Cry1Ac Protein Research Articles

Field monitoring of Helicoverpa armigera (Lepidoptera: Noctuidae) Cry1Ac insecticidal protein resistance in China (2005-2017).

Bt cotton expressing Cry1Ac can efficiently control Helicoverpa armigera, but field-evolved Bt resistance reduces its efficacy. Susceptibility of H. armigera field populations to Cry1Ac protein has been monitored since Bt cotton was commercialized in 1997 in China. Here we report the results of our continued monitoring from 2005 to 2017 to provide important guidelines for sustainably growing cotton. From 13 provinces and regions, 221 populations were sampled and measured. IC50 values (concentration producing 50% inhibition of larval development to third instar) among different strains ranged from 0.004 to 0.212 µg mL-1 , the percentage survival at a diagnostic concentration (IC99 , 1.0 µg mL-1 ) (SUR) ranged from 0% to 22.2%, and the percentage of field populations yielding survivors at diagnostic concentration (PSD) increased from 0 in 2006 and 2007 to 80% in 2015. Compared to resistance level from 1998 to 2004 and the geographic baseline in different places of China in 1997, Bt resistance of H. armigera field populations to Cry1Ac protein has increased significantly, but failure of Bt cotton has not yet been detected. © 2018 Society of Chemical Industry.

Safety of the Bacillus thuringiensis-derived Cry1A.105 protein: Evidence that domain exchange preserves mode of action and safety

The lepidopteran-active Cry1A.105 protein is a chimeric three-domain insecticidal toxin with distinct structural domains derived from the naturally occurring Cry1Ab, Cry1Ac and Cry1F proteins from the soil bacterium Bacillus thuringiensis (Bt). The X-ray crystal structure of the Cry1A.105 tryptic core at 3.0 Å resolution demonstrates its high structural similarity to the tryptic core of Cry1Ac. Bioinformatics analyses demonstrate that Cry1A.105 has no significant amino acid sequence similarity to known allergens or mammalian toxins, which is the same conclusion reached for its component domains. Like its intact donor proteins, Cry1A.105 was heat labile at temperatures ≥75 °C and degraded upon exposure to gastrointestinal proteases. No adverse effects were observed in mice when Cry1A.105 was dosed orally at 2451 mg/kg body weight. Therefore, the weight of evidence supports that Cry1A.105 is safe for human and animal consumption. These results support the conclusion that the safety of a chimeric protein for human or animal consumption can be evaluated in the context of the safety of its donor proteins.

Tolerance of DAS-444ø6-6 and DAS-444ø6-6 x DAS-81419-2 Soybeans to 2,4-D and Glyphosate in the Cerrado Region of Brazil

ABSTRACT: The DAS-444Ø6-6 soybean expresses AAD-12, 2mEPSPS and PAT enzymes to provide, respectively, tolerance to 2,4-D, glyphosate and ammonium glufosinate. This event is also associated with DAS-81419-2 expressing Cry1Ac and Cry1F proteins to provide protection against a broad spectrum of soybean lepidopteran insect species. Successful adoption by growers of these technologies relies on the traits performing in the diverse environments of Brazil, especially under adverse weather conditions. The objective of this study was to characterize the tolerance of DAS 444Ø6-6 and DAS-444Ø6-6 x DAS-81419-2 soybeans to 2,4-D and glyphosate in the Cerrado region of Brazil. Eight trials across four sites were conducted in the 2015/2016 summer rainy season using a randomized complete block design, with a factorial arrangement of treatments. Factor A was 2,4-D choline salt (456 g a.e. L-1) + glyphosate DMA salt (480 g a.e. L-1) at 975 + 1,025 and 1,950 + 2,050 g a.e. ha-1, respectively and ready-mix of (2,4-D choline salt 195 g a.e. L-1 + glyphosate DMA salt 205 g a.e. L-1) at 2,000 (975 + 1,025) and 4,000 (1,950 + 2,050) g a.e. ha-1. Factor B was application timing at V3, V6 and R2 soybean growth stages. No visible symptoms of leaf chlorosis, epinasty, or growth inhibition were observed. Slight leaf droop was observed at 1 day after treatment (DAT), but it was null at 7 DAT. Less than 6% and 13% of leaf necrosis or injury were observed at 7 DAT, respectively, at 975 + 1,025 and 1,950 + 2,050 g a.e. ha-1 of 2,4-D choline salt + glyphosate DMA salt, regardless of applied as a ready-mix or not, but it was always 2% or less at 28 DAT. Crop grain yield was not influenced by herbicide treatments, application timing and their interaction, regardless of the soybean event and experimental site. DAS-444Ø6-6 and DAS-444Ø6-6 x DAS-81419-2 soybeans showed high tolerance to 2,4-D choline salt and glyphosate DMA in the Cerrado region of Brazil.

