Protein Engineering of Vip3A in a Selected Bacillus thuringiensis Host for Consistent High Protein Production.

This study aimed to obtain reliable high Vip3A production from Bacillus thuringiensis (Bt) by modifying Vip3A to acquire higher thermostability in a suitable host. Bt117 is a great host for Vip3A production due to protein production consistency, low protease activity in culture media, and large amounts of mostly full-length protein, but it produces Vip3A with lower thermostability (Vip3Aa35). The C-terminal region of Bt117 Vip3A was replaced with that of a Vip3A with higher thermostability (Vip3Aa64 from Bt294) to generate the recombinant Bt117-Vip3Aa64-C. Like the parental strain Bt117, this strain expressed mostly full-length protein and exhibited low protease activity and similar protein expression profiles in culture media but retained greater larvicidal activity upon 37 °C storage like Bt294 Vip3Aa64. Importantly, every culture batch of Bt117-Vip3Aa64-C yielded over 200 mg/l Vip3A, which is a notable improvement over the original Vip3Aa64-producing strain Bt294 where 45% of culture batches failed to produce Vip3A at the same level. Successfully, we combined the superior qualities of two Bt strains, Bt294, which produces thermostable Vip3A but at low and inconsistent levels, and Bt117, which produces Vip3A with low thermostability but at consistently high levels. Protein engineering of Vip3A in Bt117 ultimately yielded an improved strain producing a thermostable Vip3A with reliably high protein production.

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

Comparative analysis of Spodoptera frugiperda (J. E. Smith) (Lepidoptera, Noctuidae) corn and rice strains microbiota revealed minor changes across life cycle and strain endosymbiont association.

Background: Spodoptera frugiperda (FAW) is a pest that poses a significant threat to corn production worldwide, causing millions of dollars in losses. The species has evolved into two strains (corn and rice) that differ in their genetics, reproductive isolation, and resistance to insecticides and Bacillus thuringiensis endotoxins. The microbiota plays an important role in insects' physiology, nutrient acquisition, and response to chemical and biological controls. Several studies have been carried out on FAW microbiota from larvae guts using laboratory or field samples and a couple of studies have analyzed the corn strain microbiota across its life cycle. This investigation reveals the first comparison between corn strain (CS) and rice strain (RS) of FAW during different developmental insect stages and, more importantly, endosymbiont detection in both strains, highlighting the importance of studying both FAW populations and samples from different stages. Methods: The composition of microbiota during the life cycle of the FAW corn and rice strains was analyzed through high-throughput sequencing of the bacterial 16S rRNA gene using the MiSeq system. Additionally, culture-dependent techniques were used to isolate gut bacteria and the Transcribed Internal Spacer-ITS, 16S rRNA, and gyrB genes were examined to enhance bacterial identification. Results: Richness, diversity, and bacterial composition changed significantly across the life cycle of FAW. Most diversity was observed in eggs and males. Differences in gut microbiota diversity between CS and RS were minor. However, Leuconostoc, A2, Klebsiella, Lachnoclostridium, Spiroplasma, and Mucispirilum were mainly associated with RS and Colidextribacter, Pelomonas, Weissella, and Arsenophonus to CS, suggesting that FAW strains differ in several genera according to the host plant. Firmicutes and Proteobacteria were the dominant phyla during FAW metamorphosis. Illeobacterium, Ralstonia, and Burkholderia exhibited similar abundancies in both strains. Enterococcus was identified as a conserved taxon across the entire FAW life cycle. Microbiota core communities mainly consisted of Enterococcus and Illeobacterium. A positive correlation was found between Spiroplasma with RS (sampled from eggs, larvae, pupae, and adults) and Arsenophonus (sampled from eggs, larvae, and adults) with CS. Enterococcus mundtii was predominant in all developmental stages. Previous studies have suggested its importance in FAW response to B. thuringensis. Our results are relevant for the characterization of FAW corn and rice strains microbiota to develop new strategies for their control. Detection of Arsenophonus in CS and Spiroplasma in RS are promising for the improvement of this pest management, as these bacteria induce male killing and larvae fitness reduction in other Lepidoptera species.

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

BACKGROUND: Genetically 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. RESULTS: To 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. CONCLUSIONS: This 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.

Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action.

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production.

