Identification of Crucial Modules and Genes Associated with Bt Gene Expression in Cotton.

The expression of Bacillus thuringiensis (Bt) toxins in transgenic cotton confers resistance to insect pests. However, it has been demonstrated that its effectiveness varies among cotton cultivars and different tissues. In this study, we evaluated the expression of Bt protein in 28 cotton cultivars and selected 7 cultivars that differed in Bt protein expression for transcriptome analysis. Based on their Bt protein expression levels, the selected cultivars were categorized into three groups: H (high Bt protein expression), M (moderate expression), and L (low expression). In total, 342, 318, and 965 differentially expressed genes were detected in the H vs. L, M vs. L, and H vs. M comparison groups, respectively. And three modules significantly associated with Bt protein expression were identified by weighted gene co-expression network analysis. Three hub genes were selected to verify their relationships with Bt protein expression using virus-induced gene silencing (VIGS). Silencing GhM_D11G1176, encoding an MYC transcription factor, was confirmed to significantly decrease the expression of Bt protein. The present findings contribute to an improved understanding of the mechanisms that influence Bt protein expression in transgenic cotton.

Recombinant Expression of ABCC2 Variants Confirms the Importance of Mutations in Extracellular Loop 4 for Cry1F Resistance in Fall Armyworm.

Fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive and invasive global noctuid pest. Its control is based on insecticide applications and Bacillus thuringiensis (Bt) insecticidal Cry toxins expressed in transgenic crops, such as Cry1F in Bt corn. Continuous selection pressure has resulted in populations that are resistant to Bt corn, particularly in Brazil. FAW resistance to Cry1F was recently shown to be conferred by mutations of ATP-binding cassette transporter C2 (ABCC2), but several mutations, particularly indels in extracellular loop 4 (ECL4), are not yet functionally validated. We addressed this knowledge gap by baculovirus-free insect cell expression of ABCC2 variants (and ABCC3) by electroporation technology and tested their response to Cry1F, Cry1A.105 and Cry1Ab. We employed a SYTOXTM orange cell viability test measuring ABCC2-mediated Bt toxin pore formation. In total, we tested seven different FAW ABCC2 variants mutated in ECL4, two mutants modified in nucleotide binding domain (NBD) 2, including a deletion mutant lacking NBD2, and S. frugiperda ABCC3. All tested ECL4 mutations conferred high resistance to Cry1F, but much less to Cry1A.105 and Cry1Ab, whereas mutations in NBD2 hardly affected Bt toxin activity. Our study confirms the importance of indels in ECL4 for Cry1F resistance in S. frugiperda ABCC2.

Proteomic Analysis and Promoter Modification of Bacillus thuringiensis to Improve Insecticidal Vip3A Protein Production.

Insecticidal protein Vip3A secreted from B. thuringiensis is a potential biocontrol agent for control of lepidopteran pests. Under laboratory conditions, high albeit variable Vip3A production from the local isolate Bt294 was only obtained from a much enriched TB culture medium. Proteomic analysis and strain improvement were therefore performed to improve Vip3A production. Studies indicated that the buffer capacity, carbon source, and nitrogen source are critical to efficiently produce Vip3A. Medium with lower amounts of peptone and yeast extract (compared to TB), with an additional carbon source and phosphate buffer (LB*G medium) was found to give reasonable yields of Vip3A. Proteomic analysis revealed higher expression of proteins involved in glutamate and histidine biosynthesis in cells cultured in TB compared to LB about 58 and 33 times, respectively. Experiments confirmed that glutamate supplementation could increase Vip3A production. In addition, promoter substitution with that of cry3A increased Vip3A yields by about 20-30%. Overall, very high yields of Vip3A could be obtained by culturing Bt294 (Pcry3A-vip3Aa64) in LB*G medium with glutamate supplementation.

Effects of transgenic Bacillus Thuringiensis maize (2A-7) on the growth and development in rats.

This study aimed to determine the effects of genetically modified insect-resistant maize (2A-7) on the growth and development in developing rats. Rats were fed a diet formulated with 2A-7 maize and were compared with rats fed a diet formulated with non-transgenic maize (CK group) and rats fed AIN-93G diet (BC group). 2A-7 maize was formulated into diets at ratios of 82.4% (H group) and 20.6% (L group); non-transgenic maize was formulated into diets at a ratio of 82.4%. From the first day of pregnancy, adult rats were divided into four groups and fed with the above four diets, respectively. Weaning on postnatal day 21, the diets of offspring were consistent with their parents. The results showed that body weight, hematology, serum biochemistry, organ weight, organ coefficients and allergenicity of offspring fed with 2A-7 maize were comparable with those in the CK and BC groups. In physiological and behavioral development experiments, there was no statistically significant difference among groups. Although mCry1Ab proteins were detected in organs and serum, no histopathological changes were observed among groups. In conclusion, A-7 maize cause no treatment-related adverse effects on offspring, indicating that 2A-7 maize is safe for developing rats.

