Vegetative Insecticidal Protein Research Articles

Proteomic analysis of proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Aschersonia placenta

The infection of insects by the entomopathogenic fungus Aschersonia placenta depends on conidia. To identify proteins differentially expressed in A.placenta conidia vs mycelia, we performed a comparative proteomic analysis of A.placenta using 2-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). We detected 1022 2-DE protein spots in conidia and 1049 in mycelia and analyzed 48 (13 from conidia and 35 from mycelia) using MALDI-TOF-MS. Finally, we identified 28 proteins (7 from conidia and 21 from mycelia). The identified proteins exclusive to conidia included major proteins participating in oxidation-reduction processes and vegetative insecticidal protein 1 (Vip1), a protein that is likely involved in pathogenicity. The identified proteins exclusive to mycelia were those involved in biosynthesis and metabolism, including uridine diphosphate galactopyranose mutase, which might play key roles in hyphal morphogenesis. This report provides the first proteomic analysis of different developmental stages of an Aschersonia species. Although only a small number of proteins were identified, the data represent a useful foundation for future studies concerning the molecular basis of entomopathogenicity in the species A.placenta and in the genus Aschersonia.

Screening and identification of a Bacillus thuringiensis strain S1/4 with large and efficient insecticidal activities

The bacterium Bacillus thuringiensis was recognized for its entomopathogenic activities related to Cry and Cyt proteins forming the δ-endotoxins and some extracellular activities like the vegetative insecticidal proteins (VIP) and Cry1I. These activities may act specifically against diverse organisms and some of them typically characterize each strain. Here, we screened a set of 212 B. thuringiensis strains to search the higher insecticidal activities. These strains had bipyramidal and cubic crystal morphologies and 30% of them showed PCR amplification of vip3 internal region, from which five isolates (S1/4, S17, S122, S123, and S144) showed plasmid profile variability. These five strains contained the cry1I, cry1Aa and/or cry1Ac, cry1Ab and cry2 genes, and S1/4 harbored in addition the cry1C, vip1, and vip2 genes. They produced from 25 to 46 µg δ-endotoxin per 10(7) spores. Their δ-endotoxins displayed distinct lethal concentrations 50% against either Spodoptera littoralis or Ephestia kuehniella larvae with the lowest one for S1/4, which was also active against Tuta absoluta. Fortunately, the analysis of the culture supernatants revealed that S1/4 had the higher toxicity towards these lepidopteron but it did not show any toxicity against the Tribolium castaneum coleopteran larvae; additionally, S1/4 displayed an antibacterial activity. S1/4 is a good candidate for agricultural pest control, as it is more efficient than the reference strain HD1.

Ser-Substituted Mutations of Cys Residues in Bacillus thuringiensis Vip3Aa7 Exert a Negative Effect on its Insecticidal Activity

Vegetative insecticidal proteins (VIPs), which were produced by Bacillus thuringiensis during its vegetative growth stage, display a broad insecticidal spectrum to Lepidoptera larvae. Sequence alignment of the Vip3A-type indicates that three cysteine residues were conserved in Vip3A-type proteins. To determine whether these conserved cysteine residues contributed to the insecticidal activity, the three residues were respectively substituted with serine in the Vip3Aa7 protein by site-directed mutagenesis. Bioassays using the third instar larvae of Plutella xylostella showed that the toxicity of C401S and C507S mutants were completely abolished. To find out the inactivity reason of mutants, three mutants and the wild-type Vip3Aa7 were treated with trypsin. The results indicated that the C507S mutant was rapidly cleaved and resulted in decrease of the 62kDa toxic core fragment. These results indicated that the replacement of the Cys(507) with a Ser(507) caused decrease in C507S resistance against trypsin degradation. It is suggesting a possible association between insecticidal activity and trypsin sensitivity of Vip3A proteins. This study serves a guideline for the study of Vip3A protein structure and active mechanism.

Vip3C, a Novel Class of Vegetative Insecticidal Proteins from Bacillus thuringiensis

Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm(2).

