Insecticidal Crystal Proteins Research Articles

Efficacy of Native Bacillus thuringiensis against Mosquito Vector.

Larval source management is an effective measure to control mosquito-borne diseases. Bacillus thuringiensis produces specific insecticidal crystal proteins toxic to mosquito larvae. In many parts of the South East Asian region, Bacillus thuringiensis is used for larval source management. In Nepal, larvicidal Bacillus thuringiensis is not available. The study aims to isolate larvicidal Bacillus thuringiensis from soil samples of Nepal to control mosquitoes. Native Bacillus thuringiensis was obtained from soil samples by the acetate selection method. It was identified by observing crystal protein with Coomassie Brilliant Blue stain in a light microscope. The mosquito larvae were collected from different breeding habitats. A preliminary bioassay was performed by inoculating three loopful of 48 hours culture of spherical crystal protein producing Bacillus thuringiensis in a plastic cup containing 25 larvae and 100 ml of sterile distilled water. The cup was incubated at room temperature for 24 hours to observe the mortality of larvae. Further selective bioassay was performed with the isolate which showed 100% mortality, as described above in four replicates along with the negative and positive control. Out of 1385 Bacillus thuringiensis obtained from 454 soil samples, 766 (55.30%) were spherical crystal protein producers, among them, a single strain (14P2A) showed 100% mortality against mosquito larvae. The lethal concentration doses required to kill 50% and 90% of the larval population were 32.35 and 46.77 Parts per million respectively. The native Bacillus thuringiensis produces the crystal protein effective in killing mosquito larvae. The native Bacillus thuringiensis should be included as a tool to control mosquito-borne diseases in Nepal.

A Novel Regulator PepR Regulates the Expression of Dipeptidase Gene pepV in Bacillus thuringiensis.

Bacillus thuringiensis produces insecticidal crystal proteins encoded by cry or cyt genes and targets a variety of insect pests. We previously found that a strong promoter of a DeoR family transcriptional regulator (HD73_5014) can efficiently drive cry1Ac expression in B. thuringiensis HD73. Here, we investigated the regulation of neighbor genes by HD73_5014. The HD73_5014 homologs are widely distributed in Gram-positive bacterial species. Its neighbor genes include pepV, rsuA, and ytgP, which encode dipeptidase, rRNA pseudouridine synthase and polysaccharide biosynthesis protein, respectively. The four open reading frames (ORFs) are organized to be a pepR gene cluster in HD73. RT-PCR analysis revealed that the rsuA and ytgP genes formed a transcriptional unit (rsuA-ytgP operon), while pepV formed a transcriptional unit in HD73. Promoter-lacZ fusion assays showed that the pepV and rsuA-ytgP promoters are regulated by HD73_5014. EMSA experiments showed that HD73_5014 directly binds to the pepV promoter region but not to the rusA-ytgP promoter region. Thus, the HD73_5014 transcriptional regulator, which controls the expression of the dipeptidase pepV, was named PepR (dipeptidase regulator). We also confirmed the direct regulation between PepR and PepV by the increased sensitivity to vancomycin in ΔpepV and ΔpepR mutants compared to HD73.

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

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

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.

Analysis of Synergism between Extracellular Polysaccharide from Bacillus thuringensis subsp. kurstaki HD270 and Insecticidal Proteins.

Bacillus thuringiensis (Bt) is the most widely used biopesticide worldwide and can produce several insecticidal crystal proteins and vegetative insecticidal proteins (Vips) at different growth stages. In our previous study, extracellular polysaccharides (EPSs) of Bt strain HD270 were found to enhance the insecticidal activity of Cry1Ac protoxin against Plutella xylostella (L.) and promote the binding of Cry1Ac to the intestinal brush border membrane vesicles (BBMVs). Whether the synergistic activity of Bt EPSs is common to other Cry1-type or Vip proteins is unclear, as is the potential synergistic mechanism. In this study, crude EPS-HD270 was found to increase the toxicity of Cry1-type toxins and Vip3Aa11 against different lepidopteran pests by approximately 2-fold. The purified EPS-HD270 also possessed synergistic activity against the toxicity of Cry1Ac and Vip3Aa11 against Spodoptera frugiperda (J.E. Smith) and Helicoverpa armigera (Hübner). Furthermore, we found that EPS-HD270 had a strong binding ability with Vip3Aa11 and promoted the binding of Vip3Aa11 to the BBMVs of H. armigera and S. frugiperda. Bt EPS-HD270 also protected Vip3Aa11 from proteolytic processing in larval midgut juice. Bt EPSs had universal synergistic effects on Cry1-type or Vip toxins against S. frugiperda and H. armigera. Bt EPS-HD270 exhibited synergistic activity with Vip3Aa through promotion of binding to BBMVs and protection from digestion by midgut protease. The results indicated that synergistic activity with Bt toxins was an important function of Bt EPSs, which was very different from other Bacillus spp.

