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Abstract Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Executive summary Microbes are all pervasive in their distribution and influence on the functioning and well‐being of humans, life in general and the planet. Microbially‐based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision‐makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative–the IMiLI–is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence‐based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner‐centric, not academic microbiology‐centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators–learners. As such, they will collect and analyse feedback from the end‐users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators–learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships–a global societally relevant microbiology education ecosystem–in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. Abstract The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well‐being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us–individuals/communities/nations/the human world–and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. Here, we present our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision‐making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner‐centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091–1111). Importantly, although the primary target of the freely available (CC BY‐NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity‐driven, web‐based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Abstract 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.

Plutella xylostella endures Bt toxins with no performance costs. This study reveals how, depending on the presence of the toxin, this insect modifies MAPK phosphorylation to modulate the transcription factor FTZ-F1 binding, to up- or down- regulate Bt receptors or non-receptor (resistant) paralogs.

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.

Abstract 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.

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.

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.

Summary Vip3Aa was first identified as a protein secreted during the vegetative growth phase of Bacillus thuringiensis (Bt) bacteria and which shows high insecticidal toxicity against lepidopteran insect pests (Estruch et al., 1996). Bt strains formulated as bio‐insecticides only had low amounts of Vip3Aa secreted to the medium. Here, we report that Vip3Aa proteins produced by three different Bt strains, including an industrial strain, were indeed not secreted to the culture solution when grown in sporulation medium, but were retained in the mother cell compartment. In order to further investigate the Vip3Aa secretion and location, we grew the strains in rich medium. We found that in rich medium, a fraction of Vip3Aa was secreted, suggesting that Vip3Aa secretion is nutrient‐dependent. Regardless of the growth conditions, we found that Vip3Aa retained in cell pellets exhibited high toxicity against Spodoptera frugiperda larvae. Hence, we speculate that the accumulation of Vip3Aa protein in the mother cell compartment under sporulation conditions could still be used as an efficient strategy for industrial production in commercial Bt strains.

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.

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.

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.

Bacillus thuringiensis (Bt) crops have been widely planted and the effects of Bt-crops on populations of the target and non-target insect pests have been well studied. However, the effects of Bt-crops exposure on microorganisms that interact with crop pests have not previously been quantified. Here, we use laboratory and field data to show that infection of Helicoverpa armigera with a densovirus (HaDV2) is associated with its enhanced growth and tolerance to Bt-cotton. Moreover, field monitoring showed a much higher incidence of cotton bollworm infection with HaDV2 in regions cultivated with Bt-cotton than in regions without it, with the rate of densovirus infection increasing with increasing use of Bt-cotton. RNA-seq suggested tolerance to both baculovirus and Cry1Ac were enhanced via the immune-related pathways. These findings suggest that exposure to Bt-crops has selected for beneficial interactions between the target pest and a mutualistic microorganism that enhances its performance on Bt-crops under field conditions.

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered "net like" structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

ABSTRACT Bacillus thuringiensis Cry proteins are pore-forming insecticidal toxins with specificity against different crop pests and insect vectors of human diseases. Previous work suggested that the insect host Hsp90 chaperone could be involved in Cry toxin action. Here, we show that the interaction of Cry toxins with insect Hsp90 constitutes a positive loop to enhance the performance of these toxins. Plutella xylostella Hsp90 (PxHsp90) greatly enhanced Cry1Ab or Cry1Ac toxicity when fed together to P. xylostella larvae and also in the less susceptible Spodoptera frugiperda larvae. PxHsp90 bound Cry1Ab and Cry1Ac protoxins in an ATP- and chaperone activity-dependent interaction. The chaperone Hsp90 participates in the correct folding of proteins and may suppress mutations of some client proteins, and we show here that PxHsp90 recovered the toxicity of the Cry1AbG439D protoxin affected in receptor binding, in contrast to the Cry1AbR99E or Cry1AbE129K mutant, affected in oligomerization or membrane insertion, respectively, which showed a slight toxicity improvement. Specifically, PxHsp90 enhanced the binding of Cry1AbG439D protoxin to the cadherin receptor. Furthermore, PxHsp90 protected Cry1A protoxins from degradation by insect midgut proteases. Our data show that PxHsp90 assists Cry1A proteins by enhancing their binding to the receptor and by protecting Cry protoxin from gut protease degradation. Finally, we show that the insect cochaperone protein PxHsp70 also increases the toxicity of Cry1Ac in P. xylostella larvae, in contrast to a bacterial GroEL chaperone, which had a marginal effect, indicating that the use of insect chaperones along with Cry toxins could have important biotechnological applications for the improvement of Cry insecticidal activity, resulting in effective control of insect pests. IMPORTANCE Bacillus thuringiensis took advantage of important insect cellular proteins, such as chaperones, involved in maintaining protein homeostasis, to enhance its insecticidal activity. This constitutes a positive loop where the concentrations of Hsp90 and Hsp70 in the gut lumen are likely to increase as midgut cells burst due to Cry1A pore formation action. Hsp90 protects Cry1A protoxin from degradation and enhances receptor binding, resulting in increased toxicity. The effect of insect chaperones on Cry toxicity could have important biotechnological applications to enhance the toxicity of Cry proteins to insect pests, especially those that show low susceptibility to these toxins.