Toxicity of Cry- and Vip3Aa-Class Proteins and Their Interactions against Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is one of the most important insect pests affecting corn crops worldwide. Although planting transgenic corn expressing Bacillus thuringiensis (Bt) toxins has been approved as being effective against FAW, its populations' resistance to Bt crops has emerged in different locations around the world. Therefore, it is important to understand the interaction between different Bt proteins, thereby delaying the development of resistance. In this study, we performed diet-overlay bioassays to evaluate the toxicity of Cry1Ab, Cry1Ac, Cry1B, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, Vip3Aa11, Vip3Aa19, and Vip3Aa20, as well as the interaction between Cry1Ab-, Cry1F-, Cry2Ab-, and Vip3Aa-class proteins against FAW. According to our results, the LC50 values of Bt proteins varied from 12.62 ng/cm2 to >9000 ng/cm2 (protein/diet), among which the Vip3Aa class had the best insecticidal effect. The combination of Cry1Ab and Vip3Aa11 exhibited additive effects at a 5:1 ratio. Cry1F and Vip3Aa11 combinations exhibited additive effects at 1:1, 1:2, and 5:1 ratios. The combination of Cry1F and Vip3Aa19 showed an antagonistic effect when the ratio was 1:1 and an additive effect when the ratio was 1:2, 2:1, 1:5, and 5:1. Additionally, the combinations of Cry1F and Vip3Aa20 showed antagonistic effects at 1:2 and 5:1 ratios and additive effects at 1:1 and 2:1 ratios. In addition to the above combinations, which had additive or antagonistic effects, other combinations exhibited synergistic effects, with variations in synergistic factors (SFs). These results can be applied to the establishment of new pyramided transgenic crops with suitable candidates, providing a basis for FAW control and resistance management strategies.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

Throwing Brazilian strains into the melting pot of P. xylostella resistance to Bacillus thuringiensis.

The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.

Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

Contribution of the transcription factor SfGATAe to Bt Cry toxin resistance in Spodoptera frugiperda through reduction of ABCC2 expression.

Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins.

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.

Comparison of the performance of multiple whole-genome sequence-based tools for the identification of Bacillus cereus sensu stricto biovar Thuringiensis.

The Bacillus cereus sensu stricto (s.s.) species comprises strains of biovar Thuringiensis (Bt) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential. Bt strains are identified (i) based on the production of insecticidal crystal proteins, also known as Bt toxins, or (ii) based on the presence of cry, cyt, and vip genes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on whole-genome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification of Bt was determined in reference to phenotypic microscope-based screening for the production of crystal proteins. A total of 58 diverse B. cereus sensu lato strains isolated from clinical, food, environmental, and commercial biopesticide products underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS (identification of pesticidal sequences), and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity of Bt identification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with the highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatics tools performed well overall, with IDOPS achieving high sensitivity and specificity (>0.90).IMPORTANCEStrains of Bacillus cereus sensu stricto (s.s.) biovar Thuringiensis (Bt) are used as organic biopesticides. Bt is differentiated from the foodborne pathogen Bacillus cereus s.s. by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovar Thuringiensis is necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of whole-genome sequencing (WGS)-based identification of Bt compared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, identification of pesticidal sequences performed best overall at WGS-based Bt identification.

Cross-pollination in seed-blended refuge and selection for Vip3A resistance in a lepidopteran pest as detected by genomic monitoring.

The evolution of pest resistance to management tools reduces productivity and results in economic losses in agricultural systems. To slow its emergence and spread, monitoring and prevention practices are implemented in resistance management programs. Recent work suggests that genomic approaches can identify signs of emerging resistance to aid in resistance management. Here, we empirically examined the sensitivity of genomic monitoring for resistance management in transgenic Bt crops, a globally important agricultural innovation. Whole genome resequencing of wild North American Helicoverpa zea collected from non-expressing refuge and plants expressing Cry1Ab confirmed that resistance-associated signatures of selection were detectable after a single generation of exposure. Upon demonstrating its sensitivity, we applied genomic monitoring to wild H. zea that survived Vip3A exposure resulting from cross-pollination of refuge plants in seed-blended plots. Refuge seed interplanted with transgenic seed exposed H. zea to sublethal doses of Vip3A protein in corn ears and was associated with allele frequency divergence across the genome. Some of the greatest allele frequency divergence occurred in genomic regions adjacent to a previously described candidate gene for Vip3A resistance. Our work highlights the power of genomic monitoring to sensitively detect heritable changes associated with field exposure to Bt toxins and suggests that seed-blended refuge will likely hasten the evolution of resistance to Vip3A in lepidopteran pests.

Urea Cycle of Bacillus thuringiensis Affects Its Survival under UV Stress.

