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BACKGROUND: Binding site models, derived from in vitro competition binding studies, have been widely used for predicting potential cross‐resistance among insecticidal proteins from Bacillus thuringiensis. However, because discrepancies have been found between binding data and observed cross‐resistance patterns in some insect species, new tools are required to study the functional relevance of the shared binding sites. RESULTS: Here, an in vivo approach has been applied to the competition studies to establish the functional relevance of shared binding sites as determined by in vitro competition assays. Using Cry disabled proteins as competitors in mixed protein overlay assays, we assessed the preference of Cry1Ab, Cry1Fa, and Cry1A.105 proteins for shared binding sites in vivo in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda. CONCLUSION: This study shows that in vivo and in vitro binding site competition assays can provide useful information to better ascertain whether different Cry proteins share binding sites and, consequently, whether cross‐resistance due to binding site alteration can occur. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Coffee berry borer (Hypothenemus hampei), is one of the main pests affecting coffee production worldwide. This pest feeds and reproduces inside the berry reducing its weight and quality, causing losses up to 50%. Some strains of Bacillus have plasmids that incorporate cry and cyt genes, whose products have toxic properties to insects. Bacillus thuringiensis and Bacillus pumilus have been evaluated as entomopathogenic. The objectives of this study was to determine the potential of two native Bacillus strains (B. thuringiensis Bt3971 and B. pumilus Bp4185) isolated from coffee ecosystem of Puerto Rico as biocontrol of coffee berry borer. The presence of a 12 kbp plasmid was confirmed in Bt3971 and Bp4185, and a 116 kDa protein with entomopathogenic potential from Bt3971. After 48 h of treatment, Bt3971 (A590=0.8, approximately 1010 CFU ml-1) caused 55% mortality and 82% at 72 h in a population of insects. Bp4185 strain, at 72 h caused 55% mortality (A590=1.0, approximately 1012 CFU ml-1) and 46% at 96 h (A590=0.8). Both native bacterial strains, adapted to the environmental conditions of Puerto Rico, have potential for biological control of coffee berry borer.

Reproductive organs of Bacillus thuringiensis transgenic cotton, which contribute to cotton final yield, have low insect resistant efficacy, so it is important to improve their insect resistance. This study was conducted to find out the impact of different urea spray doses on the expression of Cry1A protein in boll shell of Bt cotton (Sikang 1 and Sikang 3), and nitrogen metabolism in this process was also studied to uncover the physiological mechanism. The experiment with six urea doses was organized during peak boll stage in 2017 and 2018. The results showed that urea spray could significantly increase boll shell insecticidal protein contents in both cultivars, with the highest Bt protein content observed at 28–32 kg ha−1 urea dose. In addition, urea spray increased the contents of soluble protein and free amino acid and the activities of GS, GOGAT, GOT, and GPT, but decreased the activities of peptidase and protease in boll shell. Correlation analysis showed that the amount of boll shell Bt protein was positively correlated with levels of soluble protein and amino acid, and activities of GS, GOGAT, GOT, and GPT, but negatively correlated with peptidase and protease activities. Thus, this study demonstrated that higher protein synthesis ability and lower proteolysis ability were related to increased Bt protein content in urea-sprayed boll shell.

Reproductive organs of Bacillus thuringiensis transgenic cotton, which contribute to cotton final yield, have low insect resistant efficacy, so it is important to improve their insect resistance. This study was conducted to find out the impact of different urea spray doses on the expression of Cry1A protein in boll shell of Bt cotton (Sikang 1 and Sikang 3), and nitrogen metabolism in this process was also studied to uncover the physiological mechanism. The experiment with six urea doses was organized during peak boll stage in 2017 and 2018. The results showed that urea spray could significantly increase boll shell insecticidal protein contents in both cultivars, with the highest Bt protein content observed at 28–32 kg ha−1 urea dose. In addition, urea spray increased the contents of soluble protein and free amino acid and the activities of GS, GOGAT, GOT, and GPT, but decreased the activities of peptidase and protease in boll shell. Correlation analysis showed that the amount of boll shell Bt protein was positively correlated with levels of soluble protein and amino acid, and activities of GS, GOGAT, GOT, and GPT, but negatively correlated with peptidase and protease activities. Thus, this study demonstrated that higher protein synthesis ability and lower proteolysis ability were related to increased Bt protein content in urea-sprayed boll shell. [ABSTRACT FROM AUTHOR]

Coffee berry borer (Hypothenemus hampei), is one of the main pests affecting coffee production worldwide. This pest feeds and reproduces inside the berry reducing its weight and quality, causing losses up to 50%. Some strains of Bacillus have plasmids that incorporate cry and cyt genes, whose products have toxic properties to insects. Bacillus thuringiensis and Bacillus pumilus have been evaluated as entomopathogenic. The objectives of this study was to determine the potential of two native Bacillus strains (B. thuringiensis Bt3971 and B. pumilus Bp4185) isolated from coffee ecosystem of Puerto Rico as biocontrol of coffee berry borer. The presence of a 12 kbp plasmid was confirmed in Bt3971 and Bp4185, and a 116 kDa protein with entomopathogenic potential from Bt3971. After 48 h of treatment, Bt3971 (A590=0.8, approximately 1010 CFU ml-1) caused 55% mortality and 82% at 72 h in a population of insects. Bp4185 strain, at 72 h caused 55% mortality (A590=1.0, approximately 1012 CFU ml-1) and 46% at 96 h (A590=0.8). Both native bacterial strains, adapted to the environmental conditions of Puerto Rico, have potential for biological control of coffee berry borer. [ABSTRACT FROM AUTHOR]

