Ia gene conferring resistance to fall armyworm and boll weevil in cotton plants.

BACKGROUND: Boll weevil is a serious pest of cotton crop. Effective control involves applications of chemical insecticides, increasing the cost of production and environmental pollution. The current genetically modified Bt crops have allowed great benefits to farmers but show activity limited to lepidopteran pests. This work reports on procedures adopted for integration and expression of a cry transgene conferring resistance to boll weevil and fall armyworm by using molecular tools. RESULTS: Four Brazilian cotton cultivars were microinjected with a minimal linear cassette generating 1248 putative lines. Complete gene integration was found in only one line (T0-34) containing one copy of cry1Ia detected by Southern blot. Protein was expressed in high concentration at 45 days after emergence (dae), decreasing by approximately 50% at 90 dae. Toxicity of the cry protein was demonstrated in feeding bioassays revealing 56.7% mortality to boll weevil fed buds and 88.1% mortality to fall armyworm fed leaves. A binding of cry1Ia antibody was found in the midgut of boll weevils fed on T0-34 buds in an immunodetection assay. CONCLUSION: The gene introduced into plants confers resistance to boll weevil and fall armyworm. Transmission of the transgene occurred normally to T1 progeny. All plants showed phenotypically normal growth, with fertile flowers and abundant seeds. © 2015 Society of Chemical Industry.

Three toxins, two receptors, one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens.

Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.

A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle.

Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.

Transgenic tomato line expressing modified Bacillus thuringiensis cry1Ab gene showing complete resistance to two lepidopteran pests.

The modified truncated Bt-cry1Ab gene of Bacillus thuringiensis has been used for the development and selection of over expressing transgenic events in a commercially important variety of tomato (Solanum lycopersicum L.) by Agrobacterium-mediated leaf-disc transformation procedure. The integration and inheritance of cry1Ab gene in T0 transgenic plants and their progenies were determined by PCR, RT-PCR and Southern blot hybridization analysis. The toxin expression was monitored by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The transgenic line Ab25 E, expressing 0.47 ± 0.01% Cry1Ab toxin of total soluble protein (TSP) was finally selected in the T4 generation from the segregating population, showing 100% mortality to the second instar larvae of H. armigera and S. litura and minimal damages to leaves and fruits. Southern blot analysis data revealed single copy introgression of cry1Ab gene in highly-expressing Ab25 E transgenic line and expression of Cry1Ab toxin of molecular mass ~65 kDa was evident in Western blot analyses in transgenic plants of T4, T5 and T6 generation. Receptor binding assay performed with partially purified Cry1Ab protein from Ab25 E transgenic tomato line, confirmed efficient protein-protein interaction of Cry1Ab toxin with receptor(s) of both the insects. The higher level of Cry1Ab toxin (≈ 0.47 ± 0.01% TSP) did not affect the normal in vitro regeneration, plant development and fruit yield in this transgenic line. This high expressing Cry1Ab homozygous transgenic line can be a useful candidate in tomato breeding programmes for introgression of important agronomical traits.

Stable Bacillus thuringiensis transgene introgression from Brassica napus to wild mustard B. juncea.

Transgenic canola (Brassica napus) with a Bacillus thuringiensis cry1Ac gene and a green fluorescent protein (GFP) marker gene was used in hybridization experiments with wild Brassica juncea. Hybrid F1 and successive five backcross generations were obtained. The pod-set frequency on backcrossed B. juncea plants was over 66%, which suggested relatively high crossing compatibility between the hybrids and wild species. The seed setting in BC1 was the least of all generations tested, and then increased at the BC2 generation for which the thousand-seed weight was the highest of all generations. Seed size in backcrossed generations eventually approached that of the wild parent. The plants in all backcrossed generations were consistent with the expected 1:1 segregation ratio of the transgenes. The Bt Cry1Ac protein concentrations at bolting and flowering stages was higher compared to the 4-5-leaf and pod-formation stages. Nonetheless, the Bt toxin in the fifth backcrossing generation (BC5) was sufficient to kill both polyphagous (Helicoverpa armigera) and oligophagous (Plutella xylostella) Lepidoptera. As a consequence, the subsequent generations harboring the transgene from F1 to BC5 could have selection advantage against insect pests. The result is useful in understanding gene flow from transgenic crops and the followed transgene introgression into wild.