Transcriptomic Analysis of the Response of the Dioryctria abietella Larva Midgut to Bacillus thuringiensis 2913 Infection.

Dioryctria abietella Denis Schiffermuller (Lepidoptera: Pyralidae) is an oligophagous pest that mainly damages Pinaceae plants. Here, we investigated the effects of the Bacillus thuringiensis 2913 strain (Bt 2913), which carries the Cry1Ac, Cry2Ab, and Vip3Aa genes, on the D. abietella midgut transcriptome at 6, 12, and 24 h after infection. In total, 7497 differentially expressed genes (DEGs) were identified from the midgut transcriptome of D. abietella larvae infected with Bt 2913. Among these DEGs, we identified genes possibly involved in Bt 2913-induced perforation of the larval midgut. For example, the DEGs included 67 genes encoding midgut proteases involved in Cry/Vip toxin activation, 74 genes encoding potential receptor proteins that bind to insecticidal proteins, and 19 genes encoding receptor NADH dehydrogenases that may bind to Cry1Ac. Among the three transcriptomes, 88 genes related to metabolic detoxification and 98 genes related to immune defense against Bt 2913 infection were identified. Interestingly, 145 genes related to the 60S ribosomal protein were among the DEGs identified in the three transcriptomes. Furthermore, we performed bioinformatic analysis of zonadhesin, GST, CYP450, and CarE in the D. abietella midgut to determine their possible associations with Bt 2913. On the basis of the results of this analysis, we speculated that trypsin and other serine proteases in the D. abietella larval midgut began to activate Cry/Vip prototoxin at 6 h to 12 h after Bt 2913 ingestion. At 12 h after Bt 2913 ingestion, chymotrypsin was potentially involved in degrading the active core fragment of Vip3Aa toxin, and the detoxification enzymes in the larvae contributed to the metabolic detoxification of the Bt toxin. The ABC transporter and several other receptor-protein-related genes were also downregulated to increase resistance to Bt 2913. However, the upregulation of 60S ribosomal protein and heat shock protein expression weakened the resistance of larvae to Bt 2913, thereby enhancing the expression of NADH dehydrogenase and other receptor proteins that are highly expressed in the larval midgut and bind to activating toxins, including Cry1Ac. At 24 h after Bt 2913 ingestion, many activated toxins were bound to receptor proteins such as APN in the larval midgut, resulting in membrane perforation. Here, we clarified the mechanism of Bt 2913 infection in D. abietella larvae, as well as the larval immune defense response to Bt 2913, which provides a theoretical basis for the subsequent control of D. abietella using B. thuringiensis.

Resistance to Cry14A family Bacillus thuringiensis crystal proteins in Caenornabditis elegans operates via the nhr-31 transcription factor and vacuolar-type ATPase pathway.

Bacillus thuringiensis (Bt) has been successfully used commercially for more than 60 years for biocontrol of insect pests. Since 1996, transgenic plants expressing Bt crystal (Cry) proteins have been used commercially to provide protection against insects that predate on corn and cotton. More recently, Bt Cry proteins that target nematodes have been discovered. One of these, Cry14Ab, has been expressed in transgenic soybean plants and found to provide significant protection against the soybean cyst nematode, Heterodera glycines. However, to date there has been no description of high-level resistance to any Cry14A family protein in nematodes. Here, we describe forward genetic screens to identify such mutants using the nematode Caenorhabditis elegans. Although non-conditional screens failed to identify highly resistant C. elegans, a conditional (temperature-sensitive) genetic screen identified one mutant, bre-6(ye123) (for Bt protein resistant), highly resistant to both Cry14Aa and Cry14Ab. The mutant comes at a high fitness cost, showing significant delays in growth and development and reduced fecundity. bre-6(ye123) hermaphrodites are only weakly resistant to copper intoxication, indicating that the mutant is not highly resistant to all insults. Backcrossing-whole genome sequencing was used to identify the gene mutated in ye123 as the nuclear hormone receptor nhr-31. RNAi, DNA rescue, and CRISPR analyses confirm that resistance to Cry14Aa intoxication in bre-6(ye123) is due to mutation of nhr-31 and was renamed nhr-31(ye123). As predicted for a mutation in this gene, nhr-31(ye123) animals showed significantly reduced expression of most of the subunits of the C. elegans vacuolar ATPase (vATPase). Mutants in the vATPase subunits unc-32 and vha-7 also show resistance to Cry14Aa and/or Cry14Ab. These data demonstrate that nhr-31 and the vATPase play a significant role in the intoxication of C. elegans by Cry14A family proteins, that reduction in vATPase levels result in high resistance to Cry14A family proteins, and that such resistance comes at a high fitness cost. Based on the relative difficulty of finding resistant mutants and the fitness cost associated with the vATPase pathway, our data suggest that transgenic Cry14Ab plants may hold up well to resistance by nematode parasites.

