Retrotransposon-mediated evolutionary rewiring of a pathogen response orchestrates a resistance phenotype in an insect host.

Ongoing host-pathogen interactions can trigger a coevolutionary arms race, while genetic diversity within the host can facilitate its adaptation to pathogens. Here, we used the diamondback moth (Plutella xylostella) and its pathogen Bacillus thuringiensis (Bt) as a model for exploring an adaptive evolutionary mechanism. We found that insect host adaptation to the primary Bt virulence factors was tightly associated with a short interspersed nuclear element (SINE - named SE2) insertion into the promoter of the transcriptionally activated MAP4K4 gene. This retrotransposon insertion coopts and potentiates the effect of the transcription factor forkhead box O (FOXO) in inducing a hormone-modulated Mitogen-activated protein kinase (MAPK) signaling cascade, leading to an enhancement of a host defense mechanism against the pathogen. This work demonstrates that reconstructing a cis-trans interaction can escalate a host response mechanism into a more stringent resistance phenotype to resist pathogen infection, providing a new insight into the coevolutionary mechanism of host organisms and their microbial pathogens.

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

MAPK-mediated transcription factor GATAd contributes to Cry1Ac resistance in diamondback moth by reducing PxmALP expression.

The benefits of biopesticides and transgenic crops based on the insecticidal Cry-toxins from Bacillus thuringiensis (Bt) are considerably threatened by insect resistance evolution, thus, deciphering the molecular mechanisms underlying insect resistance to Bt products is of great significance to their sustainable utilization. Previously, we have demonstrated that the down-regulation of PxmALP in a strain of Plutella xylostella (L.) highly resistant to the Bt Cry1Ac toxin was due to a hormone-activated MAPK signaling pathway and contributed to the resistance phenotype. However, the underlying transcriptional regulatory mechanism remains enigmatic. Here, we report that the PxGATAd transcription factor (TF) is responsible for the differential expression of PxmALP observed between the Cry1Ac susceptible and resistant strains. We identified that PxGATAd directly activates PxmALP expression via interacting with a non-canonical but specific GATA-like cis-response element (CRE) located in the PxmALP promoter region. A six-nucleotide insertion mutation in this cis-acting element of the PxmALP promoter from the resistant strain resulted in repression of transcriptional activity, affecting the regulatory performance of PxGATAd. Furthermore, silencing of PxGATAd in susceptible larvae reduced the expression of PxmALP and susceptibility to Cry1Ac toxin. Suppressing PxMAP4K4 expression in the resistant larvae transiently recovered both the expression of PxGATAd and PxmALP, indicating that the PxGATAd is a positive responsive factor involved in the activation of PxmALP promoter and negatively regulated by the MAPK signaling pathway. Overall, this study deciphers an intricate regulatory mechanism of PxmALP gene expression and highlights the concurrent involvement of both trans-regulatory factors and cis-acting elements in Cry1Ac resistance development in lepidopteran insects.

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 58 kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58 kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.

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.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host.

Host-pathogen interactions are central components of ecological networks where the MAPK signaling pathways act as central hubs of these complex interactions. We have previously shown that an insect hormone modulated MAPK signaling cascade participates as a general switch to trans-regulate differential expression of diverse midgut genes in the diamondback moth, Plutella xylostella (L.) to cope with the insecticidal action of Cry1Ac toxin, produced by the entomopathogenic bacterium Bacillus thuringiensis (Bt). The relationship between topology and functions of this four-tiered phosphorylation signaling cascade, however, is an uncharted territory. Here, we carried out a genome-wide characterization of all the MAPK orthologs in P. xylostella to define their phylogenetic relationships and to confirm their evolutionary conserved modules. Results from quantitative phosphoproteomic analyses, combined with functional validations studies using specific inhibitors and dsRNAs lead us to establish a MAPK "road map", where p38 and ERK MAPK signaling pathways, in large part, mount a resistance response against Bt toxins through regulating the differential expression of multiple Cry toxin receptors and their non-receptor paralogs in P. xylostella midgut. These data not only advance our understanding of host-pathogen interactions in agricultural pests, but also inform the future development of biopesticides that could suppress Cry resistance phenotypes.

