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.

Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Type 1 secretion necessitates a tight interplay between all domains of the ABC transporter.

Type I secretion systems (T1SS) facilitate the secretion of substrates in one step across both membranes of Gram-negative bacteria. A prime example is the hemolysin T1SS which secretes the toxin HlyA. Secretion is energized by the ABC transporter HlyB, which forms a complex together with the membrane fusion protein HlyD and the outer membrane protein TolC. HlyB features three domains: an N-terminal C39 peptidase-like domain (CLD), a transmembrane domain (TMD) and a C-terminal nucleotide binding domain (NBD). Here, we created chimeric transporters by swapping one or more domains of HlyB with the respective domain(s) of RtxB, a HlyB homolog from Kingella kingae. We tested all chimeric transporters for their ability to secrete pro-HlyA when co-expressed with HlyD. The CLD proved to be most critical, as a substitution abolished secretion. Swapping only the TMD or NBD reduced the secretion efficiency, while a simultaneous exchange abolished secretion. These results indicate that the CLD is the most critical secretion determinant, while TMD and NBD might possess additional recognition or interaction sites. This mode of recognition represents a hierarchical and extreme unusual case of substrate recognition for ABC transporters and optimal secretion requires a tight interplay between all domains.

New Insights into Interactions between Mushroom Aegerolysins and Membrane Lipids.

Aegerolysins are a family of proteins that recognize and bind to specific membrane lipids or lipid domains; hence they can be used as membrane lipid sensors. Although aegerolysins are distributed throughout the tree of life, the most studied are those produced by the fungal genus Pleurotus. Most of the aegerolysin-producing mushrooms code also for proteins containing the membrane attack complex/perforin (MACPF)-domain. The combinations of lipid-sensing aegerolysins and MACPF protein partners are lytic for cells harboring the aegerolysin membrane lipid receptor and can be used as ecologically friendly bioinsecticides. In this work, we have recombinantly expressed four novel aegerolysin/MACPF protein pairs from the mushrooms Heterobasidion irregulare, Trametes versicolor, Mucidula mucida, and Lepista nuda, and compared these proteins with the already studied aegerolysin/MACPF protein pair ostreolysin A6-pleurotolysin B from P. ostreatus. We show here that most of these new mushroom proteins can form active aegerolysin/MACPF cytolytic complexes upon aegerolysin binding to membrane sphingolipids. We further disclose that these mushroom aegerolysins bind also to selected glycerophospholipids, in particular to phosphatidic acid and cardiolipin; however, these interactions with glycerophospholipids do not lead to pore formation. Our results indicate that selected mushroom aegerolysins show potential as new molecular biosensors for labelling phosphatidic acid.

Cry Toxins Use Multiple ATP-Binding Cassette Transporter Subfamily C Members as Low-Efficiency Receptors in Bombyx mori.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

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

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

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

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

Safety attributes of Pseudomonas sp. P26, an environmental microorganism with potential application in contaminated environments.

Currently, the selection of non-pathogenic microorganisms that lack clinically relevant antimicrobial resistance is crucial to bioaugmentation strategies. Pseudomonas sp. P26 (P26) is an environmental bacterium of interest due to its ability to remove aromatic compounds from petroleum, but its safety characteristics are still unknown. The study aimed to: a) determine P26 sensitivity to antimicrobials, b) investigate the presence of quinolone and ß-lactam resistance genes, c) determine the presence of virulence factors, and d) evaluate the effect of P26 on the viability of Galleria mellonella (an invertebrate animal model). P26 antimicrobial sensitivity was determined in vitro using the Kirby-Bauer agar diffusion method and the VITEK 2 automated system (BioMerieux®). Polymerase Chain Reaction was employed for the investigation of genes associated with quinolone resistance, extended-spectrum ß-lactamases, and carbapenemases. Hemolysin and protease production was determined in human blood agar and skimmed-milk agar, respectively. In the in vivo assay, different doses of P26 were injected into Galleria mellonella larvae and their survival was monitored daily. Control larvae injected with Pseudomonas putida KT2440 (a strain considered as safe) and Pseudomonas aeruginosa PA14 (a pathogenic strain) were included. Pseudomonas sp. P26 was susceptible to most evaluated antimicrobials, except for trimethoprim-sulfamethoxazole. No epidemiologically relevant genes associated with quinolone and ß-lactam resistance were identified. Hemolysin and protease production was only evidenced in the virulent strain (PA14). Furthermore, the results obtained in the in vivo experiment demonstrated that inocula less than 108 CFU/mL of P26 and P. putida KT2440 did not significantly affect larval survival, whereas larvae injected with the lowest dose of the pathogenic strain P. aeruginosa PA14 experienced instant mortality. The results suggest that Pseudomonas sp. P26 is a safe strain for its application in environmental bioremediation processes. Additional studies will be conducted to ensure the safety of this bacterium against other organisms.

A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

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.

Electro-osmotic flow in nanoconfinement: Solid-state and protein nanopores.

Electro-osmotic flow (EOF) is a phenomenon where fluid motion occurs in porous materials or micro/nano-channels when an external electric field is applied. In the particular example of single-molecule electrophoresis using single nanopores, the role of EOF on the translocation velocity of the analyte molecule through the nanopore is not fully understood. The complexity arises from a combination of effects from hydrodynamics in restricted environments, electrostatics emanating from charge decorations and geometry of the pores. We address this fundamental issue using the Poisson-Nernst-Planck and Navier-Stokes (PNP-NS) equations for cylindrical solid-state nanopores and three representative protein nanopores (α-hemolysin, MspA, and CsgG). We present the velocity profiles inside the nanopores as a function of charge decoration and geometry of the pore and applied electric field. We report several unexpected results: (a) The apparent charges of the protein nanopores are different from their net charge and the surface charge of the whole protein geometry, and the net charge of inner surface is consistent with the apparent charge. (b) The fluid velocity depends non-monotonically on voltage. The three protein nanopores exhibit unique EOF and velocity-voltage relations, which cannot be simply deduced from their net charge. Furthermore, effective point mutations can significantly change both the direction and the magnitude of EOF. The present computational analysis offers an opportunity to further understand the origins of the speed of transport of charged macromolecules in restricted space and to design desirable nanopores for tuning the speed of macromolecules through nanopores.

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.

The lipid-binding D4 domain of perfringolysin O facilitates the active loading of exogenous cargo into extracellular vesicles.

Whereas extracellular vesicles (EVs) have been engineered for cargo loading, innovative strategies for it can still be developed. Here, we describe domain 4 (D4), a cholesterol-binding domain derived from perfringolysin O, as a viable candidate for EV cargo loading. D4 and its mutants localized to the plasma membrane and the membranes of different vesicular structures in the cytoplasm, and facilitate the transport of proteins of interest (POIs) into EVs. D4-EVs were internalized by recipient cells analogous to EVs engineered with CD9. Intracellular cargo discharge from D4-EVs was successfully detected with the assistance of vesicular stomatitis virus glycoprotein. This study presents a novel strategy for recruiting POIs into EVs via a lipid-binding domain that ensures content release in recipient cells.

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.

Decoration of Burkholderia Hcp1 protein to virus-like particles as a vaccine delivery platform.

Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei. In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG1, IgG2c, and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species.