Cry1Ac Protein Research Articles

Evaluation of the strategy for insecticidal crystal encapsulation with cell wall in industrial processes.

Bacillus thuringiensis (Bt) can produce insecticidal crystal proteins during sporulation, and these are the basis of the most successful microbial insecticides in use today. However, the susceptibility of insecticidal crystal proteins to inactivation by ultraviolet (UV) radiation from sunlight causes damage to the insecticidal crystals and subsequent loss of toxicity. The deletion of the mclX gene, an unknown functional gene, can make the insecticidal crystal proteins become encapsulated by the cell wall which provides some protection against UV radiation. This study evaluates the potential of this innovative strategy during the industrial process of commercial strain KN11. Gene mclX was deleted from a commercial Bt strain KN11 successfully, and the mutant strain was cultured under different conditions, including laboratory and industrial fermentation conditions. The mother cells of the mclX mutant strain remained nonlysed after industrial processes. The deletion of mclX had no adverse effects on the production of Cry1Ac protein and no impact on the insecticidal activity. Most noteworthy, the ΔmclX mutant had improved UV resistance and insecticidal activity compared to the wild-type KN11 strain. This finding suggests that commercial strains can be genetically modified to encapsulate insecticidal crystal in Bt products using the mclX mutation strategy. This study evaluated the ability of commercial strains lacking the mclX gene to encapsulate crystals giving resistance to UV radiation. © 2024 Society of Chemical Industry.

Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

Life table study of Rachiplusia nu (Lepidoptera: Noctuidae) on different food sources and artificial diet.

Rachiplusia nu Guenée is a polyphagous species able to develop on several cultivated and non-cultivated host plants. However, basic life history information about this pest on hosts is scarce. In this study, R. nu larvae did not survive on leaves of non-Bt corn, wheat, Bt cotton that expresses proteins Cry1Ac+Cry2Ab2 or on Intacta2 Xtend soybean that expresses the Cry1A.105/Cry2Ab2/Cry1Ac proteins. Rachiplusia nu showed a viable egg-to-adult biological cycle (54%-66.3%) on non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip hosts, similar to larvae raised on the artificial diet. In addition, R. nu was unable to complete larval development on non-Bt cotton, and only 45.2% of R. nu larvae reached the pupal stage when fed leaves of intacta RR2 PRO soybean that expresses the Cry1Ac protein. Larval and pupal mass of surviving insects on Cry1Ac soybean leaves were also lower (larval: 0.104g; pupal: 0.099g) in relation to other food sources (larval: 0.165-0.189g; pupal: 0.173-0.192g). The total fecundity of R. nu on Cry1Ac soybean leaves was ≈65% lower in relation to other food sources. This fact caused ≈60% the net reproductive rate (Ro) and intrinsic rate of increase (rm) when compared to other food sources. Our findings indicate that the Cry1Ac soybean negatively affects the biological parameters of R. nu. Non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip are viable food sources for the survival and development of R. nu.

Species-specific evolution of lepidopteran TspC5 tetraspanins associated with dominant resistance to Bacillus thuringiensis toxin Cry1Ac

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have proven to be highly effective in managing some key pests. However, the evolution of resistance by the target pests threatens the sustainability of Bt crops. The L31S mutation in a tetraspanin encoded by HarmTspC5 (previously known as HaTSPAN1) has been shown to confer dominant resistance to the Bt protein Cry1Ac in Helicoverpa armigera, a globally damaging lepidopteran pest. However, the broader implications of the L31S mutation in the tetraspanins of other lepidopteran species remain unclear. The evolutionary analyses in this study indicate that TspC5s have evolved in a species-specific manner among the lepidopteran insects. To investigate the role of TspC5s in conferring dominant resistance to Cry1Ac, we used the piggyBac-based transformation system to generate four transgenic H. armigera strains that express exogenous TspC5 variants from three phylogenetically close species (Helicoverpa zea, Helicoverpa assulta and Heliothis virescens) and one phylogenetically distant species (Plutella xylostella). In comparison with the background SCD strain of H. armigera, the transgenic strains expressing HzeaTspC5-L31S, HassTspC5-L31S, or HvirTspC5-L31S exhibited significant resistance to Cry1Ac (10.0-, 21.4-, and 81.1-fold, respectively), whereas the strain expressing PxylTspC5-L27S remained susceptible. Furthermore, the Cry1Ac resistant phenotypes followed an autosomal dominant inheritance pattern and were closely linked to the introduced mutant TspC5s. These findings reveal the conserved role of TspC5s from Helicoverpa and Heliothis species in mediating the dominant resistance to Cry1Ac, and they provide crucial insights for assessing resistance risks related to mutant tetraspanins and devising adaptive resistance management strategies for these major lepidopteran pests.

