Cry Proteins Research Articles

PSMD11 modulates circadian clock function through PER and CRY nuclear translocation.

The molecular circadian clock is regulated by a transcriptional translational feedback loop. However, the post-translational control mechanisms are less understood. The NRON complex is a large ribonucleoprotein complex, consisting of a lncRNA and several proteins. Components of the complex play a distinct role in regulating protein phosphorylation, synthesis, stability, and translocation in cellular processes. This includes the NFAT and the circadian clock pathway. PSMD11 is a component of the NRON complex and a lid component of the 26S proteasome. Among the PSMD family members, PSMD11 has a more specific role in circadian clock function. Here, we used cell and biochemical approaches and characterized the role of PSMD11 in regulating the stability and nuclear translocation of circadian clock proteins. We used size exclusion chromatography to enrich the NRON complex in the cytosolic and nuclear fractions. More specifically, PSMD11 knockdown affected the abundance of PER2 and CRY2 proteins and the nuclear translocation of CRY1. This changed the relative abundance of CRY1 and CRY2 in the nucleus. Thus, this work defines the role of PSMD11 in the NRON complex regulating the nuclear translocation of circadian repressors, thereby enabling cellular circadian oscillations.

A strategy to enhance the insecticidal potency of Vip3Aa by introducing additional cleavage sites to increase its proteolytic activation efficiency

Microbially derived, protein-based biopesticides have become a vital element in pest management strategies. Vip3 family proteins from Bacillus thuringiensis have distinct characteristics from known insecticidal Cry toxins and show efficient insecticidal activity against several detrimental lepidopteran pests. They are considered to be a promising toxic candidate for the management of various detrimental pests. In this study, we found that in addition to the preliminary digestion sites lysine, there are multiple cleavage activation sites in the linker region between domain I (DI) and DII of Vip3Aa. We further demonstrated that by adding more cleavage sites between DI and DII of Vip3Aa, its proteolysis efficiency by midgut proteases can be significantly increased, and correspondingly enhance its insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera larvae. Our study promotes the understanding of the insecticidal mechanism of Vip3 proteins and illustrates an easily implementable strategy to increase the insecticidal potency of Vip3Aa. This facilitates their potential future development and efficient application for sustainable agriculture.

Different maize varieties have greater impact on Tetranychus urticae (Acari: Tetranychidae) than GE maize expressing Cry3Bb1 insecticidal protein

Genetically engineered (GE) maize expressing Bacillus thuringiensis Cry3Bb1 protein was developed to control Diabrotica spp. (Coleoptera: Chrysomelidae). However, Cry proteins have been reported to have effects on non-target arthropods. We therefore investigated whether the non-target pest Tetranychus urticae (Acari: Tetranychidae) was negatively affected by GE maize expressing the insecticidal Cry3Bb1 protein. Five treatments were used in the laboratory tests to determine the life history parameters of T. urticae on leaves of field-grown maize varieties: (1) GE maize MON 88017, (2) isogenic maize, (3) isogenic maize protected with the soil applied insecticide chlorpyrifos (Dursban 10G), and two unrelated varieties (4) Kipous and (5) PR38N86. Newly emerged T. urticae larvae were individually released on the upper surface of leaf discs placed on water saturated cotton wool. Immatures and adults survival, duration of developmental stages and female fecundity were recorded daily until T. urticae died. Age–stage, two-sex life table method and test for trends, revealed no significant differences in 13 of 18 studied parameters. The unrelated varieties Kipous and PR38N86 on one side and maize with the same genetic background, namely GE maize and isogenic maize with or without insecticide protection, on the other side, showed significant variations in male longevity, larval survival rate, preoviposition period, and fecundity. In addition to the differences between varieties, GE maize and insecticide-protected isogenic maize showed a substantial difference in age-specific fecundity, but not in the mean number of eggs laid by females.The obtained results do not indicate that consumption of Cry3Bb1 has negative effect on T. urticae and suggest that GE maize does not pose a risk with respect to the non-target mite pest T. urticae. The results may have implications for the approval and renewal of import and cultivation for GE crop in the European Union.

