Cry1Ab Protein Research Articles

Development of transgenic cotton (Narasimha) using triple gene Cry2Ab-Cry1F-Cry1Ac construct conferring resistance to lepidopteran pest

High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they are recalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing three Cry genes driven by three different promoters conferring insect resistance. The meristematic region of embryo axis explants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100 vector with three Cry gene cassettes (alpha-globulin : Cry2Ab, DECaMV35s : Cry1F and nodulin : Cry1Ac) with Npt II as a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds of kanamycin selectionmedium.About 2551T1 seedswere collected from111T0 plants and these seeds screened again with kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR, lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showed amplification of all four transgenes: Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as 395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteins accumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry genes were accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effective resistance againstHelicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentage of insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgenic cotton plants carrying triple Cry genes could be an excellent germplasmresource for the breeders for introgressions.

Food/feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/20)

Food/feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/20)

Final Health and Environmental Risk Assessment of Genetically Modified Maize MON 89034

Final Health and Environmental Risk Assessment of Genetically Modified Maize MON 89034

Final Health and Environmental Risk Assessment of Genetically Modified Maize 59122

Final Health and Environmental Risk Assessment of Genetically Modified Maize 59122

Food/Feed and Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON810 for Cultivation, Seed Production, Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/F/95/12/02)

Food/Feed and Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON810 for Cultivation, Seed Production, Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/F/95/12/02)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/21)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 59122 x 1507 x NK603 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/21)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x 59122 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/NL/2005/15)

Food/Feed and Environmental Risk Assessment of Insect-resistant and Herbicide-tolerant Genetically Modified Maize 1507 x 59122 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/NL/2005/15)

Efficacy of a cry1Ab Gene for Control of Maruca vitrata (Lepidoptera: Crambidae) in Cowpea (Fabales: Fabaceae).

Cowpea [Vigna unguiculata (L) Walp.] is an important staple legume in the diet of many households in sub-Saharan Africa. Its production, however, is negatively impacted by many insect pests including bean pod borer, Maruca vitrata F., which can cause 20-80% yield loss. Several genetically engineered cowpea events that contain a cry1Ab gene from Bacillus thuringiensis (Bt) for resistance against M. vitrata were evaluated in Nigeria, Burkina Faso, and Ghana (West Africa), where cowpea is commonly grown. As part of the regulatory safety package, these efficacy data were developed and evaluated by in-country scientists. The Bt-cowpea lines were planted in confined field trials under Insect-proof netting and artificially infested with up to 500 M. vitrata larvae per plant during bud formation and flowering periods. Bt-cowpea lines provided nearly complete pod and seed protection and in most cases resulted in significantly increased seed yield over non-Bt control lines. An integrated pest management strategy that includes use of Bt-cowpea augmented with minimal insecticide treatment for protection against other insects is recommended to control pod borer to enhance cowpea production. The insect resistance management plan is based on the high-dose refuge strategy where non-Bt-cowpea and natural refuges are expected to provide M. vitrata susceptible to Cry1Ab protein. In addition, there will be a limited release of this product until a two-toxin cowpea pyramid is released. Other than South African genetically engineered crops, Bt-cowpea is the first genetically engineered food crop developed by the public sector and approved for release in sub-Saharan Africa.

Introgression of a cry1Ab transgene into open pollinated maize and its effect on Cry protein concentration and target pest survival.

In Africa, the target pests of genetically modified Bt maize are lepidopteran stem borers, notably Busseola fusca (Lepidoptera: Noctuidae). Gene flow between Bt maize hybrids and open pollinated varieties (OPVs) that do not contain the Bt trait is highly likely in areas where both types of maize are cultivated. Consequently, introgression of the cry1Ab transgene into local OPVs will result in unknown patterns of Cry1Ab protein expression in plants during follow-up seasons when recycled seed of OPVs is planted. Too low concentrations of Cry protein in such plants may result in selection for resistant alleles and accelerate resistance evolution. The aim of this study was to determine the effects of introgression of the cry1Ab transgene into an OPV, on Cry protein concentration levels and pest survival. Bt transgene introgression was done by crossing a transgenic donor hybrid containing the cry1Ab gene with a non-Bt OPV as well as with a non-Bt near-isogenic hybrid. F1 and F2 crosses as well as back crosses were done yielding 11 genotypes (treatments). Cry1Ab protein concentrations in leaf tissue of these crosses were determined by means of ELISAs. All crosses that contained the transgene had similar or higher Cry1Ab concentrations when compared to the Bt parental hybrid, except for the Bt x OPV F1-cross that had a significantly lower Cry1Ab concentration. Survival B. fusca larvae were evaluated in assays in which larvae were reared for 14 days on whorl leaf tissue of the different treatments. Larval survival did not differ between any of the maize plant treatments which contained the Bt gene. Results suggest that Bt transgene introgression into OPVs may produce plant progenies that express Cry1Ab protein at sufficient concentrations, at last up to the F2 seed, to control B. fusca larvae. Resistance evolution is however not only influenced by the frequency of pest individuals that survive exposure to the Cry proteins but also by factors such as genetics of the pest and recipient OPV, pest biology and migration behaviour.