Detection, cloning and bioinformatics analysis of vip1/vip2 genes from local strains of Bacillus thuringiensis

Bio-insecticides based on the spore forming bacterium, Bacillus thuringiensis (Bt) have been used for commercial scale for the past 40 years. Bt is a Gram-positive soil bacterium that forms insecticidal crystal proteins (ICPs) during sporulation; it has been characterized as an insect pathogen. Vegetative insecticidal protein (VIP) is a newly discovered family of toxin protein isolated from Bt. A hundred strains of localBacillus thuringiensis were isolated from soil and dead larvae, identified by 16S rRNA and screened for the presence of vip1 and vip2 genes by polymerase chain reaction (PCR) amplification, with only four strains producing the desired bands of Vip1 and Vip2. The amplified fragments were cloned in pGEM-vector, sequenced and analyzed. The nucleotide sequences of vip1 (2.3 kb) and vip2 (1.3 kb) were given Gene-bank accession numbers: JN008908 and JN035904, respectively. Vip1 and Vip2 showed 99% homology with the previously isolated genes. Key words: Bacillus thuringiensis, vegetative insecticidal proteins (vip1 and vip2), 16S rRNA, cloning.

Vip3A Resistance Alleles Exist at High Levels in Australian Targets before Release of Cotton Expressing This Toxin

Crops engineered to produce insecticidal crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt) have revolutionised pest control in agriculture. However field-level resistance to Bt has developed in some targets. Utilising novel vegetative insecticidal proteins (Vips), also derived from Bt but genetically distinct from Cry toxins, is a possible solution that biotechnical companies intend to employ. Using data collected over two seasons we determined that, before deployment of Vip-expressing plants in Australia, resistance alleles exist in key targets as polymorphisms at frequencies of 0.027 (n = 273 lines, 95% CI = 0.019–0.038) in H. armigera and 0.008 (n = 248 lines, 0.004–0.015) in H. punctigera. These frequencies are above mutation rates normally encountered. Homozygous resistant neonates survived doses of Vip3A higher than those estimated in field-grown plants. Fortunately the resistance is largely, if not completely, recessive and does not confer resistance to the Bt toxins Cry1Ac or Cry2Ab already deployed in cotton crops. These later characteristics are favourable for resistance management; however the robustness of Vip3A inclusive varieties will depend on resistance frequencies to the Cry toxins when it is released (anticipated 2016) and the efficacy of Vip3A throughout the season. It is appropriate to pre-emptively screen key targets of Bt crops elsewhere, especially those such as H. zea in the USA, which is not only closely related to H. armigera but also will be exposed to Vip in several varieties of cotton and corn.

Fusing the vegetative insecticidal protein Vip3Aa7 and the N terminus of Cry9Ca improves toxicity against Plutella xylostella larvae

Bacillus thuringiensis insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs) have been widely used as a kind of safe bio-insecticides. A problem that has been of concern worldwide is how to improve their insecticidal activities. In this study, to determine the synergism between VIPs and ICPs effect on insecticidal activity, a construct that produces a chimeric protein of the Vip3Aa7 and the N terminus ofCry9Ca, named V3AC9C, was expressed in Escherichia coli BL21 cells. In additional experiments, the V3AC9C chimeric protein, the single Vip3Aa7, and the single N terminus of Cry9Ca were treated with trypsin. SDS-PAGE showed that the V3AC9C could be processed into two single toxins. Bioassays tested on third instar larvae of Plutella xylostella showed that the toxicity of the chimeric protein was markedly better than either of the single toxins. Interestingly, the toxicity of the chimeric protein was 3.2-fold higher than a mixture of the Vip3Aa7 and Cry9Ca toxins (mass ratio of 1:1). The synergism factor (SF) of chimeric protein containing Vip3Aa7 and Cry9Ca was calculated to be 4.79. The SF in mixture of toxins is only 1.46. Hence, the effect was more than the sum of the Vip3Aa7 and Cry9C activities. Analysis of the protein's solubility showed that the Vip3Aa7 helped the N terminus of Cry9Ca to dissolve in an alkaline buffer. It was concluded that the increase in the toxicity of the V3AC9C chimeric protein over the constituent proteins mainly resulted from this increase in solubility. These results lay a foundation for the development of a new generation of bio-insecticides and multi-gene transgenic plants.