Resistance status of lepidopteran soybean pests following large-scale use of MON 87701 × MON 89788 soybean in Brazil.

Widespread adoption of MON 87701 × MON 89788 soybean, expressing Cry1Ac Bt protein and glyphosate tolerance, has been observed in Brazil. A proactive program was implemented to phenotypically and genotypically monitor Cry1Ac resistance in Chrysodeixis includens (Walker). Recent cases of unexpected injury in MON 87701 × MON 89788 soybean were investigated and a large-scale sampling of larvae on commercial soybean fields was performed to assess the efficacy of this technology and the distribution of lepidopteran pests in Brazil. No significant shift in C. includens susceptibility to Cry1Ac was observed eight years after commercial introduction of this technology in Brazil. F2 screen results confirmed that the frequency of Cry1Ac resistance alleles remains low and stable in C. includens. Unexpected injury caused by Rachiplusia nu (Guenée) and Crocidosema aporema (Walsingham) in MON 87701 × MON 89788 soybean was detected during the 2020/21 season, and studies confirmed a genetically based alteration in their susceptibility to Cry1Ac. MON 87701 × MON 89788 soybean remains effective against Anticarsia gemmatalis (Hübner), C. includens, Chloridea virescents (Fabricius) and Helicoverpa armigera (Hübner) in Brazil. However, there is evidence of field-evolved resistance to MON 87701 × MON 89788 soybean by the secondary soybean pests R. nu and C. aporema.

Cloning and characterization of the Cry79Aa1 gene from a lepidopteran active strain of Bacillus thuringiensis.

Bacillus thuringiensis (Bt) has been used globally as a biopesticide for effective and environmentally friendly pest control. Research has intensified following the development of resistance by lepidopteran species to Bt insecticidal crystal proteins. Discovering new Bt strains with novel toxin properties which can overcome resistance is one of the strategies to improve pesticide sustainability. The genome of the Bacillus thuringiensis LTS290 strain was sequenced and assembled in 252 contigs containing a total of 6,391,328 bp. The novel cry79Aa1 gene from this strain was identified and cloned. Cry79Aa1 contains 729 amino acid residues and a molecular mass of 84.8 kDa by SDS-PAGE analysis. Cry79Aa1 was found to be active against the lepidopteran larvae of Spodoptera exigua, Helicoverpa armigera, and Plutella xylostella with LC50 values of 13.627 µg/mL, 42.8 µg/mL, and 38.086 µg/mL, respectively. However, Cry79Aa1 protein showed almost no insecticidal activity against Leguminivora glycinivorella, although some degree of growth retardation was observed.

Bacillus thuringiensis Cry4Ba Insecticidal ToxinExploits Leu615 in Its C-Terminal Domain to Interact with a Target Receptor-Aedes aegypti Membrane-Bound Alkaline Phosphatase.

In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal δ-endotoxins from Bacillus thuringiensis has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21 kDa high-purity isolated DIII fragment derived from the Cry4Ba mosquito-specific toxin was achieved via optimized preparative FPLC, allowing direct rendering analyses for binding characteristics toward its target receptor-Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP). Binding analysis via dotblotting revealed that the Cry4Ba-DIII truncate was capable of specific binding to nitrocellulose-bound Aa-mALP, with a binding signal comparable to its 65 kDa Cry4Ba-R203Q full-length toxin. Further determination of binding affinity via sandwich ELISA revealed that Cry4Ba-DIII exhibited a rather weak binding to Aa-mALP with a dissociation constant (Kd) of ≈1.1 × 10-7 M as compared with the full-length toxin. Intermolecular docking between the Cry4Ba-R203Q active toxin and Aa-mALP suggested that four Cry4Ba-DIII residues, i.e., Glu522, Asn552, Asn576, and Leu615, are potentially involved in such toxin-receptor interactions. Ala substitutions of each residue (E522A, N552A, N576A and L615A) revealed that only the L615A mutant displayed a drastic decrease in biotoxicity against A. aegypti larvae. Additional binding analysis revealed that the L615A-impaired toxin also exhibited a reduction in binding capability to the surface-immobilized Aa-mALP receptor, while two bio-inactive DII-mutant toxins, Y332A and F364A, which almost entirely lost their biotoxicity, apparently retained a higher degree of binding activity. Altogether, our data disclose a functional importance of the C-terminal domain of Cry4Ba for serving as a potential receptor-binding moiety in which DIII-Leu615 could conceivably be exploited for the binding to Aa-mALP, highlighting its contribution to toxin interactions with such a target receptor in mediating larval toxicity.

