Impact of Bt corn expressing Bacillus thuringiensis Berliner insecticidal proteins on the growth and survival of Spodoptera frugiperda larvae in Colombia.

Bioassays were conducted under controlled conditions to determine the response of Spodoptera frugiperda (J. E. Smith) larvae fed with corn materials expressing Bacillus thuringiensis (Bt) insecticidal endotoxins: (1) VT Double Pro® (VT2P) expressing Cry1A.105-Cry2Ab2 proteins and (2) VT Triple Pro® (VT3P) expressing Cry1A.105-Cry2Ab2-Cry3Bb1 proteins. The parameters assessed were: (i) mortality rate, and (ii) growth inhibition (GI) with respect to the control. To conduct this study, larvae were collected from commercial non-Bt corn fields, in four agricultural sub-regions in Colombia, between 2018 and 2020. Fifty-two populations were assessed from the field and neonate larvae from each of the populations were used for the bioassays. The study found that mortality rates in the regions for larvae fed with VT2P corn ranged from 95.1 to 100.0%, with a growth inhibition (%GI) higher than 76.0%. Similarly, mortality rate for larvae fed with VT3P corn were between 91.4 and 100.0%, with a %GI above 74.0%. The population collected in Agua Blanca (Espinal, Tolima; Colombia) in 2020, showed the lowest mortality rate of 53.2% and a %GI of 73.5%, with respect to the control. The population that exhibited the lowest %GI was collected in 2018 in Agua Blanca (Espinal, Tolima, Colombia) with a 30.2%, growth inhibition, with respect to the control. In recent years, the use of plant tissue to monitor susceptibility to fall armyworm has proven to be useful in the resistance management program for corn in Colombia determining that the FAW populations are still susceptible to Bt proteins contained in VT2P and VT3P.

Engineering of Bacillus thuringiensis Cry2Ab toxin for improved insecticidal activity.

Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC50 (0.129 µg/cm2) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm2). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin's future application.

V-ATPase E mediates Cry2Ab binding and toxicity in Helicoverpa armigera.

Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H+-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.

Baseline Susceptibility of the Field Populations of Ostrinia furnacalis in Indonesia to the Proteins Cry1A.105 and Cry2Ab2 of Bacillus thuringiensis.

Genetically modified MON 89034 corn (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins, viz. Cry1A.105 and Cry2Ab2, is a biotechnological option being considered for the management of the major corn pest in Indonesia, the Asian corn borer (Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae)). As a part of a proactive resistance-management program for MON 89034 corn in Indonesia, we assessed the baseline susceptibility of field-collected populations of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins. Dose-response bioassays using the diet-dipping method indicated that the lethal concentration (LC50) values of Cry1A.105 and Cry2Ab2 in 24 different field populations of O. furnacalis ranged from 0.006 to 0.401 µg/mL and from 0.044 to 4.490 µg/mL, respectively, while the LC95 values ranged from 0.069 to 15.233 µg/mL for Cry1A.105 and from 3.320 to 277.584 µg/mL for Cry2Ab2. The relative resistance ratios comparing the most tolerant field populations and an unselected laboratory population were 6.0 for Cry1A.105 and 2.0 for Cry2Ab2 based on their LC50 values. Some field populations were more susceptible to both proteins than the unselected laboratory population. The LC99 and its 95% fiducial limits across the field populations were calculated and proposed as candidate diagnostic concentrations. These data provide a basis for resistance monitoring in Bt Corn and further support building resistance-management strategies in Indonesia.

Reduced expression of the P-glycoprotein gene HaABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin but not Cry2Ab toxin in Helicoverpa armigera.

Rapid evolution of pest resistance to Bt insecticidal proteins presents a serious threat to the sustainable use of Bt crops. The cotton bollworm has been extensively exposed to Bt cotton worldwide and has evolved resistance in laboratory and field. Previous studies have highlighted the significant roles played by the ABC transporter proteins in Bt resistance. In this study, the ORF of HaABCB1 was cloned and analyzed. The expression of HaABCB1 was detected in all developmental stages and tissues, with the highest expression in third instar larvae stage and hindgut tissue. Compared with susceptible strain, a remarkable decrease of HaABCB1 expression in Cry1Ac resistant strain while no significant change in Cry2Ab resistant strain were found. The HaABCB1 expression reduced after susceptible larvae induced by Cry1Ac, but no obvious expression changes after Cry2Ab exposure. RNAi-mediated down-regulation of HaABCB1 could lead to a significant reduction in larval susceptibility to Cry1Ac, but not to Cry2Ab, in susceptible strain. Genetic linkage analysis confirmed that decreased expression of the HaABCB1 mediates resistance to Cry1Ac, but not Cry2Ab resistance. This knowledge contributes to better understanding of the complex molecular mechanisms underlying Bt resistance and provide theoretical foundation for the development of new strategies for pest resistance management.

Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

A Modified F2 Screen for Estimating Cry1Ac and Cry2Ab Resistance Allele Frequencies in Helicoverpa zea (Lepidoptera: Noctuidae).

Evaluating the frequency of resistance alleles is important for resistance management and sustainable use of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis. Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) is a major crop pest in the United States that has evolved practical resistance to the crystalline (Cry) proteins in Bt corn and cotton. The standard F2 screen for estimating resistance allele frequency does not work well for H. zea because successful single-pair matings are rare. In this study, we developed and implemented a modified F2 screen for H. zea that generates F1 progeny by crossing three laboratory susceptible female moths with one feral male moth instead of single-pair crosses. During 2019-2020, we used this modified method to establish 192 F2 families from 623 matings between susceptible females and feral males from Arkansas, Louisiana, Mississippi, and Tennessee. From each F2 family, we screened 128 neonates against discriminating concentrations of Cry1Ac and Cry2Ab in diet overlay bioassays. Based on these discriminating concentration bioassays, families were considered positive for resistance if at least five larvae survived to second instar, including at least one to third instar. The percentage of positive families was 92.7% for Cry1Ac and 38.5% for Cry2Ab, which yields an estimated resistance allele frequency (with 95% confidence interval) of 0.722 (0.688-0.764) for Cry1Ac and 0.217 (0.179-0.261) for Cry2Ab. The modified F2 screen developed and implemented here may be useful for future resistance monitoring studies of H. zea and other pests.

Relative fitness of susceptible and Cry1A.105/Cry2Ab2-single-/dual-protein-resistant Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) on non-Bt diet and a diet containing a low concentration of two proteins.

Helicoverpa zea (Boddie) is a destructive agricultural pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1A.105 and Cry2Ab2 are two Bt proteins expressed in a widely planted maize event MON 89034. In this study, two tests (Test-I and Test-II) were conducted to evaluate the relative fitness of Bt-susceptible and -resistant H. zea on non-Bt diet (Test-I and Test-II) and a diet containing a mix of Cry1A.105 and Cry2Ab2 at a low concentration (Test-II only). Insect populations evaluated in Test-I were two Bt-susceptible strains and three Bt-resistant strains (a single-protein Cry1A.105-, a single-protein Cry2Ab2-, and a dual-protein Cry1A.105/Cry2Ab2-resistant strains). Test-II analyzed the same two susceptible strains, three backcrossed-and-reselected Cry1A.105/Cry2Ab2-single-/dual-protein-resistant strains, and three F1 heterozygous strains. Measurements of life table parameters showed that neither the single- nor dual-protein Cry1A.105/Cry2Ab2 resistance in H. zea was associated with fitness costs under the test conditions. The single Cry protein resistances at a concentration of a mix of Cry1A.105 and Cry2Ab2 that resulted in a zero net reproductive rate for the two susceptible strains were functionally incomplete recessive or codominant, and the dual-protein resistance was completely dominant. The lack of fitness costs could be a factor contributing to the rapid revolution of resistance to the Cry proteins in this species. Data generated from this study should aid our understanding of Cry protein resistance evolution and help in refining IRM programs for H. zea.

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility.

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

Status of Cry1Ac and Cry2Ab2 resistance in field populations of Helicoverpa zea in Texas, USA.

Helicoverpa zea is a major target pest of Bt corn and Bt cotton. Field-evolved resistance of H. zea to Cry1 and Cry2 proteins has been widely reported in the United States. Understanding the frequency of resistance alleles in a target insect is critical for Bt resistance management. Despite multiple cases of practical resistance to Cry proteins having been documented in H. zea, there are no data on the current status of alleles conferring resistance to Cry1Ac and Cry2Ab2 in field populations of this pest. During 2018-2019, a total of 106 F2 families for Cry1Ac and 120 F2 families for Cry2Ab2 were established using mass mating and light trap strategy. We screened 13,568 and 15,360 neonates using a discriminatory dose of Cry1Ac and Cry2Ab2, respectively. The results showed that 93.4% and 35.0% of the F2 families could survive on the discriminatory dose of Cry1Ac and Cry2Ab2, respectively. The estimated resistance allele frequency for Cry1Ac in H. zea ranged from 0.4150 to 0.4975 and for Cry2Ab2 ranged from 0.1097 and 0.1228. These data indicate that the frequency of alleles conferring resistance to Cry1 and Cry2 proteins in H. zea in Texas are high. In addition, our data suggest the resistance to Cry1Ac and Cry2Ab2 in the screened families of H. zea varies from recessive to dominant. The information in this study provides precise estimates of Cry resistance allele frequencies in H. zea and increases our understanding of the risks to the sustainability of Bt crops.

Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity.

As a pore-forming toxin, activation, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous results revealed that the helices α4-α5 of Domain I were involved in the oligomerization of Cry2Ab, however, the key residues for Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site-directed in the helices α4-α5 of Domain I and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A could reduce the assembly of the 250 kDa oligomers, suggesting that these mutation residues might be essential for Cry2Ab oligomerization. As expected, all of these variants showed lower insecticidal activity against P. xylostella. Furthermore, we found that the pore-forming activities of these mutants also decreased when compared to wild-type Cry2Ab. Taken together, our data identified key residues for Cry2Ab oligomerization and emphasized that oligomerization was closely related to the insecticidal activity and pore-forming activity of Cry2Ab.

Paper-Based Simplified Visual Detection of Cry2Ab Insecticide from Transgenic Cottonseed Samples Using Integrated Quantum Dots-IgY Antibodies.

In the present study, an easy to use field-deployable methodology was developed for onsite detection of pesticidal crystal protein Cry2Ab from transgenic cotton crops to reduce seed adulteration. Anti Cry2Ab IgG and IgY antibodies were developed against recombinant Cry2Ab protein in New Zealand white rabbits and in white leg horn chickens, respectively. Carboxyl-functionalized CdTe quantum dots (QDs) were used as revealing probes, and nitrocellulose paper was used as an assay matrix. Recombinant Cry2Ab was generated in the lab and used for immunization of chicken and rabbits. After successful immunization and attaining the desired titer values (1:32 000 for IgY and 1:64 000 for IgG), eggs and hyperimmune sera were collected. Anti Cry2Ab IgY was purified as per the standardized protocols, and anti Cry2Ab IgG was purified using protein A affinity chromatography. Sensitivity of the generated antibodies was examined using indirect ELISA methods against recombinant Cr2Ab protein. Specificity evaluation was carried out against other Cry proteins including Cry2Ab, Cry4b, Cry4a, Cry1Ec, and Cry1Ac. Functionalized CdTe QDs were characterized for structure and shape as well as fluorescence properties using standard laboratory techniques. A field-deployable paper-based detection methodology was developed where IgG acted as the capturing antibody and IgY-linked CdTe QDs were used as revealing probes. The limit of detection (LOD) and quantification (LOQ) were found to be 2.91 ng/mL and 9.71 ng/mL, respectively. The effect of matrix interference was assessed on the different plant crude extracts of cottonseed materials.

Suppressing calcineurin activity increases the toxicity of Cry2Ab to Helicoverpa armigera.

BACKGROUND: Extensive planting of transgenetic Bacillus thuringiensis crops has driven the evolution of pest resistance to Cry1Ac. Adjustment of cropping structures has promoted further outbreak of Helicoverpa armigera in China. To control this pest, a combination of pyramiding RNA interference (RNAi) and Cry2Ab is considered a promising strategy for countering cross-resistance and enhancing the toxicity of Cry2Ab to cotton bollworm. We explored the possibility of using calcineurin (CAN) as a target RNAi gene, because it is involved in cotton bollworm responses to the toxicity of Cry2Ab. RESULTS: Cry2Ab treatment led to a significant increase in HaCAN mRNA level and HaCAN activity. Suppression of HaCAN activity due to RNAi-mediated knockdown of HaCAN increased the susceptibility of midgut cells to Cry2Ab. The increase in HaCAN activity shown by heterologous expression of HaCAN reduced the cytotoxicity of Cry2Ab to Sf9 cells. Moreover, ingestion of HaCAN-specific inhibitor FK506 increased the toxicity of Cry2Ab in larvae. Interestingly, HaCAN does not function as a Cry2Ab direct binding protein that participates in Cry2Ab toxicity. CONCLUSIONS: The results in this study provide evidence that suppression of HaCAN not only affected the development of the cotton bollworm, but also enhanced the toxicity of Cry2Ab to the pest. HaCAN is therefore an important candidate gene in cotton bollworm that can be targeted for pest control when the pest infests RNAi+Cry2Ab crops. Meanwhile, the mechanism of action of HaCAN in Cry2Ab toxicity suggested that protein dephosphorylation was involved. © 2020 Society of Chemical Industry.

