Bacillus thuringiensis Cry9Aa Insecticidal Protein Domain I Helices α3 and α4 Are Two Core Regions Involved in Oligomerization and Toxicity.

Bacillus thuringiensis Cry9 proteins show high insecticidal activity against different lepidopteran pests. Cry9 could be a valuable alternative to Cry1 proteins because it showed a synergistic effect with no cross-resistance. However, the pore-formation region of the Cry9 proteins is still unclear. In this study, nine mutations of certain Cry9Aa helices α3 and α4 residues resulted in a complete loss of insecticidal activity against the rice pest Chilo suppressalis; however, the protein stability and receptor binding ability of these mutants were not affected. Among these mutants, Cry9Aa-D121R, Cry9Aa-D125R, Cry9Aa-D163R, Cry9Aa-E165R, and Cry9Aa-D167R are unable to form oligomers in vitro, while the oligomers formed by Cry9Aa-R156D, Cry9Aa-R158D, and Cry9Aa-R160D are unstable and failed to insert into the membrane. These data confirmed that helices α3 and α4 of Cry9Aa are involved in oligomerization, membrane insertion, and toxicity. The knowledge of Cry9 pore-forming action may promote its application as an alternative to Cry1 insecticidal proteins.

JAK/STAT signaling regulated intestinal regeneration defends insect pests against pore-forming toxins produced by Bacillus thuringiensis.

A variety of coordinated host-cell responses are activated as defense mechanisms against pore-forming toxins (PFTs). Bacillus thuringiensis (Bt) is a worldwide used biopesticide whose efficacy and precise application methods limits its use to replace synthetic pesticides in agricultural settings. Here, we analyzed the intestinal defense mechanisms of two lepidopteran insect pests after intoxication with sublethal dose of Bt PFTs to find out potential functional genes. We show that larval intestinal epithelium was initially damaged by the PFTs and that larval survival was observed after intestinal epithelium regeneration. Further analyses showed that the intestinal regeneration caused by Cry9A protein is regulated through c-Jun NH (2) terminal kinase (JNK) and Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. JAK/STAT signaling regulates intestinal regeneration through proliferation and differentiation of intestinal stem cells to defend three different Bt proteins including Cry9A, Cry1F or Vip3A in both insect pests, Chilo suppressalis and Spodoptera frugiperda. Consequently, a nano-biopesticide was designed to improve pesticidal efficacy based on the combination of Stat double stranded RNA (dsRNA)-nanoparticles and Bt strain. This formulation controlled insect pests with better effect suggesting its potential use to reduce the use of synthetic pesticides in agricultural settings for pest control.

Performance insights into spray-dryer microencapsulated Bacillus thuringiensis cry pesticidal proteins with gum arabic and maltodextrin for effective pest control.

Bacillus thuringiensis (Bt) produces crystals composed mainly of Cry pesticidal proteins with insecticidal activity against pests but are highly susceptible to degradation by abiotic factors. In this sense, encapsulation techniques are designed to improve their performance and lifetime. However, the effects of polymeric matrix encapsulation such as gum arabic and maltodextrin by spray-dryer in the mechanisms of action of Bt kurstaki and Bt aizawai are unknown. We analyzed crystal solubilization, protoxin activation, and receptor binding after microencapsulation and compared them with commercial non-encapsulated products. Microencapsulation did not alter protein crystal solubilization, providing 130 kDa (Cry1 protoxin) and 70 kDa (Cry2 protoxin). Activation with trypsin, chymotrypsin, and larval midgut juice was analyzed, showing that this step is highly efficient, and the protoxins were cleaved producing similar ~ 55 to 65 kDa activated proteins for both formulations. Binding assays with brush border membrane vesicles of Manduca sexta and Spodoptera frugiperda larvae provided a similar binding for both formulations. LC50 bioassays showed no significant differences between treatments but the microencapsulated treatment provided higher mortality against S. frugiperda when subjected to UV radiation. Microencapsulation did not affect the mechanism of action of Cry pesticidal proteins while enhancing protection against UV radiation. These data will contribute to the development of more efficient Bt biopesticide formulations. KEY POINTS: • Microencapsulation did not affect the mechanisms of action of Cry pesticidal proteins produced by Bt. • Microencapsulation provided protection against UV radiation for Bt-based biopesticides. • The study's findings can contribute to the development of more efficient Bt biopesticide formulations.

