Cry Proteins Research Articles

A Modified F2 Screen for Estimating Cry1Ac and Cry2Ab Resistance Allele Frequencies in Helicoverpa zea (Lepidoptera: Noctuidae).

Evaluating the frequency of resistance alleles is important for resistance management and sustainable use of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis. Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) is a major crop pest in the United States that has evolved practical resistance to the crystalline (Cry) proteins in Bt corn and cotton. The standard F2 screen for estimating resistance allele frequency does not work well for H. zea because successful single-pair matings are rare. In this study, we developed and implemented a modified F2 screen for H. zea that generates F1 progeny by crossing three laboratory susceptible female moths with one feral male moth instead of single-pair crosses. During 2019-2020, we used this modified method to establish 192 F2 families from 623 matings between susceptible females and feral males from Arkansas, Louisiana, Mississippi, and Tennessee. From each F2 family, we screened 128 neonates against discriminating concentrations of Cry1Ac and Cry2Ab in diet overlay bioassays. Based on these discriminating concentration bioassays, families were considered positive for resistance if at least five larvae survived to second instar, including at least one to third instar. The percentage of positive families was 92.7% for Cry1Ac and 38.5% for Cry2Ab, which yields an estimated resistance allele frequency (with 95% confidence interval) of 0.722 (0.688-0.764) for Cry1Ac and 0.217 (0.179-0.261) for Cry2Ab. The modified F2 screen developed and implemented here may be useful for future resistance monitoring studies of H. zea and other pests.

Bioinformatic analysis of genetic changes CLOCK, BMAL1, CRY1, CRY2, PER1, PER2, PER3, and NPAS2 proteins in HCC patients.

Genes related to the circadian rhythm control various biological processes. The aim of this study was to comprehensively investigate the mutational and mRNA profile of core circadian rhythm genes in hepatocellular cancer (HCC) samples. In this study, the gene profile of a total of 369 patients with HCC was examined over the data obtained from the cancer genome atlas database through-cBioPortal. The effects of mutations on protein were examined by scoring the Polymorphism Phenotyping v2, Mutation Assessor, and SIFT-databases. While the association of genes with other genes was determined with the GeneMANIA-database, the association of expression levels in the genes with overall survival (OS) was evaluated with the Kaplan-Meier Plot database. As a result of the analyses, there were a total of 25 mutations. Decreased expression levels of PER1 (1.3e-05), PER3 (p=0.046), and CRY2 (p=1.8e-06) genes were found statistically associated with shorter OS. It was also found that increased expression levels of the PER2 (p=0.045) gene were associated with longer OS, and increased expression levels of the NPAS2 (p=9e-04) gene were associated with shorter OS. In particular, changes in the PER1, PER2, CRY2, and NPAS2 genes may provide possible molecular targets in chemotherapy and immunotherapy for HCC patients.

Identification of Cry toxin receptor genes homologs in a de novo transcriptome of Premnotrypes vorax (Coleoptera: Curculionidae).

The white potato worm Premnotrypes vorax (Hustache) (Coleoptera: Curculionidae) is one of the most destructive insect pests of potato crops in South America. Like many coleopteran insects, P. vorax shows low susceptibility to Cry insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). However, the presence of Cry toxin receptors in the midgut of this this insect has never been studied. The main Cry-binding proteins described in other insect species are cadherin (CAD), aminopeptidase N (APN), alkaline phosphatase (ALP) and ATP-binding cassette (ABC) transporters. In this study, we analyzed and validated a de novo assembled transcriptome of Illumina sequencing data to identify and to characterize homologs of Cry toxin receptors. We identified the protein sequences in P. vorax that show high identity with their orthologous sequences of the Cry toxin binding proteins in other coleopteran larvae such as APN, ALP, CAD and ABC transporter. This study provides preliminary identification of putative receptor genes of Cry proteins that would be useful for future studies involving biocontrol of this important potato crop pest.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton.