Transgenic cotton expressing Mpp51Aa2 does not adversely impact beneficial non-target hemiptera in the field

The modified Mpp51Aa2 (previously Cry51Aa2.834_16) insecticidal crystal protein derived from Bacillus thuringiensis (Bt) protects against feeding damage caused by targeted hemipteran and thysanopteran pests, and was transformed in cotton to produce the MON 88702 cotton event. As part of an ecological risk assessment, we evaluated the relative impact of MON 88702 on the abundance of predatory Hemiptera, compared to a conventional control. Field trials were established in 2018 at six sites within cotton production regions of the U.S. Specific arthropod populations in MON 88702 and its conventional control (DP393), grown under different insecticide regimes within a randomized complete block design, were assessed at each field site. Arthropod samples were collected 10 times, starting at early squaring and weekly thereafter, over the course of the season at each site, using vertical beat sheets, sweep nets and visual counts. Across the sites, no significant differences were detected in abundance of predatory Hemiptera (Orius spp., Geocoris spp., Nabis spp., and Zelus spp.) between unsprayed MON 88702 and the unsprayed conventional control. In contrast, a broad-spectrum insecticide treatment significantly reduced the abundance of these and other taxa. Consistent with laboratory studies, the lack of differences between unsprayed MON 88702 and the unsprayed control indicates that the cultivation of MON 88702 is unlikely to adversely impact the predatory function associated with these beneficial Hemiptera in the cotton agro ecosystem.

A sub-chronic feeding study of dual toxin insect-resistant transgenic maize (CEMB-413) on Wistar rats.

Genetically modified (GM) crops expressing insecticidal crystal proteins are widely accepted worldwide, but their commercial utilization demands comprehensive risk assessment studies. A 90-day risk assessment study was conducted on Wistar rats fed with GM maize (CEMB-413) expressing binary insect-resistant genes (cry1Ac and cry2Ab) at low (30%) and high (50%) dose along with a control diet group. The study used fifty Wistar rats randomly distributed in five treatment groups. Our study revealed that compared to controls, GM diet had no adverse effects on animal's health, including body weight, food consumption, clinical pathological parameters, serum hormone levels and histological parameters of testes and ovaries of rats. Differences were observed in transcripts levels of fertility related genes, but these were independent of treatment with GM diet.

Characterization of entomotoxic and nematotoxic genes from indigenous Bacillus thuringiensis strains and their biocontrol potential

BackgroundThe gram pod borer (Helicoverpa armigera Hubner), Bihar hairy caterpillar (Spilosoma obliqua Walker), tobacco caterpillar (Spodoptera litura Fabricius) and root knot nematode (Meloidogyne incognita (Kofoid and White) Chitwood) are the major insect pest and nematodes infesting mungbean, urdbean and other legumes. Bacillus thuringiensis (Bt) is a promising biocontrol agent that helps in managing the above pest. Deploying Bt biopesticides instead of chemical pesticides in legume cultivation enhances the soil health substantially. The present study envisages morphological, molecular cum biochemical characterization, entomotoxicity and nematotoxicity potential of four indigenous Bt strains (Ak2.IIPR, F8.IIPR, F5.IIPR and F6.IIPR) along with HD1 (standard check from BGSC).ResultsThe SEM micrograph analysis of above four Bt strains along with F1.IIPR revealed the presence of different combinations of insecticidal crystal proteins, viz. cuboidal crystal (CC), bipyramidal crystal (BC), spherical crystal (SC) and irregularly pointed crystal (IC). This was further confirmed by PCR amplification of Cry1, Cry2Aa, Cry2Ac, VIP3A and chi36 gene-specific primers and their phylogenetic analysis. F8.IIPR and Ak2.IIPR recorded the highest toxicity index against second-instar S. obliqua larvae (292.56 and 174.55), H. armigera (150.00 and 113.65) and S. litura (210.71 and 114.19) based on probit analysis. F5.IIPR (91.67%) and F8.IIPR (89.00%) recorded the highest juvenile mortality against M. incognita, followed by Ak2.IIPR (70.33%). The protein profiling of supernatant of terrific broth inoculated with F8.IIPR indicated the presence of VIP3A and Cry1I.ConclusionThe present study concluded that F8.IIPR and Ak2.IIPR were potential Bt strains isolated from Indo-Gangetic plains which is having both entomocidal and nematicidal properties. Further, these strains were deposited at national repository, ICAR-NBAIM with the following accession no.: NAIMCC-SB-065 and NAIMCC-SB-066, respectively. These strains can be formulated as microbial biopesticide.