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.

Helicoverpa armigera GATAe transcriptional factor regulates the expression of Bacillus thuringiensis Cry1Ac receptor gene ABCC2 by its interplay with additional transcription factors.

Helicoverpa armigera is a worldwide pest that has been efficiently controlled by transgenic plants expressing Bt Cry toxins. To exert toxicity, Cry toxins bind to different receptors located in larval midgut cells. Previously, we reported that GATA transcription factor GATAe activates the expression of multiple H. armigera Cry1Ac receptors in different insect cell lines. Here, the mechanism involved in GATAe regulation of HaABCC2 gene expression, a key receptor of Cry1Ac, was analyzed. HaGATAe gene silencing by RNAi in H. armigera larvae confirmed the activation role of HaGATAe on the expression of HaABCC2 in the midgut. The contribution of all potential GATAe-binding sites was analyzed by site-directed mutagenesis using Hi5 cells expressing a reporter gene under regulation of different modified HaABCC2 promoters. DNA pull-down assays revealed that GATAe bound to different predicted GATA-binding sites and mutations of the different GATAe-binding sites identified two binding sites responsible for the promoter activity. The binding site B9, which is located near the transcription initiator site, has a major contribution on HaABCC2 expression. Also, DNA pull-down assays revealed that all other members of GATA TF family in H. armigera, besides GATAe, HaGATAa, HaGATAb, HaGATAc and HaGATAd also bound to the HaABCC2 promoter and decreased the GATAe dependent promoter activity. Finally, the potential participation in the regulation of HaABCC2 promoter of several TFs other than GATA TFs expressed in the midgut cells was analyzed. HaHR3 inhibited the GATAe dependent activity of the HaABCC2 promoter, while two other midgut-related TFs, HaCDX and HaSox21, also bound to the HaABCC2 promoter region and increased the GATAe dependent promoter activity. All these data showed that GATAe induces HaABCC2 expression by binding to HaGATAe binding sites in the promoter region and that additional TFs participate in modulating the HaGATAe-driven expression of HaABCC2.

Aminopeptidase MNP-1 triggers intestine protease production by activating daf-16 nuclear location to degrade pore-forming toxins in Caenorhabditis elegans.

Pore-forming toxins (PFTs) are effective tools for pathogens infection. By disrupting epithelial barriers and killing immune cells, PFTs promotes the colonization and reproduction of pathogenic microorganisms in their host. In turn, the host triggers defense responses, such as endocytosis, exocytosis, or autophagy. Bacillus thuringiensis (Bt) bacteria produce PFT, known as crystal proteins (Cry) which damage the intestinal cells of insects or nematodes, eventually killing them. In insects, aminopeptidase N (APN) has been shown to act as an important receptor for Cry toxins. Here, using the nematode Caenorhabditis elegans as model, an extensive screening of APN gene family was performed to analyze the potential role of these proteins in the mode of action of Cry5Ba against the nematode. We found that one APN, MNP-1, participate in the toxin defense response, since the mnp-1(ok2434) mutant showed a Cry5Ba hypersensitive phenotype. Gene expression analysis in mnp-1(ok2434) mutant revealed the involvement of two protease genes, F19C6.4 and R03G8.6, that participate in Cry5Ba degradation. Finally, analysis of the transduction pathway involved in F19C6.4 and R03G8.6 expression revealed that upon Cry5Ba exposure, the worms up regulated both protease genes through the activation of the FOXO transcription factor DAF-16, which was translocated into the nucleus. The nuclear location of DAF-16 was found to be dependent on mnp-1 under Cry5Ba treatment. Our work provides evidence of new host responses against PFTs produced by an enteric pathogenic bacterium, resulting in activation of host intestinal proteases that degrade the PFT in the intestine.

Can microbial-based insecticides replace chemical pesticides in agricultural production?