Dynamics of Cry1Ac protein and soil enzyme activity in the rhizosphere of transgenic Bt oilseed rape

Dynamics of Cry1Ac protein and soil enzyme activity in the rhizosphere of transgenic Bt oilseed rape

Deletion of the 3′ End of the Introduced cry1Ac Gene Retains the Insecticidal Activity in Transgenic Cotton

The structure and expression of exogenous genes in transgenic crops are critical for the target traits. R7569 has the same exogenous insertion structure as the transgenic insect-resistant cotton MON531 but with a deletion in the 3′ end of the cry1Ac gene and the terminator region. Thus, in the present study, transcription, expression, and insecticidal activity assays were conducted to determine the function of the truncated cry1Ac gene. R7569 has a truncated cry1Ac gene with a length of 2554 bp encoding 881 amino acids, and the transcription termination site was mainly concentrated downstream of the truncated position and extended 160–270 bp from the truncated position using rapid amplification of cDNA ends (RACE). The transcript levels of the cry1Ac gene in R7569 were significantly higher than those of MON531 implants, except for during the boll stage. The content of the Cry1Ac protein in R7569 was higher than that of MON531 in the cotton leaf in all three periods. The corrected mortality rates of R7569 and MON531 against bollworms were 93.09% and 88.83%, respectively. The LC50 value of R7569 was 0.732 ng/g (dw), indicating a high level of resistance to bollworm. In this study, for the first time, we found a partial deletion of the target gene in commercially applied transgenic crops, and the partial deletion of the 3′ end of the cry1Ac gene retained a better transcription, expression level, and insecticidal activity, which can provide a specific case for the safety evaluation of transgenic crops.

STRATEGIES FOR IMPROVEMENT FOR CHARACTERIZATION AND QUALITY OF BT COTTON SEEDS

The study highlights diverse methodologies employed for seed quality assessment and characterization, including genetic, biochemical, and physiological analyses. In our present study, PCR and ELISA based techniques was adopted to identify the specific Cry1Ac gene and proteins in MECH 162 and RCH2 transgenic cotton plants. MECH 162 of 21.9ug/g and RCH2 of 17.37ug/g of seed protein was extracted. PCR analysis confirmed that gene specific CRY1Ac F/R primer as amplicon size of 550 bp for Bt gene, 600bp for NptII gene and 200bp for 35s promoter gene. The ELISA method was used for the validation of the developed assay, also ELISA needed simple equipment and took less time. Insights into the integration of these strategies into breeding programs are provided, emphasizing their crucial role in ensuring the production of high-quality Bt cotton seeds with improved agronomic performance and yield potential.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein

BackgroundGenetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood.ResultsTo investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes.ConclusionsThis study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Development of a Safe and Effective Bacillus thuringiensis-Based Nanobiopesticide for Controlling Tea Pests.

Mg(OH)2 was used as the nanocarrier of the Bacillus thuringiensis (Bt) Cry1Ac protein, and the synthesized Cry1Ac-Mg(OH)2 composites were regular and uniform nanosheets. Nano-Mg(OH)2 could effectively improve the insecticidal effect of the Cry1Ac protein toward Ectropis obliqua. It could enhance the damage degree of the Cry1Ac protein to intestinal epithelial cells and microvilli, induce and enrich the production of reactive oxygen species (ROS) in the midgut, and enhance the degradation of the Cry1Ac protein into active fragments. Furthermore, an anti-rinsing assay showed that the Cry1Ac-Mg(OH)2 composites were bound to the notch structure of the tea leaf surface. The retention of the Cry1Ac protein increased by 11.45%, and sprayed nano-Mg(OH)2 was rapidly absorbed by different tissues of tea plants. Moreover, nano-Mg(OH)2 and composites did not significantly affect non-target organisms. These results show that nano-Mg(OH)2 can serve as a safe and effective biopesticide carrier, which provides a new approach for stable and efficient Bt preparation.

Bt Soybean Cry1Ac Does Not Affect Development, Reproduction, or Feeding Behavior of Red-Banded Stink Bug Piezodorus guildinii (Hemiptera: Pentatomidae).