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.

Assessment of the role of an ABCC transporter TuMRP1 in the toxicity of abamectin to Tetranychus urticae.

The rapid evolution of pest resistance threatens the sustainable utilization of bioinsecticides such as abamectin, and so deciphering the molecular mechanisms affecting toxicity and resistance is essential for their long-term application. Historical studies of abamectin resistance in arthropods have mainly focused on mechanisms involving the glutamate-gated chloride channel (GluCl) targets, with the role of metabolic processes less clear. The two-spotted spider mite, Tetranychus urticae, is a generalist herbivore notorious for rapidly developing resistance to pesticides worldwide, and abamectin has been widely used for its control in the field. After reanalyzing previous transcriptome and RNA-seq data, we here identified an ABC transporter subfamily C gene in T. urticae named multidrug resistance-associated protein 1 (TuMRP1), whose expression differed between susceptible and resistant populations. Synergism bioassays with the inhibitor MK-571, the existence of a genetic association between TuMRP1 expression and susceptibility to abamectin, and the effect of RNA interference mediated silencing of TuMRP1 were all consistent with a direct role of this transporter protein in the toxicity of abamectin. Although ABC transporters are often involved in removing insecticidal compounds from cells, our data suggest either an alternative role for these proteins in the mechanism of action of abamectin or highlight an indirect association between their expression and abamectin toxicity.

A versatile contribution of both aminopeptidases N and ABC transporters to Bt Cry1Ac toxicity in the diamondback moth.

BACKGROUND: Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial. RESULTS: We have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a > 34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains. CONCLUSION: Our results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of action of Bt toxins, which can compensate for the absence of individual receptors, and are consistent with an interplay among diverse midgut receptors in the toxins' mechanism of action in a super pest.

MAP4K4 controlled transcription factor POUM1 regulates PxABCG1 expression influencing Cry1Ac resistance in Plutella xylostella (L.).

Deciphering the molecular mechanisms of insect resistance to Bacillus thuringiensis (Bt) based biotechnology products including Bt sprays and Bt crops is critical for the long-term application of Bt technology. Previously, we established that down-regulation of the ABC transporter gene PxABCG1, trans-regulated by the MAPK signaling pathway, contributed to high-level resistance to Bt Cry1Ac toxin in diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulatory mechanism was unknown. Herein, we identified putative binding sites (PBSs) of the transcription factor (TF) POUM1 in the PxABCG1 promoter and used a dual-luciferase reporter assay (DLRA) and yeast one-hybrid (Y1H) assay to reveal that POUM1 activates PxABCG1 via interaction with one of these sites. The expression of POUM1 was significantly decreased in the midgut tissue of Cry1Ac-resistant P. xylostella strains compared to a Cry1Ac-susceptible P. xylostella strain. Silencing of POUM1 expression resulted in reduced expression of the PxABCG1 gene and an increase in larval tolerance to Bt Cry1Ac toxin in the Cry1Ac-susceptible P. xylostella strain. Furthermore, silencing of PxMAP4K4 expression increased the expression of both POUM1 and PxABCG1 genes in the Cry1Ac-resistant P. xylostella strain. These results indicate that the POUM1 induces PxABCG1 expression, while the activated MAPK cascade represses PxABCG1 expression thus reducing Cry1Ac susceptibility in P. xylostella. This result deepens our understanding of the transcriptional regulatory mechanism of midgut Cry receptor genes and the molecular basis of the evolution of Bt resistance in insects.

Cyclosporin A acts as a novel insecticide against Cry1Ac-susceptible and -resistant Helicoverpa armigera.