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Identification of cry genes in Bacillus thuringiensis by multiplex real-time PCR

Bacillus thuringiensis is an important bacterium of the group Bacillus cereus sensu lato due to its insecticidal properties. This microorganism has high genetic variability and its strains produce different Cry toxins, known as δ-endotoxins, which are mainly responsible for its toxic effect on insects that are agricultural pests or vector human diseases. Each strain can express a variety of cry genes, out of a total of 789 cry genes described so far. The detection of these genes is very important to characterize strains, as they may indicate their toxic potential. Several methods have been used to characterize B. thuringiensis strains, but one of the most common techniques is Polymerase Chain Reaction (PCR) from primers that detect the presence of cry genes. This technique has been optimized to make real-time multiplex quantitative PCR (qPCR) assays faster, more efficient, and safer, because the presence of three genes can be detected in a single reaction. In this work, a multiplex assay was developed to identify the presence of genes from the cry1A, cry1C, and cry1F families whose respective toxins are present in both bioinsecticides, and commercial transgenic plants used to control caterpillars. Specific primers were designed to identify the families of the cited genes and the system was validated with samples that were sequenced by next-generation sequencing (NGS). The system was implemented and used to characterize 214 strains. Of these, eight were submitted to conventional PCR, and the results matched, again validating the system. Thus, the application of the proposed technique allows the reliable evaluation through this system to detect the presence of the genes of the families cry1A, cry1C, and cry1F in samples of B. thuringiensis.

Bt and G10evo-EPSPS Protein Expressed in ZDAB3 Corn Has No Impact on Nutritional Composition and Toxicological Safety

Genetically modified (GM) crops expressing insecticidal and herbicide-tolerant traits provide a new approach to agriculture production, but concerns about food safety were often raised by the public. The present research shows the findings of the nutritional assessment of ZDAB3 expressing insecticidal Cry proteins (Cry1Ab and Cry2Ab) and EPSPS protein (G10evo-EPSPS). The key nutrients and anti-nutrients of ZDAB3 maize were examined and contrasted with those of its non-transgenic control maize grown at the same locations during three planting seasons. The values for proximates, amino acids, fatty acids, minerals, vitamins, phytic acid, and trypsin inhibitor assessed for ZDAB3 were comparable to those of its non-transgenic control maize or within the range of values reported for other commercial lines. In addition, no adverse effects related to the G10evo-EPSPS protein in mammals were observed. These data indicated that the expression of Cry1Ab, Cry2Ab, and G10evo-EPSPS proteins in ZDAB3 maize does not affect the nutritional compositions, and ZDAB3 maize is equivalent to non-transgenic maize regarding those important compositions.

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

ECry1Gb.1Ig, A Novel Chimeric Cry Protein with High Efficacy against Multiple Fall Armyworm (Spodoptera frugiperda) Strains Resistant to Different GM Traits.

Spodoptera frugiperda (fall armyworm, FAW) is one of the most devastating insect pests to corn and soybean production in the Americas and is rapidly expanding its range worldwide. It is known to be hard to control either by chemical insecticide applications or by GM. Although the use of GM traits can be an effective way to control this pest, it is very rare to find native insecticidal proteins that provide the necessary level of FAW control in crop fields where FAW pressure and damage are high. Insecticidal Cry proteins sourced from Bacillus thuringiensis have been heavily utilized in the development of crops with GM traits; however, it is increasingly difficult to identify Cry proteins with unique modes of action. Protein engineering via a phylogenetically guided Cry protein domain swapping approach enabled us to discover novel chimeric Cry proteins engineered from inactive parent sequences. Some of these chimeras show excellent efficacy against key biotypes of FAW from Brazil and North America. In this study, we characterized a Cry-based chimera eCry1Gb.1Ig that is a very potent FAW toxin. eCry1Gb.1Ig showed high efficacy against multiple FAW strains that are resistant to various traits, including Cry1Fa, Vip3Aa and Cry1A.105/Cry2Ab. These results clearly indicate that the FAW strains resistant to Cry1Fa, Vip3Aa or Cry1A.105/Cry2Ab demonstrate no cross-resistance to eCry1Gb.1Ig and strongly suggest that eCry1Gb.1Ig acts through a novel mode of action compared to the existing traits. In addition to its FAW activity, eCry1Gb.1Ig has also been shown to control Chrysodeixis includens (soybean looper, SBL) and Anticarsia gemmatalis (velvetbean caterpillar, VBC), which are significant pests of soybean. When eCry1Gb.1Ig was introduced into corn and soybean crops, transgenic events showed strong efficacy against FAW, SBL and VBC, but no adverse plant phenotypes. This suggests that the in planta expression of the eCry1Gb.1Ig protein does not compromise plant growth or reproduction and can protect plants from FAW-related damage. Therefore, this valuable discovery will provide a differentiating FAW control trait that will give growers another tool to help them reduce yield loss due to FAW.