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.

Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A–C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A–C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Scarabaeoidea and Chrysomeloidea insects are agriculture-destructive coleopteran pests. Few effective Bacillus thuringiensis (Bt) insecticidal proteins against these species have been described. Bt isolate BtSU4 was found to be active against coleopteran insects. Genome sequencing revealed two new cry8 genes in BtSU4, designated as cry8Ha1 and cry8Ia1. Both genes expressed a 135 kDa protoxin forming irregular shape crystals. Bioassays performed with Cry8Ha1 protoxin showed that it was toxic to both larvae and adult stages of Holotrichia parallela, also to Holotrichia oblita adults and to Anoplophora glabripennis larvae, but was not toxic to larval stages of H. oblita or Colaphellus bowringi. The Cry8Ia1 protoxin only showed toxicity against H. parallela larvae. After activation with chymotrypsin, the Cry8Ha1 activated toxin lost its insecticidal activity against H. oblita adults and reduced its activity on H. parallela adults, but gained toxicity against C. bowringi larvae, a Chrysomeloidea insect pest that feeds on crucifer crops. The chymotrypsin activated Cry8Ia1 toxin did not show toxicity to any one of these insects. These data show that Cry8Ha1 and Cry8Ia1 protoxin and activated toxin proteins have differential toxicity to diverse coleopteran species, and that protoxin is a more robust protein for the control of coleopteran insects.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II–III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda.

Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.

For more than 20 years cotton has been the most widely sown genetically modified (GM) crop in Mexico. Its cultivation has fulfilled all requirements and has gone through the different regulatory stages. During the last 20 years, both research-institutions and biotech-companies have generated scientific and technical information regarding GM cotton cultivation in Mexico. In this work, we collected data in order to analyze the environmental and agronomic effects of the use of GM cotton in Mexico. In 1996, the introduction of Bt cotton made it possible to reactivate this crop, which in previous years was greatly reduced due to pest problems, production costs and environmental concerns. Bt cotton is a widely accepted tool for cotton producers and has proven to be efficient for the control of lepidopteran pests. The economic benefits of its use are variable, and depend on factors such as the international cotton-prices and other costs associated with its inputs. So far, the management strategies used to prevent development of insect resistance to GM cotton has been successful, and there are no reports of insect resistance development to Bt cotton in Mexico. In addition, no effects have been observed on non-target organisms. For herbicide tolerant cotton, the prevention of herbicide resistance has also been successful since unlike other countries, the onset of resistance weeds is still slow, apparently due to cultural practices and rotation of different herbicides. Environmental benefits have been achieved with a reduction in chemical insecticide applications and the subsequent decrease in primary pest populations, so that the inclusion of other technologies—e.g., use of non-Bt cotton- can be explored. Nevertheless, control measures need to be implemented during transport of the bolls and fiber to prevent dispersal of volunteer plants and subsequent gene flow to wild relatives distributed outside the GM cotton growing areas. It is still necessary to implement national research programs, so that biotechnology and plant breeding advances can be used in the development of cotton varieties adapted to the Mexican particular environmental conditions and to control insect pests of regional importance.

Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.

