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.

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.

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.

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.

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.

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.

Highly Sensitive Detection of T790 M with a Three-Level Characteristic Current by Thymine-Hg(II)-Thymine in the α-Hemolysin Nanopore.

Sensitive detection of resistance mutation T790 M is of great significance for early diagnosis and prognostic monitoring of non-small-cell lung cancer (NSCLC). In this paper, we showed a highly sensitive detection strategy for T790 M using a three-level characteristic current signal pattern in an α-hemolysin nanopore. A probe was designed that formed a C-T mismatched base pair with wild-type/P and a T-T mismatched with the T790M/P. The T790M/P produced a unique three-level characteristic current signal in the presence of mercury ions(II): first, T790M-Hg2+-P entering the vestibule of α-HL under the transmembrane potential and overhang of probe occupying the ß-barrel, then probe unzipping from the T790M/P, T790 M temporally residing inside the nanocavity due to the interaction with Hg(II), and finally T790 M passing through the ß-barrel. The blocking current distribution was concentrated with a small relative standard deviation of about 3%, and the signal peaks of T790 M and wild-type can be completely separated with a high separation resolution of more than 2.5, which achieved the highly sensitive detection of T790 M down to 0.001 pM (confidence level P 95%) with a linear range from 0.001 pM to 1 nM in human serum samples. This highly sensitive recognition strategy enables the detection of low abundance T790 M and provides a method for prognostic monitoring in NSCLC patients.

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.

ß-Hemolysin, not agrA mutation, inhibits the hemolysis of α-hemolysin in Staphylococcus aureus laboratory and clinical strains.

Staphylococcus aureus produces various hemolysins regulated by the Agr-QS system, except ß-hemolysin encoded by the gene hlb. A classical laboratory S. aureus strain RN4220 displays only the ß-hemolysin phenotype. It was suspected that the 8A mutation at the end of its agrA gene delayed the expressions of hla and RNAIII, then failed to express α- and δ-hemolysins. However, hla gene expression was detected at the later culture time without α-hemolysin phenotype, the reason for such a phenotype has not been clearly understood. We created hlb knockout and complementary mutants via homologous recombination in RN4220 and NRS049, two strains that normally produce ß-hemolysin and carry agrA mutation. We found interestingly that the presence or absence of α-hemolysin phenotype in such strains depended on the expression of ß-hemolysin instead of agrA mutations, which only inhibited δ-hemolysin expression. The hemolysis phenotype was verified by the Christie-Atkinson-Munch-Peterson (CAMP) test. Quantitative reverse transcription PCR was carried out to evaluate the relative gene expressions of hlb, hla, and RNAIII. The construction of mutants did not affect the agrA mutation status. We demonstrate that the absence of α-hemolysin in S. aureus RN4220 and NRS049 strains is attributed to their production of ß-hemolysin instead of agrA mutation. Our findings broaden the understanding of the molecular mechanisms that control hemolysin expression in S. aureus that is crucial for the development of new therapeutic strategies to combat S. aureus infections. IMPORTANCE: α-Hemolysin is a critical virulence factor in Staphylococcus aureus and its expression is largely controlled by the Agr-QS system. Nonetheless, the hemolysis phenotype and the regulation of the Agr-QS system in S. aureus still hold many mysteries. Our study finds that it is the expression of ß- hemolysin rather than the agrA mutation that inhibits the function of the α-hemolysin in an important S. aureus strain RN4220 and a clinical strain presents a similar phenotype, which clarifies the misunderstood hemolytic phenotype and mechanism of S. aureus. Our findings highlight the interactions among different toxins and their biological roles, combined with QS system regulation, which is ultimately the true underlying cause of its virulence. This emphasizes the importance of considering the collaborative action of various factors in the infection process caused by this significant human pathogen.

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.

Bacillus thuringiensis Cry9Aa Insecticidal Protein Domain I Helices α3 and α4 Are Two Core Regions Involved in Oligomerization and Toxicity.

Bacillus thuringiensis Cry9 proteins show high insecticidal activity against different lepidopteran pests. Cry9 could be a valuable alternative to Cry1 proteins because it showed a synergistic effect with no cross-resistance. However, the pore-formation region of the Cry9 proteins is still unclear. In this study, nine mutations of certain Cry9Aa helices α3 and α4 residues resulted in a complete loss of insecticidal activity against the rice pest Chilo suppressalis; however, the protein stability and receptor binding ability of these mutants were not affected. Among these mutants, Cry9Aa-D121R, Cry9Aa-D125R, Cry9Aa-D163R, Cry9Aa-E165R, and Cry9Aa-D167R are unable to form oligomers in vitro, while the oligomers formed by Cry9Aa-R156D, Cry9Aa-R158D, and Cry9Aa-R160D are unstable and failed to insert into the membrane. These data confirmed that helices α3 and α4 of Cry9Aa are involved in oligomerization, membrane insertion, and toxicity. The knowledge of Cry9 pore-forming action may promote its application as an alternative to Cry1 insecticidal proteins.