Cotton bollworm (Helicoverpa armigera) is an economically important pest, which is difficult to manage due to its biological and ecological traits, and resistance to most insecticides. Alternative compounds for the sustainable management of H. armigera are needed. As a fungal metabolite, Cyclosporin A (CsA) has not been applied in agriculture pests. Here, CsA was evaluated as a propective insecticide for H. armigera. The results showed that CsA displayed high insecticidal activity against both Cry1Ac-susceptible and -resistant populations of H. armigera. Moreover, lower concentrations of CsA had clear effects, including significantly reduced pupal weight, pupation rate, emergence rate, ovary size, female fecundity and egg hatchability. Further study confirmed that CsA suppressed calcineurin activity and the subsequent expression of endogenous antimicrobial peptide genes (APMs), leading to impaired immunity, ultimately resulting in delayed development and increased mortality. Thus, CsA treatment could control the cotton bollworm population and even showed efficacy against those with Bt resistance. In addition, the morphological changes observed in insects fed CsA with lower concentrations provide insight into insect immunity, regulation of growth and development, regulation of body color, ovary development and sexual selection under external pressure. Overall, our study provides information on biological control potential of Cry1Ac-susceptible and -resistant populations of H. armigera to develop novel bioinsecticides.

MAPK-Activated Transcription Factor PxJun Suppresses PxABCB1 Expression and Confers Resistance to Bacillus thuringiensis Cry1Ac Toxin in Plutella xylostella (L.).

Deciphering the molecular mechanisms underlying insect resistance to Cry toxins produced by Bacillus thuringiensis (Bt) is pivotal for the sustainable utilization of Bt biopesticides and transgenic Bt crops. Previously, we identified that mitogen-activated protein kinase (MAPK)-mediated reduced expression of the PxABCB1 gene is associated with Bt Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulation mechanism remains enigmatic. Here, the PxABCB1 promoter in Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella strains was cloned and analyzed and found to contain a putative Jun binding site (JBS). A dual-luciferase reporter assay and yeast one-hybrid assay demonstrated that the transcription factor PxJun repressed PxABCB1 expression by interacting with this JBS. The expression levels of PxJun were increased in the midguts of all resistant strains compared to the susceptible strain. Silencing of PxJun expression significantly elevated PxABCB1 expression and Cry1Ac susceptibility in the resistant NIL-R strain, and silencing of PxMAP4K4 expression decreased PxJun expression and also increased PxABCB1 expression. These results indicate that MAPK-activated PxJun suppresses PxABCB1 expression to confer Cry1Ac resistance in P. xylostella, deepening our understanding of the transcriptional regulation of midgut Cry receptor genes and the molecular basis of insect resistance to Bt Cry toxins. IMPORTANCE The transcriptional regulation mechanisms underlying reduced expression of Bt toxin receptor genes in Bt-resistant insects remain elusive. This study unveils that a transcription factor PxJun activated by the MAPK signaling pathway represses PxABCB1 expression and confers Cry1Ac resistance in P. xylostella. Our results provide new insights into the transcriptional regulation mechanisms of midgut Cry receptor genes and deepen our understanding of the molecular basis of insect resistance to Bt Cry toxins. To our knowledge, this study identified the first transcription factor that can be involved in the transcriptional regulation mechanisms of midgut Cry receptor genes in Bt-resistant insects.

A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins.

In 1998 a nomenclature for the growing list of pesticidal proteins from Bacillus thuringiensis (Bt) was derived based solely on protein sequence comparisons. This nomenclature was widely adopted and provided a robust framework for the naming and classification of the proteins. The success of these proteins in integrated pest management schemes prompted an increased effort to find others with improved or more diverse activities. These discovery activities led to the characterization of proteins from a wider range of bacteria and with a variety of different protein folds. Since most of these new proteins were grouped together as Cry proteins it became apparent that the existing nomenclature had limitations in representing the diverse range of proteins that had been identified. This revised nomenclature retains the basic principles of the 1998 version but provides specific mnemonics to represent different structural groups. For the purposes of consistency, the vast majority of the proteins have either retained their name or have a new name that clearly references the previous one. Other pesticidal proteins not previously included in the nomenclature have been incorporated into this version.

A cis-Acting Mutation in the PxABCG1 Promoter Is Associated with Cry1Ac Resistance in Plutella xylostella (L.).