Bt soybean cultivation is increasing worldwide. The Cry1Ac protein expressed in Bt soybean efficiently controls several lepidopteran pests. The stink bug, Piezodorus guildinii (Westwood), a major pest for soybean in the Americas, is not controlled by Bt crops, although possible sub-lethal effects may occur. Even if there were no negative effects for sting bug, ingesting toxins could affect its bio-controllers. We tested through ELISA detection if P. guildinii ingests Cry1Ac from Bt soybean and possible effects on its development, reproduction, survival, and feeding behavior. Biological traits were evaluated under controlled conditions of nymphs and adults feeding on pods of near-isogenic cultivars DM5958iPRO (Bt) and DM59i (non-Bt). Feeding behavior was recorded using an AC-DC electropenetrography (EPG) device. Results indicated that P. guildinii ingested the Cry1Ac protein; however, nymphal period and accumulated survival percentage did not differ between cultivars. Feeding on Bt soybean pods did not affect fecundity (i.e., number of egg masses and eggs/female) nor egg viability. Different feeding behaviors were only detected on the pathway phase (stylet penetration into plant tissue), which was more pronounced in the Bt cultivar. However, the total duration of the feeding activities on seeds was numerically higher (ca. 2X) on Bt plants compared to non-Bt. This is the first study to demonstrate that P. guildinii does ingest the Cry1Ac protein and excrete it without being absorbed, probably explaining the lack of direct adverse effects on its biological parameters. EPG could indicate that Bt soybean plants might be less palatable than non-Bt to red-banded stink bug.

Random Mutagenesis to Enhance the Toxicity of Bacillus thuringiensis Cry Proteins Against Earias Vittella (F.)

Insecticidal proteins derived from Bacillus thuringiensis (Bt) are widely utilized in a variety of insect control applications,including sprays and transgenic crops.The development of resistance in pests, on the other hand, can lessen the effectiveness of Bttoxins. In this study, we made efforts to enhance the toxicity of two cry proteins Cry1Ac and Cry2Aa through random mutagenesis against cotton bollworm (Earias vitella), one of the most destructive cotton pests in Pakistan. Random mutagenesis is an important tool for elucidating protein structure-function relationships and for modifying proteins to enhance or change their characteristics. We focused on whole cry proteins for random mutagenesis througherror-prone PCR and constructed a recombinant library of cry proteins. Sequence analysis of eight mutants showed the mutations of 34 different nucleotides in Cry1Ac and Cry2Aa genes. All mutants were spared for toxicity bioassays against 2nd instar larvae of spotted bollworm. Cry1Ac mutant RM1AcM4 (D242E) and Cry2Aa mutants RM2AaM2 (T354A, T492R, F511L, G585E, D606Y) showed enhanced toxicity as compared to proteins without mutation. These two mutants comprise the mutations in domain-II of cry proteins important in specificity determining regions on midgut receptors in insect pests.

20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis ISPC-12: Purification, characterization, solution scattering and structural analysis

The 20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis (Bti) has been identified as an essential molecular chaperone in the enhancement of Cry11Aa and Cyt1Aa toxins production and their bio-crystallization. Additionally, P20 plays a vital role in suppressing the toxic effect of Cyt toxin on the host bacterium and also enhances insecticidal activity of Cry1Ac protein. Thus, the function of P20 is more specific than that of the chaperones. However, P20 is poorly investigated and insufficiently characterized. In the present study, we recombinantly expressed p20 from local isolate Bti ISPC-12 in heterologous bacterium E. coli and P20 protein was purified to homogeneity. Detailed biochemical and biophysical characterization provides crucial insights about in-vitro behavior as well as spatial conformations of P20 protein. Further, structural modelling and analysis provides insights into three-dimensional organization of the protein and shows that P20 is a non-toxic member of cytolytic (Cyt) toxin family similar to Cyt1Ca, with presence of conserved cytolysin fold. Additionally, solution scattering reveals that P20 is present as a dimer in the solution and probable dimeric assembly of P20 is presented. The findings reported here reveal engaging facts about P20 thereby advancing our understanding about this protein, which will expedite future studies.