Preliminary Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON 89034 for Cultivation (EFSA/GMO/BE/2011/90)

Preliminary Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON 89034 for Cultivation (EFSA/GMO/BE/2011/90)

Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON810 for Cultivation, Seed Production, Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/F/95/12/02)

Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON810 for Cultivation, Seed Production, Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/F/95/12/02)

Performance of Field-Collected Spodoptera frugiperda (Lepidoptera: Noctuidae) Strains Exposed to Different Transgenic and Refuge Maize Hybrids in Argentina.

Spodoptera frugiperda (J. E. Smith) is one of the major pests of maize in Argentina. The main tool for its control is the use of genetically modified maize hybrids expressing Bacillus thuringiensis (Bt) insecticidal proteins. Maize growers in Argentina initially controlled this pest with Bt maize that expressed a single Bt protein (Cry1Ab or Cry1F). Currently it is necessary to plant maize cultivars that produce two Bt proteins to achieve the satisfactory control. Recently, Cry1F field-evolved resistant populations of this species were confirmed in Argentina. The objective of this study was to evaluate the performance of S. frugiperda field-collected strains on different Bt and non-Bt maize hybrids. Strains were collected from non-Bt maize (T1), Agrisure TDMax (T2), Agrisure Viptera (T3), Agrisure Viptera 3110 (T4), Genuity VT Triple Pro (T5), and Power Core (T6). Three experiments were performed to 1) determine the survivorship and reproduction of field-collected larvae (F0) from Bt maize hybrids, 2) evaluate Cry1F resistance using an F1 screen, and 3) assess the performance of F1 strains on different maize hybrids. In the F0, the survivorship from larva to adult ranged from 0 to 63%. We obtained adults from only the T1, T2, T5, and T6 strains with no significant differences in the reproductive parameters. Continuously rearing F1 larvae on their collected hosts affected larval duration, which was significantly shorter for a known-laboratory Bt-susceptible strain than the field-collected strains. Our results support the existence of Cry1F-resistance alleles in S. frugiperda field populations in Argentina.

Short communication: The rate of release of Cry1Ab protein from Bt maize leaves into water

Transgenic Bt maize plants are genetically modified to contain genes of Bacillus thuringiensis that encode for δ-endotoxins (Cry1Ab protein) that have insecticidal properties. These endotoxins target certain lepidopteran pests of maize. There are several entry routes by which Cry proteins enter the aquatic ecosystem in which aquatic organisms are exposed to these proteins. The main route is through plant debris such as leaves, stalks and postharvest detritus that are transported by means of run-off, rain and wind. While several studies have been conducted on the fate of Cry1Ab protein in terrestrial ecosystems, little is known of the release rates of Cry1Ab proteins from maize plant tissues that end up in aquatic ecosystems. In this study, leaves of Bt-maize and its isoline were submerged in containers filled with deionised or borehole water for a period of 16 days, and kept at 3 different temperatures (10±1, 21±1 and 30±1°C). Samples were collected at 1, 2, 4, 8, 16, 24, 48, 96, 192 and 384 h post submersion and analysed for Cry protein content using enzyme-linked immunosorbent assay (ELISA). The release of Cry protein from submerged maize leaves was influenced by temperature, and duration of immersion. An increase in Cry protein levels in the water was observed from the first hour onwards in both water types until the end of the experiment. The highest concentration of Cry protein was found at 30°C. This study showed that temperature and time period influence the release rate of Cry proteins from dried leaf matter into the aquatic environment.

Response of the bacterial community of Propylea japonica (Thunberg) to Cry2Ab protein