Cry41-Related Mutants against Myzus persicae Based on Its Interaction with Cathepsin B

Cry toxins produced by Bacillus thuringiensis (Bt) are toxic to Lepidoptera, Coleoptera, and Diptera, but display very low activity against aphids. Recently, Cry41-related toxin was found to show moderate toxicity against Myzus persicae with the 50% lethal concentration (LC50) of 32.7 μg/mL, and its mode of action was proposed to be the acceleration of the apoptosis of aphid cells by enhancing Cathepsin B activity. This study focused on constructing Cry41-related mutants against M. persicae based on its interaction with Cathepsin B. First, eight key interacting residues in Cry41-related toxin were identified using alanine scanning and site-directed saturation mutagenesis. Subsequently, the positive mutant Cry41-7M protein (mutations of Gly48, Ile59, Lys364, Gln367, Gln377, Tyr378, and Ser400 to Tyr, Ala, Arg, Lys, Lys, Lys, and Ala in Cry41-related toxin, respectively) and the negative mutant Cry41-6A protein (mutations of Gly48, Lys364, Gln367, Gln377, and Tyr378 to Ala and mutation of Pro453 to Glu in Cry41-related toxin) were constructed, expressed, and purified. We then found that Cry41-7M protein performed slightly stronger than Cry41-related toxin in enhancing the enzymatic activity of Cathepsin B, whereas Cry41-6A protein did not affect Cathepsin B activity. A further bioassay showed that the mortality caused by Cry41-7M protein (LC50 = 19.144 μg/mL) was marginally higher than that of Cry41-related toxin, while Cry41-6A protein caused a decreased mortality (LC50 = 42.478 μg/mL). These results are expected to open new avenues for improving Cry aphidicidal activity.

Key amino acids residues enhance the ability of CpcR to activate cry gene expression in Bacillus thuringiensis

Typical Bacillus thuringiensis (Bt) produces one or more parasporal crystals composed of insecticidal Cry proteins during the sporulation, and the parasporal crystals and spores are produced from the same cell. Strain Bt LM1212 is different from typical Bt strains in that its crystals and spores are produced in different cells. Previous studies have found that the cell differentiation process of Bt LM1212 is related to the transcription factor CpcR which activates the cry-gene promoters. In addition, CpcR could activate the Bt LM1212 cry35-like gene promoter (P35) when introduced in the heterologous HD73- strain. It was shown that P35 was only activated in non-sporulating cells. In this study, the peptidic sequences of CpcR homologous proteins found in other strains of the Bacillus cereus group were used as references to identify two key amino acid sites for CpcR activity. The function of these amino acids was investigated by measuring P35 activation by CpcR in strain HD73-. These results will lay a foundation for the optimization of the insecticidal protein expression system in non-sporulating cells.

Bacillus thuringiensis Cyt Proteins as Enablers of Activity of Cry and Tpp Toxins against Aedes albopictus.

Aedes albopictus is a species of mosquito, originally from Southeast Asia, that belongs to the Culicidae family and the Dipteran insect order. The distribution of this vector has rapidly changed over the past decade, making most of the temperate territories in the world vulnerable to important human vector-borne diseases such as dengue, yellow fever, zika or chikungunya. Bacillus thuringiensis var. israeliensis (Bti)-based insecticides represent a realistic alternative to the most common synthetic insecticides for the control of mosquito larvae. However, several studies have revealed emerging resistances to the major Bti Crystal proteins such as Cry4Aa, Cry4Ba and Cry11Aa, making the finding of new toxins necessary to diminish the exposure to the same toxicity factors overtime. Here, we characterized the individual activity of Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa against A. albopictus and found a new protein, Cyt1A-like, that increases the activity of Cry11Aa more than 20-fold. Additionally, we demonstrated that Cyt1A-like facilitates the activity three new Bti toxins: Cry53-like, Cry56A-like and Tpp36-like. All in all, these results provide alternatives to the currently available Bti products for the control of mosquito populations and position Cyt proteins as enablers of activity for otherwise non-active crystal proteins.

In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Magnitude and Extent of Helicoverpa zea Resistance Levels to Cry1Ac and Cry2Ab2 across the Southeastern USA.

