Cry Proteins Research Articles

BPPRC database: a web-based tool to access and analyse bacterial pesticidal proteins.

Pesticidal proteins derived from the bacterium Bacillus thuringiensis, have provided the bases for a diverse array of pest management tools ranging from natural products used in organic agriculture, to modern biotechnological approaches. With advances in genome sequencing technologies and protein structure determination, an increasing number of pesticidal proteins from myriad bacterial species have been identified. The Bacterial Pesticidal Protein Resource Center (BPPRC) has been established to provide informational and analytical resources on the wide range of pesticidal proteins derived from bacteria that have potential utility for arthropod management. In association with a revised nomenclature for these proteins, BPPRC contains a database that allows users to browse and download sequences. Users can search the database for the best matches to sequences of interest and can incorporate their own sequences into basic informatic analyses. These analyses include the ability to draw and export guide trees from either whole protein sequences or, in the case of the three-domain Cry proteins, from individual domains. The associated website also provides a portal for users to submit protein sequences for naming. The BPPRC provides a single authoritative source of information to which all stakeholders can be referred including academics, government regulatory bodies and research and development personnel in the industrial sector. The database provides information on more than 1060 pesticidal proteins derived from 13 species of bacteria, including insecticidal activities for a subset of these proteins. Database URL: www.bpprc.org and www.bpprc-db.org/

Expression of thurincin H, ChiA74 and Cry proteins at the sporulation phase in Bacillus thuringiensis HD1.

The objective of this study was to produce thurincin H, ChiA74 and Cry proteins together using Bacillus thuringiensis subsp. kurstaki HD1 as a heterologous host. pSTAB-ThurH and pSTAB-ChiA74 constructs were designed to produce thurincin H and chitinase, respectively, at the sporulation phase. They were transformed into Bt HD1 generating the recombinant strains HD1/pSTAB-ThurH and HD1/pSTAB-ThurH/pSTAB-ChiA74. Antimicrobial and chitinolytic activity tests were performed with recombinant strains. Both strains were able to produce thurincin H up to 72h with antibacterial activity of ~4000Umg-1 . The HD1/pSTAB-ThurH/pSTAB-ChiA74 strain also showed chitinolytic activity of ~23mUmg-1 at 72h. All B. thuringiensis strains exhibited crystal formation at 72, and 96h. In addition, the application of thurincin H in corn seeds increased the germination percentage and root length by 7% and 10%, respectively. We showed that is possible to produce three proteins of biotechnological interest at the sporulation stage in B. thuringiensis, which two of them (thurincin H, and ChiA74) are naturally expressed in the vegetative stage. These results form the basis for developing of a biocontrol and biostimulator product that can be used as an alternative for chemical application.

Targeting delta-endotoxin (Cry1Ac) of Bacillus thuringiensis to subcellular compartments increases the protein expression, stability, and biological activity.

Evolving insect resistance to delta-endotoxins can be delayed by using a few strategies like high dosage, refugia, and gene stacking which require the expression of delta-endotoxins at sufficiently high levels to kill the resistant insects. In this study, we comparatively analyzed the efficacy of targeting truncated cry1Ac protein to the cytoplasm, endoplasmic reticulum (ER), and chloroplast to obtain high protein expression. mRNA and protein profiling of cry1Ac showed that both ER and chloroplast are efficient targets for expressing high levels of truncated cry1Ac. A maximum of 0.8, 1.6, and 2.0% cry1Ac of total soluble protein were obtained when the truncated cry1Ac was expressed in the cytoplasm, routed through ER, and targeted to the chloroplast. We further showed that not only the protein content but also the biological activity of truncated cry1Ac increases by sub-cellular targeting and the biological activity is slightly greater in the ER routed transgenic lines by conducting different bioassays on Helicoverpa armigera. Using native Western analysis, we demonstrated that the truncated cry1Ac protein could exist as oligomers in plant cells and this oligomerization capability is low in the cytoplasm. In conclusion, routing of delta endotoxins through ER is the first choice to obtain high protein expression and bioactivity.

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.