Mepiquat chloride increases Cry1Ac protein content by regulating the carbon and amino acid metabolism of Bt cotton under high temperature and drought stress

The effects of mepiquat chloride (DPC) on Cry1Ac protein contents in Bacillus thuringiensis (Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and high-efficiency cotton cultivation. This study was undertaken on the Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, China. The potted cotton plants were exposed to high temperature and drought stress, and 20 mg L−1 DPC and water (CK) were sprayed on cotton plants. Seven days after treatment, Cry1Ac protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed. DESeq was used for differential gene analysis. Under the DPC treatment, in contrast with the water treatment (CK), Cry1Ac protein content increased by 4.7-11.9%. α-Ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased. Transcriptome analysis revealed that there were 7542 upregulated genes and 10449 downregulated genes for DPC vs. CK. GO and KEGG analyses showed that differentially expressed genes were mainly involved in biological processes, such as amino acid metabolism and carbon metabolism. Genes coding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly upregulated. DPC increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operating rate of the glycolytic pathway, the citric acid cycle. It also significantly up-regulated the genes encoding glutamate synthase and pyrrolidine-5-carboxylic acid dehydrogenase, and down-regulated the genes encoding glutamine synthetase as well as up-regulated the genes encoding glutamate oxaloacetate transaminase and N-acetylglutamate synthetase. Therefore, the synthesis ability of aspartic acid, glutamic acid, pyruvate, and arginine was increased by DPC, and then Cry1Ac protein contents were increased by regulating carbon and amino acid metabolism.

Development of a bioassay method to test activity of cry insecticidal proteins against Diatraea spp. (Lepidoptera: Crambidae) sugarcane stem borers.

The genus Diatraea (Lepidoptera: Crambidae) includes stem borers representing the most critical sugarcane pests in the Americas. Colombia's most widely distributed and damaging Diatraea species include Diatraea saccharalis, D. indigenella, D. busckella, and D. tabernella. The reduced efficacy of biological tools commonly used in controlling several species highlights the importance of evaluating alternative management strategies, such as transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The selection of optimal Bt insecticidal proteins for Diatraea control depends on bioassays with purified Bt proteins. Because there is no described artificial diet for borer species other than D. saccharalis and availability of most purified Bt toxins is restricted, this study aimed at developing a bioassay method using fresh corn tissue and providing proof of concept by testing susceptibility to the Cry1Ac insecticidal protein from Bt. Toxicity was evaluated with a single Cry1Ac dose applied directly to corn discs. Stem borer mortality after seven days was higher than 90% for all four tested Diatraea species, while control mortality was below 8%. In addition, we observed that Cry1Ac caused more than 90% weight inhibition in all survivors and delayed development. These results validate the use of this method to determine mortality and growth inhibition due to the consumption of the Cry1Ac protein in each of the Diatraea species. Furthermore, this method could be used to assess other entomopathogenic substances to control these insect pests.

Transgenic sugarcane expressing Bacillus thuringiensis Cry1Ac protein offers new possibilities for controlling the giant borer, Telchin licus (Drury), a pest difficult to control

The giant borer, Telchin licus (Drury), is an important insect pest of sugarcane in Central and South America. This pest is difficult to control with chemical or biological control approaches due to the larvae behavior of hiding in the deepest parts of the plants. Manual scavenging and skewer killing have been the main approaches for monitoring and control of T. licus in Brazil, the world's largest sugarcane producer. Recently, Brazil launched the first commercial use of transgenic sugarcane expressing Bacillus thuringiensis (Bt) proteins to control the sugarcane borer, Diatraea saccharalis (F.), a primary insect pest of sugarcane in the Americas. In this study, five independent field/greenhouse tests were conducted to evaluate the larval survival of and plant injury by T. licus on five non-Bt and five Bt sugarcane varieties containing a single cry1Ac gene. The multiple field/greenhouse tests showed that Cry1Ac sugarcane was highly effective to control T. licus; and the high control efficiency was consistent across varieties, from immature to mature plant stages, as well as for F0 generation plants and F1 ratoons. In addition, diet-incorporated bioassays were performed to determine the susceptibility of three field-collected T. licus populations in Brazil to diet treated with purified Cry1Ac protein or with a 25-fold dilution of Cry1Ac sugarcane stalk tissue. The diet bioassays exhibited that the field T. licus populations were also susceptible to the purified Cry1Ac protein, as well as to diet treated with the 25-fold dilution of stalk tissue. The results of this study provide compelling evidence that the single-protein Cry1Ac sugarcane varieties offer new possibilities for managing the T. licus, a pest that is difficult to control with other current available technologies.