Propylea japonica is a very important predator in agricultural ecosystems, which could be exposed to Bt protein. In this study, the bacterial community of P. japonica fed with normal food and food containing Cry2Ab protein was characterized for the first time using qPCR and high-throughput sequencing approaches. Results showed no effect of Cry2Ab on P. japonica development and reproduction. The most abundant bacterial phylum was Firmicutes, and the most abundant genus was Staphylococcus. The total bacteria copy number was not significantly different across four larval stages. Bacteria species composition was gathered more closely in feed on sucrose solution (sucrose-fed) than in larvae only fed on pea aphid (aphid-fed), the diversity indices of some operational taxonomic unit (OTU) were significantly different between sucrose-fed and aphid-fed samples. Different instar larval stages of P. japonica fed with sucrose solution containing Cry2Ab Bt protein and found no effect on microbial community composition and total bacteria copy numbers. However, effects on relative abundance of microbes, copy numbers of Corynebacterium 1 and Glutamicibacter arilaitensis were observed significantly lower in Bt-fed first and fourth larval stages. Low and high concentrations of Cry2Ab protein altered the microbial abundance relative to sucrose-fed P. japonica and copy numbers of G. arilaitensis and Staphylococcus xylosus were significantly lower in Bt-fed samples than control sucrose-fed. Our results are the first report showing that feeding on Cry2Ab protein does not alter microbial species composition in P. japonica, but effects gene copy number of some dominant bacteria. Further investigations are needed to assess the effect of copy number change on P. japonica.

Homozygosis of Bt locus increases Bt protein expression and the control of Spodoptera frugiperda (Lepidoptera: Noctuidae) in maize hybrids

Spodoptera frugiperda (J.E. Smith) is the main pest of maize in Brazil and its control has been targeted by Bt maize hybrids. Generally, Bt maize hybrids possess the transgenic locus in a hemizygous condition, that is, containing only one copy of the Bt allele. However, studies have shown that maize hybrids with an additional transgenic allele, namely, in a homozygous state, increases transgenic protein expression. Our aim in this study was to evaluate whether the transgenic event TC1507 x MON89034 x NK603 in homozygosis increases Bt protein expression levels and, consequently, reduces S. frugiperda leaf-feeding injury and larval survival which affects maize grain yield. Leaf-feeding injury of S. frugiperda was 29% lower on homozygous hybrids relative to their isogenic hemizygous versions. Isogenic homozygous and hemizygous hybrids did not differ in grain yield in this study. S.frugiperda survivorship on homozygous hybrids was significantly lower than on their hemizygous isogenic versions (16.9% and 38.5%, respectively). Homozygous hybrids presented higher Cry1F, Cry1A.105, and Cry2Ab2 protein expression levels relative to their isogenic hemizygous versions (approximately 1.5-, 2.0-, and 2.5-fold, respectively). The Bt maize event TC1507 x MON89034 x NK603 in a homozygous state increases Bt protein expression levels and the control of S. frugiperda. Therefore, the deployment of homozygous transgenic maize hybrids to farmers is more desirable than the hemizygous versions.

Assessing the Potential for Interaction in Insecticidal Activity Between MON 87751 × MON 87701 Produced by Conventional Breeding.

Pyramiding (combining) of plant incorporated protectants (PIPs) with insecticidal activity in genetically engineered crops is a strategy used to improve efficacy as well as delay potential resistance for a specific group of targets. In some countries, a regulatory risk assessment is required for breeding “stacks” expressing multiple PIPs and these countries may require an assessment of potential interaction among the PIPs. This study evaluated whether combining soybean events MON 87551 and MON 87701 results in a toxicological interaction that effects a species that is controlled by each event. MON 87751 coexpresses the Cry1A.105 and Cry2Ab2 proteins and MON 87701 expresses the Cry1Ac protein. EC50 values for MON 87751 and MON 87701 were comparable in diet-incorporation bioassays using corn earworm (Lepidoptera: Noctuidae, Helicoverpa zea) and the observed combined activity of the stack was consistent with predictions of additivity (i.e., no interaction). Under the concentration and response addition models, predicted and observed median effect levels differed by <10%. These results demonstrate independent action at the median effect level between the insecticidal activity of MON 87751 and MON 87701. Taken together, no interaction between these PIPs and acceptable margins of safety for the individual proteins to nontarget organisms, it is appropriate to bridge back to the risk assessments for the individual products that demonstrated environmental safety of stack products containing both MON 87751 and MON 87701.

Effects of transgenic Bt rice lines with single Cry1Ab and fused Cry1Ab/Cry1Ac on the abundance dynamics and community diversity of soil mites

ABSTRACTRisk assessment and ecological effects are a concern of transgenic rice. In this study, the effects of three lines of Bt rice expressing single Cry1Ab protein (Kemingdao: cv. KMD) and fused Cry1Ab/Cry1Ac proteins (Huahui 1: cv. HH1; Bt Shanyou 63: cv. Bt-SY63) on soil mite community were investigated under field conditions. Transgenic treatment (cv. Bt-rice vs. non-Bt rice) significantly decreased the community index of Margalef’s richness (SR) of soil mite in KMD, and significantly increased the abundance and the community indexes of Shannon diversity (H’), Pielou’s evenness (J) and SR of soil mite in Bt-SY63 (p < 0.05). Transgene type (cv. KMD vs. HH1) did not significantly affect the composition, abundance and the community index (p > 0.05). Breeding type (cv. HH1 vs. Bt-SY63) significantly changed the composition and markedly improved the abundance and the J value in Bt-SY63 (p < 0.05). However, seasonal and inter-annual changes were apparent, and the Bt effects were not consistent at all for the different plant backgrounds over a 2-year period. These results suggested that the cultivation of Bt rice has a significant but transient effect on soil mite community.