Synthesis and characterization of cry protein coated zinc oxide nanocomposites and its assessment against bacterial biofilm and mosquito vectors

Mosquitoes need to be eradicated as they can spread deadly diseases. Cry toxic proteins from Bacillus and zinc oxide nanoparticles also can tremendously control pest and bacterial pathogens. With this reference, the Ac-ZnO NPs was effectively synthesized using Acorus calamus rhizomes extract where after incorporated with bacterial cry toxic protein (Btp) to produce Btp-Ac-ZnO nanocomposites. The XRD and FTIR, disclose the crystalline form with an average size of 17.47 nm and the possible biomolecules of Btp-Ac-ZnO NCs. SEM and TEM make known the well agglomerated and cone shape of Btp-Ac-ZnO NCs. The NCs show concentration-dependent antioxidant activity. Btp-Ac-ZnO NCs drastically arrest the formation of biofilm by the pathogenic bacteria such as E. faecalis, S. aureus, P. aeruginosa, and P. vulgaris at 100 μg/mL. All the above, the Btp-Ac-ZnO NCs exhibits superior larvicidal activity against three mosquito vectors namely Ae. aegypti, An. stephensi and Cx. quinquefasciatus with LC50 values of 43.76, 39.60 and 37.13 μg/mL respectively. Besides, the biological enzymes are significantly reduced in the treated larvae than that of untreated one, which indicates the effect of Btp-Ac-ZnO NCs. Since, the Btp-Ac-ZnO NCs could be utilized against the pathogenic bacteria, and its biofilm structure, and also in the vector control sectors.

Helicoverpa zea (Lepidoptera: Noctuidae) feeding incidence and survival on Bt maize in relation to maize in the landscape.

BackgroundCharacterizing Helicoverpa zea (Boddie) damage to maize (Zea mays L.) in relation to the spatiotemporal composition of Bt crops is essential to understand how landscape composition affects H. zea abundance. To examine this relationship, paired Bt (expressing Cry1A.105 + Cry2Ab2) and non‐Bt maize plots were sampled across North and South Carolina during 2017–2019. Kernel damage and larval exit holes were measured following larval development. To understand how maize abundance surrounding sample sites related to feeding damage and larval development, we quantified maize abundance in a 1 km buffer surrounding the sample site and examined the relationship between local maize abundance and kernel damage and larval exit holes.ResultsAcross the years and locations, damage in Bt maize was widespread but significantly lower than in non‐Bt maize, indicating that despite the widespread occurrence of resistance to Cry toxins in maize, Bt maize still provides a measurable reduction in damage. There were negative relationships between kernel injury and ears with larval exit holes in both Bt and non‐Bt maize and the proportion of maize in the landscape during the current year.ConclusionDespite the widespread occurrence of resistance to Cry toxins in maize, this resistance is incomplete, and on average Bt maize continues to provide a measurable reduction in damage. We interpret the negative relationship between abundance of maize within 1 km of the sample location and maize infestation levels, as measured by kernel damage and larval exit holes, to reflect dispersion of the ovipositing moth population over available maize within the local landscape. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis

Due to their different specificity, the use of Vip3 proteins from Bacillus thuringiensis in combination with the conventionally used Cry proteins in crop protection is being essential to counteract the appearance of insect resistance. Therefore, understanding the mode of action of Vip3 proteins is crucial for their better application, with special interest on the binding to membrane receptors as the main step for specificity. Derived from in vitro heterologous competition binding assays using 125I-Vip3A and other Vip3 proteins as competitors, it has been shown that Vip3 proteins share receptors in Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). In this study, using 125I-Vip3Aa, we have first extended the in vitro competition binding site model of Vip3 proteins to Spodoptera littoralis. With the aim to understand the relevance (in terms of toxicity) of the binding to the midgut sites observed in vitro on the insecticidal activity of these proteins, we have performed in vivo competition assays with S. littoralis larvae, using disabled mutant (non-toxic) Vip3 proteins as competitors for blocking the toxicity of Vip3Aa and Vip3Af. The results of the in vivo competition assays confirm the occurrence of shared binding sites among Vip3 proteins and help understand the functional role of the shared binding sites as revealed in vitro.