Bt corn hybrids expressing mCry3A and eCry3.1Ab Proteins protect corn roots against western corn rootworm injury.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is one of the most serious pests of corn (Zea mays L.) In 2017 and 2018, studies were conducted in fields with and without known unexpected root injury to Cry3Bb1, to determine root protection by Bt corn hybrids expressing both mCry3A and eCry3.1Ab insecticidal crystal proteins, and hybrids expressing either mCry3A or eCry3.1Ab only against the WCR root injury. Node injury was evaluated using the Iowa State University 0-3 node-injury scale (NIS), and the consistency of root protection was also determined. In 2017, with medium to high larval feeding pressure, the Bt corn hybrids expressing both mCry3A and eCry3.1Ab in the breeding stack, molecular stack, and Bt corn hybrid expressing eCry3.1Ab only, sustained low node injury compared with Bt corn hybrid expressing mCry3A only, and the non-Bt corn. In 2018, with low larval feeding pressure in most of the locations, node injury was not different for the Bt and Non-Bt corn hybrids. Across all locations in both years, the Bt corn hybrids expressing both mCry3A and eCry3.1Ab provided better and consistent node injury protection. Bt corn hybrids expressing both mCry3A and eCry3.1Ab proteins provided better root protection and consistency than the Bt corn hybrid expressing mCry3A only, and non-Bt. Therefore, stacking of Bt traits will be the best option for managing insect resistance. © 2023 Society of Chemical Industry.

Application and Development of Bt Insect Resistance Genes in Rice Breeding

As pests are an important factor in reducing crop yields, pest control is an important measure in preventing reductions in crop yields. With the aim of ending the use of chemical pesticides, biological control and genetically modified methods are now considered more reasonable pest control strategies. The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins that have specific toxicity to lepidopteran insects, and so it has been applied as a microbial insecticide in the control of crop pests for several decades. With the development of plant genetic engineering, Bt genes encoding insecticidal crystal protein have been introduced into many crop species for pest control. This article indicates that, after years of experiments and research, Bt transgenic rice is close to becoming a commercial insect-resistant rice, and many studies have shown that transgenic rice has pronounced abilities in the control of pests such as yellow stem borers (Scirpophaga incertulas, YSB), striped stem borers (Chilo suppressalis, SSB), and rice leaf rollers (Cnaphalocrocis medinalis, RLR); moreover, it does not obviously differ from non-transgenic rice in terms of safety. This paper suggests that transgenic Bt rice has application potential and commercial value.

Whole genome sequencing of a novel Bacillus thuringiensis isolated from Assam soil

