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The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized control of some major pests. However, more than 25 cases of field-evolved practical resistance have reduced the efficacy of transgenic crops producing crystalline (Cry) Bt proteins, spurring adoption of alternatives including crops producing the Bt vegetative insecticidal protein Vip3Aa. Although practical resistance to Vip3Aa has not been reported yet, better understanding of the genetic basis of resistance to Vip3Aa is urgently needed to proactively monitor, delay, and counter pest resistance. This is especially important for fall armyworm ( Spodoptera frugiperda ), which has evolved practical resistance to Cry proteins and is one of the world's most damaging pests. Here, we report the identification of an association between downregulation of the transcription factor gene SfMyb and resistance to Vip3Aa in S. frugiperda . Results from a genome-wide association study, fine-scale mapping, and RNA-Seq identified this gene as a compelling candidate for contributing to the 206-fold resistance to Vip3Aa in a laboratory-selected strain. Experimental reduction of SfMyb expression in a susceptible strain using RNA interference (RNAi) or CRISPR/Cas9 gene editing decreased susceptibility to Vip3Aa, confirming that reduced expression of this gene can cause resistance to Vip3Aa. Relative to the wild-type promoter for SfMyb , the promoter in the resistant strain has deletions and lower activity. Data from yeast one-hybrid assays, genomics, RNA-Seq, RNAi, and proteomics identified genes that are strong candidates for mediating the effects of SfMyb on Vip3Aa resistance. The results reported here may facilitate progress in understanding and managing pest resistance to Vip3Aa.

Evolution of resistance to Cry proteins in multiple pest insects has been threatening the sustainable use of Bacillus thuringiensis (Bt)-transgenic crops. Better understanding about the mechanism of resistance to Cry proteins in insects is needed. Our preliminary study reported that the transcription of HaABCC3 was significantly decreased in a near-isogenic line (LFC2) of a Cry1Ac-resistant strain (LF60) of the global pest Helicoverpa armigera. However, the causality between HaABCC3 downregulation and resistance to Cry1Ac remains to be verified, and the regulatory mechanism underlying the HaABCC3 downregulation is still unclear. In this study, our data showed that both HaABCC3 and HaABCC3 downregulation were genetically linked to resistance to Cry1Ac in LF60. However, no InDels were observed in the coding sequence of HaABCC3 from LF60. Furthermore, F 1 offspring from the cross of LF60 and a HaABCC2/3-knockout mutant exhibited moderate resistance to Cry1Ac toxin; this indicated that the high resistance to Cry1Ac toxin in LF60 may have resulted from multiple genetic factors, including HaABCC2 mis-splicing and HaABCC3 downregulation. Results from luciferase reporter assays showed that promoter activity of HaABCC3 in LF60 was significantly lower than that in the susceptible strain, which indicated that HaABCC3 downregulation was likely mediated by promoter variation. Consistently, multiple variations of the GATA- or FoxA-binding sites in the promoter region of HaABCC3 were identified. Collectively, all results in this study suggested that the downregulation of HaABCC3 observed in the H. armigera LF60 strain, which is resistant to Cry1Ac, may be mediated by a cis-regulatory mechanism., (© 2022 Institute of Zoology, Chinese Academy of Sciences.)

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.

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

Background: The fall armyworm Spodoptera frugiperda is a major agricultural pest that has invaded the East Hemisphere since 2016, generating a serious threat to food security worldwide including Africa and Asia. The Cry toxins produced by Bacillus thuringiensis (Bt) have been shown to be effective against this insect pest. In different insect ABC transporters (ABCC2 or ABCC3) have been shown to be involved as receptors of some Cry1 toxins. Here we analyzed the role of SfABCC2 and SfABCC3 in the toxicity of Cry1Fa and Cry1Ab toxins in this insect pest., Results: Two S. frugiperda SfABCC2 and SfABCC3 knockout strains, coding for potential functional Bt receptors, were created using CRISPR/Cas9 genome editing system. Both knockout strains showed resistance to both Cry1Fa and Cry1Ab toxins compared with the susceptible strain. SfABCC2 knockout strain showed higher resistance to both Cry toxins than SfABCC3 knockout strain, suggesting a major role of SfABCC2 in the mode of action of these Cry toxins. In addition, expression of SfABCC2 and SfABCC3 genes in Trichoplusia ni Hi5 cells also increased the susceptibility to Cry1Ab and Cry1Fa toxins, in agreement with the genome editing results. The double knockout of SfABCC2 and SfABCC3 strain was not viable in contrast to other lepidopteran species. Furthermore, we report here that SfABCC2 or SfABCC3 knockout strains increased their susceptibility to abamectin and spinosad insecticides., Conclusion: We provide functional evidence that in S. frugiperda these two ABCC transporters serve as receptors of Bt Cry1Fa and Cry1Ab toxins. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Although it is well known that Bacillus thuringiensis Cry toxins kill insect pest by disrupting midgut cells of susceptible larvae through their pore formation activity, it is not clear what intracellular events are triggered after pore formation on the cell membrane of the target cells. Here we analyzed the role of Cry toxins on autophagy activation using several cell lines as models as well as in Helicoverpa armigera larvae. The selected insect cell lines (Hi5, Sl-HP and Sf9) were susceptible to activated Cry1Ca toxin, but only Sl-HP cells were also susceptible to activated Cry1Ac toxin. In contrast, the mammalian cell line 293 T was not susceptible to Cry1Ac or to Cry1Ca. Results show that Cry toxins induced autophagy only in the susceptible cell lines as shown by the analysis of the changes in the ratio of Atg8-PE to Atg8 and by formation of autophagosome dots containing Atg8-PE. The Cry1Ac enhanced autophagy in the midgut tissue of H. armigera larvae. Silencing expression of specific genes by RNAi assays confirmed that the autophagy induced by activated Cry toxins was dependent on AMPK and JNK pathways. Finally, inhibition of autophagy in the cell lines by specific inhibitors or RNAi assays resulted in delayed cell death triggered by Cry toxins, suggesting that the increased autophagy activity observed after toxin intoxication may contribute to cell death., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Evolution of resistance by pests reduces the benefits of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Here we analyzed resistance to Bt toxin Cry1Ac in a field-derived strain of pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that the r14 allele of the pink bollworm cadherin gene (PgCad1) has a 234-bp insertion in exon 12 encoding a mutant PgCad1 protein that lacks 36 amino acids in cadherin repeat 5 (CR5). A strain homozygous for this allele had 237-fold resistance to Cry1Ac, 1.8-fold cross-resistance to Cry2Ab, and developed from neonate to adult on Bt cotton producing Cry1Ac. Inheritance of resistance to Cry1Ac was recessive and tightly linked with r14. PgCad1 transcript abundance in midgut tissues did not differ between resistant and susceptible larvae. Toxicity of Cry1Ac to transformed insect cells was lower for cells expressing r14 than for cells expressing wild-type PgCad1. Wild-type PgCad1 was transported to the cell membrane, whereas PgCad1 produced by r14 was not. In larval midgut tissue, PgCad1 protein occurred primarily on the brush border membrane only in susceptible larvae. The results imply r14 mediates pink bollworm resistance to Cry1Ac by reduced translation, increased degradation, and/or mislocalization of cadherin.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are cultivated extensively, but rapid evolution of resistance by pests reduces their efficacy. We report a 3,370-bp insertion in a cadherin gene associated with resistance to Bt toxin Cry1Ac in the pink bollworm (Pectinophora gossypiella), a devastating global cotton pest. We found the allele (r15) harboring this insertion in a field population from China. The insertion is a miniature inverted repeat transposable element (MITE) that contains two additional transposons and produces two mis-spliced transcript variants (r15A and r15B). A strain homozygous for r15 had 290-fold resistance to Cry1Ac, little or no cross-resistance to Cry2Ab, and completed its life cycle on Bt cotton producing Cry1Ac. Inheritance of resistance was recessive and tightly linked with r15. For transformed insect cells, susceptibility to Cry1Ac was greater for cells producing the wild-type cadherin than for cells producing the r15 mutant proteins. Recombinant cadherin protein occurred on the cell surface in cells transformed with the wild-type or r15A sequences, but not in cells transformed with the r15B sequence. The similar resistance of pink bollworm to Cry1Ac in laboratory- and field-selected insects from China, India and the U.S. provides a basis for developing international resistance management practices.

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. In some previously studied strains of three major lepidopteran pests, resistance to Bt toxin Cry1Ac is associated with mutations disrupting the extracellular or cytoplasmic domains of cadherin proteins that bind Cry1Ac in the midgut of susceptible larvae. Here we report the first case of a cadherin transmembrane mutation associated with insect resistance to Bt. We discovered this mutation in a strain of the devastating global cotton pest, the pink bollworm (Pectinophora gossypiella), derived from a field population in the Yangtze River Valley of China. The mutant allele analyzed here has a 207 base pair deletion and encodes a cadherin protein lacking its transmembrane domain. Relative to a susceptible strain, a strain homozygous for this allele had 220-fold resistance to Cry1Ac and 2.1-fold cross-resistance to Cry2Ab. On transgenic cotton plants producing Cry1Ac, no susceptible larvae survived, but the resistant strain completed its life cycle. Inheritance of resistance to Cry1Ac was autosomal, recessive and tightly linked with the cadherin gene. Transportation of cadherin protein to the cell membrane and susceptibility to Cry1Ac occurred in transfected insect cells expressing the wild type cadherin allele, but not in transfected insect cells expressing the mutant cadherin allele. The results imply that the mutant allele analyzed here confers resistance to Cry1Ac by disrupting cellular trafficking of cadherin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm ( Helicoverpa armigera ), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

Although many insect cell lines derived from various tissues are available, it is unclear whether endogenous receptors of Bacillus thuringiensis (Bt) crystal toxins are expressed in these cell lines. In the present study, we demonstrated that the ovaries-derived Spodoptera litura Sl-HP cell line was susceptible to activated Cry1Ac although larvae of S. litura are not susceptible to the toxin. Assays of the transcriptome revealed that thirteen ATP-binding cassette transporter genes (ABC) were expressed at different levels in this cell line. Of these, the SlABCC3 shared 52-55% amino acid sequence identity with the known Bt toxin receptor ABCC2. RNAi-mediated knockdown targeting SlABCC3 significantly decreased the susceptibility of Sl-HP cells to activated Cry1Ac. Over-expression of the gene strongly increased the susceptibility of Trichoplusia ni Hi5 cells to the toxin. Not only was SlABCC3 comparable to the heterologously expressed Helicoverpa armigera Hacadherin on the receptor-mediated cytotoxicity of activated Cry1Ac to Hi5 cells, but also SlABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of activated Cry1Ac. These results suggested that Bt toxin receptors-expressing insect cell lines can be used as an alternative model for evaluating cytotoxicity of Bt toxins and studying their mechanisms of action., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

BACKGROUND: With the increasing incidence of pest resistance to transgenic crops producing Bacillus thuringiensis (Bt) proteins in the field, elucidating the molecular basis of resistance is important for monitoring, delaying and countering pest resistance. Previous work revealed that mutation or down‐regulated expression of the cadherin gene (PgCad1) is associated with pink bollworm (Pectinophora gossypiella) resistance to Cry1Ac, and 20 mutant PgCad1 alleles (r1‐r20) were characterized. Here, we tested the hypothesis that the ABC transporter PgABCC2 is a functional receptor for the Bt toxin Cry1Ac and that a mutation is associated with resistance. RESULTS: We identified and characterized the first resistance allele (rC2) of PgABCC2 in the laboratory‐selected Cry1Ac‐resistant strain AQ‐C2 of pink bollworm. The rC2 allele had a one‐base deletion in exon20, resulting in a frameshift and the introduction of a premature stop codon. This resulting PgABCC2 protein had a truncated C‐terminus, including the loss of the NBD2 domain. AQ‐C2 exhibited 20.2‐fold greater resistance to Cry1Ac than the susceptible strain, and its inheritance of Cry1Ac resistance was recessive and genetically linked to PgABCC2. When produced in cultured insect cells, recombinant wild‐type and rC2 mutant PgABCC2 proteins localized within the cell plasma membrane, although substantial cytoplasmic retention was also observed for the mutant protein, while the mutant PgABCC2 caused a 13.9‐fold decrease in Cry1Ac toxicity versus the wild‐type PgABCC2. CONCLUSIONS: PgABCC2 is a functional receptor of Cry1Ac and the loss of its carboxyl terminus (including its NBD2 domain) confers low‐level resistance to Cry1Ac in both larvae and in cultured cells. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Helicoverpa armigera is a polyphagous destructive lepidopteran pest with strong Bacillus thuringiensis (Bt) resistance. Cholesterol, a vital component for insect growth, can only be obtained from food, and its transfer and metabolism are regulated by sterol carrier protein-2 (SCP-2). This study examined whether H. armigera SCP-2 (HaSCP-2) gene expression, involved in cholesterol absorption, can be silenced by nanocarrier fluorescent nanoparticle-RNA interference (FNP-RNAi) by larval feeding and whether the silencing affected H. armigera development. Fluorescence microscopy showed that nanoparticle-siRNA was distributed in Ha cells and the larval midgut. FNP-HaSCP-2 siRNA suppressed HaSCP-2 expression by 52.5% in H.armigera Ha cells. FNP can effectively help deliver siRNA into cells, protect siRNA, and is not affected by serum. FNP-siRNA in vivo biological assays showed that HaSCP-2 transcript levels were inhibited by 70.19%, 68.16%, and 67.66% in 3rd, 4th, and 5th instar larvae, leading to a decrease in the cholesterol level in the larval and prepupal fatbodies. The pupation rate and adult emergence were reduced to 26.0% and 56.52%, respectively. This study demonstrated that FNP could deliver siRNA to cells and improve siRNA knockdown efficiency. HaSCP-2 knockdown by FNP-siRNA in vivo hindered H. armigera growth and development. FNP could enhance RNAi efficiency to achieve pest control by SCP-2-targeted FNP-RNAi. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Insecticidal proteins from Bacillus thuringiensis (Bt) are widely used to control insect pests, but their efficacy is reduced when pests evolve resistance. We report on a novel allele (r16) of the cadherin gene (PgCad1) in pink bollworm (Pectinophora gossypiella) associated with resistance to Bt toxin Cry1Ac, which is produced by transgenic cotton. The r16 allele isolated from a field population in China has 1545 base pairs of a degenerate transposon inserted in exon 20 of PgCad1, which generates a mis-spliced transcript containing a premature stop codon. A strain homozygous for r16 had 300-fold resistance to Cry1Ac, 2.6-fold cross-resistance to Cry2Ab, and completed its life cycle on transgenic Bt cotton producing Cry1Ac. Inheritance of Cry1Ac resistance was recessive and tightly linked with r16. Compared with transfected insect cells expressing wild-type PgCad1, cells expressing r16 were less susceptible to Cry1Ac. Recombinant cadherin protein was transported to the cell membrane in cells transfected with the wild-type PgCad1 allele, but not in cells transfected with r16. Cadherin occurred on brush border membrane vesicles (BBMVs) in the midgut of susceptible larvae, but not resistant larvae. These results imply that the r16 allele mediates Cry1Ac resistance in pink bollworm by interfering with the localization of cadherin. [ABSTRACT FROM AUTHOR]

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

Abstract Bacillus thuringiensis Cry toxins exert their toxicity by forming membrane pores after binding with larval midgut membrane proteins known as receptors. Spodoptera litura and Spodoptera frugiperda belong to the same genus, but S. litura is tolerant to Cry1Ac, while S. frugiperda is susceptible. The mechanism involved in the differential toxicity of Cry1Ac to these insect species is not understood. Amino acid sequences analysis of ABCC2, a well-recognized Cry1Ac receptor, from both species showed high sequence identity. Hi5 insect cells expressing SfABCC2 from S. frugiperda were 65-fold more susceptible than those expressing the SlABCC2 from S. litura. Substitution of fragments, point mutations and deletions between the ABCC2 of the two species revealed that ABCC2 amino acid Q125 from SfABCC2 or E125 from SlABCC2 was key factor for the differential Cry1Ac toxicity to Hi5 cells expressing these receptors. Consistently with this, cells expressing Helicoverpa armigera HaABCC2Q122-GFP, were more susceptible to Cry1Ac than cells expressing HaABCC2E122-GFP mutant. Q125 or E125 is located in a predicted exposed loop 1 region of ABCC2 indicating that this region could be important for Cry1Ac binding. These findings identified a single amino acid residue located in loop 1 of ABCC2 transporter as responsible for the different levels of susceptibility to Cry1Ac among various lepidopteran species. Graphical abstract Image 1 Highlights • Spodoptera frugiperda larvae are susceptible to Cry1Ac, but Spodoptera litura larvae are very tolerant to this toxin. • There are 43 differential amino acid residues between their ABCC2 proteins. • Cytotoxicity of Cry1Ac mediated by the two ABCC2 proteins has significant difference. • The 125th amino acid residue (Q or E) of ABCC2 is linked with the differential susceptibility of insect cells to Cry1Ac. [ABSTRACT FROM AUTHOR]

Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in Sl-HP cells that are sensitive to Cry1Ac toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in Sl-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced Cry1Ac-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase , aminopeptidase or cadherin . Silencing of FOXA gene expression also resulted in a Cry1Ac-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing Cry1Ac protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the Cry1Ac-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae. [ABSTRACT FROM AUTHOR]

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

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that kill insect pests. Insect specificity of Cry toxins relies on their binding to larval gut membrane proteins such as cadherin and ATP-binding cassette (ABC) transporter proteins. Mutations in ABC transporters have been implicated in high levels of resistance to Cry toxins in multiple pests. Spodoptera frugiperda is an insect pest susceptible to Cry1Fa and Cry1Ab toxins while Mythimna separata is tolerant to Cry1Fa and less susceptible to Cry1Ab. Here, we analyzed the potential role of ABCC2 in determining the susceptibility of S. frugiperda to Cry1Fa and Cry1Ab, by expressing SfABCC2 or MsABCC2 in Hi5 insect cell line and by the systematic replacements of extracellular loops (ECLs) between these two proteins. Expression of SfABCC2 in Hi5 conferred susceptibility to both Cry1Fa and Cry1Ab, in contrast to the expression of MsABCC2 that mediated low toxicity to Cry1Ab and no toxicity to Cry1Fa in agreement with their larvicidal toxicities. The SfABCC2 and MsABCC2 amino acid sequences showed differential residues among ECL1, ECL2, ECL4 and ECL6 loops, while ECL3 and ECL5 share the same primary sequence. The exchange of ECLs between SfABCC2 and MsABCC2 demonstrated that ECL4 and ECL2 contribute to Cry1Fa toxicity, where ECL4 plays a major role. The medium region (named M2) of ECL4 was identified as the most important region of SfABCC2 involved in Cry1Fa toxicity as shown by point mutations in this region. These findings will be helpful to understand the mechanisms of action of Bt toxins in S. frugiperda. [Display omitted] • Spodoptera frugiperda is susceptible to Cry1Fa, and Mythimna separata is not. • SfABCC2 could mediate cytotoxicity of Cry1Fa, and MsABCC2 could not. • SfABCC2 ECL4 played major role and ECL2 played minor role in mediating cytotoxicity of Cry1Fa. • Multiple amino acid residues of SfABCC2 ECL4 were involved in mediating cytotoxicity of Cry1Fa. [ABSTRACT FROM AUTHOR]

The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are powerful tools for insect control. Cry toxin receptors such as cadherin (CAD), ABCC2 transporter and alkaline phosphatase (ALP), located on insect midgut cells, are needed for Cry toxicity. Although insect cell lines are useful experimental models for elucidating toxin action mechanism, most of them show low expression of Cry-receptors genes. The GATA transcription factor family plays important roles in regulating development and differentiation of intestine stem cells. Here, we investigated whether GATAs transcription factors are involved in the expression of Cry1Ac-receptors genes, using multiple insect cell lines. Four GATA genes were identified in the transcriptome of the midgut tissue from the lepidopteran larvae Helicoverpa armigera. These HaGATA genes were transiently expressed in three lepidopteran cell lines, Spodoptera frugiperda Sf9, H. armigera QB-Ha-E5 and Trichoplusia ni Hi5. Analysis of transcription activity using transcriptional gene-fusions showed that only H. armigera GATAe (HaGATAe) significantly increased the transcription of CAD, ABCC2 and ALP receptors genes in all insect cell lines. Key DNA regions for HaGATAe regulation were identified in the promoter sequence of these Cry-receptors genes by using promoter deletion mapping. The transient expression of HaGATAe in these cell lines, conferred sensitivity to Cry1Ac toxin, although in Hi5 cells the susceptibility to Cry1Ac was lower than in other two cell lines. High sensitivity to Cry1Ac correlated with simultaneous transcription of ABCC2 and CAD genes in Sf9 and QB-Ha-E5 cells. Our results reveal that HaGATAe enhances transcription of several lepidopteran Cry1Ac receptor genes in cultured insect cells. Image 1 • GATAe increased activities of the promoters. • GATAe enhanced expression of the genes. • GATAe induced susceptibility of insect cells to Bt Cry1Ac. [ABSTRACT FROM AUTHOR]