Bio controlled efficacy of Bacillus thuringiensis cry protein protection against tomato fruit borer Helicoverpa armigera in a laboratory environment

Bio-pesticides that utilize beneficial microbial agents represent a new option that can potentially reduce reliance on chemical pesticides. To facilitate control of crop pests, this study includes both selection and characterization of Bacillus thuringiensis strains based on Cry gene content and toxicity evaluation. The result is an effective and non-hazardous polyphagous lepidopteran pest controller, Helicoverpa armigera. To achieve this objective, we selected ten soil bacteria samples selected from 10 different agricultural regions in Queretaro State, Mexico. Certainly, 65 and 130 kDa protein bands were observed in SDS-PAGE analysis of spore-crystal mixtures. Efficacy of bio-pesticides, 4 g/L effective water yielded maximum mortality; a mean of 64.88% of tomato fruit borer larvae were eliminated. The PCR sequences of bacterial strain were characterized and showed 98–99% similarity shows the distance from comparison of the 16S rRNA gene sequences of Bacillus thuringiensis.

Molecular characterization and functional analysis of Cry toxin receptor-like genes from the model insect Galleria mellonella.

Crystal (Cry) toxins produced from the soil bacterium, Bacillus thuringiensis (Bt), have gained worldwide attention for long due to their insecticidal potential. A number of receptor proteins located on the epithelial cells of the larval midgut were shown to be crucial for Cry intoxication in different insect pests belonging to order Lepidoptera, Diptera and Coleoptera. A beehive pest, Galleria mellonella, serves as an excellent insect model for biochemical research. However, information on the Cry receptor-like genes in G. mellonella is limited. In the present study, the full-length sequences of four putative Cry receptor genes (ABC transporter, alkaline phosphatase, aminopeptidase N and cadherin) were cloned from G. mellonella. All these receptor genes were substantially upregulated in the midgut tissue of fourth-instar G. mellonella larvae upon early exposure (6h) to a sub-lethal dose of Cry1AcF toxin. Oral and independent delivery of bacterially-expressed dsRNAs corresponding to four receptor genes in G. mellonella suppressed the transcription of target receptors which in turn significantly reduced the larval sensitivity to Cry1AcF toxin. As the laboratory populations of G. mellonella develop Bt resistance in a relatively short time, molecular characterization of Cry receptor genes in G. mellonella performed in the present study may provide some useful information for future research related to the genetic basis of Bt resistance in the model insect.

Characterization of insecticidal Cry1Cb2 protein from Bacillus thuringiensis toxic to Myzus persicae (Sulzer)

The toxins produced by Bacillus thuringiensis (Bt) are well known for their insecticidal activity against Lepidoptera, Diptera and Coleoptera; however, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. We describe the aphicidal effect of Cry toxin from Bt strain GP919 against one of the most pernicious hemipterans in the agricultural environment, Myzus persicae. The mortality bioassay shows that the strain cause mortality rates above 80% at concentration of 10 ng/µl with a LC50 of 9.01 ng/µl; whereas it showed no lethal toxicity against the lepidopteran Spodoptera frugiperda. The mayor protein (∼130 kDa) expressed by this strain was subjected to purification, solubilization and trypsin digestion, the band of ∼ 65 kDa which was obtained from trypsin digestion was purified by ion-exchange chromatography and was used to feed the aphid. The bioassay shows mortality rates above 85% at concentration of 10 ng/µl and the LC50 was 6.58 ng/µl. The resulting fragment from the digestion was identified by mass spectrometry and the candidate protein showed an overall 100% amino acid sequence identity to the reported Cry1Cb2 (WP 033698561.1) protein from Bt. Koch’s postulated also was carried out with the GP919 strain and also, we document the signs of infection caused by this strain. This is the first report of a Cry1Cb2 protein that is toxic to a sucking insect and this protein may become a promising environmentally friendly tool for the control of M. persicae and possible also for other sap sucking insect pests.