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

Removal of an Aminopeptidase N From Midgut Brush Border Does Not Affect Susceptibility of Spodoptera litura (Lepidoptera: Noctuidae) Larvae to Four Insecticidal Proteins of Bacillus thuringiensis (Bacillales: Bacillaceae).

Spodoptera litura is one of the most destructive lepidopteran insects of cabbages and cauliflowers in the world. Cry1 and Vip3 toxins from Bacillus thuringiensis have been reported to show toxicity in multiple lepidopteran insects. Binding of toxic molecules to specific receptors on the midgut epithelial cells is known to be a key step in the action mode of Bt toxins. Aminopeptidase N (APN) -like proteins have been reported to be binding sites of multiple Cry toxins in the midgut of Cry susceptible insects. In the present study, we identified six midgut APNs by analysis of the genome and midgut transcriptome of S. litura. CRISPR/Cas9 mediated gene-knockout system was utilized to mutate the GPI-anchor signal peptide at the C terminus of SlAPN1. SlAPN1 was verified to be removed from the midgut brush border membrane vesicles of a homozygous knockout strain of S. litura (SlAPN1-KO). Bioassay results indicated that susceptibility of the SlAPN1-KO strain to Cry1Aa, Cry1Ac, Cry1Ca, and Vip3Aa toxins was close to that of the wild-type strain of S. litura. RT-qPCR results showed that the transcriptional level of SlAPN2-6 was not up-regulated after knockout of the SlAPN1. Results in this study indicated that the SlAPN1 did not play a critical role in the pathway of toxicity of Cry1Aa, Cry1Ac, Cry1Ca, and Vip3Aa toxins in S. litura.

Hemocyte response to treatment of susceptible and resistant Asian corn borer (Ostrinia furnacalis) larvae with Cry1F toxin from Bacillus thuringiensis

Midgut receptors have been recognized as the major mechanism of resistance to Cry proteins in lepidopteran larvae, while there is a dearth of data on the role of hemocyte's response to Cry intoxication and resistance development. We aimed at investigating the role of circulating hemocytes in the intoxication of Cry1F toxin in larvae from susceptible (ACB-BtS) and resistant (ACB-FR) strains of the Asian corn borer (ACB), Ostrinia furnacalis. Transcriptome and proteome profiling identified genes and proteins involved in immune-related (tetraspanin and C-type lectins) and detoxification pathways as significantly up-regulated in the hemocytes of Cry1F treated ACB-FR. High-throughput in vitro assays revealed the binding affinity of Cry1F with the tetraspanin and C-type lectin family proteins. We found significant activation of MAPKinase (ERK 1/2, p38α, and JNK 1/2) in the hemocytes of Cry1F treated ACB-FR. In testing plausible crosstalk between a tetraspanin (CD63) and downstream MAPK signaling, we knocked down CD63 expression by RNAi and detected an alteration in JNK 1/2 level but a significant increase in susceptibility of ACB-FR larvae to Cry1F toxin. Information from this study advances a change in knowledge on the cellular immune response to Cry intoxication and its potential role in resistance in a lepidopteran pest.

Expression of a mosquito larvicidal gene in chloroplast and nuclear compartments of Chlamydomonas reinhardtii

As a part of the search for environment-friendly biocontrol of mosquito-borne diseases, mosquito larvicidal potential of Bacillus thuringiensis subsp. jegathesan (Btj) Cry toxins is explored for toxins with increased toxicity. Safe delivery of the Cry toxins to mosquito larvae in aquatic habitats is a major concern. This is because in water bodies Bacillus thuringiensis (Bt) protein formulations degrade by sunlight, can sink down and get adsorbed by the silt. So, because of its short persistence the toxin requires repeated applications at the given site. Therefore, an upcoming approach is incorporating the Bt toxins in Chlamydomonas reinhardtii (C. reinhardtii) because it is a food of mosquito larvae in water and its molecular toolkit is well investigated for foreign gene expression. The present work aimed to compare the feasibility of C. reinhardtii chloroplast and nuclear compartments for stable expression of Cry11Ba toxin as this is the most toxic Btj protein to date, lethal to different mosquito species. With chloroplast expression of cry11Ba gene we were able to generate marker-free C. reinhardtii strain stably expressing Cry11Ba protein and demonstrating mortality against Aedes aegypti larvae. Moreover, for nuclear expression linking the cry11Ba gene to zeocin via foot and mouth disease virus (FMDV) 2A peptide resulted in the selection of transformants with increased cry11Ba mRNA expression levels by semi-quantitative reverse transcriptase PCR. Obtained results lay a foundation for the C. reinhardtii chloroplast expression system to be used for genetic engineering with Bt toxins which possess enhanced toxicity.