Utility of Cry1Ja for Transgenic Insect Control.

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

Death-Associated LIM-Only Protein Reduces Cry1Ac Toxicity by Sequestration of Cry1Ac Protoxin and Activated Toxin in Helicoverpa armigera.

The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP (H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.

Efficient control of root-knot nematodes by expressing Bt nematicidal proteins in root leucoplasts.

Root-knot nematodes (RKNs) are plant pests that infect the roots of host plants. Bacillus thuringiensis (Bt) nematicidal proteins exhibited toxicity to nematodes. However, the application of nematicidal proteins for plant protection is hampered by the lack of effective delivery systems in transgenic plants. In this study, we discovered the accumulation of leucoplasts (root plastids) in galls and RKN-induced giant cells. RKN infection causes the degradation of leucoplasts into small vesicle-like structures, which are responsible for delivering proteins to RKNs, as observed through confocal microscopy and immunoelectron microscopy. We showed that different-sized proteins from leucoplasts could be taken up by Meloidogyne incognita female. To further explore the potential applications of leucoplasts, we introduced the Bt crystal protein Cry5Ba2 into tobacco and tomato leucoplasts by fusing it with a transit peptide. The transgenic plants showed significant resistance to RKNs. Intriguingly, RKN females preferentially took up Cry5Ba2 protein when delivered through plastids rather than the cytosol. The decrease in progeny was positively correlated with the delivery efficiency of the nematicidal protein. In conclusion, this study offers new insights into the feeding behavior of RKNs and their ability to ingest leucoplast proteins, and demonstrates that root leucoplasts can be used for delivering nematicidal proteins, thereby offering a promising approach for nematode control.

A Shared Receptor Suggests a Common Ancestry between an Insecticidal Bacillus thuringiensis Cry Protein and an Anti-Cancer Parasporin.

Cry toxins, produced by the bacterium Bacillus thuringiensis, are of significant agronomic value worldwide due to their potent and highly specific activity against various insect orders. However, some of these pore-forming toxins display specific activity against a range of human cancer cells whilst possessing no known insecticidal activity; Cry41Aa is one such toxin. Cry41Aa has similarities to its insecticidal counterparts in both its 3-domain toxic core structure and pore-forming abilities, but how it has evolved to target human cells is a mystery. This work shows that some insecticidal Cry toxins can enhance the toxicity of Cry41Aa against hepatocellular carcinoma cells, despite possessing no intrinsic toxicity themselves. This interesting crossover is not limited to human cancer cells, as Cry41Aa was found to inhibit some Aedes-active Cry toxins in mosquito larval assays. Here, we present findings that suggest that Cry41Aa shares a receptor with several insecticidal toxins, indicating a stronger evolutionary relationship than their divergent activities might suggest.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti.

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58 kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58 kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.

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.

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.

Individual transmembrane domains of SfABCC2 from Spodoptera frugiperda do not serve as functional Cry1F receptors.

The fall armyworm (Spodoptera frugiperda) is a major global pest causing severe damage to various crops, especially corn. Transgenic corn producing the Cry1F pesticidal protein from the bacterium Bacillus thuringiensis (Cry1F corn) showed effectiveness in controlling this pest until S. frugiperda populations at locations in North and South America evolved practical resistance. The mechanism for practical resistance involved disruptive mutations in an ATP binding cassette transporter subfamily C2 gene (SfABCC2), which serves as a functional Cry1F receptor in the midgut cells of susceptible S. frugiperda. The SfABCC2 protein contains two transmembrane domains (TMD1 and TMD2), each with a cytosolic nucleotide (ATP) binding domain (NBD1 and NBD2, respectively). Previous reports have demonstrated that disruptive mutations in TMD2 were linked with resistance to Cry1F, yet whether the complete SfABCC2 structure is needed for receptor functionality or if a single TMD-NBD protein can serve as functional Cry1F receptor remains unknown. In the present study, we separately expressed TMD1 and TMD2 with their corresponding NBDs in cultured insect cells and tested their Cry1F receptor functionality. Our results show that the complete SfABCC2 structure is required for Cry1F receptor functionality. Moreover, binding competition assays revealed that Cry1F specifically bound to SfABCC2, whereas neither SfTMD1-NBD1 nor SfTMD2-NBD2 exhibited any significant binding. These results provide insights into the molecular mechanism of Cry1F recognition by SfABCC2 in S. frugiperda, which could facilitate the development of more effective insecticidal proteins.