After resistance is first detected, continued resistance monitoring can inform decisions on how to effectively manage resistant populations. We monitored for resistance to Cry1Ac (2018 and 2019) and Cry2Ab2 (2019) from southeastern USA populations of Helicoverpa zea. We collected larvae from various plant hosts, sib-mated the adults, and tested neonates using diet-overlay bioassays and compared them to susceptible populations for resistance estimates. We also compared LC50 values with larval survival, weight and larval inhibition at the highest dose tested using regression, and found that LC50 values were negatively correlated with survival for both proteins. Finally, we compared resistance rations between Cry1Ac and Cry2Ab2 during 2019. Some populations were resistant to Cry1Ac, and most were resistant to CryAb2; Cry1Ac resistance ratios were lower than Cry2Ab2 during 2019. Survival was positively correlated with larval weight inhibition for Cry2Ab. This contrasts with other studies in both the mid-southern and southeastern USA, where resistance to Cry1Ac, Cry1A.105, and Cry2Ab2 increased over time and was found in a majority of populations. This indicates that cotton expressing Cry proteins in the southeastern USA was at variable risk for damage in this region.

Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila.

Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions.

Targeted innovative design of Bt Cry toxin insecticidal mimics

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton.

The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major global pest of cotton. Current management practices include chemical insecticides, cultural strategies, sterile insect releases, and transgenic cotton producing crystalline (Cry) protein toxins of the bacterium Bacillus thuringiensis (Bt). These strategies have contributed to the eradication of P. gossypiella from the cotton-growing areas of the United States and northern Mexico. However, this pest has evolved resistance to Bt cotton in Asia, where it remains a critical pest, and the benefits of using transgenic Bt crops have been lost. A complete annotated reference genome is needed to improve global Bt resistance management of the pink bollworm. We generated the first chromosome-level genome assembly for pink bollworm from a Bt-susceptible laboratory strain (APHIS-S) using PacBio continuous long reads for contig generation, Illumina Hi-C for scaffolding, and Illumina whole-genome re-sequencing for error correction. The pseudo-haploid assembly consists of 29 autosomes and the Z sex chromosome. The assembly exceeds the minimum Earth BioGenome Project quality standards, has a low error rate, is highly contiguous at both the contig and scaffold levels (L/N50 of 18/8.26 MB and 14/16.44 MB, respectively), and is complete, with 98.6% of lepidopteran single-copy orthologs represented without duplication. The genome was annotated with 50% repeat content and 14,107 protein-coding genes, further assigned to 41,666 functional annotations. This assembly represents the first publicly available complete annotated genome of pink bollworm and will serve as the foundation for advancing molecular genetics of this important pest species.

NMR-Based Metabolomics: A New Paradigm to Unravel Defense-Related Metabolites in Insect-Resistant Cotton Variety through Different Multivariate Data Analysis Approaches.

Cotton (Gossypium hirsutum) is an economically important crop and is widely cultivated around the globe. However, the major problem of cotton is its high vulnerability to biotic and abiotic stresses. It has been around three decades since the cotton plant was genetically engineered with genes encoding insecticidal proteins (mainly Cry proteins) with an aim to protect it against insect attack. Several studies have been reported on the impact of these genes on cotton production and fiber quality. However, the metabolites responsible for conferring resistance in genetically modified cotton need to be explored. The current work aims to unveil the key metabolites responsible for insect resistance in Bt cotton and also compare the conventional multivariate analysis methods with deep learning approaches to perform clustering analysis. We aim to unveil the marker compounds which are responsible for inducing insect resistance in cotton plants. For this purpose, we employed 1H-NMR spectroscopy to perform metabolite profiling of Bt and non-Bt cotton varieties, and a total of 42 different metabolites were identified in cotton plants. In cluster analysis, deep learning approaches (linear discriminant analysis (LDA) and neural networks) showed better separation among cotton varieties compared to conventional methods (principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLSDA)). The key metabolites responsible for inter-class separation were terpinolene, α-ketoglutaric acid, aspartic acid, stigmasterol, fructose, maltose, arabinose, xylulose, cinnamic acid, malic acid, valine, nonanoic acid, citrulline, and shikimic acid. The metabolites which regulated differently with the level of significance p < 0.001 amongst different cotton varieties belonged to the tricarboxylic acid cycle (TCA), Shikimic acid, and phenylpropanoid pathways. Our analyses underscore a biosignature of metabolites that might involve in inducing insect resistance in Bt cotton. Moreover, novel evidence from our study could be used in the metabolic engineering of these biological pathways to improve the resilience of Bt cotton against insect/pest attacks. Lastly, our findings are also in complete support of employing deep machine learning algorithms as a useful tool in metabolomics studies.