Extensive use of chemical insecticides to control insect pests in agriculture has improved yields and production of high-quality food products. However, chemical insecticides have been shown to be harmful also to beneficial insects and many other organisms like vertebrates. Thus, there is a need to replace those chemical insecticides by other control methods in order to protect the environment. Insect pest pathogens, like bacteria, viruses or fungi, are interesting alternatives for production of microbial-based insecticides to replace the use of chemical products in agriculture. Organic farming, which does not use chemical pesticides for pest control, relies on integrated pest management techniques and in the use of microbial-based insecticides for pest control. Microbial-based insecticides require precise formulation and extensive monitoring of insect pests, since they are highly specific for certain insect pests and in general are more effective for larval young instars. Here, we analyse the possibility of using microbial-based insecticides to replace chemical pesticides in agricultural production.

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.

Insect chaperones Hsp70 and Hsp90 cooperatively enhance toxicity of Bacillus thuringiensis Cry1A toxins and counteract insect resistance.

Bacillus thuringiensis (Bt) produces different insecticidal proteins effective for pest control. Among them, Cry insecticidal proteins have been used in transgenic plants for the control of insect pests. However, evolution of resistance by insects endangers this technology. Previous work showed that the lepidopteran insect Plutella xylostella PxHsp90 chaperone enhanced the toxicity of Bt Cry1A protoxins by protecting them from degradation by the larval gut proteases and by enhancing binding of the protoxin to its receptors present in larval midgut cells. In this work, we show that PxHsp70 chaperone also protects Cry1Ab protoxin from gut proteases degradation, enhancing Cry1Ab toxicity. We also show that both PxHsp70 and PxHsp90 chaperones act cooperatively, increasing toxicity and the binding of Cry1Ab439D mutant, affected in binding to midgut receptors, to cadherin receptor. Also, insect chaperones recovered toxicity of Cry1Ac protein to a Cry1Ac-highly resistant P. xylostella population, NO-QAGE, that has a disruptive mutation in an ABCC2 transporter linked to Cry1Ac resistance. These data show that Bt hijacked an important cellular function for enhancing its infection capability, making use of insect cellular chaperones for enhancing Cry toxicity and for lowering the evolution of insect resistance to these toxins.

A major conformational change of N-terminal helices of Bacillus thuringiensis Cry1Ab insecticidal protein is necessary for membrane insertion and toxicity.

Pore forming toxins rely on oligomerization for membrane insertion to kill their targets. Bacillus thuringiensis produces insecticidal Cry-proteins composed of three domains that form pores that kill the insect larvae. Domain I is involved in oligomerization and membrane insertion, whereas Domains II and III participate in receptor binding and specificity. However, the structural changes involved in membrane insertion of these proteins remain unsolved. The most widely accepted model for membrane insertion, the 'umbrella model', proposed that the α-4/α-5 hairpin of Domain I swings away and is inserted into the membrane. To determine the topology of Cry1Ab in the membrane, disulfide bonds linking α-helices of Domain I were introduced to restrict their movement. Disulfide bonds between helices α-2/α-3 or α-3/α-4 lost oligomerization and toxicity, indicating that movement of these helices is needed for insecticidal activity. By contrast, disulfide bonds linking helices α-5/α-6 did not affect toxicity, which contradicts the 'umbrella model'. Additionally, Föster resonance energy transfer closest approach analyses measuring distances of different points in the toxin to the membrane plane and collisional quenching assays analysing the protection of specific fluorescent-labeled residues to the soluble potassium iodide quencher in the membrane inserted state were performed. Overall, the data show that Domain I from Cry1Ab may undergo a major conformational change during its membrane insertion, where the N-terminal region (helices α-1 to α-4) participates in oligomerization and toxicity, probably forming an extended helix. These data break a paradigm, showing a new 'folding white-cane model', which better explains the structural changes of Cry toxins during insertion into the membrane.

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis.

Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

A single transcription factor facilitates an insect host combating Bacillus thuringiensis infection while maintaining fitness.