No Adverse Effects of Stacked Bacillus thuringiensis Maize on the Midge Chironomus riparius

Material from genetically engineered maize producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) may enter aquatic ecosystems and expose nontarget organisms. We investigated the effects on life table parameters of the midge Chironomus riparius (Diptera: Chironomidae) of SmartStax maize leaves, which contain six different Cry proteins targeting Lepidoptera and Coleoptera pests, in two plant backgrounds. For midge development and emergence, 95% confidence intervals for the means of six conventional maize lines (Rheintaler, Tasty Sweet, ES‐Eurojet, Planoxx, EXP 258, and EXP 262), were used to capture the natural range of variation. For reproduction, lowest and highest means were used. The natural range of variation allows one to judge whether observed effects between Bt maize and the closest non‐Bt comparator are likely to be of biological relevance. No adverse effects on C. riparius were observed with any Bt maize line compared with the respective non‐Bt counterpart. Development time was shorter when females were fed Bt maize than when they were fed non‐Bt maize, but this effect was not considered adverse. Development time, emergence ratio, sex ratio, and larvae/egg rope measured for Bt maize were within the natural range of variation. Fecundity for the Bt lines was equal to or higher than that for the conventional lines. Future risk assessment studies may consider plant background effects and the natural range of variation to judge the relevance of observed differences between particular genetically engineered and non‐genetically engineered plants. Environ Toxicol Chem 2022;41:1078–1088. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Recombinant Expression of ABCC2 Variants Confirms the Importance of Mutations in Extracellular Loop 4 for Cry1F Resistance in Fall Armyworm.

Fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive and invasive global noctuid pest. Its control is based on insecticide applications and Bacillus thuringiensis (Bt) insecticidal Cry toxins expressed in transgenic crops, such as Cry1F in Bt corn. Continuous selection pressure has resulted in populations that are resistant to Bt corn, particularly in Brazil. FAW resistance to Cry1F was recently shown to be conferred by mutations of ATP-binding cassette transporter C2 (ABCC2), but several mutations, particularly indels in extracellular loop 4 (ECL4), are not yet functionally validated. We addressed this knowledge gap by baculovirus-free insect cell expression of ABCC2 variants (and ABCC3) by electroporation technology and tested their response to Cry1F, Cry1A.105 and Cry1Ab. We employed a SYTOXTM orange cell viability test measuring ABCC2-mediated Bt toxin pore formation. In total, we tested seven different FAW ABCC2 variants mutated in ECL4, two mutants modified in nucleotide binding domain (NBD) 2, including a deletion mutant lacking NBD2, and S. frugiperda ABCC3. All tested ECL4 mutations conferred high resistance to Cry1F, but much less to Cry1A.105 and Cry1Ab, whereas mutations in NBD2 hardly affected Bt toxin activity. Our study confirms the importance of indels in ECL4 for Cry1F resistance in S. frugiperda ABCC2.

A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System.

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day–night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator’s period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep–wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.

Herbivorous Juvenile Grass Carp (Ctenopharyngodon idella) Fed with Genetically Modified MON 810 and DAS-59122 Maize Varieties Containing Cry Toxins: Intestinal Histological, Developmental, and Immunological Investigations.

Feeding experiments with juvenile grass carp (Ctenopharyngodon idella) fed with genetically modified maize MON 810 or DAS-59122 dried leaf biomass were carried out with 1-, 3- and 6-month exposures. Dosages of 3–7 μg/fish/day Cry1Ab or 18-55 μg/fish/day Cry34Ab1 toxin did not cause mortality. No difference occurred in body or abdominal sac weights. No differences appeared in levels of inorganic phosphate, calcium, fructosamine, bile acids, triglycerides, cholesterol, and alanine and aspartame aminotransferases. DAS-59122 did not alter blood parameters tested after 3 months of feeding. MON 810 slightly decreased serum albumin levels compared to the control, only in one group. Tapeworm (Bothriocephalus acheilognathi) infection changed the levels of inorganic phosphate and calcium. Cry34Ab1 toxin appeared in blood (12.6 ± 1.9 ng/mL), but not in the muscle. It was detected in B. acheilognathi. Cry1Ab was hardly detectable in certain samples near the limit of detection. Degradation of Cry toxins was extremely quick in the fish gastrointestinal tract. After 6 months of feeding, only mild indications in certain serum parameters were observed: MON 810 slightly increased the level of apoptotic cells in the blood and reduced the number of thrombocytes in one group; DAS-59122 mildly increased the number of granulocytes compared to the near-isogenic line.