Bacillus thuringiensis (Bt) is widely used to produce biological pesticides. However, its persistence is limited because of ultraviolet (UV) rays. In our previous study, we found that exogenous intermediates of the urea cycle were beneficial to Bt for survival under UV stress. To further explore the effect of the urea cycle on the resistance mechanism of Bt, the rocF/argG gene, encoding arginase and argininosuccinate synthase, respectively, were knocked out and recovered in this study. After the target genes were removed, respectively, the urea cycle in the tested Bt was inhibited to varying degrees. The UV stress test showed that the urea cycle disorder could reduce the resistance of Bt under UV stress. Meanwhile, the antioxidant enzyme activities of Bt were also decreased to varying degrees due to the knockout of the target genes. All of these results revealed that the urea cycle can metabolically regulate the stress resistance of Bt.

Knockdown of MAPK p38-linked genes increases the susceptibility of Chilo suppressalis larvae to various transgenic Bt rice lines.

Bacillus thuringiensis (Bt) toxins have provided exceptional control of agricultural insect pests, however, over reliance on the proteins would potentially contribute to the development of field tolerance. Developing new sustainable insect pest control methods that target the mechanisms underlying Bt tolerance can potentially support the Bt control paradigm while also providing insights into basic insect physiology. The MAPK p38 pathway is strongly associated with Bt tolerance in Chilo suppressalis, a major pest of rice. To gain insights into how this pathway impacts tolerance, high-throughput screening of C. suppressalis larval midguts initially identified eight novel target genes. Increased larval sensitivity to the transgenic cry1Ca rice strain T1C-19 was observed following RNA interference-mediated knockdown of four of the genes, Cscnc, Csgcp, Cszfp26 and CsZMYM1. Similar enhanced sensitivity to the TT51 (expressing Cry1Ab/1Ac) and T2A-1 (expressing Cry2Aa) transgenic rice lines occurred when Cszfp26 and CsZMYM1 were knocked down. All four target genes are downstream of the MAPK p38 pathway but do not participate in negative feedback loop of the pathway. These results implicate Cscnc, Csgcp, Cszfp and CsZMYM1 in the C. suppressalis transgenic cry1Ca rice tolerance mechanism regulated by MAPK p38. These findings further enhance our understanding of the MAPK p38-dependent molecular mechanisms underlying Bt tolerance in C. suppressalis and open new avenues of tolerance management to develop.

Resistance management and integrated pest management insights from deployment of a Cry3Bb1+ Gpp34Ab1/Tpp35Ab1 pyramid in a resistant western corn rootworm landscape.

In Nebraska USA, many populations of western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, now exhibit some level of resistance to all corn rootworm-active Bacillus thuringiensis Berliner (Bt) proteins expressed in commercial hybrids. Therefore, a study was conducted in northeast Nebraska from 2020-2022 to reevaluate current corn rootworm management options in continuous maize (consecutive planting for ≥2 years). Results from on-farm experiments to evaluate a standard soil-applied insecticide (Aztec® 4.67G) in combination with non-rootworm Bt or rootworm-active Bt pyramided maize (Cry3Bb1 + Gpp34Ab1/Tpp35Ab1) are reported within the context of WCR Bt resistance levels present. Corrected survival from Bt pyramid single-plant bioassays (<0.3, 0.3-0.49, >0.5) was used to place populations into 3 resistance categories. Variables evaluated included root injury, adult emergence, proportion lodged maize, and grain yield. Key results: A composite analysis of all populations across resistance levels indicated that addition of soil insecticide to Bt pyramid significantly reduced adult emergence and lodging but did not significantly increase root protection or yield. Within and among resistance category analyses of root injury revealed that the Bt pyramid remained highly efficacious at any non-rootworm Bt root injury level when resistance was absent or low. When corrected survival was >0.3, mean Bt pyramid root injury tracked more closely in a positive linear fashion with mean non-rootworm Bt root injury (rootworm density x level of resistance interaction). Similar trends were obtained for adult emergence but not yield. Mean Bt pyramid root injury rating was <0.75 in most populations with Bt resistance, which contributed to no significant yield differences among categories. Results are discussed within the context of IPM:IRM tradeoffs and the need to reduce WCR densities in this system to decrease the impact of the density x resistance interaction to bridge use of current pyramids with new technologies introduced over the next decade.

Diverse genetic basis of Vip3Aa resistance in five independent field-derived strains of Helicoverpa zea in the US.

BACKGROUND: Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long-term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field-derived H. zea strains (M1-RR, AC4-RR, R2-RR and R15-RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70-RR). RESULTS: Maternal effects and sex linkage were absent, and the effective dominance level (DML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 µg cm-2, in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1-RR and LT#70-RR; another shared by R2-RR and R15-RR; and a distinct one for AC4-RR. CONCLUSION: Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.