Removal of early fruit branches as potential regulator of Cry1Ac, antioxidants, senescence and yield in Bt. cotton

Excessive use of insecticides, degradation of Cry1Ac protein, insect resistance and premature senescence in cotton are serious threats to the sustainable cotton production. An experiment was conducted to manage these adversities and to determine dependence of morphological attribute on physiochemical responses. Experiments were conducted at University of Agriculture Faisalabad, Pakistan over two years (2015, 2016) and two locations. The experiment was laid out in randomized complete block design under factorial arrangement. Treatments were comprised of branch removal viz. R0 = No removal of early fruit branches; R1 = Removal of two early fruit branches (REFB) and genotypes V1 = MNH-886 and V2 = FH-142. Results revealed that Cry1Ac (33%) and nitrogen concentration (16%) were increased under REFB compared to no REFB over years and locations. Chlorophyll a and b contents were improved by 11 and 6% under REFB compared to no REFB over two localities and years. While, SOD, POD and CAT activities were increased by 13, 13 and 10%, respectively under REFB compared to no REFB. Nodes above white flower (5 days), nodes for first fruiting branch and seed cotton yield were enhanced by 14, 15 and 17%, respectively under REFB compared to no REFB over years and locations. Number of insect damaged bolls were decreased by 28% under REFB compared to control over years and locations. More promising responses were recorded for MNH-886 compared to FH-142 over REFB, years and locations. Conclusively, all physiochemical and agronomic attributes were improved, and senescence and insect attack were decreased in both genotypes over the spatial and temporal variations. Significant and strong correlation of physiochemical attributes with morphological attributes were recorded over years and locations.

Dynamics of Bt cotton Cry1Ac protein content under an alternating high temperature regime and effects on nitrogen metabolism

This study was conducted to investigate the effects of alternating high temperature on Cry1Ac protein content on Bt cotton cultivars Sikang 1 (SK-1, a conventional cultivar) and Sikang 3 (SK-3, a hybrid cultivar). In 2011 and 2012, cotton plants were subjected to high temperature treatments ranging from 32 to 40°C in climate chambers to investigate the effects of high temperature on boll shell insecticidal protein expression. The experiments showed that significant decline of the boll shell insecticidal protein was detected at temperatures higher than 38°C after 24 h. Based on the results, the cotton plants were treated with the threshold temperature of 38°C from 6:00 a.m. to 6:00 p.m. followed by a normal temperature of 27°C during the remaining night hours (DH/NN) in 2012 and 2013. These treatments were conducted at peak boll growth stage for both cultivars in study periods of 0, 4, 7, and 10 d. Temperature treatment of 32°C from 6:00 a.m. to 6:00 p.m. and 27°C in the remaining hours was set as control. The results showed that, compared with the control, after the DH/NN stress treatment applied for 7 d, the boll shell Cry1Ac protein content level was significantly decreased by 19.1 and 17.5% for SK-1 and by 15.3 and 13.7% for SK-3 in 2012 and 2013, respectively. Further analysis of nitrogen metabolic physiology under DH/NN showed that the soluble protein content and the glutamic pyruvic transaminase (GPT) activities decreased slightly after 4 d, and then decreased sharply after 7 d. The free amino acid content and the protease content increased sharply after 7 d. The changes in SK-1 were greater than those in SK-3. These results suggest that under DH/NN stress, boll shell Cry1Ac protein content decline was delayed. Reduced protein synthesis and increased protein degradation in the boll shell decreased protein content, including Bt protein, which may reduce resistance to the cotton bollworm.