Baseline susceptibility of cabbage butterfly, Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae) to Bacillus thuringiensis (Berliner) Cry toxins in Meghalaya

BackgroundCabbage butterfly, Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae), is one of the most important pests of cabbage and other cruciferous crops and accounts for > 40% yield reduction in the crops. An investigation on the baseline susceptibility of P. brassicae to Bacillus thuringiensis (Bt) Cry toxins in Meghalaya was evaluated for future exploitation in Bt resistance monitoring. Two different Cry toxins, Bt Cry1C and Cry2Ab, were screened against 11 different field populations of P. brassicae from the state of Meghalaya. LC50 was evaluated based on the response of larval mortality of P. brassicae using the leaf-dip bioassay method.ResultsThe baseline-susceptibility tests conducted on P. brassicae in 11 different field populations from Meghalaya revealed that Smit population strains seemed to show less tolerance to both the Bt Cry toxins (Cry1C and Cry2Ab). Compared to the Cry1C toxin, Cry2Ab was found more potent against P. brassicae. The median lethal concentrations, LC50 72 h, varied from 0.535 to 1.725 µg/ml for Cry2Ab and 0.546–1.803 µg/ml for Cry1C toxin. The screening using leaf-dip bioassay resulted in a tolerance ratio of 3.3-fold and 3.2-fold for Cry1C and Cry2Ab, respectively. The most tolerant strains of P. brassicae from Umiam and Pepbah regions were observed to show discriminating concentrations of 19.30 µg/ml for Cry1C and 24.03 µg/ml for Cry2Ab (LC99, 72 h).ConclusionsThe Cry2Ab toxin was found to be more virulent than Cry1C toxin for P. brassicae. Certain candidate discriminating concentrations for Cry1C and Cry2Ab can be used as benchmarks for future resistance monitoring of P. brassicae to Bt Cry toxins.

Resistance Allele Frequency of Helicoverpa zea to Vip3Aa Bacillus thuringiensis Protein in the Southeastern U.S.

Helicoverpa zea is a major target pest of Bt crops expressing Cry and/or Vip3Aa proteins in the U.S.A. Widespread practical resistance of H. zea to the Cry1 and Cry2 proteins makes Vip3Aa the only effective Bt protein against this pest. Understanding the frequency of resistance alleles against Vip3Aa in field populations of H. zea is crucial for resistance management and the sustainability of Vip3Aa technology. Using a modified F2 screen method by crossing susceptible laboratory female moth with feral male moth of H. zea, we successfully screened a total of 24,576 neonates from 192 F2 families of H. zea collected from Arkansas, Louisiana, Mississippi, and Tennessee during 2019-2020. We found five F2 families containing ≥3rd instar survivors on the diagnostic concentration of 3.0 µg/cm2 Vip3Aa39. Dose-response bioassays confirmed the high levels of Vip3Aa resistance in these F2 families, with an estimated resistance ratio of >909.1-fold relative to the susceptible strain. The estimated resistance allele frequency against Vip3Aa in H. zea for these four southern states is 0.0155 with a 95% CI of 0.0057-0.0297. These data should provide critical information for understanding the risks of Vip3Aa resistance in H. zea and help design appropriate resistance management strategies for the sustainability of the Vip3Aa technology.

RNAi-based knockdown of candidate gut receptor genes altered the susceptibility of Spodoptera frugiperda and S. litura larvae to a chimeric toxin Cry1AcF.