Large-scale assessment of lepidopteran soybean pests and efficacy of Cry1Ac soybean in Brazil.

The soybean technology MON 87701 × MON 89788, expressing Cry1Ac and conferring tolerance to glyphosate, has been widely adopted in Brazil since 2013. However, pest shifts or resistance evolution could reduce the benefits of this technology. To assess Cry1Ac soybean performance and understand the composition of lepidopteran pest species attacking soybeans, we implemented large-scale sampling of larvae on commercial soybean fields during the 2019 and 2020 crop seasons to compare with data collected prior to the introduction of Cry1Ac soybeans. Chrysodeixis includens was the main lepidopteran pest in non-Bt fields. More than 98% of larvae found in Cry1Ac soybean were Spodoptera spp., although the numbers of Spodoptera were similar between Cry1Ac soybean and non-Bt fields. Cry1Ac soybean provided a high level of protection against Anticarsia gemmatalis, C. includens, Chloridea virescens and Helicoverpa spp. Significant reductions in insecticide sprays for lepidopteran control in soybean were observed from 2012 to 2019. Our study showed that C. includens and A. gemmatalis continue to be primary lepidopteran pests of soybean in Brazil and that Cry1Ac soybean continues to effectively manage the target lepidopteran pests. However, there was an increase in the relative abundance of non-target Spodoptera spp. larvae in both non-Bt and Cry1Ac soybeans.

Characterization of a novel Bacillus thuringiensis toxin active against Aedes aegypti larvae.

Some Bacillus thuringiensis (Bt) strains produce dipteran-active toxins and can control larval mosquitoes. We identified a novel mosquitocidal toxin named Xpp81Aa1 with the thioredoxin domain from Bt strain HSY204. This toxin has very little sequence similarity to the three-domain Cry toxin and Cyt toxins and has significant toxicity to Aedes aegypti larvae. A safety assessment indicated that the Xpp81Aa1 toxin has no cytocidal activity against red blood cells and did not induce allergic reactions. The Xpp81Aa1 toxin exhibited a synergistic effect in combination with Cry2Aa and Cry4Aa protein toxins. Thus, the Xpp81Aa1 toxin could be a good candidate for mosquito control applications to reduce the mosquito-borne disease.

A cis-Acting Mutation in the PxABCG1 Promoter Is Associated with Cry1Ac Resistance in Plutella xylostella (L.).

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.

Transition Phase Regulator AbrB Positively Regulates the sip1Ab1 Gene Expression in Bacillus thuringiensis.

Bacillus thuringiensis secreted insecticidal proteins (Sip) are a secretion that is toxic to coleopteran pests. However, the transcriptional mechanism of sip genes is still unknown. The transcriptional regulation of the sip1Ab1 gene and the expression of the Sip1Ab1 protein were investigated in this study. The results demonstrated that the secretion of the Sip1Ab1 protein in HD73 was almost the same as that in the original QZL38 strain during the transition phase. Analysis of the ß-galactosidase activities of sip1Ab1-lacZ in both the HD73 and abrB mutant strains indicated that the transcription of sip1Ab1 is dependent on AbrB. Electrophoretic mobility shift assays showed that AbrB could bind with the sip1Ab1 promoter, and two binding sites of AbrB in the region of the promoter of sip1Ab1 were determined by DNase I footprinting assays. All of the above-described results proved that AbrB positively regulates the sip1Ab1 gene. IMPORTANCE Bacillus thuringiensis Sip proteins are secreted insecticidal toxins that are toxic to coleopteran pests. In this study, we investigated the transcriptional mechanism of the sip gene and showed strong evidence that Sip1Ab1 is secreted in the transition phase and that AbrB, a transition phase regulator that is usually a repressor, positively and directly regulates sip1Ab1. Reports of AbrB positive regulation are rare, even in Bacillus subtilis. To the best of our knowledge, no toxic gene has been reported to be positively regulated by AbrB in Bacillus species.

Baseline Susceptibility and Laboratory Selection of Resistance to Bt Cry1Ab Protein of Chinese Populations of Yellow Peach Moth, Conogethes punctiferalis (Guenée).