Efficacy of Cry1Ac protein against gypsy moth and fall webworm in transgenic poplar (Populus davidiana × Populus bolleana) by bioassay

A modified Bacillus thuringiensis (Bt) cry1Ac gene was introduced into poplar ‘Shanxin’ (Populus davidiana × Populus bolleana) by Agrobacterium-mediated transformation. Seventeen cry1Ac transgenic lines were regenerated. The integration and expression of the cry1Ac gene in these transgenic lines were analyzed by polymerase chain reaction (PCR) and reverse-transcription quantitative PCR, respectively. Variable amounts of cry1Ac mRNA accumulated in different transgenic plants. The expression of Cry1Ac toxin protein in transgenic plants was analyzed by enzyme-linked immunosorbent assay, the concentration ranged from 0.12 to 21.00 μg g−1. The concentration of Cry1Ac protein was highly consistent with mRNA amount in each transgenic line. Leaf section bioassays were conducted using the larvae of both gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). Those plants, except lines 10 and 11, caused rapid mortality of all gypsy moth and fall webworm larvae with almost no defoliation, while lines 10 and 11, with Cry1Ac protein concentrations of 0.12 and 0.76 μg g−1, respectively, provided relatively weak insect protection. Results of insect bioassays indicate that Cry1Ac protein expression over 1.3 μg g−1 consistently produced high insect mortality for both species of moths. The correlation between concentration of Cry1Ac protein and insect mortality is important to understand for managing insect pests with transgenic plants. Transgenic poplars with high insect resistance will be useful tools for managing Lepidoptera pests of these trees.

Defoliation of Soybean Expressing Cry1Ac by Lepidopteran Pests.

Lepidoptera, stink bugs, and weevils are important pests in soybean. For lepidopteran control, insecticides and seed treatments are used. As an alternative, Bt soybean was developed to control primary pests of Lepidoptera such as Rachiplusia nu (Guenée) (Noctuidae), Chrysodeixis includens (Walker) (Noctuidae), Anticarsia gemmatalis Hübner (Erebidae), Helicoverpa gelotopoeon (Dyar) (Noctuidae), and Crocidosema aporema (Walsingham) (Tortricidae). However, the use of transgenic plants, and the resulting reduction of insecticide against target pests, may allow other pest species to become more prevalent in agricultural environments. Soybean expressing Cry1Ac against different lepidopteran nontarget and target insect pests was evaluated, and its performance was compared with non-Bt soybean with seed treatment. The treatments were Bt soybean, non-Bt soybean with seed treatment (Fortenza® diamide insecticide, Syngenta, Buenos Aires, Argentina), and non-Bt soybean without seed treatment. Larvae of H. gelotopoeon, Spodoptera albula (Walker) (Noctuidae), Spodoptera cosmiodes (Walker) (Noctuidae), Spodoptera eridania (Stoll) (Noctuidae), and Spodoptera frugiperda (J. E. Smith) (Lep.: Noctuidae) were used. The plants of each treatment were infested with larvae of each species, and the percentage of leaf damage produced by each species was recorded. The results showed that Bt soybean provided control of H. gelotopoeon and had a suppressive effect on S. frugiperda and S. albula. However, S. eridania and S. cosmiodes were not susceptible to the Cry1Ac protein in MON 87701 × MON 89788 soybean when evaluated by greenhouse infestation. Considering the performance of each species using non-Bt soybean without seed treatment, S. eridania would represent a potential risk in soybean crops.

Evaluation of Cry1Ac and Cry2Aa Toxin Binding to Two Important Beneficial Cotton Field Insects, Harmonia axyridis and Orius similis.