A multitude of Cry toxins (secreted by Bacillus thuringiensis or Bt) has been deployed globally either via transgenic mean or bio-pesticidal formulations in order to manage insect pests. However, Bt resistance development in insects is emerging as a major concern. To avoid this problem, multiple gene pyramiding or protein-engineered chimeric toxin-based strategy has been analyzed. In the present study, one such chimeric toxin Cry1AcF (contain the swapped domains of Cry1Ac and Cry1F) was used to investigate its in vivo pathogenesis process in lepidopteran pests Spodoptera frugiperda and S. litura. A number of biochemical and molecular analysis were performed. Oral ingestion of Cry1AcF caused greater toxicity in S. frugiperda than S. litura with larvae displaying increased hemolymph melanization. Histopathology of the midgut transverse sections exhibited Cry1AcF-induced extensive gut damage in both the test insects followed by cytotoxicity in terms of reduced hemocyte numbers and viability. Elevated hemolymph phenoloxidase activity indicated the immune-stimulatory nature of Cry1AcF. In order to analyze the role of gut receptor proteins in Cry1AcF intoxication in test insects, we performed RNAi-mediated silencing using bacterially-expressed dsRNAs of individual receptor-encoding genes including CAD, ABCC2, ALP1 and APN. Target-specific induced downregulation of receptor mRNAs differentially altered the insect susceptibility to Cry1AcF toxin in our study. The susceptibility of ALP1 and APN dsRNA pre-treated S. frugiperda was considerably decreased when treated with Cry1AcF in LD50 and LD90 doses, whereas susceptibility of CAD and ABCC2 dsRNA pre-treated S. litura was significantly reduced when ingested with Cry1AcF in different doses. CAD/ABCC2-silenced S. frugiperda and ALP1/APN-silenced S. litura were vulnerable to Cry1AcF alike of control larvae. In conclusion, our results indicate ALP1/APN and CAD/ABCC2 as the functional receptor for Cry1AcF toxicity in S. frugiperda and S. litura, respectively.

Utilization of a strong promoter combined with the knockout of protease genes to improve the yield of Vip3Aa in Bacillus thuringiensis BMB171.

Vip3Aa is an insecticidal protein secreted by some Bacillus thuringiensis strains during vegetative growth. It has excellent insecticidal activity, its mechanism of action is different from that of Cry protein, and it can delay the development of pest resistance. To date, Vip3Aa has been widely used in genetically modified Bt crops. However, the secretion of Vip3Aa by industrial production strains is usually very low. Moreover, most of the Vip3Aa in the medium is degraded by proteases, limiting its application as a biopesticide. We report a novel constitutive strong promoter from B. thuringiensis, Prsi , which directs the abundant expression of vip3Aa in B. thuringiensis BMB171. Furthermore, to reduce the degradation of Vip3Aa caused by proteases, we constructed B. thuringiensis mutants in which different protease genes were knocked out. We found that the degradation of Vip3Aa was greatly inhibited and its yield was significantly improved in a mutant that lacked all three protease genes. Our results provide a new strategy to enhance the production of Vip3Aa in B. thuringiensis and have reference value for the research and development of novel bioinsecticides. © 2023 Society of Chemical Industry.

Protection of HSP60 in Myzus persicae Treated with Cry7Ab4 Toxin

Heat shock proteins (HSPs) are essential for the survival and development of animals under various stresses. However, little is known about the function of HSPs in insects response to Bacillus thuringiensis (Bt) toxins treatment. Here, we investigated the role of HSP60 in Myzus persicae (M. persicae) treated with an active Cry7Ab4 toxic core. First, we demonstrated the insensitivity of M. persicae to the Cry7Ab4 toxic core through a membrane capsule method. Then, using protein pull-down assay, several putative Cry7Ab4-binding proteins, including HSP60, were identified in an M. persicae nymph. P-loop GTPase Obg-like ATPase-1 (OLA1) was also found to be a Cry2Ab12-binding protein (unpublished data). Subsequent enzymatic and RT-qPCR assays revealed that highly expressed HSP60 removed the enhanced OLA1 activity caused by Cry7Ab4. ELISA analysis confirmed the binding interactions between Cry7Ab4, HSP60, and OLA1. Interestingly, a combination of ELISA and molecular docking analysis further suggested that HSP60 could block the binding interaction between Cry7Ab4 and OLA1 via higher affinity with Cry7Ab4. Besides, the Jun N-terminal kinase (JNK) pathway was found to be activated. Overall, we proposed the model that HSP60 protects M. persicae from Cry7Ab4 toxin. The study implies that HSP60 can be a crucial factor in insect defense against Cry toxins.