Inheritance of Bacillus thuringiensis Cry2Ab2 protein resistance in Helicoverpa zea (Lepidoptera: Noctuidae).

BACKGROUND: The corn earworm, Helicoverpa zea (Boddie), is a major target pest of pyramided Bt corn and cotton in the United States. Field-evolved practical resistance to Cry1 and Cry2 proteins in H. zea has been documented in multiple locations in the United States. Understanding the genetic basis of Bt resistance is essential in developing insect resistance management (IRM) strategies for the sustainable use of the Bt crop technology. In this study, we characterized the genetic bases of Cry2Ab2 resistance in H. zea using diet-overlay bioassays with two different forms of Cry2Ab2 protein. RESULTS: Laboratory bioassays using a Cry2Ab2-resistant (RR) strain, a susceptible (SS) strain, as well as cross and backcross strains, revealed that resistance to Cry2Ab2 was autosomally inherited and controlled by more than one locus. In diet bioassays, the dominance of Cry2Ab2 resistance in H. zea varied from incompletely recessive to incompletely dominant across all tested Cry2Ab2 concentrations of either Bt corn leaf powder or solubilized protein. On leaf tissue of TwinLink cotton (expressing Cry1Ab and Cry2Ae), Cry2Ab2 resistance in H. zea was completely dominant. CONCLUSION: These results have significant implications for understanding the widespread field-evolved resistance of H. zea against Cry1 and Cry2 proteins in Bt corn and cotton and should be useful in developing effective IRM strategies for H. zea. © 2020 Society of Chemical Industry.

ATP-Binding Cassette Subfamily A Member 2 is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, but not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins.

: Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239 cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1F, Cry1Ca, Cry1Da, or Cry9Aa toxins.

Usage of Graphene Oxide in Fluorescence Quenching-Linked Immunosorbent Assay for the Detection of Cry2Ab Protein Present in Transgenic Plants.

Graphene oxide-based sensor technologies in various detection platforms have been adopted in multiple dimensions. Most of the applications in combination with other materials such as gold, silver, enzymes, and so forth are read as electrical, electrochemical, impedance, and fluorescence signals. We report the development of a novel and simple fluorescence quenching-based immunoassay platform that provides quantitative binding sites for the Cry2Ab protein content present in the transgenic cotton (Gossypium hirsutum) plant. In this assay, the graphene oxide-conjugated anti-Cry2Ab antibody serves as the binding site for the analyte Cry2Ab protein, which forms a complex with a second anti-Cry2Ab fluorescein isothiocyanate (FITC)-conjugated antibody. This complex acts as the reaction center of this platform where the graphene oxide quenches the fluorescence signal of the FITC molecule. This microtiter plate-based method currently works at a sensitivity of 0.78 ng /ml, which can further be improved.

Synthesis and Characterization of Cry2Ab-AVM Bioconjugate: Enhanced Affinity to Binding Proteins and Insecticidal Activity.

Bacillus thuringiensis insecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4"-O-succinyl avermectin (AVM) to synthesize Cry2Ab-AVM bioconjugate. It was found that Cry2Ab-AVM showed higher insecticidal activity against Plutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab-AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in the P. xylostella cadherin (PxCR10-11), with a much higher affinity (dissociation equilibrium constant KD = 3.44 nM) than Cry2Ab30 (KD = 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab-AVM ligand docking into the PxCR10-11 is a potential mechanism to enhance the binding affinity of Cry2Ab-AVM to PxCR10-11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management.

Mutation of ABC transporter ABCA2 confers resistance to Bt toxin Cry2Ab in Trichoplusia ni.