Yellow Peach Moth (YPM), Conogethes punctiferalis (Guenée), is one of the most destructive maize pests in the Huang-Huai-Hai summer maize region of China. Transgenic Bacillus thuringiensis (Bt) maize provides an effective means to control this insect pest in field trials. However, the establishment of Bt resistance to target pests is endangering the continued success of Bt crops. To use Bt maize against YPM, the baseline susceptibility of the local populations in the targeted areas needs to be verified. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC50 values ranged from 0.35 to 2.38 ng/cm2 over the two years of the collection, and the difference between the most susceptible and most tolerant populations was sevenfold. The upper limit of the LC99 estimates of six pooled populations produced >99% larval mortality for representative eight populations collected in 2020 and was designated as diagnostic concentrations for monitoring susceptibility in YPM populations in China. Hence, we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Although the resistant ratio was generally low, YPM potentially could evolve resistance to Cry1Ab. The potential developmentof resistance by target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.

Hetero-oligomerization of Bacillus thuringiensis Cry1A proteins enhance binding to the ABCC2 transporter of Spodoptera exigua.

The ATP binding cassette (ABC) transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. By labeling the Cry1Aa protein, we have found that virtually all of the binding is in an oligomeric state, a conformation that allowed higher levels of specific binding that could not be achieved by the monomeric protein on its own. Furthermore, we have observed that Cry1A proteins can hetero-oligomerize in the presence of the transporter, which is reflected in an increase in binding and toxicity to SeABCC2-expressing cells. This synergism can be one of the reasons why B. thuringiensis co-expresses different Cry1 proteins that can apparently have similar binding preferences. The results from in vitro competition and ex vivo competition showed that Cry1Aa, Cry1Ab and Cry1Ac share functional binding sites. By using Cry1Ab-Cry1Ac chimeras, the presence of domain I from Cry1A proteins was revealed to be critical for oligomer formation.

Bacillus thuringiensis chimeric proteins Cry1A.2 and Cry1B.2 to control soybean lepidopteran pests: New domain combinations enhance insecticidal spectrum of activity and novel receptor contributions.

Two new chimeric Bacillus thuringiensis (Bt) proteins, Cry1A.2 and Cry1B.2, were constructed using specific domains, which provide insecticidal activity against key lepidopteran soybean pests while minimizing receptor overlaps between themselves, current, and soon to be commercialized plant incorporated protectants (PIP's) in soybean. Results from insect diet bioassays demonstrate that the recombinant Cry1A.2 and Cry1B.2 are toxic to soybean looper (SBL) Chrysodeixis includens Walker, velvetbean caterpillar (VBC) Anticarsia gemmatalis Hubner, southern armyworm (SAW) Spodoptera eridania, and black armyworm (BLAW) Spodoptera cosmioides with LC50 values < 3,448 ng/cm2. Cry1B.2 is of moderate activity with significant mortality and stunting at > 3,448 ng/cm2, while Cry1A.2 lacks toxicity against old-world bollworm (OWB) Helicoverpa armigera. Results from disabled insecticidal protein (DIP) bioassays suggest that receptor utilization of Cry1A.2 and Cry1B.2 proteins are distinct from each other and from current, and yet to be commercially available, Bt proteins in soy such as Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A. However, as Cry1A.2 contains a domain common to at least one commercial soybean Bt protein, resistance to this common domain in a current commercial soybean Bt protein could possibly confer at least partial cross resistance to Cry1A2. Therefore, Cry1A.2 and Cry1B.2 should provide two new tools for controlling many of the major soybean insect pests described above.

A Bacillus thuringiensis Cry protein controls soybean cyst nematode in transgenic soybean plants.

Plant-parasitic nematodes (PPNs) are economically important pests of agricultural crops, and soybean cyst nematode (SCN) in particular is responsible for a large amount of damage to soybean. The need for new solutions for controlling SCN is becoming increasingly urgent, due to the slow decline in effectiveness of the widely used native soybean resistance derived from genetic line PI 88788. Thus, developing transgenic traits for controlling SCN is of great interest. Here, we report a Bacillus thuringiensis delta-endotoxin, Cry14Ab, that controls SCN in transgenic soybean. Experiments in C. elegans suggest the mechanism by which the protein controls nematodes involves damaging the intestine, similar to the mechanism of Cry proteins used to control insects. Plants expressing Cry14Ab show a significant reduction in cyst numbers compared to control plants 30 days after infestation. Field trials also show a reduction in SCN egg counts compared with control plants, demonstrating that this protein has excellent potential to control PPNs in soybean.

Detection of alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio Iso-Seq.

Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date, the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 h. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi-exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.

Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein.

Pigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane.

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin-membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.

CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab.

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system.

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.