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.

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.

Peptide mediated, enhanced toxicity of a bacterial pesticidal protein against southern green stink bug.

The damage caused by stink bugs that feed on agricultural crops accounts for such significant losses that transgenic plant resistance to stink bugs would be highly desirable. As the level of toxicity of the Bacillus thuringiensis-derived, ETX/Mtx2 pesticidal protein Mpp83Aa1 is insufficient for practical use against the southern green stink bug Nezara viridula, we employed two disparate approaches to isolate peptides NvBP1 and ABP5 that bind to specific proteins (alpha amylase and aminopeptidase N respectively) on the surface of the N. viridula gut. Incorporation of these peptides into Mpp83Aa1 provided artificial anchors resulting in increased gut binding, and enhanced toxicity. These peptide-modified pesticidal proteins with increased toxicity provide a key advance for potential future use against N. viridula when delivered by transgenic plants to mitigate economic loss associated with this important pest.

Streamlined phage display library protocols for identification of insect gut binding peptides highlight peptide specificity.

Phage display libraries have been used to isolate insect gut binding peptides for use as pathogen transmission blocking agents, and to provide artificial anchors for increased toxicity of bacteria-derived pesticidal proteins. Previously, phage clones displaying enriched peptides were sequenced by Sanger sequencing. Here we present a streamlined protocol for identification of insect gut binding peptides, using insect-appropriate feeding strategies, with next generation sequencing and tailored bioinformatics analyses. The bioinformatics pipeline is designed to eliminate poorly enriched and false positive peptides, and to identify peptides predicted to be stable and hydrophilic. In addition to developing streamlined protocols, we also sought to address whether candidate gut binding peptides can bind to insects from more than one order, which is an important consideration for safe, practical use of peptide-modified pesticidal proteins. To this end, we screened phage display libraries for peptides that bind to the gut epithelia of two pest insects, the Asian citrus psyllid, Diaphorina citri (Hemiptera) and beet armyworm, Spodoptera exigua (Lepidoptera), and one beneficial insect, the western honey bee, Apis mellifera (Hymenoptera). While unique peptide sequences totaling 13,427 for D. citri, 89,561 for S. exigua and 69,053 for A. mellifera were identified from phage eluted from the surface of the insect guts, final candidate pools were comprised of 53, 107 and 1423 peptides respectively. The benefits of multiple rounds of biopanning, along with peptide binding properties in relation to practical use of peptide-modified pesticidal proteins for insect pest control are discussed.

Extraoral digestion: outsourcing the role of the hemipteran midgut.

Extraoral digestion allows for breakdown of dietary components before they reach the midgut for final enzymatic degradation and absorption. In the Hemiptera, this is achieved by the secretion of enzyme-rich fluids from the salivary gland, with the combination of protein and mRNA from these tissues termed the sialome. Separate channels within the hemipteran stylets allow for secretion of saliva and ingestion of predigested material in a non-reflux mechanism. Both feeding mode and diet type influence the composition of the hemipteran sialome, as illustrated by 1) differences in protease abundance between hematophagous and predatory heteropteran sialomes, 2) diet specific aminopeptidase-N genes among aphid biotypes, and 3) adaptation-induced sialome variation in related cicada populations. Despite challenges associated with incomplete genome annotation, -omics analysis of the sialomes of diverse hemipteran species will enhance understanding of both sialome function and the evolution of extraoral digestion within the order.

Proteases and nucleases across midgut tissues of Nezara viridula (Hemiptera:Pentatomidae) display distinct activity profiles that are conserved through life stages.

The southern green stink bug, Nezara viridula is a polyphagous pest of commercially important crops during both nymph and adult stages. This insect has recently transitioned from a secondary agricultural pest to one of primary concern. Novel management solutions are needed due to the limited effectiveness of current control strategies. We performed biochemical and transcriptomic analyses to characterize digestive enzymes in the salivary glands and along midgut tissues of N. viridula nymphs and adults fed on sweet corn. The digestive profiles were more distinct between midgut regions (M1 to M3) than between life stages. Aminopeptidase and chymotrypsin activities declined from the M1 (anterior) toward the M3 midgut region. Cysteine protease activity was higher in the M2 and M3 regions than in M1. Differences in sensitivity to chymotrypsin inhibitors between midgut regions suggest that distinct genes or isoforms are expressed in different regions of the gut. In nymphs, DNA and RNA degradation was higher in M1 than in M3. Adult nuclease activity was low across all midgut regions, but high in salivary glands. The differences in protease activities are reflected by transcriptomic data and functional enrichment of GO terms. Together, our results show that different regions of the digestive tract of N. viridula have specific and distinct digestive properties, and increase our understanding of the physiology of this organism.

