Expression Of Insecticidal Proteins Research Articles

Optimizing Bacillus thuringiensis (Bt) protein concentration in cotton: Coordinated regulation of exogenous amino acids and EDTA to reduce spatiotemporal variability in boll and leaf toxin

During the boll formation stage, cotton bolls exhibit the lowest expression of insecticidal proteins. Insect resistance varies notably among different organs, posing challenges for controlling cotton bollworms. Consequently, a strategy was designed in the 2020-2021 cotton growing season to coordinate the enhancement of protein synthesis and the attenuation of degradation. Two Bt cultivars of G. hirsutum, namely the hybrid Sikang 3 and the conventional Sikang 1, were used as test materials. Three treatments were implemented: CK (the control), T1 (amino acids), and T2 (amino acids and EDTA). These treatments were applied at the peak flowering period. The results show that, in comparison to the CK group, Bt protein content significantly increased both cotton bolls and their subtending leaves under T1 and T2 treatments. The maximum increase observed was 67.5% in cotton bolls and 21.7% in leaves. Moreover, the disparity in Bt protein content between cotton bolls and their subtending leaves notably decreased by 31.2%. Correlation analysis suggests that the primary physiological mechanisms for augmenting Bt protein content involve increased protein synthesis and decreased protein catabolism, independent of Bt gene expression levels. Stepwise regression and path analysis reveal that elevating soluble protein content and transaminase activity, while reducing catabolic enzyme activity, are instrumental in enhancing Bt protein content. Consequently, the coordinated regulation of amino acids and EDTA emerges as a strategy capable of improving the overall resistance of Bt cotton and mitigating spatiotemporal variations in Bt toxin concentrations in both cotton bolls and leaves.

Intermittent induction of LEA peptide by lactose enhances the expression of insecticidal proteins in Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis (Bt) have been extensively applied in agriculture to substitute the use of chemical insecticides. We have previously reported the use of a coexpression system in which late embryogenesis abundant (LEA) peptides under the control of the lac promoter increase the expression of insecticidal proteins in Bt. The use of lactose to induce the expression of LEA peptides may be a desirable alternative to isopropyl β‐D‐thiogalactopyranoside, the most frequently used inducer for recombinant protein expression. In this study we investigated the use of lactose as an inducer for optimal protein expression. We observed enhanced insecticidal Cry protein expression by applying a simple technique based on intermittent induction, and then optimized concentration and the point of induction time from the 11th h to the 15th h. Our data suggest that intermittent induction of lactose might be a new technique for the enhancement of bacterial protein expression.

Recovery Characteristics of Cry1Ac Endotoxin Expression and Related Physiological Mechanisms in Bt Transgenic Cotton Squares after High-Temperature Stress Termination

High-temperature stress reduces the endotoxin expression of the insecticidal gene (Cry1Ac) in transgenic Bacillus thuringiensis (Bt) cotton and affects nitrogen metabolism. However, its effects on Cry1Ac endotoxin expression after high-temperature stress termination remain unclear. In order to investigate the effect of high-temperature stress on the expression of insecticidal proteins in Bt cotton squares, the conventional Sikang-1 cultivar and hybrid Sikang-3 cultivar were used as the experimental materials. The potted cotton plants in the squaring stage were moved to an artificial climatic chamber at 38 °C for 72 h and 96 h in 2017 and 2018, respectively, and plants were moved to the climate chamber where the control cotton plants were located (at 27 °C). Then, cotton squares were collected to measure the Bt protein concentration and nitrogen metabolism physiology at 0, 12, 24, 48, 72 and 96 h after high-temperature stress termination, respectively. The Cry1Ac endotoxin expression of the squares could be recovered to the corresponding control level and a longer recovery time was required as the high-temperature stress period increased. Therefore, the recovery degree of Cry1Ac endotoxin expression of cotton squares can be predicted according to the duration of high-temperature stress, which may provide a reference for the rational control of Helicoverpa armigera and related pests in cotton production.

Engineering of Cry3Bb1 provides mechanistic insights toward countering western corn rootworm resistance.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an economically important pest of corn (maize) in North America and Europe. Current management practices for WCR involve transgenic expression of insecticidal proteins to minimize larval feeding damage to corn roots. The evolution of resistant WCR populations to transgenic corn expressing insecticidal proteins (e.g. Cry3Bb1, Gpp34Ab1/Tpp35Ab1) necessitates efforts to discover and deploy new modes of action for WCR control. Here, we tested the hypothesis that the addition of short peptides selected for binding to the WCR gut would restore insecticidal activity of Cry3Bb1 to resistant insects. Phage display technology coupled with deep sequencing was used to identify peptides selected for binding to WCR brush border membrane vesicles and to recombinant putative receptors aminopeptidase and cadherin. The binding and specificity of selected peptides was confirmed by ELISA and pull-down assays, and candidate gut surface binding partners were identified. Although production of 284 novel Cry3Bb1 variants with these peptides did not restore activity against resistant WCR in artificial diet bioassays, 112 variants were active against susceptible insects. These results provided insights for the mechanism of Cry3Bb1 activity and toward engineering a new mode-of-action via receptor re-targeting in the context of protein structure and function.