Bacillus thuringiensis Cry1Ac toxin binds to midgut proteins, as cadherin (CAD) and ABCC2 transporter, to form pores leading to larval death. In cell lines, co-expression of CAD and ABCC2 enhance Cry1Ac toxicity significantly, but the mechanism remains elusive. Here, we show that the expression of Helicoverpa armigera CAD (HaCAD-GFP) in Hi5 cells induces susceptibility to Cry1Ac and enhanced Cry1Ac toxicity when co-expressed with H. armigera ABCC2 (HaABCC2-GFP), since Cry1Ac toxicity increased 735-fold compared to Hi5 cells expressing HaCAD-GFP alone or 28-fold compared to HaABCC2-GFP alone. In contrast, the expression of the Spodoptera litura CAD (SlCAD-GFP) in Hi5 cells did not induce susceptibility to Cry1Ac nor it potentiated Cry1Ac toxicity with HaABCC2-GFP. To identify the CAD regions involved in the enhancement of Cry1Ac toxicity with ABCC2, the different CAD domains were replaced between SlCAD-GFP and HaCad-GFP proteins, and cytotoxicity assays were performed in Hi5 cells in the absence or presence of HaABCC2-GFP. The HaCAD toxin-binding region (TB), specifically the CAD repeat-11, was necessary to enhance Cry1Ac toxicity with ABCC2. We propose that CAD TB is involved in recruiting Cry1Ac to localize it in a good position for its interaction with the ABCC2, resulting in efficient toxin membrane insertion enhancing Cry1Ac toxicity.

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control.

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is the most destructive insect pest of corn in China. Susceptibility to the Cry1F toxin derived from Bacillus thuringiensis has been demonstrated for ACB, suggesting the potential for Cry1F inclusion as part of an insect pest management program. Insects can develop resistance to Cry toxins, which threatens the development and use of Bt formulations and Bt crops in the field. To determine possible resistance mechanisms to Cry1F, a Cry1F-resistant colony of ACB (ACB-FR) that exhibited more than 1700-fold resistance was established through selection experiments after 49 generations of selection under laboratory conditions. The ACB-FR strain showed moderate cross-resistance to Cry1Ab and Cry1Ac of 22.8- and 26.9-fold, respectively, marginally cross-resistance to Cry1Ah (3.7-fold), and no cross-resistance to Cry1Ie (0.6-fold). The bioassay responses of progeny from reciprocal F1 crosses to different Cry1 toxin concentrations indicated that the resistance trait to Cry1Ab, Cry1Ac and Cry1F has autosomal inheritance with no maternal effect or sex linked. The effective dominance (h) of F1 offspring was calculated at different concentrations of Cry1F, showing that h decreased as concentration of Cry1F increased. Finally, the analysis of actual and expected mortality of the progeny from a backcross (F1 × resistant strain) indicated that the inheritance of the resistance to Cry1F in ACB-FR was due to more than one locus. The present study provides an understanding of the genetic basis of Cry1F resistance in ACB-FR and also shows that pyramiding Cry1F with Cry1Ah or Cry1Ie could be used as a strategy to delay the development of ACB resistance to Bt proteins.

Insecticidal proteins from Bacillus thuringiensis (Bt) are used extensively in sprays and transgenic crops for pest control, but their efficacy is reduced when pests evolve resistance. Better understanding of the mode of action of Bt toxins and the mechanisms of insect resistance is needed to enhance the durability of these important alternatives to conventional insecticides. Mode of action models agree that binding of Bt toxins to midgut proteins such as cadherin is essential for toxicity, but some details remain unresolved, such as the role of toxin oligomers. In this study, we evaluated how Bt toxin Cry1Ac and its genetically engineered counterpart Cry1AcMod interact with brush border membrane vesicles (BBMV) from resistant and susceptible larvae of Pectinophora gossypiella (pink bollworm), a global pest of cotton. Compared with Cry1Ac, Cry1AcMod lacks 56 amino acids at the amino-terminus including helix α-1; previous work showed that Cry1AcMod formed oligomers in vitro without cadherin and killed P. gossypiella larvae harboring cadherin mutations linked with >1000-fold resistance to Cry1Ac. Here we found that resistance to Cry1Ac was associated with reduced oligomer formation and insertion. In contrast, Cry1AcMod formed oligomers in BBMV from resistant larvae. These results confirm the role of cadherin in oligomerization of Cry1Ac in susceptible larvae and imply that forming oligomers without cadherin promotes toxicity of Cry1AcMod against resistant P. gossypiella larvae that have cadherin mutations.

