Comparison of in vitro and in vivo binding site competition of Bacillus thuringiensis Cry1 proteins in two important maize pests.

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.

Bacillus thuringiensis chimeric proteins Cry1A.2 and Cry1B.2 to control soybean lepidopteran pests: New domain combinations enhance insecticidal spectrum of activity and novel receptor contributions.

Two new chimeric Bacillus thuringiensis (Bt) proteins, Cry1A.2 and Cry1B.2, were constructed using specific domains, which provide insecticidal activity against key lepidopteran soybean pests while minimizing receptor overlaps between themselves, current, and soon to be commercialized plant incorporated protectants (PIP's) in soybean. Results from insect diet bioassays demonstrate that the recombinant Cry1A.2 and Cry1B.2 are toxic to soybean looper (SBL) Chrysodeixis includens Walker, velvetbean caterpillar (VBC) Anticarsia gemmatalis Hubner, southern armyworm (SAW) Spodoptera eridania, and black armyworm (BLAW) Spodoptera cosmioides with LC50 values < 3,448 ng/cm2. Cry1B.2 is of moderate activity with significant mortality and stunting at > 3,448 ng/cm2, while Cry1A.2 lacks toxicity against old-world bollworm (OWB) Helicoverpa armigera. Results from disabled insecticidal protein (DIP) bioassays suggest that receptor utilization of Cry1A.2 and Cry1B.2 proteins are distinct from each other and from current, and yet to be commercially available, Bt proteins in soy such as Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A. However, as Cry1A.2 contains a domain common to at least one commercial soybean Bt protein, resistance to this common domain in a current commercial soybean Bt protein could possibly confer at least partial cross resistance to Cry1A2. Therefore, Cry1A.2 and Cry1B.2 should provide two new tools for controlling many of the major soybean insect pests described above.

Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI ß; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

Author Correction: A transgenic approach for controlling Lygus in cotton.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bacillus thuringiensis Cry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms, Spodoptera frugiperda (J.E. Smith).

Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

Disabled insecticidal proteins: A novel tool to understand differences in insect receptor utilization.

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.

Characterization of the Activity Spectrum of MON 88702 and the Plant-Incorporated Protectant Cry51Aa2.834_16.

The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysanopteran insect pests in cotton was characterized by selecting and screening multiple pest and non-pest species, based on representation of ecological functional groups, taxonomic relatedness (e.g. relationship to species where activity was observed), and availability for effective testing. Seven invertebrate orders, comprising 12 families and 17 representative species were screened for susceptibility to Cry51Aa2.834_16 protein and/or the ability of the protein to protect against feeding damage in laboratory, controlled environments (e.g. greenhouse/growth chamber), and/or field studies when present in cotton plants. The screening results presented for Cry51Aa2.834_16 demonstrate selective and limited activity within three insect orders. Other than Orius insidiosus, no activity was observed for Cry51Aa2.834_16 against several groups of arthropods that perform key ecological roles in some agricultural ecosystems (e.g. pollinators, decomposers, and natural enemies).

The sequence, structural, and functional diversity within a protein family and implications for specificity and safety: The case for ETX_MTX2 insecticidal proteins.

The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common ß-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although ß-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the "relatedness"). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).

A transgenic approach for controlling Lygus in cotton.

Lygus species of plant-feeding insects have emerged as economically important pests of cotton in the United States. These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton varieties resulting in economic losses and increased application of insecticide. Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity against Lygus spp. However, transgenic cotton plants expressing this protein did not exhibit effective protection from Lygus feeding damage. Here we employ various optimization strategies, informed in part by protein crystallography and modelling, to identify limited amino-acid substitutions in Cry51Aa2 that increase insecticidal activity towards Lygus spp. by >200-fold. Transgenic cotton expressing the variant protein, Cry51Aa2.834_16, reduce populations of Lygus spp. up to 30-fold in whole-plant caged field trials. One transgenic event, designated MON88702, has been selected for further development of cotton varieties that could potentially reduce or eliminate insecticide application for control of Lygus and the associated environmental impacts.

The food and environmental safety of Bt crops.