Microbial Toxins in Insect and Nematode Pest Biocontrol.

Invertebrate pests, such as insects and nematodes, not only cause or transmit human and livestock diseases but also impose serious crop losses by direct injury as well as vectoring pathogenic microbes. The damage is global but greater in developing countries, where human health and food security are more at risk. Although synthetic pesticides have been in use, biological control measures offer advantages via their biodegradability, environmental safety and precise targeting. This is amply demonstrated by the successful and widespread use of Bacillus thuringiensis to control mosquitos and many plant pests, the latter by the transgenic expression of insecticidal proteins from B. thuringiensis in crop plants. Here, I discuss the prospects of using bacterial and fungal toxins for pest control, including the molecular basis of their biocidal activity.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.

Engineering Chloroplasts for High-Level Constitutive or Inducible Transgene Expression.

Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., for expression of insecticidal proteins) and molecular farming (e.g., for expression of pharmaceutical proteins and industrial enzymes). Over the past decades, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (e.g., subunit vaccines and protein antibiotics) and industrial enzymes. Although in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression, and highlight possible optimization strategies to alleviate problems with poor expression levels. I also discuss available techniques for inducible expression of chloroplast transgenes.

Impact of Various Bt Cotton Traits and the Application of an Insecticide on the Within Plant Distribution of Helicoverpa zea (Lepidoptera: Noctuidae) Larvae and Injured Floral Structures

Previous studies have indicated that the expression of insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) in cotton can have a significant influence on the behavior of bollworm larvae (Helicoverpa zea, Lepidoptera:Noctuidae). This suggests that the particular Bt protein produced by a cotton variety may need to be considered when determining the most ideal scouting methods to utilize for bollworm. NonBt, WideStrike (producing Cry1Ac + Cry1F Bt insecticidal proteins), and Bollgard II (Cry1Ac + Cry2Ab) cotton varieties were planted and either treated with an insecticide or left untreated. The presence of H. zea larvae and their feeding injury were recorded according to their location in the canopy and type of floral structure where they were found. Results from comparison of larval and injury distributions indicated no significant differences between the different cotton varieties tested, and that insecticide treatment had minimal impact on this distribution. Larval size was generally associated with location in the canopy, suggesting that larvae tend to move towards the middle of the canopy as they age. The effect of different Bt cotton technologies appears to associate with how quickly larvae move to preferred feeding sites rather than their preference for particular feeding sites. These results suggest that scouting methods could be standardized independently of the presence of a Bt cotton trait or previous insecticide application. Focusing scouting efforts on the middle portion of the canopy (i.e., nodes 6-9) should increase the detection of small larvae and ‘fresh’ injury and be less influenced by previous insecticide applications.

In-planta expression of insecticidal proteins provides protection against lepidopteran insects

The development of advanced biotechnological control strategies opens a new era of environment friendly pest management. The current study is part of such an effort, in which we developed a control strategy based on gene pyramiding that confers broad-spectrum resistance against lepidopteran (Helicoverpa armigera and Spodoptera litura) and hemipteran (Myzus persicae, Phenacoccus solenopsis, and Bemisia tabaci) insect pests. Previously, we reported a double gene construct expressing Hvt and lectin in tobacco (Nicotiana tabacum) plants under phloem specific promoters which confers resistance against hemipteran insects. Here we extended our studies by evaluating the advanced generation of these tobacco plants expressing hvt-lectin against lepidopteran insects. Tobacco plants expressing both toxins were tested against H. armigera and S. litura. Insect bioassay results showed 100% mortality of H. armigera within 48–72 hours and 100% mortality of S. litura within 72–96 hours. Our results suggest that the use of both toxins as a gene pyramiding strategy to control both lepidopteran and hemipterans insects on commercial basis to reduce the use of chemical pesticides.

A strong early acting wound-inducible promoter, RbPCD1pro, activates cryIAc expression within minutes of wounding to impart efficient protection against insects.

SummaryThe expression of insecticidal proteins under constitutive promoters in transgenic plants is fraught with problems like developmental abnormalities, yield drag, expression in unwanted tissues, and seasonal changes in expression. RbPCD1pro, a rapid, early acting wound‐inducible promoter from rose that is activated within 5 min of wounding, was isolated and characterized. Wounding increased transcript levels up to 150 and 500 folds within 5 and 20 min coupled with high translation as seen by histochemical GUS enzyme activity within 5–20 min. RbPCD1pro was activated by both sucking and chewing insects and showed wound‐inducible expression in various aerial tissues of plants representing commercially important dicot and monocot families. The promoter showed no expression in any vegetative tissue except upon wounding. Functionality of RbPCD1pro was tested by its ability to drive expression of the insecticidal protein gene cryIAc in transgenic Arabidopsis and tomato. Strong wound‐inducible CryIAc expression was observed in both plants that increased 100–350 fold (Arabidopsis) and 280–600 fold (tomato) over the unwounded background within 5 min and over 1000–1600 fold within 20 min. The unwounded background level was just 3–6% of the CaMV35S promoter while wound‐induced expression was 5–27 folds higher than the best CaMV35S line in just 5 min and 80‐fold higher in 20 min. Transgenic plants showed strong resistance even to larger fourth instar larvae of H. armigera and no abnormalities in development and general plant growth. This is one of the earliest acting promoters with wide biotechnological application across monocot and dicot plants.

Effect of NaCl-stressed Bacillus thuringiensis (Bt) cotton on the feeding behaviors and nutritional parameters of Helicoverpa armigera.

Saline-alkali soil is an arable land resource on which transgenic Bacillus thuringiensis (Bt) cotton has been planted on a large scale in accordance with food security strategies. There are, however, concerns about the insecticidal effects of Bt cotton on target insect pests. In this study, a Bt cotton variety, GK19, and its nontransgenic parent variety, Simian-3, were used as experimental models for investigating the effect of the expression of exogenous insecticidal proteins in Bt cotton under NaCl stress on the feeding behavior and nutritional parameters of Helicoverpa armigera. The results showed that the expression of exogenous insecticidal proteins in GK19 was significantly inhibited under NaCl stress. However, the feeding, crawling, resting and spinning down behavior of the 5th instar H. armigera larvae on GK19 Bt cotton, as well as the amount of food consumed and feces produced by these larvae, did not markedly differ under different NaCl concentrations. In contrast, the mean relative growth rate (MRGR), relative growth rate (RGR), approximate digestibility (AD), efficiency of conversion of ingested food (ECI) and efficiency of conversion of digested food (ECD) of the larvae markedly decreased in response to NaCl stress. Under the same concentration of NaCl, the nutritional parameters of the bollworm larvae on GK19 Bt cotton or Simian-3 nontransgenic cotton were different. However, the interaction between salt stress and cotton variety had no significant effect on the feeding behavior or nutritional parameters of H. armigera larvae. These results may provide a scientific basis for determining the effect of exogenous insecticidal protein expression in Bt cotton under NaCl stress on H. armigera and can therefore be useful for the effective application of Bt cotton in saline-alkali soils to prevent and control H. armigera.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80–100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25–80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Engineering Dual Begomovirus-Bemisia tabaci Resistance in Plants.

The whitefly Bemisia tabaci is an important pest of many economically important crops and the vector of begomoviruses (family Geminiviridae). Recently, the expression of insecticidal proteins and/or toxins or double-stranded (ds)RNA homologous to B. tabaci genes has been demonstrated to provide the plant with protection against B. tabaci and the viruses that it transmits.

Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses.

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

Do genetically modified plants impact arbuscular mycorrhizal fungi?

The development and use of genetically modified plants (GMPs), as well as their ecological risks have been a topic of considerable public debate since they were first released in 1996. To date, no consistent conclusions have been drawn dealing with ecological risks on soil microorganisms of GMPs for the present incompatible empirical data. Arbuscular mycorrhizal fungi (AMF), important in regulating aboveground and underground processes in ecosystems, are the most crucial soil microbial community worthy of being monitored in ecological risks assessment of GMPs for their sensitivity to environmental alterations (plant, soil, climatic factor etc.). Based on current data, we suggest that there is a temporal-spatial relevance between expression and rhizosphere secretion of anti-disease and insecticidal proteins (e.g., Bt-Bacillus thuringiensis toxins) in and outer roots, and AMF intraradical and extraradical growth and development. Therefore, taking Bt transgenic plants (BTPs) for example, Bt insecticidal proteins constitutive expression and rhizosphere release during cultivation of BTPs may damage some critical steps of the AMF symbiotic development. More important, these processes of BTPs coincide with the entire life cycle of AMF annually, which may impact the diversity of AMF after long-term cultivation period. It is proposed that interactions between GMPs and AMF should be preferentially studied as an indicator for ecological impacts of GMPs on soil microbial communities. In this review, advances in impacts of GMPs on AMF and the effect mechanisms were summarized, highlighting the possible ecological implications of interactions between GMPs and AMF in soil ecosystems.

