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

BackgroundAlthough 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.ResultsIn 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.ConclusionsThis 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

The Regulation Landscape of MAPK Signaling Cascade for Thwarting \u3ci\u3eBacillus thuringiensis\u3c/i\u3e 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

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 \u3e 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

Structural changes of the Cry1Ac oligomeric pre-pore from Bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion. Biochemistry 45

ABSTRACT: The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation. The major determinants of the Bacillus thuringiensis (Bt) insecticidal properties are the δ-endotoxins, also known as Cry and Cyt proteins (1). It is widely accepted that the primary action of Cry toxins is to lyse midgut epithelial cells in their target insects by forming lytic pores in the apical microvilli membrane (1-3). Upon crystal ingestion by the susceptible larvae, the solubilized inactive protoxins are cleaved by midgut proteases yielding 60-70 kDa protease resistant proteins (4). The activated toxin binds to specific receptors on the brush border membrane of the midgut columnar cells The crystal structures of several Cry toxins have been solved (7-10). The seven R-helices that form N-terminal domain I, have been implicated in the pore formation. Domain II consists of three antiparallel -sheets with exposed loop regions, whereas domain III is a -sandwich. Domains II and III are important in receptor recognition (7-10). The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor epitopes and carbohydrate molecules. In the case of the lepidopteran insect Manduca sexta, at least two Cry1A-binding proteins, a cadherin-like protein (Bt-

The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles on 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 β-sheet. Regarding to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet allowing insertion of β-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 in 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, N-terminal region is able to induce aggregation of Cyt1Aa toxin in solution. Finally, Cterminal domain composed mainly of β-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 in the interaction of the toxin with the lipid membrane.Research was funded in part through grants from the National Institutes of Health, 1R01 AI066014, DGAPA/UNAM IN218608 and IN210208-N, CONACyT U48631-Q 478. IRdE received a José Castillejo postdoctoral grant, and a mobility grant for teaching and research staff of UPNA, Spain

MAPK-Mediated Transcription Factor GATAd Contributes to Cry1Ac Resistance in Diamondback Moth by Reducing \u3ci\u3ePxmALP\u3c/i\u3e 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