Transgenic crops expressing Cry toxins are effective and considered environmentally friendly alternatives to synthetic pesticides, but assessment of environmental risks of their application on nontarget organisms is ongoing. The main risk is the transfer of Cry toxins to natural enemies through the food chain. There is reported evidence supporting that Cry toxins can be detected in the body and gut of some natural enemy insects. Considering that binding of Cry toxins to insect proteins is an essential step in the intoxication process, this work was conducted to evaluate interactions between Cry1Ac and Cry2Aa toxins with proteins from larvae/nymphs and adults of two important predatory natural enemies in cotton fields, Harmonia axyridis and Orius similis. Results support the absence of Cry1Ac or Cry2Aa binding proteins in immature stages of H. axyridis and O. similis, as well as in imaginal stage of H. axyridis. One same binding band about 70 kDa was found in imaginal total protein of O. similis when probed with the two Cry proteins, with the best match to Hsc70 of O. sauteri in the Uniprot database. However, nonspecific binding was verified by following competitive binding assays between the two Cry proteins and imaginal total protein of O. similis. From these results, we may infer that Cry1Ac and Cry2Aa have no likely detrimental effects on H. axyridis and O. similis.

Significant increase in toxicity of the Cry1Ac1 protein of Bacillus thuringiensis by the exchange of two amino acids in its domain III

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed high biochemical and genetic similarities with the HD-73 standard strain, besides expressing only a Cry1Ac protein: Cry1Ac39. However, LBIT-1200 was 6.4 and 9.5 times more toxic against Manduca sexta and Trichoplusia ni larvae, respectively, than HD-73, which expresses the Cry1Ac1 protein. Sequencing of Cry1Ac1 and Cry1Ac39 proteins showed only four amino acid differences, including two amino acid replacements in domain III, as compared to the rest of Cry1Ac proteins reported to date: N547Y and R602G. These two amino acid substitutions in domain III were introduced in the Cry1Ac1 protein from HD-73 through site directed mutagenesis. Three mutants were obtained: two single and one double mutant, which were expressed in an acrystaliferous B. thuringiensis strain, as well as the native Cry1Ac1 and Cry1Ac39 toxins. Bipyramidal crystals produced by the single mutant strains were larger than the original Cry1Ac1 protein; however, the high toxicity levels previously observed in the strain LBIT-1200 were restored in the three mutants, when tested against M. sexta and T. ni. The substitution N547Y seems to have a higher contribution to LBIT-1200 toxicity.

Bio-safety of Helicoverpa-resistant transgenic chickpea with Bacillus thuringiensis genes in the environment

With the advent of recombinant DNA technology, it has become possible to clone and insert genes into the crop plants to confer resistance to insect pests. Genes conferring resistance to insect pests have been inserted into several crops, and efforts are underway to develop transgenic chickpea with resistance to pod borer, Helicoverpa armigera(Hubner) – which causes an estimated loss of over $500 million annually. However, concerns have been raised regarding the impact of genetically engineered plants on the non-target organisms, gene flow, and biosafety of the food derived from genetically engineered plants. There is no issue related to gene flow involving transgenic chickpea as most ofthe studies have indicated little or <1% pollen flow to the neighboring plants. The effects of transgenic plants on the activity and abundance of natural enemies vary across crops, and the insect species involved. In general, there are no major adverse effects of genetically modified insect-resistant crops on the generalist predators such as Cheilomenes sexmaculatus, Coccinella septempunctata, and Chrysoperla carnea, while some adverse effects have been observed on the host specific parasitoids such as Campoletis chlorideae, which are largely due to early mortality of the host larvae or poor nutritional quality of the host, rather than direct toxicity of the transgene products to the natural enemies. Wherever the transgenic crops have shown adverse effects on the natural enemies, these effects may still be far lower than those of the broad-spectrum pesticides commonly used for pest management. While no specific studies have beencarried out on the biosafety of food derived from transgenic chickpea due to unavailability of events with optimum gene expression for controlling H. armigera, there is enough information on the acute and chronic toxicity of Bt Cry proteins (Cry1Ab, Cry1Ac, and CryIIa), which have also been deployed in commercial transgenic maize and cotton. The issue of biosafety of the food derived from chickpea could also be addressed based on the principle of nutritional equivalence as and when the transgenic events are available for testing. Insect-resistant transgenic crops would play a significant role in pest management in future, and lead to a major reduction in insecticide sprays. Production andrelease of transgenic chickpea could be based on the guidelines laid down by the concerned agencies internationally, and the experience gained with other transgenic crops for improving food security and environment conservation.

Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280FRGSAQGIEGS290, 368RRPFNIGINNQQ379 and 436FRSGFSNSSVSIIR449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368RRPFNIGINNQQ379. This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865NITIHITDTNNK876, a specific recognition site in insect receptor Bt-R1, to 368RRPFNIGINNQQ379. The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368RRPFNIGINNQQ379. This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

Subchronic toxicity study in rats evaluating genetically modified DAS-81419-2 soybean

A 90-day feeding study in rats was conducted to evaluate the subchronic oral toxicity of genetically modified (GM) DAS-81419-2 soybean. Wistar rats were fed with diets containing toasted soybean meal produced from DAS-81419-2 soybean grain that expresses the Cry1F, Cry1Ac, and Pat proteins or containing conventional soybean at doses of 30.0%, 15.0%, 7.5%, or 0% (control group) for 90 consecutive days. The general behavior, body weight and food consumption were observed. At the middle and end of the experiment, blood, serum, and urine samples were collected for biochemical assays. At the conclusion of the study, the internal organs were weighed and histopathological examination was completed. The rats exhibited free movement and shiny coats without any abnormal symptoms or abnormal secretions in their noses, eyes, or mouths. There were no adverse effects on body weight in GM soybean groups and conventional soybean groups. No biological differences in hematological, biochemical, or urine indices were observed. No significant differences in relative organ weights were detected between the experimental groups and the control group. No histopathological changes were observed. Under the conditions of this study, DAS-81419-2 soybean did not cause any treatment-related effects in Wistar rats following 90 days of dietary administration.

Development of transgenic tobacco lines with pyramided insect resistant genes.

Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with cry1Ac and cry2A genes under the control of 35S promoter was used to infect leaf discs of both cultivars; plasmid also contained uidA within the T-DNA region for earlier screening of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively. PCR results confirmed the integration of cry1Ac, cry2A, uidA, and nptII genes in 40 plants of Basma and 16 plants of Nail. ELISA results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 µg/g of fresh tissue. Bioassay results with potato tuber moth (Phthorimea operculella Zeller) showed significant mortality of the targeted pest on primary transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion. The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.

A General Safety Assessment for Purified Food Ingredients Derived From Biotechnology Crops: Case Study of Brazilian Sugar and Beverages Produced From Insect-Protected Sugarcane.

Insect-protected sugarcane that expresses Cry1Ab has been developed in Brazil. Analysis of trade information has shown that effectively all the sugarcane-derived Brazilian exports are raw or refined sugar and ethanol. The fact that raw and refined sugar are highly purified food ingredients, with no detectable transgenic protein, provides an interesting case study of a generalized safety assessment approach. In this study, both the theoretical protein intakes and safety assessments of Cry1Ab, Cry1Ac, NPTII, and Bar proteins used in insect-protected biotechnology crops were examined. The potential consumption of these proteins was examined using local market research data of average added sugar intakes in eight diverse and representative Brazilian raw and refined sugar export markets (Brazil, Canada, China, Indonesia, India, Japan, Russia, and the USA). The average sugar intakes, which ranged from 5.1 g of added sugar/person/day (India) to 126 g sugar/p/day (USA) were used to calculated possible human exposure. The theoretical protein intake estimates were carried out in the “Worst-case” scenario, assumed that 1 μg of newly-expressed protein is detected/g of raw or refined sugar; and the “Reasonable-case” scenario assumed 1 ng protein/g sugar. The “Worst-case” scenario was based on results of detailed studies of sugarcane processing in Brazil that showed that refined sugar contains less than 1 μg of total plant protein /g refined sugar. The “Reasonable-case” scenario was based on assumption that the expression levels in stalk of newly-expressed proteins were less than 0.1% of total stalk protein. Using these calculated protein intake values from the consumption of sugar, along with the accepted NOAEL levels of the four representative proteins we concluded that safety margins for the “Worst-case” scenario ranged from 6.9 × 105 to 5.9 × 107 and for the “Reasonable-case” scenario ranged from 6.9 × 108 to 5.9 × 1010. These safety margins are very high due to the extremely low possible exposures and the high NOAELs for these non-toxic proteins. This generalized approach to the safety assessment of highly purified food ingredients like sugar illustrates that sugar processed from Brazilian GM varieties are safe for consumption in representative markets globally.