Potential Risk of Pollen from Genetically Modified MON 810 Maize Containing Cry1Ab Toxin to Protected Lepidopteran Larvae in the Pannonian Biogeographical Region-A Retrospective View.

Simple SummaryThe Cry1Ab toxin content in different plant organs is highly variable by genetic events and cultivars. This applies particularly to the pollen, which is the main route of exposure of protected lepidopteran larvae. Thus, uncertainties appear regarding safety assessments on the basis of analytical and biological studies: (a) genetic events and cultivars produce various Cry1Ab toxin contents; thus, risk analyses based on single pollen counts may be erroneous; (b) analytical problems have been identified explaining the high variability of the documented pollen toxin content; (c) stinging nettle patches subject to maize pollen sedimentation are frequent nearby maize field edges, where protected nymphalid larvae may feed; (d) substantial maize pollen sedimentation (300–600 pollen grains/cm2 in the dominant wind direction) occurs on the leaves of stinging nettle; (e) July and August are the critical months for the young larvae of Nymphalis io, which are the most sensitive for pollen containing Cry1Ab toxin; (f) the exposure of these larvae to maize pollen containing >100 ng of Cry1Ab toxin/g results in <40% mortality and extended developmental times in younger stages. This is a definite hazard, which is a sufficient legal ground for habitat conservation of this protected species in Hungary.A credible risk analysis of maize pollen containing Cry1Ab toxin must include the assessment of (i) pollen production and its Cry1 toxin content; (ii) distribution of the pollen grains in the surroundings; (iii) pollen-catching capacity of the weeds on field edges; (iv) the lifestyle of protected lepidopteran larvae living on weeds; (v) Cry1 toxin sensitivity of non-target caterpillars; and (vi) Cry1 toxin resistance of individual non-target populations. The concentration range of 5–4300 ng Cry1Ab toxin/g dry pollen determined in MON 810 pollen batches is too diverse for handling it as a single set in any mathematical modeling. Within the work carried out mainly with the DK-440 BTY cultivar, the seed samples officially received from the variety owner produced significantly different (250–470 vs. 5–15 ng/g) Cry1Ab toxin concentrations in the pollen. Nymphalis io L1-L3 larvae were nearly six times more sensitive for Dipel than Nymphalis c-album. Feeding on the back side and in a leaf nest, Vanessa atalanta may be subject to lower pollen exposures. N. io larvae may actively attempt to avoid patches with high pollen contamination. Cry1Ab toxin resistance also partially emerged in N. io populations reared in the Pannonian Biogeographical Region (Hungary).

Engineered resistance and risk assessment associated with insecticidal and weeds resistant transgenic cotton using wister rat model

Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and weeds is a promising tool to save crop from losses. Transgenic cotton variety, VH-289, with double Bt and cp4EPSPS genes under the control of 35S promoter was used for the expression analyses and biosafety studies. The transgenic cotton plants were screened through PCR amplification of fragments, 1.7 kb for Cry1Ac, 582 bp for Cry2A and 250 bp for cp4EPSPS; which confirmed the presence of all genes transformed in transgenic cotton. The Cry1Ac + Cry2A and cp4EPSPS proteins were quantified through ELISA in transgenic cotton plants. The Glyphosate assay performed by spraying 1900 mL per acre of glyphosate Roundup further confirmed complete survival of transgenic cotton plants as compared to the non-transgenic cotton plants and all weeds. Similarly, insect infestation data determined that almost 99% insect mortality was observed in controlled field grown transgenic cotton plants as compared to the non-transgenic control plants. Evaluation of effect of temperature and soil nutrients availability on transgene expression in cotton plants was done at two different cotton growing regions, Multan and Lahore, Pakistan and results suggested that despite of higher temperature in Multan field, an increased level of Cry and cp4EPSPS proteins was recorded due to higher soil organic matter availability compared to Lahore field. Before commercialization of any transgenic variety its biosafety study is mandatory so, a 90 days biosafety study of the transgenic cotton plants with 40% transgenic cottonseeds in standard diet showed no harmful effect on wister rat model when studied for liver function, renal function and serum electrolyte.

Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli.

Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and spectacular socioeconomic gains. To attain the enhanced level of protein expression, the cry genes have to be extensively modified for RNA stability and translation efficiency in the plant systems. However, such modifications in nucleotide sequences make it difficult to express the cry genes in Escherichia coli because of the presence of E. coli rare codons. Induction of gene expression through the T7 promoter/lac operator system results in high levels of transcription but limits the availability of activated tRNA corresponding to rare codons that leads to translation stalling at ribosomes. In the present study, an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)/rifampicin combination-based approach was adopted to induce transcription of cry genes through T7 promoter/lac operator while simultaneously inhibiting the transcription of host genes through rifampicin. The results show that the IPTG/rifampicin combination leads to high-level expression of four plant codon-optimized cry genes (cry2Aa, cry1F, cry1Ac, and cry1AcF). Northern blot analysis of the cry gene expressing E. coli samples showed that the RNA expression level in the IPTG-induced samples was higher as compared to that in the IPTG/rifampicin-induced samples. Diet overlay insect bioassay of IPTG/rifampicin-induced Cry toxins with Helicoverpa armigera larvae showed bioactivity (measured as LC50) similar to the previous studies. The experiment has proved that recombinant synthetic gene (plant codon-optimized gene) with the combination of Rifampicin which inhibits DNA-dependent bacterial RNA polymerase and reduces the excessive baggage of translational machinery of the bacterial cell triggers the production of synthetic protein. Purification of protein using high pH buffer increases the solubility of the protein. Further, LC50 analysis shows no reduction of protein activity leads to protein stability. Further, purified cry toxin protein can be used for crop protection against pests and a purified form of the synthetic protein can be used for antibody production and perform the immunoassay for the identification of the transgenic plant. The crystallographic structure of synthetic protein could be used for interaction study with another insect to see insecticidal activity.

An Overview on Insect Tolerance Transgenic Crops

Although transgenic crops have enhanced insect pest control, their effectiveness has diminished due to pest resistance. Humans examined global monitoring data from the very first two decades of genetically engineered crops, with each occurrence representing one pest species' response to one Bacillus thuringiensis insecticidal protein in one country (Bt). Pest tolerance to transgenic crops' Bt crystalline (Cry) proteins has increased from three cases in 2005 to sixteen cases in 2016. Hereditary change that is bug-free innovation is fastgrowing, with enormous exploration effort in both the commercial and legislative regions. Although the bacterium Bacillus thuringiensis is the most widely advertised bug-safe transgenic plant, a wide range of traits from higher plants, particularly those producing stomachrelated chemical inhibitors or lectins, have also been introduced into crop cultivars. So far, the quality of opposition from various microbes and species has only been used in a few circumstances.

Activation of Bacillus thuringiensis Cry1I to a 50\xa0kDa stable core impairs its full toxicity to Ostrinia nubilalis

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products.Key points• Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis• Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis• Protoxin and its final processed forms compete for the same functional binding sites

An Integrative Analysis of Transcriptomics and Proteomics Reveals Novel Insights into the Response in the Midgut of Spodoptera frugiperda Larvae to Vip3Aa.

The insecticidal Vip3 proteins, secreted by Bacillus thuringiensis (Bt) during its vegetative growth phase, are currently used in Bt crops to control insect pests, and are genetically distinct from known insecticidal Cry proteins. Compared with Cry toxins, the mechanisms of Vip3 toxins are still poorly understood. Here, the responses of Spodoptera frugiperda larvae after Vip3Aa challenge are characterized. Using an integrative analysis of transcriptomics and proteomics, we found that Vip3Aa has enormous implications for various pathways. The downregulated genes and proteins were mainly enriched in metabolic pathways, including the insect hormone synthesis pathway, whereas the upregulated genes and proteins were mainly involved in the caspase-mediated apoptosis pathway, along with the MAPK signaling and endocytosis pathways. Moreover, we also identified some important candidate genes involved in apoptosis and MAPKs. The present study shows that exposure of S. frugiperda larvae to Vip3Aa activates apoptosis pathways, leading to cell death. The results will promote our understanding of the host response process to the Vip3Aa, and help us to better understand the mode of action of Vip3A toxins.