N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.</p

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.</p

Optimizing pyramided transgenic Bt crops for sustainable pest management

Transgenic crop pyramids producing two or more Bacillus thuringiensis (Bt) toxins that kill the same insect pest have been widely used to delay evolution of pest resistance. To assess the potential of pyramids to achieve this goal, we analyze data from 38 studies that report effects of ten Bt toxins used in transgenic crops against 15 insect pests. We find that compared with optimal low levels of insect survival, survival on currently used pyramids is often higher for both susceptible insects and insects resistant to one of the toxins in the pyramid. Furthermore, we find that cross-resistance and antagonism between toxins used in pyramids are common, and that these problems are associated with the similarity of the amino acid sequences of domains II and III of the toxins, respectively. This analysis should assist in future pyramid design and the development of sustainable resistance management strategies

Throwing Brazilian strains into the melting pot of P. xylostella resistance to Bacillus thuringiensis

The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.</p

Throwing Brazilian strains into the melting pot of P. xylostella resistance to Bacillus thuringiensis

The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.</p

Group selection as a basis for screening mutagenized libraries of public goods (bacillus thuringiensis cry toxins)

The pesticidal toxins of Bacillus thuringiensis (Bt) supply the active proteins for genetically modified insect-resistant crops. There is therefore keen interest in finding new toxins, or improving known toxins, in order to increase the mortality of various targets. The production and screening of large libraries of mutagenized toxins are among the means of identifying improved toxins. Since Cry toxins are public goods, and do not confer advantages to producers in competition, conventional directed evolution approaches cannot be used here. Instead, thousands of individual mutants have to be sequenced and assayed individually, a costly and time-consuming process. In this study, we tested a group selection-based approach that could be used to screen an uncharacterized pool of Cry toxin mutants. This involved selecting for infectivity between subpopulations of Bt clones within metapopulations of infected insects in three rounds of passage. We also tested whether additional mutagenesis from exposure to ethyl methanesulfonate could increase infectivity or supply additional Cry toxin diversity during passage. Sequencing of pools of mutants at the end of selection showed that we could effectively screen out Cry toxin variants that had reduced toxicity with our group selection approach. The addition of extra mutagenesis during passage decreased the efficiency of selection for infectivity and did not produce any additional novel toxin diversity. Toxins with loss-of-function mutations tend to dominate mutagenized libraries, and so a process for screening out these mutants without time-consuming sequencing and characterization steps could be beneficial when applied to larger libraries. IMPORTANCE Insecticidal toxins from the bacterium Bacillus thuringiensis are widely exploited in genetically modified plants. This application creates a demand for novel insecticidal toxins that can be used to better manage resistant pests or control new or recalcitrant target species. An important means of producing novel toxins is via high-throughput mutagenesis and screening of existing toxins, a lengthy and resource-intensive process. This study describes the development and testing of an efficient means of screening a test library of mutagenized insecticidal toxins. Here, we showed that it is possible to screen out loss-of-function mutations with low infectivity within a pool without the need to characterize and sequence each mutant individually. This has the potential to improve the efficiency of processes used to identify novel proteins

Genetics and mechanism of resistance to deltamethrin in a field population of Spodoptera litura (Lepidoptera: Noctuidae)

BACKGROUND: Spodoptera litura (F.) causes enormous losses in many economically important crops. The genetics of insecticide resistance has been extensively studied in several insect pests, but there is a lack of information on S. litura. Therefore, the genetics and mechanisms of the resistance of S. litura to deltamethrin were investigated. RESULTS: Bioassays at generation G1 gave resistance ratios of 9, 5, 41, 52 and 49 for deltamethrin, cypermethrin, profenofos, chlorpyrifos and triazofos respectively, when compared with the susceptible Lab-PK strain. Bioassays at G4 with a deltamethrin-selected population (Delta-SEL) showed that selection gave resistance ratios of 63 and 7 for deltamethrin when compared with the Lab-PK and UNSEL strains respectively. Cross-resistance to other insecticides tested was observed in the selected population. A notable feature of the Delta-SEL strain was that resistance to deltamethrin, cypermethrin, profenofos and chlorpyrifos did not decline over the course of five generations. Synergism tests with microsomal oxidase (MO) and esterase-specific inhibitors indicated that the deltamethrin resistance was associated with MO and, possibly, esterase activity. Reciprocal crosses between the Delta-SEL and Lab-PK strains indicated that resistance was autosomal and incompletely dominant. A direct test of monogenic inheritance suggested that resistance to deltamethrin was controlled by more than one locus. CONCLUSION: Stability and dominance of resistance and cross-resistance suggest that insecticides with different modes of action should be recommended to reduce pyrethroid selection pressure. Copyright © 2007 Society of Chemical Industr

Contribution of the individual components of the d-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis

The delta-endotoxin crystal of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis contains four major delta-endotoxins. Expression systems were devised to synthesize each of the four toxins at concentrations at which they formed inclusion bodies in an acrystalliferous mutant of Bacillus thuringiensis. The relative activities of these inclusions were then determined against Aedes aegypti larvae. Bioassays of mixtures of the individual toxins revealed a number of synergistic interactions which explained in part why the native crystal is considerably more toxic than any of the individual toxins

A shared receptor suggests a common ancestry between an insecticidal Bacillus thuringiensis cry protein and an anti-cancer parasporin

Cry toxins, produced by the bacterium Bacillus thuringiensis, are of significant agronomic value worldwide due to their potent and highly specific activity against various insect orders. However, some of these pore-forming toxins display specific activity against a range of human cancer cells whilst possessing no known insecticidal activity; Cry41Aa is one such toxin. Cry41Aa has similarities to its insecticidal counterparts in both its 3-domain toxic core structure and pore-forming abilities, but how it has evolved to target human cells is a mystery. This work shows that some insecticidal Cry toxins can enhance the toxicity of Cry41Aa against hepatocellular carcinoma cells, despite possessing no intrinsic toxicity themselves. This interesting crossover is not limited to human cancer cells, as Cry41Aa was found to inhibit some Aedes-active Cry toxins in mosquito larval assays. Here, we present findings that suggest that Cry41Aa shares a receptor with several insecticidal toxins, indicating a stronger evolutionary relationship than their divergent activities might suggest.</p

Development of an online genome sequence comparison resource for bacillus cereus sensu lato strains using the efficient composition vector method

An automated method was developed for differentiating closely related B. cereus sensu lato (s.l.) species, especially biopesticide Bacillus thuringiensis, from other human pathogens, B. anthracis and B. cereus sensu stricto (s.s.). In the current research, four typing methods were initially compared, including multi-locus sequence typing (MLST), single-copy core genes phylogenetic analysis (SCCGPA), dispensable genes content pattern analysis (DGCPA) and composition vector tree (CVTree), to analyze the genomic variability of 23 B. thuringiensis strains from aizawai, kurstaki, israelensis, thuringiensis and morrisoni serovars. The CVTree method was the best option to be used for typing B. thuringiensis strains since it proved to be the fastest method, whilst giving high-resolution data about the strains. In addition, CVTree agrees well with ANI-based method, revealing the relationship between B. thuringiensis and other B. cereus s.l. species. Based on these data, an online genome sequence comparison resource was built for Bacillus strains called the Bacillus Typing Bioinformatics Database to facilitate strain identification and characterization