Co-expression of Cocculus hirsutus trypsin inhibitor with Cry protein reduces resistant development in targeted insects along with complete mortality

Crop losses due to various insects are a global problem. For decades, several types of research have been ongoing on this issue with limited success due to co-evolving insect pests. In this study, the modified Cocculus hirsutus trypsin inhibitor (ChTI), a serine proteinase inhibitor gene encoding a natural plant defense protein, inhibiting the digestive proteases in the midgut of phytophagous insects, was expressed alone and pyramided with the Bt-toxin Cry1Ab, to reduce resistance development against two herbivorous insects, Helicoverpa armigera and Spodoptera litura with complete insect mortality. In-silico analysis revealed the structural and conformational stability of ChTI protein. Furthermore, 80 % weight loss and reduced larval growth were observed in both insects challenged with 200 µg of the purified ChTI protein. The ChTI protein trypsin inhibitor activity was observed up to 5.5 U/mg expressed in E. coli, 3.0–3.5 U/mg in transgenic Arabidopsis plants, and up to 2.5–3.0 U/mg in transgenic tomato plants. Transgenic plants (Arabidopsis and tomato) were developed by pyramiding ChTI, and cry1Ab genes showed 100 % mortality against both insects while the transgenic plants expressing the ChTI gene alone showed up to 70 % mortality without yield penalty. Transcriptomic analysis of the transgenic plants further revealed that other pathogenesis-related defense proteins were also differently regulated.

Bio-fabricated zinc oxide and cry protein nanocomposites: Synthesis, characterization, potentiality against Zika, malaria and West Nile virus vector's larvae and their impact on non-target organisms

The role of mosquito vectors in spreading disastrous diseases to living organisms, especially to humans is inevitable and undeniable. The impacts of the available chemical and synthetic insecticides on non-specific organisms as well as on nature are being the reason behind the search for target-specific, biocompatible and eco-friendly alternatives. The Madhuca longifolia seed extract and cry proteins from Bacillus thuringiensis-based nanocomposites (Cp-Ml-ZnO NCs) were produced to conquer the above-mentioned issues. The Cp-Ml-ZnO NCs (100 μg/mL) expressed better scavenging potentiality on 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radicals than Ml seed extract and Ml-ZnO NPs. The susceptibility of tested vector larvae to the Cp-Ml-ZnO NCs was Ae. aegypti ˃An. stephensi ˃ Cx. quinquefasciatus along with LC50-27.73, 34.81, and 42.54 μg/mL concentration. The target specificity and biocompatibility of Cp-Ml-ZnO NCs were authenticated by the results obtained by evaluating the efficacy on D. similis, A. salina, P. reticulata, G. affinis, and RBCs of goat blood. Thus the Cp-Ml-ZnO NCs could be adopted for the control of vector larvae.

Characterizing the lethal and sub-lethal effects of genetically modified soybean expressing Cry1A.105, Cry2Ab2, and Cry1Ac insecticidal proteins against Spodoptera species (Lepidoptera: Noctuidae) in Brazil.

MON 87701 × MON 89788 × MON 87751 × MON 87708 soybean, that expresses Cry1A.105, Cry2Ab2, and Cry1Ac insecticidal proteins and confers tolerance to glyphosate and dicamba, is a potential tool for managing Spodoptera species in soybean fields in Brazil. In this study, we characterized the lethal and sub-lethal effects of Cry1A.105/Cry2Ab2/Cry1Ac soybean against Spodoptera species and genotypes of Spodoptera frugiperda resistant and susceptible to Cry1 and Cry2 proteins. These evaluations were also conducted with MON 87701 × MON 89788 soybean, which expresses Cry1Ac protein. Cry1A.105/Cry2Ab2/Cry1Ac soybean caused high lethality in neonates of Spodoptera cosmioides and Spodoptera albula. However, it showed low lethality in S. frugiperda genotypes homozygous for resistance to Cry1 and Cry2 proteins but reduced their population growth potential. No relevant lethal effects of Cry1Ac soybean were detected in the Spodoptera species and genotypes evaluated. Spodoptera frugiperda genotypes heterozygous for Cry1 and Cry2 resistance were controlled by Cry1A.105/Cry2Ab2/Cry1Ac soybean, with no insects developing into adults. This Bt soybean also caused intermediate mortality of neonates of Spodoptera eridania (60%-83%) but no surviving larvae developed to adulthood, resulting in population suppression. Cry1A.105/Cry2Ab2/Cry1Ac soybean caused high mortality of S. cosmioides, S. albula, and S. frugiperda genotypes susceptible to Cry1 and Cry2 and heterozygous for Cry1 and Cry2 resistance. This Bt soybean also suppressed population growth of S. eridania but had minimal impact on S. frugiperda homozygous for resistance to Cry1 and Cry2 proteins. Cry1Ac soybean had minimal impact on all Spodoptera species and genotypes evaluated. © 2022 Society of Chemical Industry.