Transport and instream removal of the Cry1Ab protein from genetically engineered maize is mediated by biofilms in experimental streams.

The majority of maize planted in the US is genetically-engineered to express insecticidal properties, including Cry1Ab protein, which is designed to resist the European maize borer (Ostrinia nubilalis). After crop harvest, these proteins can be leached into adjacent streams from crop detritus left on fields. The environmental fate of Cry1Ab proteins in aquatic habitats is not well known. From June-November, we performed monthly short-term additions of leached Cry1Ab into four experimental streams with varying benthic substrate to estimate Cry1Ab transport and removal. At the start of the experiments, when rocks were bare, we found no evidence of Cry1Ab removal from the water column, but uptake steadily increased as biofilm colonized the stream substrate. Overall, Cry1Ab uptake was strongly predicted by measures of biofilm accumulation, including algal chlorophyll a and percent cover of filamentous algae. Average Cry1Ab uptake velocity (vf = 0.059 ± 0.009 mm s-1) was comparable to previously reported uptake of labile dissolved organic carbon (DOC; mean vf = 0.04 ± 0.008 mm s-1). Although Cry1Ab has been shown to rapidly degrade in stream water, benthic biofilms may decrease the distance proteins are transported in lotic systems. These results emphasize that once the Cry1Ab protein is leached, subsequent detection and transport through agricultural waterways is dependent on the structure and biology of receiving stream ecosystems.

Susceptibility of Corn Earworm (Lepidoptera: Noctuidae) to Cry1A.105 and Cry2Ab2 in North and South Carolina.

The corn earworm, Helicoverpa zea (Boddie), is managed in corn and cotton in the United States primarily using transgenic cultivars that produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). However, increasing reports of resistance to one or more Bt proteins threaten the continued efficacy of Bt traits. To better understand the development of resistance of H. zea to Bt corn and cotton in the southeastern United States, we monitored for resistance to Cry1A.105 and Cry2Ab2 among 22 field populations of H. zea collected in non-Bt and Bt corn expressing Cry1A.105 + Cry2Ab2 during 2017 and 2018. Colonies were established in the laboratory and progeny were screened in diet-overlay bioassays to purified Cry1A.105 and Cry2Ab2 proteins. Compared with two susceptible laboratory colonies, all 14 field colonies tested with Cry1A.105 were highly resistant, with resistance ratios (RRs) ranging from 13.5 to >4,000. For Cry2Ab2, 19 colonies were tested and RRs ranged from 0.26 to 33.7. Field populations were significantly more susceptible to Cry2Ab2 than Cry1A.105. We documented variability in F0 and F1 pupal weight and developmental rates of natural populations of H. zea, but observed no significant correlation with susceptibility to either Cry1A.105 or Cry2Ab2. Our results expand on the recent reports of H. zea resistance to Cry1A and Cry2A proteins and will aid in the design and deployment of future pyramided crops in the United States.

Toxicity of Bacillus thuringiensis-Derived Pesticidal Proteins Cry1Ab and Cry1Ba against Asian Citrus Psyllid, Diaphorina citri (Hemiptera).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera), is an important pest of citriculture. The ACP vectors a bacterium that causes huanglongbing (HLB), a devastating and incurable disease of citrus. The bacterium Bacillus thuringiensis (Bt) produces multiple toxins with activity against a diverse range of insects. In efforts to provide additional control methods for the ACP vector of HLB, we identified pesticidal proteins derived from Bt for toxicity against ACP. The trypsin proteolytic profiles of strain-derived toxins were characterized. Strain IBL-00200, one of six strains with toxins shown to have basal activity against ACP was selected for liquid chromatography-mass spectrometry (LC-MS/MS) identification of the individual Cry toxins expressed. Toxicity assays with individual toxins derived from IBL-00200 were then performed. The activated form of the Cry toxins Cry1Ab and Cry1Ba were toxic to ACP with LC50 values of approximately 120 µg/mL. Disruption of the midgut epithelium was associated with the toxicity of both the IBL-00200-derived toxin mixture, and with Cry1Ba. With further optimization of the efficacy of Cry1Ab and Cry1Ba, these toxins may have practical utility against ACP. Bt toxins with activity against ACP may provide an additional tool for management of ACP and the associated HLB disease, thereby providing a more sustainable and environmentally benign approach than repeated application of broad-spectrum insecticides.