Insecticidal and Potato Growth Stimulation Activity of Bacillus thuringiensis kurstaki HD-1

Bacillus thuringiensis (Bt) produces Cry toxins against pest insects. Cry proteins are conformed by domains related to pore formation and recognition of protein receptors. Plant-induced systemic resistance (ISR) is triggered due to pest attack, it could be activated by Bacillus sp. Tecia solanivora (Ts) is a potato pest, susceptible to Cry1Ac and Cry1B proteins. This paper indicates the endorsement of Bt kurstaki HD-1 (BtkHD1) in relation to Ts control (Cry1Ac and Cry1B proteins), potato growth promotion, and plant ISR due to pests related to the BtkHD1-potato system. To ensure that ongoing quality control of BtkHD1 was maintained, crystal synthesis (microscopy), cry1 genes presence, and Cry protein production were checked. Bioassays Ts larvae and potato plantlets and an in silico analysis of the hybrid Cry1Ac-Cry1Ba protein and potato ISR related to the BtkHD1 infl uence were performed. Bioassay on Ts larvae shows an LC50 of 536 ng/cm2 of diet. A potato growth promotion assay revealed the effect of BtkHD1 on the length and dry weight of stems. The prospective analysis took into account relevant factors affecting the biological function of the hybrid protein focused on domain II. In silico identification of 15 BtkHD1 proteins and 68 potato proteins related to plant ISR due to pests was completed. This project serves to validation of toxicity on Ts larvae and potato growth effect based on BtkHD1, including a forward analysis of the hybrid Cry1Ac1-Cry1Ba1, and proteins associated with this strain and potato for eliciting plant ISR due to pests.

The mechanism of Cry41-related toxin against Myzus persicae based on its interaction with Buchnera-derived ATP-dependent 6-phosphofructokinase.

Myzus persicae (Hemiptera: Aphididae) is one of the most notorious pests of many crops worldwide. Most Cry toxins produced by Bacillus thuringiensis show very low toxicity to M. persicae; however, a study showed that Cry41-related toxin had moderate toxic activity against M. persicae. In our previous work, potential Cry41-related toxin-binding proteins in M. persicae were identified, including cathepsin B, calcium-transporting ATPase, and Buchnera-derived ATP-dependent 6-phosphofructokinase (PFKA). Buchnera is an endosymbiont present in almost all aphids and it provides necessary nutrients for aphid growth. This study investigated the role of Buchnera-derived PFKA in Cry41-related toxicity against M. persicae. In this study, recombinant PFKA was expressed and purified, and in vitro assays revealed that PFKA bound to Cry41-related toxin, and Cry41-related toxin at 25 μg ml-1 significantly inhibited the activity of PFKA. In addition, when M. persicae was treated with 30 μg ml-1 of Cry41-related toxin for 24 h, the expression of dnak, a single-copy gene in Buchnera, was significantly decreased, indicating a decrease in the number of Buchnera. Our results suggest that Cry41-related toxin interacts with Buchnera-derived PFKA to inhibit its enzymatic activity and likely impair cell viability, resulting in a decrease in the number of Buchnera, and finally leading to M. persicae death. These findings open up new perspectives in our understanding of the mode of action of Cry toxins and are useful in helping improve Cry toxicity for aphid control. © 2023 Society of Chemical Industry.

Toxicity Evaluation and Genetic Improvement of Bacillus thuringiensis Isolated from Different Regions in Assiut, Egypt against Mosquito Larvae

This study aims to evaluate the toxicity and genetic improvement of Bacillus thuringiensis isolates. Isolates were obtained from soil, insect and water samples from different regions of Assiut, Egypt for biological control of mosquito larvae. B. thuringiensis colonies were identified based on morphological and then by PCR which detect the Cry toxic genes in the isolates. Bioassays were performed to evaluate the toxicity of different strains of B. thuringiensis against mosquito larvae such as (Culex spp). In general, 36 B. thuringiensis isolates were obtained (31 from soil, 4 from insects, and 1 from water). And they were all toxic to mosquito larvae with different mortality percentages from 7 to 97% after 48 hours. Isolate Am2 recorded the highest mortality percentage 97% and Mn3 lowest mortality percentage 7%. PCR revealed that Am2 isolate which caused the highest mortality encodes three different types of Cry toxins, Cyt1AA, Cry1Ac and Cry2Aa. This isolate Am2 was examined by scanning electron microscopy to observe the shape of the Cry proteins. The results showed that the Am2 isolate contained of spherical and cuboidal toxic proteins. Then UV-mutagenesis was performed on the Am2 isolate to improve its toxicity. Out of 30 obtained UV-mutants, only one mutant showed improvement in the mortality of mosquito larvae since it caused a mortality rate of 100%. The results of the present study revealed the larvicidal efficacy of B. thuringiensis (Am2) isolate found in the soil of Assiut, could be used in biological control program of mosquito larvae.