Genome profiling of an indigenous Bacillus thuringiensis isolate, T405 toxic against the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

In this study, we present the molecular and insecticidal characteristics of an indigenous Bt isolate T405 toxic against the maize fall armyworm (FAW), Spodoptera frugiperda. The presence of cry1, cry2 (cry2Aa &cry2Ab) and vip3A1 genes in T405 was confirmed. The SDS-PAGE gel analysis confirmed the occurrence of Cry and Vip proteins with molecular masses of 130, ∼88 and 65 kDa in T405. LC50 estimates of T405 and HD1 were 161.37 and 910.73 μg ml−1 for neonates whereas, 412.29 and 1014.95 μg ml−1 correspondingly for 2nd instar FAW larvae. Scanning Electron Microscopy depicted the existence of bipyramidal, spherical and cubic crystals in T405 spore suspension. The whole genome sequencing and assembly of T405 produced a total of 563 scaffolds with a genome size of 6,673,691 bp. The BLAST similarity search showed that 12 plasmids were distributed in this genome. Genome annotation revealed the presence of 6174 protein coding genes, 13 rRNA and 98 tRNA, in which 6126 genes were completely annotated for their functions through sequence similarity search, domains/motifs identification and gene ontology studies. Further analysis of these genes identified the presence of many insecticidal toxin protein coding genes viz., cry1Ac32, cry1Ab9, cry1Aa6, cry1Ac5, cry1Aa18, cry1Ab8, cry1Ab11, cry2Aa9, cry1Ia40, cry2Aa9, cry1Ia40, cry2Ab35, cyt, vip3Aa7 and tpp80Aa and several additional virulence assisted factors viz., immune inhibitor A, phospholipase C, sphingomyelinase, cell wall hydrolases, chitinase, hemolysin XhlA and seven urease subunit coding genes (ureA, ureB, ureC, ureD, ureE, ureF, ureG) in the annotated genome.

Phage-displayed nanobody-based fluorescence-linked immunosorbent assay for the detection of Cry3Bb toxin in corn

Cry toxins have been widely used as biopesticides and applied in transgenic crops for the control of pests, raising public concerns for unforeseen environmental and health risks. In this study, five phage-displayed nanobodies against Cry3Bb toxin were selected from a naive nanobody library by screening. Using purified phage-displayed nanobody as capture antibody, a novel double-antibody sandwich fluorescence-linked immunosorbent assay (FLISA) based on quantum dot beads (QBs) was established for the determination of Cry3Bb toxin. The developed method exhibited a linear working range of 31.25–500 ng/mL and a low detection limit of 8.45 ng/mL. The recoveries of Cry3Bb toxin from spiked corn samples were ranging from 93.1% to 116.8% with a coefficient of variation lower than 9.4%. Molecular modeling results showed that key amino acids in the CDR3 region of nanobody P27 combine with Cry3Bb toxin through hydrogen bonds and hydrophobic interactions. These results indicated that the phage-displayed nanobody-based QBs-FLISA may provide a possible tool for detecting Cry3Bb toxin in corn.

Resistance of Cabbage Loopers to Bacillus thuringiensis (Bt) Toxin Cry1F and to Dual-Bt Toxin WideStrike Cotton Plants.

The Cry proteins from Bacillus thuringiensis (Bt) are major insecticidal toxins in formulated Bt sprays and are expressed in genetically engineered Bt crops for insect pest control. However, the widespread application of Bt toxins in the field imposes strong selection pressure on target insects, leading to the evolution of insect resistance to the Bt toxins. Identification and understanding of mechanisms of insect resistance to Bt toxins are an important approach for dissecting the modes of action of Bt toxins and providing knowledge necessary for the development of resistance management technologies. In this study, cabbage looper (Trichoplusia ni) strains resistant to the transgenic dual-Bt toxin WideStrike cotton plants, which express Bt toxins Cry1Ac and Cry1F, were selected from T. ni strains resistant to the Bt formulation Bt-DiPel. The WideStrike-resistant T. ni larvae were confirmed to be resistant to both Bt toxins Cry1Ac and Cry1F. From the WideStrike-resistant T. ni, the Cry1F resistance trait was further isolated to establish a T. ni strain resistant to Cry1F only. The levels of Cry1F resistance in the WideStrike-resistant and the Cry1F-resistant strains were determined, and the inheritance of the Cry1F-resistant trait in the two strains was characterized. Genetic association analysis of the Cry1F resistance trait indicated that the Cry1F resistance in T. ni isolated in this study is not shared with the Cry1Ac resistance mechanism nor is it associated with a mutation in the ABCC2 gene, as has so far been reported in Cry1F-resistant insects. IMPORTANCE Insecticidal toxins from Bacillus thuringiensis (Bt) are highly effective for insect control in agriculture. However, the widespread application of Bt toxins exerts strong selection for Bt resistance in insect populations. The continuing success of Bt biotechnology for pest control requires the identification of resistance and understanding of the mechanisms of resistance to Bt toxins. Cry1F is an important Bt toxin used in transgenic cotton, maize, and soybean varieties adopted widely for insect control. To understand the mode of action of Cry1F and mechanisms of Cry1F resistance in insects, it is important to identify Cry1F-specific resistance and the resistance mechanisms. In this study, Trichoplusia ni strains resistant to commercial "WideStrike" cotton plants that express Bt toxins Cry1Ac and Cry1F were selected, and a Cry1F-specific resistant strain was isolated. The isolation of the novel Cry1F-specific resistance in the T. ni provided an invaluable biological system to discover a Cry1F-specific novel resistance mechanism.

Characterization and expression of prohibitin during the mexican bean weevil (Zabrotes subfasciatus, Boheman, 1833) larvae development.

Prohibitin (PHB) is a highly conserved eukaryotic protein complex involved in multiple cellular processes. In insects, PHB has been identified as a potential target protein to insecticidal molecules acting as a receptor of PF2 insecticidal lectin in the midgut of Zabrotes subfasciatus larvae (bean pest) and Cry protein of Bacillus thuringiensis in Leptinotarsa decemlineata (Colorado potato beetle). This study aimed to characterize the structural features of Z. subfasciatus prohibitin (ZsPHB) by homology modeling and evaluate its expression and tissue localization at different stages of larval development both at the transcript and protein levels. The samples were collected from eggs and larvae of different developmental stages. The immunodetection of ZsPHB was done with anti-PHB1 and confirmed by LC-MS/MS analysis. Gene expression analysis of ZsPHB1 and ZsPHB2 was performed by RT-qPCR, and immunohistochemistry with FITC-labeled anti-PHB1. Results showed that ZsPHBs exhibit distinctive characteristics of the SPFH protein superfamily. The transcript levels suggest a coordinated expression of ZsPHB1 and ZsPHB2 genes, while ZsPHB1 was detected in soluble protein extracts depending on the stage of development. Histological examination showed ZsPHB1 is present in all larval tissues, with an intense fluorescence signal observed at the gut. These results suggest a physiologically important role of PHB during Z. subfasciatus development and show its regulation occurs at the transcriptional and post-transcriptional levels. This is the first characterization of PHB in Z. subfasciatus.

Quantum-Dot-Bead-Based Fluorescence-Linked Immunosorbent Assay for Sensitive Detection of Cry2A Toxin in Cereals Using Nanobodies.

In this study, a quantum-dot-bead (QB)-based fluorescence-linked immunosorbent assay (FLISA) using nanobodies was established for sensitive determination of the Cry2A toxin in cereal. QBs were used as the fluorescent probe and conjugated with a Cry2A polyclonal antibody. An anti-Cry2A nanobody P2 was expressed and used as the capture antibody. The results revealed that the low detection limit of the developed QB-FLISA was 0.41 ng/mL, which had a 19-times higher sensitivity than the traditional colorimetric ELISA. The proposed assay exhibited a high specificity for the Cry2A toxin, and it had no evident cross-reactions with other Cry toxins. The recoveries of Cry2A from the spiked cereal sample ranged from 86.6–117.3%, with a coefficient of variation lower than 9%. Moreover, sample analysis results of the QB-FLISA and commercial ELISA kit correlated well with each other. These results indicated that the developed QB-FLISA provides a potential approach for the sensitive determination of the Cry2A toxin in cereals.