Members of the insect ATP binding cassette transporter subfamily C2 (ABCC2) in several moth species are known as receptors for the Cry1Ac insecticidal protein from Bacillus thuringiensis (Bt). Mutations that abolish the functional domains of ABCC2 are known to cause resistance to Cry1Ac, although the reported levels of resistance vary widely depending on insect species. In this study, the function of the ABCC2 gene as a putative Cry1Ac receptor in Helicoverpa zea, a major pest of over 300 crops, was evaluated using CRISPR/Cas9 to progressively eliminate different functional ABCC2 domains. Results from bioassays with edited insect lines support that mutations in ABCC2 were associated with Cry1Ac resistance ratios (RR) ranging from 7.3- to 39.8-fold. No significant differences in susceptibility to Cry1Ac were detected between H. zea with partial or complete ABCC2 knockout, although the highest levels of tolerance were observed when knocking out half of ABCC2. Based on >500–1000-fold RRs reported in similar studies for closely related moth species, the low RRs observed in H. zea knockouts support that ABCC2 is not a major Cry1Ac receptor in this insect.

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori ; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [ BmABCC2_S ] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [ BmABCC2_R(+ 234 Y) ] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+ 234 Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins. [ABSTRACT FROM AUTHOR]

For almost half a century, the structure of the full-length Bacillus thuringiensis ( Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability. [ABSTRACT FROM AUTHOR]

Abstract: Large amounts of genetically modified grains producing Bacillus thuringiensis (Bt) toxins have been imported to Korea. Therefore, the establishment of a risk assessment system for evaluating the potential impacts of imported Bt maize on non-target insects is important. Before evaluating the environmental impacts of Bt grains of unknown origin, Cry protein types must first be identified in test Bt grains. Cry toxins of imported Bt maize grains were analyzed by ELISA. Because all tested Bt maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as the non-target insect species. A domestic maize strain that showed few differences in nutritional composition compared to the Bt maize grain was used as the alternative non-Bt control. Slightly increased survival rate and head capsule width of Bt maize-fed T. molitor were observed, indicating that Bt maize has no sub-chronic adverse effects on T. molitor. An ELISA test revealed that concentrations of Cry1A toxins slowly increased in the body of T. molitor when the insects were fed Bt maize. Such substantial amounts of Cry toxins remaining in the alimentary tract of larvae indicate that Cry toxins can be transferred to the higher trophic level of predatory insects. However, no Cry proteins were detected in the hemolymph of the Bt maize-fed larvae, suggesting that there is little possibility of Cry toxin exposure via T. molitor to the higher endoparasitoids. The risk assessment strategies and protocols established in this study may also be applicable to other imported Bt crops in Korea. [Copyright &y& Elsevier]

Members of the insect ATP binding cassette transporter subfamily C2 (ABCC2) in several moth species are known as receptors for the Cry1Ac insecticidal protein from Bacillus thuringiensis (Bt). Mutations that abolish the functional domains of ABCC2 are known to cause resistance to Cry1Ac, although the reported levels of resistance vary widely depending on insect species. In this study, the function of the ABCC2 gene as a putative Cry1Ac receptor in Helicoverpa zea, a major pest of over 300 crops, was evaluated using CRISPR/Cas9 to progressively eliminate different functional ABCC2 domains. Results from bioassays with edited insect lines support that mutations in ABCC2 were associated with Cry1Ac resistance ratios (RR) ranging from 7.3- to 39.8-fold. No significant differences in susceptibility to Cry1Ac were detected between H. zea with partial or complete ABCC2 knockout, although the highest levels of tolerance were observed when knocking out half of ABCC2. Based on >500–1000-fold RRs reported in similar studies for closely related moth species, the low RRs observed in H. zea knockouts support that ABCC2 is not a major Cry1Ac receptor in this insect. [ABSTRACT FROM AUTHOR]

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R1, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R1 was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells. [ABSTRACT FROM AUTHOR]

Cry1Ab is one of the most studied insecticidal proteins produced by Bacillus thuringiensis during sporulation. Structurally, this protoxin has been divided in two domains: the N-terminal toxin core and the C-terminal portion. Although many studies have addressed the biochemical characteristics of the active toxin that corresponds to the N-terminal portion, there are just few reports studying the importance of the C-terminal part of the protoxin. Herein, we show that Cry1Ab protoxin has a unique natural cryptic endotoxic property that is evident when their halves are expressed individually. This toxic effect of the separate protoxin domains was found against its original host B. thuringiensis, as well as to two other bacteria, Escherichia coli and Agrobacterium tumefaciens. Interestingly, either the fusion of the C-terminal portion with the insecticidal domain-III or the whole N-terminal region reduced or neutralized such a toxic effect, while a non-Cry1A peptide such as maltose binding protein did not neutralize the toxic effect. Furthermore, the C-terminal domain, in addition to being essential for crystal formation and solubility, plays a crucial role in neutralizing the toxicity caused by a separate expression of the insecticidal domain much like a dot/anti-dot system. [Copyright &y& Elsevier]

A genetically altered variant of Cry9Ca from Bacillus thuringiensis shows high potency against the spruce budworm, Choristoneura fumiferana Clemens. Its activity, as measured by feeding inhibition in frass-failure assays, is estimated to be four to seven times greater than B. thuringiensis subsp. kurstaki HD-1, the strain currently used in commercial products to control this insect. Bioassays against budworm of mixtures of the modified Cry9Ca and two of the Cry1A endotoxin proteins produced by HD-1 show neither synergism nor antagonism. Experiments with brush border membrane vesicles from budworm midgut revealed that Cry9Ca and the Cry1A toxins share a common binding site and that bound Cry9Ca can be displaced from the membrane to some extent by the Cry1A toxins. However, it is uncertain whether the binding site is actually the receptor molecule or a membrane protein associated with pore formation. [Copyright &y& Elsevier]

For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability., (© 2014 The Protein Society.)