Cry toxin expression in different plant parts of Bt cotton at different phenological stages

BackgroundCompared to Bollgard-I, the utilization of Cry2Ab protein in Bollgard-II cotton cultivars enhances insect control. Field and laboratory studies demonstrated reduction in the numbers of bollworm-infested terminals, squares, and bolls in Bollgard-II cotton lines when compared to both Bollgard-I and non-Bollgard cotton cultivars. This indicates that the combination of Cry2Ab with Cry1Ac increased the overall expression of proteins. The expression of Cry protein varied across different plant parts, such as leaves, bracts, squares, and bolls. As the season progresses, the expression of Cry protein decreased in these plant parts. Leaves exhibited the highest levels of Cry protein expression, followed by squares, flowers, and bolls. Variation in the expression levels of delta endotoxins in different plant parts was one of the contributing factors to the survival of pest populations on Bt cotton.ResultsUsing a commercially available QL 96 ELISA plate kit, the concentration of delta endotoxin in various plant parts at different phenological stages was determined in twelve BG-II cotton hybrids, namely Ajeet-155, JKCH-2245, RCH-3863, NCS-866, MRC-7373, JKCH-99, MRC-7387, NCEH-21, ANKR-3324, NCSI-1904, and NCHB 9902. Cry1Ac and Cry2Ab protein levels were determined from samples of flowers and fruiting parts (Rind, locule, seed, locule wall and seed) at 40, 75, 100 and 125 days after sowing (DAS) over two consecutive years 2018–2019 and 2019–2020. Cry1Ac protein content and expression was the highest at 100DAS in locule, seed and rind; followed by rind and locule wall and seed in green bolls at 125 DAS; followed by seed, locule, rind and flowers at 75 DAS; followed by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry2Ab protein content and expression was the highest at 125DAS in locule wall and seed and rind, followed by seed, locule and rind in green bolls at 100 DAS, followed by seed, locule, rind and flowers at 75 DAS, then by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry1Ac protein expression was less in comparison with Cry2Ab.ConclusionsThe research findings indicate that the locule of BG-II cotton plants exhibited the highest expression levels of Cry1Ac and Cry2Ab proteins, followed by the seeds, rind, locule wall, and flowers. These results provide valuable insights into the distribution of Cry protein expression in different plant parts, which can contribute to a better understanding of insect control in Bt cotton cultivars.

Mechanisms of feeding cessation in Helicoverpa armigera larvae exposed to Bacillus thuringiensis Cry1Ac toxin

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been applied in sprayable formulations and expressed in transgenic crops for the control of pests in the field. When exposed to Bt proteins insect larvae display feeding cessation, yet the mechanism for this phenomenon remains unknown. In this study, we investigated the feeding behavior and underlying mechanisms of cotton bollworm (Helicoverpa armigera) larvae after exposure to the Cry1Ac protein from Bt. Three H. armigera strains were studied: the susceptible SCD strain, the C2/3-KO strain with HaABCC2 and HaABCC3 knocked out and high-level resistance to Cry1Ac (>15,000-fold), and the SCD-KI strain with a T92C point mutation in tetraspanin (HaTSPAN1) and medium-level resistance to Cry1Ac (125-fold). When determining the percentage of insects that continued feeding after various exposure times to Cry1Ac, we observed quick cessation of feeding in larvae from the susceptible SCD strain, whereas larvae from the C2/3-KO strain did not display feeding cessation. In contrast, larvae from the SCD-KI strain rapidly recovered from the initial feeding cessation. Histopathological analyses and qRT-PCR in midguts of SCD larvae after Cry1Ac exposure detected serious epithelial damage and significantly reduced expression of the neuropeptide F gene (NPF) and its potential receptor gene NPFR, which are reported to promote insect feeding. Neither epithelial damage nor altered NPF and NPFR expression appeared in midguts of C2/3-KO larvae after Cry1Ac treatment. The same treatment in SCD-KI larvae resulted in milder epithelial damage and subsequent repair, and a decrease followed by an initial increase in NPF and NPFR expression. These results demonstrate that the feeding cessation response to Cry1Ac in cotton bollworm larvae is closely associated with midgut epithelial damage and downregulation of NPF and NPFR expression. This information provides clues to the mechanism of feeding cessation in response to Bt intoxication and contributes to the mode of action of the Cry1Ac toxin in target pests.

Performance of cotton expressing Cry1Ac, Cry1F and Vip3Aa19 insecticidal proteins against Helicoverpa armigera, H. zea and their hybrid progeny, and evidence of reduced susceptibility of a field population of H. zea to Cry1 and Vip3Aa in Brazil.