Flowerlike Mg(OH)2 Cross-Nanosheets for Controlling Cry1Ac Protein Loss: Evaluation of Insecticidal Activity and Biosecurity.

Bacillus thuringiensis (Bt) can produce Cry proteins during the sporulation phase, and Cry protein is effective against lepidopteran, coleopteran, and dipteran insects and nematodes. However, Cry protein tends to be discharged into soil and nontarget plants through rainwater runoff, leading to reduced effective period toward target insects. In the present study, nano-Mg(OH)2 (magnesium hydroxide nanoparticles, MHNPs) were synthesized to control the loss of Cry1Ac protein and deliver protein to Helicoverpa armigera (Lepidoptera: Noctuidae). The results showed that Cry1Ac protein could be loaded onto MHNPs through electrostatic adsorption, and both MHNPs and Cry protein were stable during the adsorption process. Meanwhile, the Cry1Ac-loaded MHNPs could remain on the surface of cotton leaves, resulting in enhanced adhesion of Cry1Ac protein by 59.50% and increased pest mortality by 75.00%. Additionally, MHNPs could be slowly decomposed by acid medium and MHNPs showed no obvious influence on cotton, Bt, Escherichia coli, and H. armigera. Therefore, MHNPs could serve as an efficient nanocarrier for delivery of Cry1Ac protein and be used as a potential adjuvant for biopesticide in agricultural applications.

Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis.

In this study, a sporulation-specific gene (tentatively named cwlC) involved in mother cell lysis in Bacillus thuringiensis was characterized. The encoded CwlC protein consists of an N-terminal N-acetylmuramoyl-l-alanine amidase (MurNAc-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from Escherichia coli were able to directly bind to and digest the B. thuringiensis cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is an N-acetylmuramoyl-l-alanine amidase. Results of transcriptional analyses indicated that cwlC is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σK). Deletion of cwlC completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase for B. thuringiensis mother cell lysis during sporulation. Engineered B. thuringiensis strains targeting cwlC, which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.IMPORTANCE Mother cell lysis has been well studied in Bacillus subtilis, which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis in Bacillus thuringiensis CwlC of B. thuringiensis only shows 9 and 21% sequence identity with known B. subtilis mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ between B. subtilis and B. thuringiensis The cwlC gene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.

Establishment of a dietary exposure assay for evaluating the toxicity of insecticidal compounds to Apolygus lucorum (Hemiptera: Miridae).

With the commercialization of transgenic cotton that expresses Bt (Bacillus thuringiensis) insecticidal proteins, mirid bugs have become key pests in cotton and maize fields in China. Genetically engineered (GE) crops for controlling mirids are unavailable owing to a lack of suitable insecticidal genes. In this study, we developed and validated a dietary exposure assay for screening insecticidal compounds and for assessing the potential effects of insecticidal proteins produced by GE plants on Apolygus lucorum, one of the main mirid pests of Bt cotton and Bt maize. Diets containing potassium arsenate (PA) or the cysteine protease inhibitor E-64 were used as positive controls for validating the efficacy of the dietary exposure assay. The results showed that with increasing concentrations of PA or E-64, A.lucorum larval development time was prolonged and adult weight and fecundity were decreased, suggesting that the dietary exposure assay was useful for detecting the toxicity of insecticidal compounds to A.lucorum. This assay was then used to assess the toxicity of Cry1Ab, Cry1Ac, Cry1F, Cry2Aa, and Cry2Ab proteins, which have been transformed into several crops, against A.lucorum. The results showed that A.lucorum did not show a negative effect by feeding on an artificial diet containing any of the purified Cry proteins. No significant changes in the activities of digestive, detoxifying, or antioxidant enzymes were detected in A.lucorum that fed on a diet containing Cry proteins, but A.lucorum fitness was reduced when the insect fed on a diet containing E-64 or PA. These results demonstrate that A.lucorum is not sensitive to the tested Cry proteins and that the dietary exposure assay is useful for evaluating the toxicity of insecticidal compounds to this species.

A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis.