N-Terminal α-Helices in Domain I of Bacillus thuringiensis Vip3Aa Play Crucial Roles in Disruption of Liposomal Membrane.

Bacillus thuringiensis Vip3 toxins form a tetrameric structure crucial for their insecticidal activity. Each Vip3Aa monomer comprises five domains. Interaction of the first four α-helices in domain I with the target cellular membrane was proposed to be a key step before pore formation. In this study, four N-terminal α-helix-deleted truncations of Vip3Aa were produced and, it was found that they lost both liposome permeability and insecticidal activity against Spodoptera litura. To further probe the role of domain I in membrane permeation, the full-length domain I and the fragments of N-terminal α-helix-truncated domain I were fused to green fluorescent protein (GFP), respectively. Only the fusion carrying the full-length domain I exhibited permeability against artificial liposomes. In addition, seven Vip3Aa-Cry1Ac fusions were also constructed by combination of α-helices from Vip3Aa domains I and II with the domains II and III of Cry1Ac. Five of the seven combinations were determined to show membrane permeability in artificial liposomes. However, none of the Vip3Aa-Cry1Ac combinations exhibited insecticidal activity due to the significant reduction in proteolytic stability. These results indicated that the N-terminal helix α1 in the Vip3Aa domain I is essential for both insecticidal activity and liposome permeability and that domain I of Vip3Aa preserved a high liposome permeability independently from domains II-V.

Involvement of miR-8510a-3p in response to Cry1Ac protoxin by regulating PxABCG3 in Plutella xylostella.

Overuse of insecticides has accelerated the evolution of insecticide resistance and created serious environmental concerns worldwide, thus incentivizing development of alternative methods. Bacillus thuringiensis (Bt) is an insecticidal bacterium that has been developed as a biopesticide to successfully control multiple species of pests. It operates by secreting several insect toxins such as Cry1Ac. However, metabolic resistance based on ATP-binding cassette (ABC) transporters may play a crucial role in the development of metabolic resistance to Bt. Here, we characterized an ABCG gene from the agricultural pest Plutella xylostella (PxABCG3) and found that it was highly expressed in a Cry1Ac-resistant strain, up-regulated after Cry1Ac protoxin treatment. Binding miR-8510a-3p to the coding sequence (CDS) of PxABCG3 was then confirmed by a luciferase reporter assay and RNA immunoprecipitation. miR-8510a-3p agomir delivery markedly reduced PxABCG3 expression in vivo and consequently decreased the tolerance of P. xylostella to Cry1Ac, while reduction of miR-8510a-3p significantly increased PxABCG3 expression, accompanied by an increased tolerance to Cry1Ac. Our results suggest that miR-8510a-3p could potentially be used as a novel molecular target against P. xylostella or other lepidopterans, providing novel insights into developing effective and environmentally friendly pesticides.

Toxic Effects of Bt-(Cry1Ab+Vip3Aa) Maize on Storage Pest Paralipsa gularis (Zeller).