Maintaining fitness during pathogen infection is vital for host survival as an excessive response can be as detrimental as the infection itself. Fitness costs are frequently associated with insect hosts countering the toxic effect of the entomopathogenic bacterium Bacillus thuringiensis (Bt), which delay the evolution of resistance to this pathogen. The insect pest Plutella xylostella has evolved a mechanism to resist Bt toxins without incurring significant fitness costs. Here, we reveal that non-phosphorylated and phosphorylated forms of a MAPK-modulated transcription factor fushi tarazu factor 1 (FTZ-F1) can respectively orchestrate down-regulation of Bt Cry1Ac toxin receptors and up-regulation of non-receptor paralogs via two distinct binding sites, thereby presenting Bt toxin resistance without growth penalty. Our findings reveal how host organisms can co-opt a master molecular switch to overcome pathogen invasion with low cost, and contribute to understanding the underlying mechanism of growth-defense tradeoffs during host-pathogen interactions in P. xylostella.

Bacillus thuringiensis Cry1Ac Protoxin and Activated Toxin Exert Differential Toxicity Due to a Synergistic Interplay of Cadherin with ABCC Transporters in the Cotton Bollworm.

Bacillus thuringiensis Cry proteins are used worldwide for insect control. It was proposed that Cry-protoxins must be converted into activated toxin by proteases to bind midgut cell proteins to kill insects. However, Cry-protoxins also bind to midgut proteins and kill insects that have evolved resistance to activated toxins suggesting an independent toxicity pathway. Cadherin (CAD) and ABCC transporters are recognized as important receptors for Cry proteins. Here we constructed different Helicoverpa armigera mutations in these receptors by CRISPR/Cas9. HaCAD-KO mutant showed much higher resistance to Cry1Ac activated toxin than to Cry1Ac protoxin. In contrast, the HaABCC2-M and HaABCC3-M mutants showed higher resistance to Cry1Ac-protoxin than to activated toxin. However, in the double HaABCC2/3-KO mutant, very high levels of resistance were observed to both Cry1Ac protoxin and activated toxin, supporting that both ABC transporters have redundant functions for these two proteins. In addition, Hi5 cells transfected with HaCAD were susceptible only to the activated toxin but not to protoxin. In contrast, both forms of Cry1Ac were similarly toxic to Hi5 cells expressing HaABCC2 or HaABCC3. Co-expression of HaCAD with HaABCC2 or HaABCC3 revealed a more important synergistic effect for activated toxin compared to protoxin. Overall, our results show that toxicity of Cry1Ac activated toxin involves synergistic interplay of HaCAD with ABCC transporters, while the Cry1Ac protoxin toxicity is mainly mediated by ABCC transporters with little participation of HaCAD. These data help to understand the mode of action of Cry proteins that will be relevant to enhance efficacy and durability of Bt-crops. IMPORTANCE Better understanding of the mode of action of Bacillus thuringiensis toxins is beneficial for the sustainable application of Bt crops. It is generally accepted that Cry-protoxins need to be activated by proteases to bind with midgut cell proteins and exert toxicity against insects. Here, we provide new insights into the toxic pathway of Cry proteins in the cotton bollworm. First, our results demonstrate that Cry1Ac protoxin is able to exert cytotoxicity against the insect cells expressing ABCC transporters. Second, we reveal that CAD plays a critical role in the different toxicity of protoxin and toxin by facilitating a synergistic interplay with ABCC transporters. Our results provide in vivo and in vitro experimental evidence supporting that Cry1Ac protoxin exerts toxicity against H. armigera via different steps from that of toxin. These new findings on the mode of action of Cry proteins could be beneficial for efficacy enhancement and durability of Bt-crops.

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.

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.

Whole Genome Sequencing Analysis of Bacillus thuringiensis GR007 Reveals Multiple Pesticidal Protein Genes.