Insecticidal proteins from Bacillus thuringiensis (Bt) are the primary recombinant proteins expressed in transgenic crops (Bt-crops) to confer insect resistance. Development of resistance to Bt toxins in insect populations threatens the sustainable application of Bt-crops in agriculture. The Bt toxin Cry2Ab is a major insecticidal protein used in current Bt-crops, and resistance to Cry2Ab has been selected in several insects, including the cabbage looper, Trichoplusia ni. In this study, the Cry2Ab resistance gene in T. ni was mapped to Chromosome 17 by genetic linkage analyses using a whole genome resequencing approach, and was then finely mapped using RNA-seq-based bulked segregant analysis (BSA) and amplicon sequencing (AmpSeq)-based fine linkage mapping to a locus containing two genes, ABCA1 and ABCA2. Mutations in ABCA1 and ABCA2 in Cry2Ab resistant T. ni were identified by both genomic DNA and cDNA sequencing. Analysis of the expression of ABCA1 and ABCA2 in T. ni larvae indicated that ABCA2 is abundantly expressed in the larval midgut, but ABCA1 is not a midgut-expressed gene. The mutation in ABCA2 in Cry2Ab resistant T. ni was identified to be an insertion of a transposon Tntransib in ABCA2. For confirmation of ABCA2 as the Cry2Ab-resistance gene, T. ni mutants with frameshift mutations in ABCA1 and ABCA2 were generated by CRISPR/Cas9 mutagenesis. Bioassays of the T. ni mutants with Cry2Ab verified that the mutations of ABCA1 did not change larval susceptibility to Cry2Ab, but the ABCA2 mutants were highly resistant to Cry2Ab. Genetic complementation test of the ABCA2 allele in Cry2Ab resistant T. ni with an ABCA2 mutant generated by CRISPR/Cas9 confirmed that the ABCA2 mutation in the Cry2Ab resistant strain confers the resistance. The results from this study confirmed that ABCA2 is essential for the toxicity of Cry2Ab in T. ni and mutation of ABCA2 confers the resistance to Cry2Ab in the resistant T. ni strain derived from a Bt resistant greenhouse population.

Field-evolved resistance of Helicoverpa zea (Boddie) to transgenic maize expressing pyramided Cry1A.105/Cry2Ab2 proteins in northeast Louisiana, the United States.

The corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), is a major target pest of pyramided Bt maize and cotton in the U.S. In 2017 and 2018, notable ear damage and larval survival of H. zea were observed on pyramided Cry1A.105/Cry2Ab2 maize in some fields in northeast Louisiana, U.S. The objective of this study was to determine if the field control problem was due to resistance development to the Bt proteins in plants. A total of 15 H. zea populations were collected from Bt and non-Bt maize plants in 2017 and 2018 in multiple locations in Louisiana, Florida, and Georgia. Diet-overlay bioassays showed that LC50s of Cry1A.105 and Cry2Ab2 for populations collected from the areas with control problems in northeast Louisiana were as much as >1623- and 88-fold greater than that of a susceptible strain, respectively. In addition, two field trials in 2018 validated that Cry1A.105/Cry2Ab2 maize failed in managing natural H. zea populations, while Bt maize containing Vip3A was effective in northeast Louisiana. Results of the study documented that the observed field control problems of Cry1A.105/Cry2Ab2 maize against H. zea in northeast Louisiana were due to resistance development of the insect to the Bt proteins in plants. This is the first documentation of field-evolved resistance to pyramided Bt maize in a target insect species in southern U.S. However, susceptibility levels to Cry1A.105 and Cry2Ab2 varied greatly among populations collected from the three states, suggesting uneven distributions of the resistance in the region.

Stability of expression of Cry1Ac and Cry2Ab2 proteins in Bollgard-II hybrids at different stages of crop growth in different genotypes across cropping seasons and multiple geographies.

Bollgard-II cotton expressing Cry1Ac and Cry2Ab2 insecticidal proteins has been commercially cultivated in India since 2006 to control bollworms. These genes were introgressed into parental germplasm of numerous hybrids. Therefore, it is imperative that these insecticidal proteins are expressed in sufficient quantities in different tissues, throughout the season irrespective of genetic background or environmental conditions for effective performance. Here, we document results of a comprehensive study on pattern of expression of Bt proteins across different stages of crop growth in > 2000 cotton hybrids (Gossypium hirsutum), across 12 cropping seasons tested in the Northern, Southern or Central zones in India, in terminal leaf, pre-candle square and boll epicarp tissues. Statistical analysis of variability using Linear mixed effect model was used to estimate factors contributing to variability in expression of Bt proteins. For Cry1Ac, variability was maximally contributed by genotype × season × plant growth stage effect in terminal leaves and boll epicarp, while season effect drove variability in pre-candle square. In Cry2Ab2, season effect drove variability in three tissue types. Pre-candle square tissue had most variability in expression of both proteins followed by terminal leaf and boll epicarp. Further, expression of Bt proteins in 234 G. hirsutum × G. barbadense hybrids showed similar expression patterns as intra specific hybrids though there was a significant difference in expression levels. Cry2Ab2 was expressed in significantly higher amounts when genes were in homozygous state. Bt proteins were also found to be expressed in varied amounts in different tissues and were expressed even when hybrids were grown at sub-optimal temperatures.