5-aminoimidazole-4-carboxamide nucleotide (AICAR) is a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli. In this work, the effect of AICAriboside (AICAr), the precursor of AICAR on the expression of the Sinorhizobium meliloti fixNOQP operon encoding the symbiotic terminal oxidase cbb3, was analyzed. AICAr reduced the microaerobic induction levels of fixN-lacZ and fixT-lacZ gene fusions 18- and sevenfold respectively, and both genes were activated by the transcriptional activator FixK. A fixK-lacZ fusion presented 14-fold-reduced induction levels in microaerobic cell cultures in the presence of AICAr. AICAr also reduced three-fold the microaerobic expression levels of the nifA-lacZ fusion, whose expression as well as that of fixK is controlled by the two-component system FixL-FixJ. In contrast, AICAr had no effect on the expression levels of a hemA-lacZ fusion. These data suggest that AICAr prevents fixNOQP induction by the inhibition of fixK transcription.

Among the complexities in the regulation of nitrogen fixation in the Rhizobiaceae are reiteration of regulatory components as well as variant roles for each component between species. For Rhizobium etli CFN42, we reported that the symbiotic plasmid (pCFN42d) contains a key regulatory gene (fixKd) and genes for a symbiotic cytochrome oxidase (fixNOQPd). Here we discuss the occurrence of reiteration of these genes (fixKf and fixNOQPf) and the finding of an unusual fixL homolog on a plasmid previously considered cryptic (pCFN42f). The structure of the deduced FixL polypeptide is suggestive of a fusion of the receiver and transmitter modules of a two-component regulatory system as described in R. leguminosarum bv. viciae VF39. Gene fusion analysis, coupled with mutation of each regulatory element, revealed that free-living expression of FixKf was dependent fully on FixL. In contrast, synthesis of FixKd was not detected under the conditions tested. The FixKf protein is needed for microaerobic expression of both fixN reiterations, whereas the FixKd protein appears to be dispensable. Interestingly, expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription of fixNf was suppressed in the absence of FixL but expression of fixNd still showed significant levels. This suggests the existence of a FixL-independent mechanism for expression of the fixNd reiteration. Surprisingly, mutations in fixL, fixKd, or fixKf (either singly or in combination) did not alter symbiotic effectiveness. A mutation in fixNd (but not in fixNf) was, however, severely affected, indicating a differential role for these reiterations in nitrogen fixation.

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibited higher nitrogenase activity and total nitrogen content (68% and 14 to 53%, respectively) than bean plants inoculated with the R. etli wild type. These results suggest that the synthesis of VHb in engineered R. etli stimulated the respiratory efficiency of free-living rhizobia, and also probably of symbiotic bacteroids, thus leading to higher levels of symbiotic nitrogen fixation.

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p

BackgroundBacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt) indicating that oligomer formation is a key step in toxicity of Cry toxins.Methodology/principal findingsThe DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt) probably by competition of binding with toxin receptors.Conclusions/significanceWe show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.

Bacillus thuringiensis produces crystal toxins known as Cry that are highly selective against important agricultural and human health-related insect pests. Cry proteins are pore-forming toxins that interact with specific receptors in the midgut cell membrane of susceptible larvae making pores that cause osmotic shock, leading finally to insect death. In the case of pore-forming toxins that are specific to mammalian cells, death responses at low doses may induce apoptosis or pyroptosis, depending on the cell type. The death mechanism induced by Cry toxins in insect midgut cells is poorly understood. Here, we analyze the caspases expression by RT-PCR analysis, showing that the initial response of Manduca sexta midgut cells after low dose of Cry1Ab toxin administration involves a fast and transient accumulation of caspase-1 mRNA, suggesting that pyroptosis was activated by Cry1Ab toxin as an initial response but was repressed later. In contrast, caspase-3 mRNA requires a longer period of time of toxin exposure to be activated but presents a sustained activation, suggesting that apoptosis may be a cell death mechanism induced also at low dose of toxin.

BACKGROUND:Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins. CONCLUSIONS/SIGNIFICANCE:This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms.