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Previous studies have confirmed HaCad (cadherin), HaABCC2 and HaABCC3 are functional receptors of Bt toxin Cry1Ac in cotton bollworm, Helicoverpa armigera. Aminopeptidase N1 (APN1) has been suggested as a putative receptor in several lepidopteran insects including H. armigera through evidence from RNAi‐based gene silencing approaches. In the current study, we tested the role of APNs in the mode of action of Bt toxins using CRISPR/Cas9‐mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible SCD strain of H. armigera to establish three homozygous knockout strains. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera (see pages 440–448). Photo by Yi‐Dong Wu.     

Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system

Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera.     

Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera

A functional assessment of Bacillus thuringiensis (Bt) toxin receptors in the midgut of lepidopteran insects will facilitate understanding of the toxin mode of action and provide effective strategies to counter the development of resistance. In this study, we produced anti-aminopeptidase (APN) and anti-cadherin sera with purified Cry1Ac toxin-binding APN or cadherin fragments from Heliocoverpa armigera. Antisera were evaluated for their effects on Cry1Ac toxicity through bioassays. Our results indicated that both the anti-APN and anti-cadherin sera reduced Cry1Ac toxicity in vivo, although cadherin antiserum reduced toxicity more than APN antiserum. These results suggest that both APN and cadherin are involved in Cry1Ac intoxication of H. armigera, evidence that the pore formation model may be representative of Cry1Ac toxin mode of action in this insect.     

CRISPR/Cas9 mediated genome editing of Helicoverpa armigera with mutations of an ABC transporter gene HaABCA2 confers resistance to Bacillus thuringiensis Cry2A toxins

High levels of resistance to Bt toxin Cry2Ab have been identified to be genetically linked with loss of function mutations of an ABC transporter gene (ABCA2) in two lepidopteran insects, Helicoverpa armigera and Helicoverpa punctigera. To further confirm the causal relationship between the ABCA2 gene (HaABCA2) and Cry2Ab resistance in H. armigera, two HaABCA2 knockout strains were created from the susceptible SCD strain with the CRISPR/Cas9 genome editing system. One strain (SCD-A2KO1) is homozygous for a 2-bp deletion in exon 2 of HaABCA2 created by non-homologous end joining (NHEJ). The other strain (SCD-A2KO2) is homozygous for a 5-bp deletion in exon 18 of HaABCA2 made by homology-directed repair (HDR), which was produced to mimic the r2 resistance allele of a field-derived Cry2Ab-resistant strain from Australia. Both knockout strains obtained high levels of resistance to both Cry2Aa (>120-fold) and Cry2Ab (>100-fold) compared with the original SCD strain, but no or very limited resistance to Cry1Ac (<4-fold). Resistance to Cry2Ab in both knockouts is recessive, and genetic complementary tests confirmed Cry2Ab resistance alleles are at the same locus (i.e. HaABCA2) for the two strains. Brush border membrane vesicles (BBMVs) of midguts from both knockout strains lost binding with Cry2Ab, but maintained the same binding with Cry1Ac as the SCD strain. In vivo functional evidence from this study demonstrates knockout of HaABCA2 confers high levels of resistance to both Cry2Aa and Cry2Ab, confirming that HaABCA2 plays a key role in mediating toxicity of both Cry2Aa and Cry2Ab against H. armigera.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

Proteomic analysis of BBMV in Helicoverpa armigera midgut with and without Cry1Ac toxin treatment

The crystal proteins of Bacillus thuringiensis (Bt) are widely used for insect control. Helicoverpa armigera is the model insect for Bt studies. In this study, brush border membrane vesicle (BBMV) proteins from fifth instar larvae of Helicoverpa armigera were prepared, and proteomic approaches based on two-dimensional (2D) gel electrophoresis and mass spectrometry were used to elucidate changes in BBMV proteins with and without Cry1Ac toxin treatment. Sixty-one protein bands separated by 1D electrophoresis were cut out from the gel for tryptic digestion and were detected with molecular mass spectrometry (ESI-Q-TOF) and High Capacity Ion Trap Ultra (HCT Ultra). BBMV proteins of interest separated by 2D electrophoresis were excised and digested with trypsin, and the resulting peptides were analyzed by mass spectrometry. Mass fingerprints were compared with the non-redundant NCBI Metazoa (Animals) database. We found a noticeable increase in the level of aminopeptidase N (APN) that is important for detoxification reactions. Additionally, a significant decrease in the level of trypsin-like protease is important during early responses and adaptation of the insect to Bt and exposure to its toxins. Furthermore, the increase in V-ATPase subunits indicate elevated cellular energy profile which is necessary to combat toxin stress. The increased level of actin in larvae provides immediate protection by strengthening the midgut epithelium and enhancing cellular defenses in the tissue. This study presents the differences in the BBMV proteins of Helicoverpa armigera with and without Cry1Ac toxin treatment, and provides a theoretical basis for research on the mechanism of action of Bt toxin.     

Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation

Helicoverpa armigera is one of the most harmful pests in China. Although it had been successfully controlled by Cry1A toxins, some H. armigera populations are building up resistance to Cry1A toxins in the laboratory. Vip3A, secreted by Bacillus thuringiensis, is another potential toxin against H. armigera. Previous reports showed that activated Vip3A performs its function by inserting into the midgut brush border membrane vesicles (BBMV) of susceptible insects. To further investigate the binding of Vip3A to BBMV of H. armigera, the full-length Vip3Aa10 toxin expressed in Escherichia coli was digested by trypsin or midgut juice extract, respectively. Among the fragments of digested Vip3Aa10, only a 62 kDa fragment (Vip3Aa10-T) exhibited binding to BBMV of H. armigera and has insecticidal activity. Moreover, this interaction was specific and was not affected by the presence of Cry1Ab toxin. Binding of Vip3Aa10-T to BBMV resulted in the formation of an ion channel. Unlike Cry1A toxins, Vip3Aa10-T was just slightly associated with lipid rafts of BBMV. These data suggest that although activated Vip3Aa10 specifically interacts with BBMV of H. armigera and forms an ion channel, the mode of action of it may be different from that of Cry1A toxins.     

Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the Cry1A class of Bt toxins have been identified: an aminopeptidase N (APN-1) and a 270 kDa anionic glycoconjugate (BTR-270). Studies have shown that APN-1 has a relatively weak affinity and a very narrow specificity to Cry1Ac, the only Cry1A toxin that it binds. In contrast, BTR-270 binds all toxins that are active against L. dispar larvae, and the affinities for these toxins to BTR-270 correlate positively with their respective toxicities. In this study, an immunohistochemical approach was coupled with fluorescence microscopy to localize APN-1 and BTR-270 in paraffin embedded midgut sections of L. dispar larvae. The distribution of cadherin and alkaline phosphatase in the gut tissue was also examined. A strong reaction indicative of polyanionic material was detected with alcian blue staining over the entire epithelial brush border, suggesting the presence of acidic glycoconjugates in the microvillar matrix. The Cry1A toxin-binding sites were confined to the apical surface of the gut epithelial cells with intense labeling of the apical tips of the microvilli. APN-1, BTR-270, and alkaline phosphatase were found to be present exclusively along the brush border microvilli along the entire gut epithelium. In contrast, cadherin, detected only in older gypsy moth larvae, was present both in the apical brush border and in the basement membrane anchoring the midgut epithelial cells. The topographical relationship between the Bt Cry toxin-binding molecules BTR-270 and APN-1 and the Cry1A toxin-binding sites that were confined to the apical brush border of the midgut cells is consistent with findings implicating their involvement in the mechanism of the action of Bt Cry toxins.     

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: identification of proteins binding the delta-endotoxin, Cry1Ac of Bacillus thuringiensis

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) delta-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.     

Expression of Cry1Aa in cassava improves its insect resistance against Helicoverpa armigera

Lepidopteran insects affect cassava production globally, especially in intercropping system. The expression of Cry toxins in transgenic crops has contributed to an efficient control of insect pests, leading to a significant reduction in chemical insecticide usage. Helicoverpa armigera is a Lepidopteran pest that feeds on a wide range of plants like cotton and cassava. In the present study, transgenic cassava plants over-expressing Cry1Aa, which we named as Bt cassava, were developed and used to evaluate its efficacy against H. armigera as a model. Insect feeding assays were carried out to test the effects of Bt cassava leaves on the development and survival of H. armigera. Significant reduction (P < 0.05) in the survival and weight were detected on larvae fed with Bt cassava leaves in comparison with those fed with wild-type cassava leaves. The higher expression of Cry1Aa in transgenic cassava caused the lethal effect in larvae, in contrast to the normal growth and development of adults and pupation observed when fed with wild-type leaves. Morphological observation on the larval midguts showed that the consumption of Bt cassava affected the gut integrity of H. armigera. The columnar cells of the midgut epithelium were dramatically damaged and showed loose or disordered structure. Their cytoplasms become highly vacuolated and contained disorganized microvilli. Our study demonstrated that the transgenic cassava expressing the Cry1Aa is effective in controlling H. armigera. Our Bt transgenic cassava plant would provide a long-term beneficial effect on all crops in intercropping system, which in-turn, will be profitable to the farmers.     

Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with ¹²⁵I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with ¹²⁵I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K_d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R_t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.     

Role of alkaline phosphatase in insecticidal action of Cry1Ac against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> larvae

Cry1Ac δ-endotoxin produced by Bacillus thuringiensis (Bt) is used as a bio-pesticide for the control of Helicoverpa armigera. Aminopeptidases N (APN) and alkaline phosphatase (ALP) play critical roles in its action against H. armigera larvae. The binding of Cry1Ac with brush border membrane vesicle (BBMV) proteins was increased with the larval development although the sensitivity of larvae to δ-endotoxins decreased. There was higher expression of ALP than APN in early instar larvae with a ~10-fold higher affinity of Cry1Ac towards ALP than to APN. Binding to a specific receptor is therefore more important for the insecticidal activity rather than overall binding to the BBMV proteins. ALP might play a major role in toxicity as compared to APN.     

Compatibility of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding

δ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.     

Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

Background

Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F₂ screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.

Methodology/Principal Findings

Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with ¹²⁵I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in ¹²⁵I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.

Conclusion/Significance

This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.