Effect of Temperature on the Fitness of a Vip3A Resistant Population of <I>Heliothis virescens</I> (Lepidoptera: Noctuidae)

Microbial insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) have become increasingly important for pest management. In addition to crystal (Cry) insecticidal protein toxins formed during sporulation, vegetative insecticidal protein (Vip) toxins can be produced during the vegetative phase. Resistance to Cry toxins has been reported in laboratory- and field-selected populations of various Lepidoptera species and several studies have identified fitness costs associated with Cry toxin resistance. Here, fitness costs are examined in the first insect population to be reported with resistance to a Vip toxin, a laboratory-selected Vip3A-resistant subpopulation of the tobacco budworm, Heliothis virescens (L.) (Vip-Sel). The Vip-Sel population showed reduced survival to adult eclosion compared with an unselected subpopulation at all test temperatures, including the culture temperature (25 degrees C). Vip3A resistance was also associated with reduced egg viability and mating success and a lower intrinsic rate of population increase (r(m)) at temperatures below (20 degrees C) and above (30 degrees C) the optimal laboratory culture temperature. The latter findings agree with previous studies, that fitness costs associated with resistance are usually greater under stressful conditions. Such data can help predict the impact of fitness costs on the rate of development of resistance in the field and in the development of resistance management strategies that more fully exploit fitness costs.

Detection and Identification of Vegetative Insecticidal Proteins vip3 Genes of Bacillus thuringiensis Strains Using Polymerase Chain Reaction-High Resolution Melt Analysis

In this study, vegetative insecticidal proteins vip3 genes from Bacillus thuringiensis strains were detected based on polymerase chain reaction-high resolution melt (PCR-HRM) analysis. A pair of primers was designed according to the conservative sequences in 150 bp region of the known vip3 subfamily. The 150 bp regions of difference vip3 genes have only a few nucleotide difference vip3 genes were detected in 8 of 11 standard B. thuringiensis strains, and vip3Aa genes, vip3Af genes and vip3Ba gene can be distinguished as different melting curves by this method. The results demonstrate the utility of the HRM assay for mutant screening using vip3 gene. The PCR-HRM method may be a valuable and reliable tool for specific detection and identification of vip3 genes.

Screening, Diversity and Partial Sequence Comparison of Vegetative Insecticidal Protein (vip3A) Genes in the Local Isolates of Bacillus thuringiensis Berliner

Characterization, direct sequencing of the PCR amplicon and phylogenetic relationship was done to discover a novel Vip protein genes of the Bt isolates, to improve the prospects for insect control, more Vip proteins should be sought out and researched to predict their insecticidal activity. Characterization was based on direct sequencing of PCR amplicon using primers specific to vip3A gene was presented here. 12 out of 18 isolates screened were positive for vip gene-specific primers. Homology search for the partial sequences using BLAST showed that 11 isolates had high similarity to vip3Aa gene and only one fragment with vip3Ae gene (25-100% at nucleotide and amino acid level). Phylogenetic analysis showed that the gene sequences were responsible for geographic separation for divergence within vip genes, consistent with the evaluation of distinct bacterial population. Despite the geographical distances, strains harbouring vip genes have originated from common ancestors may significantly contribute to control resistant insect pests. Some strains have evolved to be quite distinct and others remain as members of closely related groups. The reported method is a powerful tool to find novel Vip3A proteins from large-scale Bt strains which is effective in terms of time and cost. Further the Vip proteins produced by different strains of B. thuringiensis are unique in terms of the sequence divergence and hence may also differ in their insecticidal activities.

Development of ELISA for the detection of transgenic vegetative insecticidal protein in GM crops/produce.