The genetically modified cotton DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 expressing Cry1Ac, Cry1F and Vip3Aa19 from Bacillus thuringiensis Berliner (Bt) has been cultivated in Brazil since the 2020/2021 season. Here, we assessed the performance of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton expressing Cry1Ac, Cry1F and Vip3Aa19 against Helicoverpa armigera (Hübner), Helicoverpa zea (Boddie), and their hybrid progeny. We also carried out evaluations with DAS-21023-5 × DAS-24236-5 cotton containing Cry1Ac and Cry1F. In leaf-disk bioassays, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 was effective in controlling neonates from laboratory colonies of H. armigera, H. zea and the hybrid progeny (71.9%-100% mortality). On floral bud bioassays using L2 larvae, H. zea presented complete mortality, whereas H. armigera and the hybrid progeny showed <55% mortality. On DAS-21023-5 × DAS-24236-5 cotton, the mortality of H. armigera on leaf-disk and floral buds ranged from 60% to 73%, whereas mortality of hybrids was <46%. This Bt cotton caused complete mortality of H. zea larvae from a laboratory colony in the early growth stages, but mortalities were <55% on advanced growth stages and on floral buds. In field studies conducted from 2014 to 2019, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton was also effective at protecting plants against H. armigera. In contrast, a population of H. zea collected in western Bahia in 2021/2022 on Bt cotton expressing Cry1 and Vip3Aa proteins, showed 63% mortality after 30 d, with insects developing into fifth and sixth instars, on DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton. We conclude that H. armigera, H. zea, and their hybrid progeny can be managed with DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton; however we found the first evidence in Brazil of a significant reduction in the susceptibility to DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton of a population of H. zea collected from Bt cotton in Bahia in 2021/2022.

Insect chaperones Hsp70 and Hsp90 cooperatively enhance toxicity of Bacillus thuringiensis Cry1A toxins and counteract insect resistance.

Bacillus thuringiensis (Bt) produces different insecticidal proteins effective for pest control. Among them, Cry insecticidal proteins have been used in transgenic plants for the control of insect pests. However, evolution of resistance by insects endangers this technology. Previous work showed that the lepidopteran insect Plutella xylostella PxHsp90 chaperone enhanced the toxicity of Bt Cry1A protoxins by protecting them from degradation by the larval gut proteases and by enhancing binding of the protoxin to its receptors present in larval midgut cells. In this work, we show that PxHsp70 chaperone also protects Cry1Ab protoxin from gut proteases degradation, enhancing Cry1Ab toxicity. We also show that both PxHsp70 and PxHsp90 chaperones act cooperatively, increasing toxicity and the binding of Cry1Ab439D mutant, affected in binding to midgut receptors, to cadherin receptor. Also, insect chaperones recovered toxicity of Cry1Ac protein to a Cry1Ac-highly resistant P. xylostella population, NO-QAGE, that has a disruptive mutation in an ABCC2 transporter linked to Cry1Ac resistance. These data show that Bt hijacked an important cellular function for enhancing its infection capability, making use of insect cellular chaperones for enhancing Cry toxicity and for lowering the evolution of insect resistance to these toxins.

Molecular Genetic Basis of Lab- and Field-Selected Bt Resistance in Pink Bollworm.

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) control some important insect pests. However, evolution of resistance by pests reduces the efficacy of Bt crops. Here we review resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella, one of the world's most damaging pests of cotton. Field outcomes with Bt cotton and pink bollworm during the past quarter century differ markedly among the world's top three cotton-producing countries: practical resistance in India, sustained susceptibility in China, and eradication of this invasive lepidopteran pest from the United States achieved with Bt cotton and other tactics. We compared the molecular genetic basis of pink bollworm resistance between lab-selected strains from the U.S. and China and field-selected populations from India for two Bt proteins (Cry1Ac and Cry2Ab) produced in widely adopted Bt cotton. Both lab- and field-selected resistance are associated with mutations affecting the cadherin protein PgCad1 for Cry1Ac and the ATP-binding cassette transporter protein PgABCA2 for Cry2Ab. The results imply lab selection is useful for identifying genes important in field-evolved resistance to Bt crops, but not necessarily the specific mutations in those genes. The results also suggest that differences in management practices, rather than genetic constraints, caused the strikingly different outcomes among countries.