BackgroundBacillus thuringiensis (Bt) is a gram-positive ubiquitous saprophytic bacterium that produces proteins (Crystal protein, Vegetative insecticidal protein, and Secreted insecticidal protein) toxic to insects during its growth cycle. In the present study, the whole genome of a locally isolated B. thuringiensis strain BA04 was sequenced to explore the genetic makeup and to identify the genes responsible to produce insecticidal proteins including the virulence factors. The strain was isolated from the soil sample of the Kaziranga National Park, Assam, North-Eastern part of India (Latitude: 26°34′39.11''N and Longitude: 93°10′16.04''E).ResultsThe whole genome sequencing (WGS) of the BA04 strain revealed that it has a circular genome of size 6,113,005 bp with four numbers of plasmids. A total of 6,111 genes including two novel crystal protein-encoding genes (MH753362.1 and MH753363.1) were identified. The BLASTn analysis of MH753362.1 showed 84% similarities (maximum identity) with Cry1Ia (KJ710646.1) gene, whereas MH753363.1 exhibited 66% identity with Insecticidal Crystal Protein (ICP)-6 gene (KM053257.1). At the protein level, MH753362.1 and MH753363.1 shared 79% identity with Cry1Ia (AIW52613.1) and 40% identity with Insecticidal Crystal Protein (ICP)-6 (AJW76687.1) respectively. Three-dimensional structures of these two novel protein sequences revealed that MH753362.1 have 48% structural similarity with Cry8ea1 protein, whereas MH753363.1 showed only 20% structural similarity with Cry4Aa protein.Apart from these insecticidal genes, the strain was also found to contain virulence and virulence-associated factors including the antibiotic resistance genes and Clustered regularly interspaced short palindromic repeat (CRISPR) sequences.ConclusionThis is the first report on the whole genome sequence of Bt strain BA04 isolated from Assam, a North-Eastern state of India. The WGS of strain BA04 unveils the presence of two novel types of insecticidal crystal protein-encoding genes which can be used for the development of insect-resistant transgenic crops. Additionally, the strain could be used for the formulations of effective biopesticides. The WGS provides the fastest and cheapest platform for a better understanding of the genetic makeup of a strain and helps to explore the role of virulence genes in pathogenicity against the insect host.

Creation of an Industrial Bacillus thuringiensis Strain With High Melanin Production and UV Tolerance by Gene Editing.

Bacillus thuringiensis produces insecticidal crystal proteins (ICPs) which exhibit strong insecticidal toxicity. But when used in the field, ICPs would be destroyed by ultraviolet (UV) radiation in sunlight, thus decreasing the insecticidal activity and shortening the persistence. To improve the duration of B. thuringiensis preparations, we endowed a highly toxic industrial B. thuringiensis HD-1 with UV tolerance by making it produce melanin, a pigment that absorbs UV radiation. In B. thuringiensis, melanin is derived from homogentisate (HGA), an intermediate in the tyrosine pathway. And the absence of homogentisate-1,2-dioxygenase (HmgA) will lead to the formation of melanin. In this study, we used the CRISPR/Cas9 system to knock out the hmgA gene and obtained a melanin-producing mutant HD-1-ΔhmgA from strain HD-1. The melanin yield by mutant HD-1-ΔhmgA reached 3.60 mg/mL. And the anti-UV test showed that melanin serves as a protection to both the organism and the ICPs. After UV irradiation for 3 h, mutant HD-1-ΔhmgA still had an 80% insecticidal activity against the cotton bollworm, Helicoverpa armigera, while the control line only had about 20%. This study creates a light-stable biopesticide prototype based on a classic industrial strain that can be applied directly and takes the melanin-producing strain as a concept to improve the preparation validity.

Genetic transformation of common beans (Phaseolus vulgaris L.) through Agrobacterium tumefaciens carrying Cry1Ab gene.

Seed beetles are one of the most important causes of yield loss in bean production. It is essential to develop resistant varieties in the fight against these pests. Agrobacterium-based gene transformation is the most widely used breeding method worldwide to develop insect-resistant varieties. Embryonic axes and plumule explants were obtained from Agrobacterium tumefciens treated mature zygotic embryos of low and high raw protein-based common bean cultivars Akman 98 and Karacaşehir 90. Agrobacterium tumefaciens contained a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt Cry1Ab) controlled by the 35S promoter and NOS terminator sequences. The transformation event was genotype and explant dependent. The plumule explants could not withstand kanamycin-based selection pressure and died. It was possible to get two transgenic plants using embryonic axis explants of low protein cultivar Akman 98. These results were validated using GUS analysis, PCR, RT-PCR, bioassay analysis, and ELISA test from the samples taken from T0 and T1 generations. Bioassay tests showed that these plants were protected from the damage of legume seed insects (Bruchus spp.). The results are very encouraging and may help in producing better transgenic common bean germplasm leading to safe agriculture and reducing environmental pollutions.