Bacillus thuringiensis (Bt) are soil ubiquitous bacteria. They produce a great variability of insecticidal proteins, where certain of these toxins are used worldwide for pest control. Through their adaptation to diverse ecosystems, certain Bt strains have acquired genetic mobile elements by horizontal transfer, harboring genes that encode for different virulent factors and pesticidal proteins (PP). Genomic characterization of Bt strains provides a valuable source of PP with potential biotechnological applications for pest control. In this work, we have sequenced the complete genome of the bacterium Bt GR007 strain that is toxic to Spodoptera frugiperda and Manduca sexta larvae. Four replicons (one circular chromosome and three megaplasmids) were identified. The two largest megaplasmids (pGR340 and pGR157) contain multiple genes that codify for pesticidal proteins: 10 cry genes (cry1Ab, cry1Bb, cry1Da, cry1Fb, cry1Hb, cry1Id, cry1Ja, cry1Ka, cry1Nb, and cry2Ad), two vip genes (vip3Af and vip3Ag), two binary toxin genes (vpa2Ac and vpb1Ca), five genes that codify for insecticidal toxin components (Tc's), and a truncated cry1Bd-like gene. In addition, genes that codify for several virulent factors were also found in this strain. Proteomic analysis of the parasporal crystals of GR007 revealed that they are composed of eight Cry proteins. Further cloning of these genes for their individual expression in Bt acrystalliferous strain, by means of their own intrinsic promoter showed expression of seven Cry proteins. These proteins display differential toxicity against M. sexta and S. frugiperda larvae, where Cry1Bb showed to be the most active protein against S. frugiperda larvae and Cry1Ka the most active protein against M. sexta larvae.

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.

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance.

Larvicides based on the bacteria Bacillus thuringiensis svar. israelensis (Bti) and Lysinibacillus sphaericus are effective and environmentally safe compounds for the control of dipteran insects of medical importance. They produce crystals that display specific and potent insecticidal activity against larvae. Bti crystals are composed of multiple protoxins: three from the three-domain Cry type family, which bind to different cell receptors in the midgut, and one cytolytic (Cyt1Aa) protoxin that can insert itself into the cell membrane and act as surrogate receptor of the Cry toxins. Together, those toxins display a complex mode of action that shows a low risk of resistance selection. L. sphaericus crystals contain one major binary toxin that display an outstanding persistence in field conditions, which is superior to Bti. However, the action of the Bin toxin based on its interaction with a single receptor is vulnerable for resistance selection in insects. In this review we present the most recent data on the mode of action and synergism of these toxins, resistance issues, and examples of their use worldwide. Data reported in recent years improved our understanding of the mechanism of action of these toxins, showed that their combined use can enhance their activity and counteract resistance, and reinforced their relevance for mosquito control programs in the future years.

Synergistic resistance of Helicoverpa armigera to Bt toxins linked to cadherin and ABC transporters mutations.

Resistance evolution of target pests reduces efficacy of Bacillus thuringiensis Cry toxins used in insect-pest control. Mutations in Cadherin (CAD) or ATP-binding cassette (ABC) transporters genes are linked to Cry resistance in different pests. Also, it has been shown that ABCC2 and CAD have synergistic interaction on Cry toxicity when co-express in cell lines, which we confirmed here by Helicoverpa armigera HaABCC2 and HaCAD expression in Hi5 cells. To confirm that CAD and ABC transporters interact in vivo, we constructed nearly H. armigera isogenic lines such as LFC2 and 96CAD strains, linked to HaABCC2 and HaCAD mutations that showed 512- and 396-fold Cry1Ac resistance-ratios, respectively. Interestingly, Fusion-1 strain linked to both HaABCC2 and HaCAD mutations, showed 6273-fold resistance-ratio, significantly higher than the single mutant strains. To confirm the interaction of HaABCC2 and CAD in Cry1Ac resistance, we analyzed the Cry1Ac susceptibility in CRISPR/Cas9 knockdown strains, C2-KO (ABCC2-gene knockout-strain) and CAD-KO (CAD-gene knockout-strain), that showed 112- and 531-fold Cry1Ac resistance-ratios, respectively. However, the resistance-ratio of Fusion-2 strain obtained from crossing C2-KO and CAD-KO strains, was only 816-fold. The analysis of HaABCC3 gene transcript levels showed nearly 4-fold lower expression in LFC2 and Fusion-1 strains compared to the susceptible strain, suggesting that additional mutations in these strains resulted in low HaABCC3 expression, which contribute to their enhanced Cry1Ac resistance. Our data show that the CAD and ABCC2/ABCC3 interact synergistically to induce high Cry1Ac resistance in H. armigera. These results can be helpful for Bt resistance monitoring and pest management.