Making effective use of existing data for case‐by‐case risk assessments of genetically engineered crops

AbstractMany crops in developing countries suffer devastating attacks from insect pests. Expression of insecticidal proteins in genetically engineered (GE) crops is a potentially powerful means of controlling such pests. Potentially harmful effects of these crops on non‐target organisms (NTOs) is of major concern as many of those provide important ecological functions such as pest regulation. Consequently, the likelihood of adverse effects of insect‐resistant GE crops on NTOs is assessed case‐by‐case as part of environmental risk assessments that inform regulatory decision‐making. While risk assessments should be rigorous, it is vital that regulatory barriers do not unnecessarily restrict or prevent the application of genetic engineering to important crops in those countries. Efficient regulatory decision‐making should make effective use of published information on the biology and ecology of the crop in the country where approval is sought, along with regulatory data produced for GE insect‐resistant crops that have received regulatory approvals elsewhere. Just as the risks are assessed for each GE crop individually, the amount of new regulatory data required for a GE crop should vary between crops depending on the amount of existing data and the severity of the perceived risks: new data should be collected only if existing data do not corroborate identified risk hypotheses with sufficient certainty. In this paper, we illustrate how such an approach could work using risks to NTOs from insect‐resistant GE pigeonpea in India as an example.

Field Resistance of Bacillus thuringiensis Berliner Transformed Maize to Fall Armyworm (Lepidoptera: Noctuidae) and Southwestern Corn Borer (Lepidoptera: Crambidae) Leaf Feeding

The fall armyworm, Spodoptera frugiperda (J. E. Smith), and the southwestern corn borer, Diatraea grandiosella (Dyar), can cause economic damage to maize, Zea mays L., grown in the southeastern United States. Maize hybrids are commercially available that have been transformed to express insecticidal crystalline proteins from Bacillus thuringiensis (Bt) Berliner. The field efficacy of seven Bt hybrids were tested for control of leaf-feeding fall armyworm and southwestern corn borer. All Bt hybrids performed better than their conventional near-isolines for control of both insects. In general, the Bt hybrids provided intermediate resistance to the fall armyworm and near immunity to the southwestern corn borer. Based on larval establishment and weights, the fall armyworm was more tolerant of the insecticidal proteins expressed by the Bt hybrids than the southwestern corn borer. There was no difference in expression of insecticidal proteins among the Bt hybrids. Bt hybrids should be advantageous for the production of maize in areas that are affected by southwestern corn borer. The moderate level of resistance in the Bt hybrids to fall armyworm should be further examined to determine if amplifying the expression of insecticidal proteins or integrating other control methods along with the use of current Bt hybrid maize is needed to protect the crop from yield reduction by this pest.

Enhanced expression of insecticidal crystal proteins in wild Bacillus thuringiensis strains by a heterogeneous protein P20.

P20 is a small chaperone-like protein encoded by a cry11A operon in Bacillus thuringiensis subsp. israelensis (Bti); it is essential to Cyt1Aa expression. In this report, the gene P20 was transformed into wild strains of subsp. kurstaki to raise the yield of crystal proteins. As a result, larger crystals were produced by the transformant than by the wild control, and most were in the form of a big bipyramid (average 2.4 microm long); some were irregular because of too high expression, while the spores turned out to be small spheroids unlike the long rods in the wild strains. SDS-PAGE analysis confirmed that Cry1A protoxin production was doubled by P20, but no increase of Cry2A production was observed. Besides, P20 caused obvious changes not only in bacterial morphology, but in the sporulation process as well. Further investigation discovered that a serious degradation happened to Cry1A in vivo of the wild strains, and reconfirmed that P20 was effective in preventing the degradation. Our results suggest that P20 is useful in engineered strain construction with enhanced protein expression.

Agrobacterium-mediated transformation of Brassica campestris ssp. Parachinensis with synthetic Bacillus thuringiensis cry1Ab and cry1Ac genes.

An effective plant regeneration procedure and a gene transfer system via Agrobacterium tumefaciens were developed in Brassica campestris ssp. parachinensis. Hypocotyls from 5-day-old seedlings with 2 days pre-culture were infected with Agrobacterium strain MOG301 harboring a binary vector containing a synthetic Bacillus thuringiensis (B.t.) cry1Ab or cry1Ac gene with full codon-modification. After culture and selection on MS medium supplemented with 4.0 mg/l BAP, 2.0 mg/l NAA, 70 μM AgNO3 and 50 mg/l kanamycin, a number of kanamycin-resistant plantlets were regenerated. PCR and Southern blotting analysis were used to identify and characterize the transgenic plants with the integrated cry1Ab or cry1Ac gene. Western blotting analysis of the transgenic plants confirmed the expression of insecticidal proteins encoded by cry1Ab or cry1Ac. Subsequent bioassay with larvae of the Diamondback moth, Plutella xylostella, demonstrated that the transgenic plants were resistant to feeding damage.