The RNA Chaperone Protein Hfq Regulates the Characteristic Sporulation and Insecticidal Activity of Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is one of the most widely used bio-insecticides at present. It can produce many virulence factors and insecticidal crystal proteins during growth and sporulation. Hfq, on the other hand, is a bacterial RNA chaperone that can regulate the function of different kinds of RNAs, thereby affecting various bacterial phenotypes. To further explore the physiological functions of Hfq in Bt, we took BMB171 as the starting strain, knocked out one, two, or three hfq genes in its genome in different combinations, and compared the phenotypic differences between the deletion mutant strains and the starting strain. We did observe significant changes in several phenotypes, including motility, biofilm formation, sporulation, and insecticidal activity against cotton bollworm, among others. Afterward, we found through transcriptome studies that when all hfq genes were deleted, 32.5% of the genes in Bt were differentially transcribed, with particular changes in the sporulation-related and virulence-related genes. The above data demonstrated that Hfq plays a pivotal role in Bt and can regulate its various physiological functions. Our study on the regulatory mechanism of Hfq in Bt, especially the mining of the regulatory network of its sporulation and insecticidal activity, could lay a theoretical foundation for the better utilization of Bt as an effective insecticide.

PHB2 affects the virulence of Vip3Aa to Sf9 cells through internalization and mitochondrial stability

ABSTRACT The vegetative insecticidal proteins (Vip3A) secreted by some Bacillus thuringiensis (Bt) strains during vegetative growth are regarded as a new generation of insecticidal toxins. Like insecticidal crystal proteins, they are also used in transgenic crops to control pests. However, their insecticidal mechanisms are far less defined than those of insecticidal crystal protein. Prohibitin 2 (PHB2) is a potential Vip3Aa binding receptor identified from the membrane of Sf9 cells in our previous work. In this paper, we demonstrated the interaction between Vip3Aa and PHB2 using pull-down, dot blotting, microscale thermophoresis, and co-immunoprecipitation assays. PHB2 is distributed on the cell membrane and in the cytoplasm, and the co-localization of PHB2 and Vip3Aa was observed in Sf9 cells using a confocal laser scanning microscope. Moreover, PHB2 could interact with scavenger receptor-C via its SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain. Downregulation of phb2 expression reduced the degree of internalization of Vip3Aa, exacerbated Vip3Aa-mediated mitochondrial damage, and increased Vip3Aa toxicity to Sf9 cells. This suggested that PHB2 performs two different functions: Acting as an interacting partner to facilitate the internalization of Vip3Aa into Sf9 cells and maintaining the stability of mitochondria. The latter has a more important influence on the virulence of Vip3Aa.

The Combination Analysis Between Bacillus thuringiensis Sip1Ab Protein and Brush Border Membrane Vesicles in Midgut of Colaphellus bowringi Baly.

The secretory insecticidal protein Sip1Ab and crystal protein Cry8Ca from Bacillus thuringiensis (Bt) are widely recognized for their coleopteran insecticidal activities. It is worthwhile to investigate the insecticidal mechanisms of these two proteins against Colaphellus bowringi Baly, which is a serious pest of cruciferous vegetables in China and other Asian countries. To that end, the genes encoding the Sip1Ab and Cry8Ca proteins were amplified from the strain QZL38 genome, then expressed in Escherichia coli, after which bioassays were conducted in C. bowringi larvae. After feeding these two proteins, the histopathological changes in the midguts of C. bowringi larvae were observed using transmission electron microscopy (TEM), and the Brush Border Membrane Vesicle (BBMV) was extracted for competition binding assays. TEM showed that ingestion of Sip1Ab caused a significant reduction in growth of the larvae, disruption of midgut microvilli, and expansion of intercellular spaces. Competition binding assays demonstrated that Sip1Ab bound to C. bowringi BBMV with a high binding affinity. However, a mixture of the two proteins in equal proportions showed no significant difference in insecticidal activity from that of Sip1Ab. These results could provide a molecular basis for the application of Sip1Ab in coleopteran insect control and contribute to the study of the Sip1Ab insecticidal mechanism as well.

Cloning and Characterization of Cry1 and Cry2 Genes from Indigenous Bacillus thuringiensis Isolates

Insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are toxic to specific insect pests. They are used as alternatives to chemicals for controlling insect pests using transgenic plants and biopesticides. Three strains of Bacillus thuringiensis expressing cry1 and cry2 genes were identified in soil samples collected from different locations in India. The full-length cry1, cry1I, and cry2 type genes (3.5, 2.1, and 1.9 kb, respectively) were cloned and sequenced. The full-length clones of the three genes were sub-cloned in the E. coli expression vector, and expressed proteins were analysed. The expression of cry1, cry1I, and cry2 resulted in 132, 81, and 70 kDa proteins, respectively. The expressed proteins were tested against secondinstar larvae of Helicoverpa armigera. The expressed proteins of cry1 and cry2- type from BRI-86 exhibited the highest toxicity to H. armigera larvae with an LC50 of 2.34 and 0.2 µg/ml diet, respectively. The LC50 exhibited by the test proteins was two and four-fold lower than the reference Btk HD-1 proteins. Thus, the spore preparation or the genes can be used individually or in combination to control insect pests.