Paralipsa gularis (Zeller) is a storage pest; however, in recent years it has evolved into a considerable maize pest during the late growth stage in the border region between China and other Southeast Asian countries. Bt transgenic insect-resistant maize is an effective measure in controlling a wide range of lepidopteran pests, but there is a lack of research on the toxic effects of storage pests. We tested the toxicity of Bt-Cry1Ab, Vip3Aa, and their complex proteins against P. gularis via bioassay and investigated the efficiency of Bt-(Cry1Ab+Vip3Aa) maize in controlling P. gularis during the late growth stage of maize in the period 2022-2023. The bioassay results show that the susceptibilities of P. gularis to the two Bt proteins and their complex proteins were significantly different. The LC50 values of DBNCry1Ab ("DBN9936" event), DBNVip3Aa ("DBN9501" event), DBN Cry1Ab+Vip3Aa ("DBN3601T" event), and Syngenta Cry1Ab+Vip3Aa ("Bt11" event × "MIR162" event) were 0.038 µg/g, 0.114 µg/g, 0.110 µg/g, and 0.147 µg/g, and the GIC50 values were 0.014 µg/g, 0.073 µg/g, 0.027 µg/g, and 0.026 µg/g, respectively. Determination of the expression content of the insecticidal protein in different tissues of Bt-(Cry1Ab+Vip3Aa) maize shows that the total Bt protein content in different tissues was in the following order: stalk > bract > cob > kernel. However, the bioassay results show that the mortalities of P. gularis feeding on Bt-(Cry1Ab+Vip3Aa) maize in different tissues at different growth stages were all above 93.00%. The field trial indicates that the occurrence density of larvae and plant damage rate for conventional maize were 422.10 individuals/100 plants and 94.40%, respectively, whereas no larvae were found on Bt-(Cry1Ab+Vip3Aa) maize. In summary, this study implies that Bt-(Cry1Ab+Vip3Aa) maize has a high potential for control of P. gularis, providing a new technical measure for the management of the pest.

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

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

RNA m6 A Methylation Suppresses Insect Juvenile Hormone Degradation to Minimize Fitness Costs in Response to A Pathogenic Attack.

Bioinsecticides and transgenic crops based on the bacterial pathogen Bacillus thuringiensis (Bt) can effectively control diverse agricultural insect pests, nevertheless, the evolution of resistance without obvious fitness costs has seriously eroded the sustainable use of these Bt products. Recently, it has been discovered that an increased titer of juvenile hormone (JH) favors an insect host (Plutella xylostella) to enhance fitness whilst resisting the Bt pathogen, however, the underlying regulatory mechanisms of the increased JH titer are obscure. Here, the involvement of N6 -methyladenosine (m6 A) RNA modification in modulating the availability of JH in this process is defined. Specifically, it is found that two m6 A methyltransferase subunit genes, PxMettl3 and PxMettl14, repress the expression of a key JH-degrading enzyme JH esterase (JHE) to induce an increased JH titer, mitigating the fitness costs associated with a robust defense against the Bt pathogen. This study identifies an as-yet uncharacterized m6 A-mediated epigenetic regulator of insect hormones for maintaining fitness during pathogen defense and unveils an emerging Bt resistance-related m6 A methylation atlas in insects, which further expands the functional landscape of m6 A modification and showcases the pivotal role of epigenetic regulation in host-pathogen interactions.

Cell wall-localized Bt protein endows rice high resistance to Lepidoptera pests.

BACKGROUND: The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP-Cry1Ca-CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects. RESULTS: The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single-copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 µg g-1 in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages. CONCLUSION: The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.

Synergism of Cry1Ca toxicity by gut resident Enterococcus spp. in the rice stem borer, Chilo suppressalis.

The bacterium Bacillus thuringiensis (Bt) is the most economically successful biopesticide to date, and Bt insecticidal proteins are produced in transgenic crops for pest control. However, relevant details in the Bt-mediated killing process remain undefined. In our previous research, we observed reduced larval susceptibility to Bt Cry1Ca in Chilo suppressalis, a major rice pest in China, after gut microbiota elimination. Here, we tested the hypothesis that gut microbiota, particularly abundant Enterococcus spp., influences C. suppressalis susceptibility to Cry1Ca. We isolated and identified four Enterococcus spp. from C. suppressalis gut microbiota and evaluated their impact on Cry1Ca toxicity. Among the four Enterococcus spp. identified, three of them (E. casseliflavus, E. faecalis, and E. mundtii) dramatically increased larval mortality when introduced in axenic C. suppressalis challenged with Cry1Ca. Gut epithelial damage by Cry1Ca promoted the translocation of Enterococcus spp. from the gut lumen into the hemocoel, where they proliferated and induced larval melanization and hemocyte apoptosis. Our combined findings demonstrate that the presence of specific gut microbiota can greatly affect susceptibility to Cry1Ca through melanization and apoptosis of hemocytes. Better understanding of the Bt intoxication process guides the development of bio-enhancers for Bt-based microbial biopesticides and potential improvement of transgenic crops.

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.