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.

Differential proteolytic activation of the Bacillus thuringiensis Cry41Aa parasporin modulates its anticancer effect.

Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells) cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made in the N-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore, we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60, respectively) which we hypothesize occlude a binding motif.

Identification of Aedes aegypti specificity motifs in the N-terminus of the Bacillus thuringiensis Cry2Aa pesticidal protein.

One advantage of using the Cry proteins of Bacillus thuringiensis as pesticides is their relatively narrow spectrum of activity, thus reducing the risk of non-target effects. Understanding the molecular basis of specificity has the potential to help us design improved products against emerging pests, or against pests that have developed resistance to other Cry proteins. Many previous studies have associated specificity with the binding of the Cry protein, particularly through the apical regions of domain II, to particular receptors on the midgut epithelial cells of the host insect. We have previously found that the specificity of Cry2A proteins against some insects is associated with domain I, which is traditionally associated with pore-formation but not receptor binding. In this work we identify four amino acids in the N-terminal region that, when mutated, can confer activity towards Aedes aegypti to Cry2Ab, a protein known to lack this toxicity. Intriguingly these amino acids are located in the region (amino acids 1-49) that is believed to be removed during proteolytic activation of the Cry protein. We discuss how the motifs containing these amino acids might be involved in the toxic process.

The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts.

Understanding how certain protein toxins from the normally insecticidal bacterium Bacillus thuringiensis (Bt) target human cell lines has implications for both the risk assessment of products containing these toxins and potentially for cancer therapy. This understanding requires knowledge of whether the human cell active toxins work by the same mechanism as their insecticidal counterparts or by alternative ones. The Bt Cry41Aa (also known as Parasporin3) toxin is structurally related to the toxins synthesised by commercially produced transgenic insect-resistant plants, with the notable exception of an additional C-terminal ß-trefoil ricin domain. To better understand its mechanism of action, we developed an efficient expression system for the toxin and created mutations in regions potentially involved in the toxic mechanism. Deletion of the ricin domain did not significantly affect the activity of the toxin against the human HepG2 cell line, suggesting that this region was not responsible for the mammalian specificity of Cry41Aa. Various biochemical assays suggested that unlike some other human cell active toxins from Bt Cry41Aa did not induce apoptosis, but that its mechanism of action was consistent with that of a pore-forming toxin. The toxin induced a rapid and significant decrease in metabolic activity. Adenosine triphosphate depletion, cell swelling and membrane damage were also observed. An exposed loop region believed to be involved in receptor binding of insecticidal Cry toxins was shown to be important for the activity of Cry41Aa against HepG2 cells.

Cry78Aa, a novel Bacillus thuringiensis insecticidal protein with activity against Laodelphax striatellus and Nilaparvata lugens.

Transgenic plants expressing insecticidal proteins originating from Bacillus thuringiensis (Bt) have successfully been used to control lepidopteran and coleopteran pests with chewing mouthparts. However, only a handful of Bt proteins have been identified that have bioactivity against sap sucking pests (Hemiptera), including aphids, whiteflies, plant bugs and planthoppers. A novel Bt insecticidal protein with significant toxicity against a hemipteran insect pest is described here. The gene encoding the 359 amino acid, 40.7 kDa protein was cloned from strain C9F1. After expression and purification of the toxin, its median lethal concentration (LC50) values against Laodelphax striatellus and Nilaparvata lugens were determined as 6.89 µg/mL and 15.78 µg/mL respectively. Analysis of the toxin sequence revealed the presence of both Toxin_10 and Ricin_B_Lectin domains.

Structural classification of insecticidal proteins - Towards an in silico characterisation of novel toxins.

The increasing rate of discovery of new toxins with potential for the control of invertebrate pests through next generation sequencing, presents challenges for the identification of the best candidates for further development. A consideration of structural similarities between the different toxins suggest that they may be functionally less diverse than their low sequence similarities might predict. This is encouraging from the prospective of being able to use computational tools to predict toxin targets from their sequences, however more structure/function data are still required to reliably inform such predictions.