Transcription and Activity of Digestive Enzymes of Nezara viridula Maintained on Different Plant Diets.

Nezara viridula is a polyphagous stink bug that feeds on crops of economic importance such as corn, soybean and cotton. To increase understanding of the ability of this pest insect to feed on such diverse cropping systems, we analyzed the impact of an exclusive diet of corn or green bean on the enzymatic activity and transcriptomic profile of digestive enzymes. Growth rate and survival were reduced when insects were reared exclusively on green bean compared to corn. However, the overall protease and nuclease activity profiles were comparable between the two treatments. Distinct differences in inhibitor sensitivity and activity were seen in some cases, particularly for serine proteases in some regions of the midgut. The transcription profiles from N. viridula fed on corn versus green bean were distinct on principal component analysis of RNA-seq data. While specific transcripts differentially transcribed according to diet and across several tissues were identified, a large number of these transcripts remain unannotated. Further annotation for identification of these genes will be important for improved understanding of the remarkable polyphagy of N. viridula.

Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus thuringiensis.

BACKGROUND: Although much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion. RESULTS: In this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses. CONCLUSIONS: This study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae.

Membrane binding and oligomer membrane insertion are necessary but insufficient for Bacillus thuringiensis Cyt1Aa toxicity.

Bacillus thuringiensis Cyt proteins are pore-forming toxins that have insecticidal activity mainly against dipteran insects. However, certain Cyt proteins have toxicity to some insect orders, but not toxicity of Cyt1Aa against lepidopteran larvae has been found. Insect specificity has been proposed to rely in specific binding to certain lipids on the brush border membrane of midgut cells since no protein receptors have been described so far. To determine the molecular basis of Cyt1Aa insect specificity we compared different steps of Cyt1Aa mode of action in a susceptible insect as the dipteran Aedes aegypti and also in the non-susceptible lepidopteran Manduca sexta. Our data shows that the lack toxicity of Cyt1Aa to M. sexta larvae does not rely on protoxin processing, membrane binding interaction, and oligomerization of Cyt1Aa since these steps were similar in the two insect species analyzed.

Oligomerization is a key step in Cyt1Aa membrane insertion and toxicity but not necessary to synergize Cry11Aa toxicity in Aedes aegypti larvae.

Bacillus thuringiensis produces insecticidal Cry and Cyt proteins that are toxic to different insect orders. In addition, Cyt toxins also display haemolytic activity. Both toxins are pore-forming proteins that form oligomeric structures that insert into the target membrane to lyse cells. Cyt toxins play an important role in mosquitocidal activity since they synergize Cry toxins and are able to overcome resistance to Cry toxins. Cry and Cyt toxins interact by specific epitopes, and this interaction is important to induce the synergistic activity observed. It was proposed that Cyt toxins do not interact with protein receptors but directly interacting with the specific midgut cell lipids. Here, we analysed if oligomerization and membrane insertion of Cyt1Aa are necessary steps to synergize Cry11Aa toxicity. We characterized Cyt1Aa helix α-C mutants that were affected in oligomerization, in membrane insertion and also in haemolytic and insecticidal activities. However, these mutants were still able to synergize Cry11Aa toxicity indicating these steps are independent events of Cyt1Aa synergistic activity. Furthermore, the data indicate that formation of stable Cyt1Aa-oligomeric structure is a key step for membrane insertion, haemolysis and insecticidal activity.

Domains II and III of Bacillus thuringiensis Cry1Ab toxin remain exposed to the solvent after insertion of part of domain I into the membrane.

Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism.

Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop ß6-αE K198A, E204A and ß7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.

The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles in toxin oligomerization and pore formation.

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a ß-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the ß-sheet, allowing insertion of ß-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of ß-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.