Bacillus thuringiensis (Bt) microbial pesticides have a 50-year history of safety in agriculture. Cry proteins are among the active insecticidal ingredients in these pesticides, and genes coding for Cry proteins have been introduced into agricultural crops using modern biotechnology. The Cry gene sequences are often modified to enable effective expression in planta and several Cry proteins have been modified to increase biological activity against the target pest(s). Additionally, the domains of different but structurally conserved Cry proteins can be combined to produce chimeric proteins with enhanced insecticidal properties. Environmental studies are performed and include invertebrates, mammals, and avian species. Mammalian studies used to support the food and feed safety assessment are also used to support the wild mammal assessment. In addition to the NTO assessment, the environmental assessment includes a comparative assessment between the Bt crop and the appropriate conventional control that is genetically similar but lacks the introduced trait to address unintended effects. Specific phenotypic, agronomic, and ecological characteristics are measured in the Bt crop and the conventional control to evaluate whether the introduction of the insect resistance has resulted in any changes that might cause ecological harm in terms of altered weed characteristics, susceptibility to pests, or adverse environmental impact. Additionally, environmental interaction data are collected in field experiments for Bt crop to evaluate potential adverse effects. Further to the agronomic and phenotypic evaluation, potential movement of transgenes from a genetically modified crop plants into wild relatives is assessed for a new pest resistance gene in a new crop. This review summarizes the evidence for safety of crops containing Cry proteins for humans, livestock, and other non-target organisms.

Xentrivalpeptides A-Q: depsipeptide diversification in Xenorhabdus.

Seventeen depsipeptides, xentrivalpeptides A-Q (1-17), have been identified from an entomopathogenic Xenorhabdus sp. Whereas the structure of xentrivalpeptide A (1) was determined after its isolation by NMR spectroscopy and the advanced Marfey's method, the structures of all other derivatives were determined using a combination of stable isotope labeling and detailed MS analysis.

Cotton plants expressing a hemipteran-active Bacillus thuringiensis crystal protein impact the development and survival of Lygus hesperus (Hemiptera: Miridae) nymphs.

The plant bugs Lygus hesperus Knight (Hemiptera: Miridae) and L. lineolaris (Palisot de Beauvois) have emerged as economic pests of cotton in the United States. These hemipteran species are refractory to the insect control traits found in genetically modified commercial varieties of cotton. In this article, we report the isolation and characterization of a 35 kDa crystal protein from Bacillus thuringiensis, designated TIC807, which causes reduced mass gain and mortality of L. hesperus and L. lineolaris nymphs when presented in an artificial diet feeding assay. Cotton plants expressing the TIC807 protein were observed to impact the survival and development of L. hesperus nymphs in a concentration-dependent manner. These results, demonstrating in planta activity of a Lygus insecticidal protein, represent an important milestone in the development of cotton varieties protected from Lygus feeding damage.

Phenotypic variation and host interactions of Xenorhabdus bovienii SS-2004, the entomopathogenic symbiont of Steinernema jollieti nematodes.

Xenorhabdus bovienii (SS-2004) bacteria reside in the intestine of the infective-juvenile (IJ) stage of the entomopathogenic nematode, Steinernema jollieti. The recent sequencing of the X. bovienii genome facilitates its use as a model to understand host - symbiont interactions. To provide a biological foundation for such studies, we characterized X. bovienii in vitro and host interaction phenotypes. Within the nematode host X. bovienii was contained within a membrane bound envelope that also enclosed the nematode-derived intravesicular structure. Steinernema jollieti nematodes cultivated on mixed lawns of X. bovienii expressing green or DsRed fluorescent proteins were predominantly colonized by one or the other strain, suggesting the colonizing population is founded by a few cells. Xenorhabdus bovienii exhibits phenotypic variation between orange-pigmented primary form and cream-pigmented secondary form. Each form can colonize IJ nematodes when cultured in vitro on agar. However, IJs did not develop or emerge from Galleria mellonella insects infected with secondary form. Unlike primary-form infected insects that were soft and flexible, secondary-form infected insects retained a rigid exoskeleton structure. Xenorhabdus bovienii primary and secondary form isolates are virulent towards Manduca sexta and several other insects. However, primary form stocks present attenuated virulence, suggesting that X. bovienii, like Xenorhabdus nematophila may undergo virulence modulation.

Control of coleopteran insect pests through RNA interference.

Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.

Discovery and characterization of Sip1A: A novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae.

Bioassay screening of Bacillus thuringiensis culture supernatants identified strain EG2158 as having larvicidal activity against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Ion-exchange fractionation of the EG2158 culture supernatant resulted in the identification of a protein designated Sip1A (secreted insecticidal protein) of approximately 38 kDa having activity against Colorado potato beetle (CPB). An oligonucleotide probe based on the N-terminal sequence of the purified Sip1A protein was used to isolate the sip1A gene. The sequence of the Sip1A protein, as deduced from the sequence of the cloned sip1A gene, contained 367 residues (41,492 Da). Recombinant B. thuringiensis and Escherichia coli harboring cloned sip1A produced Sip1A protein which had insecticidal activity against larvae of CPB, southern corn rootworm (Diabrotica undecimpunctata howardi), and western corn rootworm (Diabrotica virgifera virgifera).

Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte.

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.