JAK/STAT signaling regulated intestinal regeneration defends insect pests against pore-forming toxins produced by Bacillus thuringiensis.

A variety of coordinated host-cell responses are activated as defense mechanisms against pore-forming toxins (PFTs). Bacillus thuringiensis (Bt) is a worldwide used biopesticide whose efficacy and precise application methods limits its use to replace synthetic pesticides in agricultural settings. Here, we analyzed the intestinal defense mechanisms of two lepidopteran insect pests after intoxication with sublethal dose of Bt PFTs to find out potential functional genes. We show that larval intestinal epithelium was initially damaged by the PFTs and that larval survival was observed after intestinal epithelium regeneration. Further analyses showed that the intestinal regeneration caused by Cry9A protein is regulated through c-Jun NH (2) terminal kinase (JNK) and Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. JAK/STAT signaling regulates intestinal regeneration through proliferation and differentiation of intestinal stem cells to defend three different Bt proteins including Cry9A, Cry1F or Vip3A in both insect pests, Chilo suppressalis and Spodoptera frugiperda. Consequently, a nano-biopesticide was designed to improve pesticidal efficacy based on the combination of Stat double stranded RNA (dsRNA)-nanoparticles and Bt strain. This formulation controlled insect pests with better effect suggesting its potential use to reduce the use of synthetic pesticides in agricultural settings for pest control.

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin.

Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae.

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

PoreGlow: A split green fluorescent protein-based system for rapid detection of Listeria monocytogenes.

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 µg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.

Draft genome of neotropical Bacillus thuringiensis UFT038 and its potential against lepidopteran soybean pests.

Bacillus thuringiensis (Bt) is known for its Cry and Vip3A pesticidal proteins with high selectivity to target pests. Here, we assessed the potential of a novel neotropical Bt strain (UFT038) against six lepidopteran pests, including two Cry-resistant populations of fall armyworm, Spodoptera frugiperda. We also sequenced and analyzed the genome of Bt UFT038 to identify genes involved in insecticidal activities or encoding other virulence factors. In toxicological bioassays, Bt UFT038 killed and inhibited the neonate growth in a concentration-dependent manner. Bt UFT038 and HD-1 were equally toxic against S. cosmioides, S. frugiperda (S_Bt and R_Cry1 + 2Ab populations), Helicoverpa zea, and H. armigera. However, larval growth inhibition results indicated that Bt UFT038 was more toxic than HD-1 to S. cosmioides, while HD-1 was more active against Chrysodeixis includens. The draft genome of Bt UFT038 showed the cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Besides this, genes encoding the virulence factors (inhA, plcA, piplC, sph, and chi1-2) and toxins (alo, cytK, hlyIII, hblA-D, and nheA-C) were also identified. Collectively, our findings reveal the potential of the Bt UFT038 strain as a source of insecticidal genes against lepidopteran pests, including S. cosmioides and S. frugiperda.

Regulation of PhoB on biofilm formation and hemolysin gene hlyA and ciaR of Streptococcus agalactiae.

PhoB is a response regulator protein that plays a key role in the PhoBR two-component signal transduction system. In this study, we used transcriptome and proteomics techniques to evaluate the detect the gene network regulated by PhoB of Streptococcus agalactiae. The results showed that expression of biofilm formation and virulence-related genes were changed after phoB deficiency. Crystal violet and CLSM assay confirmed that the deletion of the phoB increased the thickness of S. agalactiae biofilm. The results of lacZ reporter and the bacterial one-hybridization method showed that PhoB could directly bind to the promoter regions of hemolysin A and ciaR genes but not to the promoter regions of cylE and hemolysin III. Through the construction of an 18-base pair deoxyribose nucleic acid (DNA) random fragment library and the bacterial one-hybridization system, it was found that the conservative sequence of PhoB binding was TTGGAGAA(G/T). Our research has uncovered the virulence potential of the PhoBR two-component system of S. agalactiae. The findings of this study provide the theoretical foundation for in-depth research on the pathogenic mechanism of S. agalactiae.

Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin.

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.

[Prokaryotic expression and biological activities of the hemolysin BL subunit of a pathogenic Bacillus cereus of cattle origin].

Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.