Synergism of Bacillus thuringiensis Toxin Cry1Ac by a Fragment of Toxin-Binding Polycalin from Plutella xylostella.

The continued success of pest control using insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) in transgenic plants was threatened by the evolution of resistance. Previous studies suggested that polycalin from Plutella xylostella could bind to Cry1Ac toxin as a potential receptor. In this study, a fragment of P. xylostella polycalin (Pxpolycalinf, G2209-A2942) containing a carboxyl-terminal GPI-anchored signal peptide was cloned and expressed. Purified Pxpolycalinf retained the binding ability to Cry1Ac and synergized Cry1Ac toxicity to the third larvae of P. xylostella in bioassays. Moreover, the polyclonal antibody of Pxpolycalinf decreased the Cry1Ac activity after being fed together with normal food. Further, the ELISA results showed the concentration-dependent binding of Pxpolycalinf to P. xylostella brush border membrane vesicles (BBMV). Spodoptera frugiperda 9 (Sf9) cells expressing Pxpolycalinf were not susceptive to Cry1Ac, whereas Pxpolycalinf increased Cry1Ac cytotoxicity to Sf9 cells expressing P. xylostella ATP-dependent binding cassette transporter C2 (PxABCC2). Immunolocalization presented the binding of Pxpolycalinf to the Sf9 cell membrane, and ELISA showed the concentration-dependent binding of Pxpolycalinf to Sf9 cell extraction. These results here provide the first evidence that a fragment of P. xylostella polycalin, a potential receptor of Cry1Ac, synergizes Cry1Ac toxicity to P. xylostella larvae and Sf9 cells expressing PxABCC2.

Cloning and characterization of the Cry79Aa1 gene from a lepidopteran active strain of Bacillus thuringiensis

Bacillus thuringiensis (Bt) has been used globally as a biopesticide for effective and environmentally friendly pest control. Research has intensified following the development of resistance by lepidopteran species to Bt insecticidal crystal proteins. Discovering new Bt strains with novel toxin properties which can overcome resistance is one of the strategies to improve pesticide sustainability. The genome of the Bacillus thuringiensis LTS290 strain was sequenced and assembled in 252 contigs containing a total of 6,391,328 bp. The novel cry79Aa1 gene from this strain was identified and cloned. Cry79Aa1 contains 729 amino acid residues and a molecular mass of 84.8 kDa by SDS-PAGE analysis. Cry79Aa1 was found to be active against the lepidopteran larvae of Spodoptera exigua, Helicoverpa armigera, and Plutella xylostella with LC50 values of 13.627 µg/mL, 42.8 µg/mL, and 38.086 µg/mL, respectively. However, Cry79Aa1 protein showed almost no insecticidal activity against Leguminivora glycinivorella, although some degree of growth retardation was observed.

Structure characteristics and function of a novel extracellular polysaccharide from Bacillus thuringiensis strain 4D19

Bacillus thuringiensis (Bt) are entomopathogenic bacteria that produce different kinds of insecticidal proteins. However, studies on Bt exopolysaccharides are lacking. Here, we aimed to explore the characteristics and insecticidal synergism of EPS, an exopolysaccharide from Bt strain 4D19. The molecular weight of EPS-2 was 58.0 kDa, which consisted of mannose (44.2%), GlcN (35.5%), D-GalN (8.0%), glucose (5.5%), arabinose (5.1%), galactose (0.9%), Man-UA (0.3%) and Glc-UA (0.2%). The toxicity of insecticidal proteins against Plutella xylostella was increased by adding EPS. EPS-2 bound to Cry1Ac protoxin and promoted the binding of Cry1Ac protoxin to the gut membrane of P. xylostella, but did not bind to activated toxins. These results suggested that EPS-2 may bind to the protoxin C-terminal region to enhance insecticidal activity. Our findings indicated that Bt strains produce exopolysaccharide to enhance the toxicity of insecticidal crystal proteins, which could be applied in biopesticide research and product development.