Bacillus thuringiensis bacteria show insecticidal activities that rely upon the production of insecticidal crystal proteins, which are encoded by cry or cyt genes and can target a variety of insect pests. It has been shown that cry1Ac is the only cry gene in B. thuringiensis subsp. kurstaki HD73 (B. thuringiensis HD73) and its expression is controlled by both σE and σK. Here, we report a novel σE-dependent strong promoter of a non-cry gene (HD73_5014), which can direct strong cry1Ac gene expression in B. thuringiensis HD73. We constructed an E. coli-B. thuringiensis shuttle vector (pHT315-P 5014 -1Ac) for cry1Ac gene expression, using the HD73_5014 gene promoter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis showed that expression of the cry1Ac gene directed by the HD73_5014 gene promoter was at the same level as that directed by the previously known strongest cry promoter, P cry8E . However, this strain did not form typical bipyramidal crystals in mother cells, as observed by transmission electron microscopy and atomic force microscope. The strain with Cry1Ac protein expression under the control of the HD73_5014 gene promoter (P 5014 -cry1Ac) showed insecticidal activity against Plutella xylostella similar to that under the control of the orf1cry8E gene promoter (P cry8E -cry1Ac). Collectively, these results suggest that the HD73_5014 gene promoter, as a non-cry gene promoter, would be an efficient transcriptional element for cry gene expression. These data also show the possibility for improving Cry production by searching for transcriptional elements in not only cry genes, but also non-cry genes.

Domain III of Cry1Ac Is Critical to Binding and Toxicity against Soybean Looper (Chrysodeixis includens) but Not to Velvetbean Caterpillar (Anticarsia gemmatalis).

Insecticidal proteins Cry1Ac and Cry2Ac7 from the bacterium Bacillus thuringiensis (Bt) belong to the three-domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control the larvae of the soybean looper (Chrysodeixis includens) and the velvet bean caterpillar (Anticarsia gemmatalis). The specificity of Cry1Ac is determined by loops extending from domain II and regions of domain III in the three-dimensional structure of the toxin. In this study, we constructed a hybrid toxin (H1.2Ac) containing domains I and II of Cry1Ac and domain III of Cry2Ac7, in an attempt to obtain a protein with enhanced toxicity compared to parental toxins. Bioassays with H1.2Ac revealed toxicity against the larvae of A. gemmatalis but not against C. includens. Saturation binding assays with radiolabeled toxins and midgut brush border membrane vesicles demonstrated no specific H1.2Ac binding to C. includens, while binding in A. gemmatalis was specific and saturable. Results from competition binding assays supported the finding that Cry1Ac specificity against A. gemmatalis is mainly dictated by domain II. Taken together, these distinct interactions with binding sites may help explain the differential susceptibility to Cry1Ac in C. includens and A. gemmatalis, and guide the design of improved toxins against soybean pests.

Soil incubation studies with Cry1Ac protein indicate no adverse effect of Bt crops on soil microbial communities

Bt crops that are transgenic crops engineered to produce Bt toxins which occur naturally with Bacillus thuringiensis (Bt) have been widely planted and its environmental risk assessment has been heavily debated. The effects of Bt crops on soil microbial communities are possible through changing the quantity and quality of C inputs and potential toxic activity of Bt protein on soil organisms. To date, the direct effects of Bt protein on soil microorganisms is unclear. Here we added Cry1Ac, one of the most commonly used Bt protein in Bt crops, to the soil and monitored changes in soil bacterial, fungal and archaeal diversities and community structures using ribosomal DNA-fingerprinting method, as well as their population sizes by real-time PCR over a 100-day period. Despite the fact that variations were observed in the indices of evenness, diversity and population sizes of bacteria, fungi and archaea with different Cry1Ac addition rates up to 100ngg−1 soil, the indices of soil microbial diversities and evennesses did not significantly shift with Cry1Ac protein addition, nor did population sizes change over time. The diversities of the dominant bacteria, fungi and archaea were not significantly changed, given Cry1Ac protein addition rates over a period of 100 days. These results suggested that Bt protein derived by cultivations of transgenic Bt crops is unlikely to cause transient or even persisting significant changes in soil microorganisms in field.