In the process of the development of insect-resistant genetically modified (GM) crops and also to evaluate the consistency in the expression of toxin under field conditions, immunological assays are commonly being used. An immunoassay was developed to support the labelling of vegetative insecticidal protein (Vip3A)-based GM produce. The developed ELISA for the measurement of Vip3A is a triple antibody sandwich procedure utilising a polyclonal capture antibody (mouse anti-Vip3A) and a polyclonal detection antibody (rabbit anti-Vip3A) followed by use of a third HRP-conjugated anti-species antibody (goat anti-rabbit IgG). The limit of detection limit of the ELISA assay was 16 ng ml(-1) with a linear quantification range from approximately 31 to 500 ng ml(-1) of Vip3A protein. Furthermore, the assay was in-house validated with GM brinjal samples. The assay was specific, sensitive and reproducible, which can be helpful to detect and track down the spread of unapproved and intentionally/unintentionally released GM produce harbouring Vip protein.

Co-expression and Synergism Analysis of Vip3Aa29 and Cyt2Aa3 Insecticidal Proteins from Bacillus thuringiensis

Vegetative insecticidal protein (Vip3) from Bacillus thuringiensis shows high activity against lepidopteran insects. Cytolytic δ-endotoxin (Cyt) also has high toxicity to dipteran larvae and synergism with other crystal proteins (Cry), but synergism between Cyt and Vip3 proteins has not been tested. We analyzed for synergism between Cyt2Aa3 and Vip3Aa29. Both cyt2Aa3 and vip3Aa29 genes were co-expressed in Escherichia coli strain BL21 carried on vector pCOLADuet-1. Vip3Aa29 showed insecticidal activity against Chilo suppressalis and Spodoptera exigua, with 50% lethal concentration (LC(50)) at 24.0 and 36.6 μg ml(-1), respectively. It could also inhibit Helicoverpa armigera growth, with 50% inhibition concentration at 22.6 μg ml(-1). While Cyt2Aa3 was toxic to Culex quinquefasciatus (LC(50): 0.53 μg ml(-1)) and Chironomus tepperi (LC(50): 36 μg ml(-1)), it did not inhibit C. suppressalis, S. exigua, and H. armigera. However, the co-expression of Cyt2Aa3 and Vip3Aa29 showed synergistic effect on C. suppressalis and S. exigua, and the individual activities were strengthened 3.35- and 4.34-fold, respectively. The co-expression had no synergism against C. tepperi and H. armigera, but exerted some antagonistic effect on Cx. quinquefasciatus. The synergism between Cyt2Aa and Vip3Aa was thus discovered for the first time, which confirmed that Cyt toxin can enhance the toxicity of other toxins against some non-target insects. By synergism analysis, the effectiveness of microbial insecticides can be verified.

Isolation, cloning, and overexpression of vip3Aa gene isolated from a local Bacillus thuringiensis

Vegetative insecticidal protein (Vip) is a newly discovered family of toxin protein isolated from Bacillus thuringiensis (Bt). An 88.5-kDa Vip3Aa protein was secreted by a local strain of the bacterium during the vegetative growth phase. The full length of the coding region ‘2.3 kbp’ of the vip3Aa gene was isolated from plasmid DNA, cloned in pGEM-T vector and finally cloned in pQE-30 expression vector. Nucleotide sequence revealed 98% homology with that of the previously isolated genes. Expression of the vip3Aa in Escherichia coli was carried out and the expressed protein was detected in the concentrated supernatant, not in the pellet. This indicated that vip3Aa is secreted into the culture medium. Expressed protein was purified, blotted, and assayed against the cotton leaf worm Spodoptera littoralis. The LC50 was found to be 142.4 µ/mL while the LC50 was 90 ppm for the wild strain. These results suggest the use of either the isolated Bt strains or the expressed vip3Aa in an integrated pest management program against lepidopteran insect pests.

Rapid detection of vip1-type genes from Bacillus cereus and characterization of a novel vip binary toxin gene

A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance.

Molecular Characterization of a Novel Vegetative Insecticidal Protein from Bacillus thuringiensis Effective Against Sap-Sucking Insect Pest

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on LC(50) values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.

A Quantitative Immunopolymerase Chain Reaction Method for Detection of Vegetative Insecticidal Protein in Genetically Modified Crops

Vegetative insecticidal protein (Vip) is being employed for transgenic expression in selected crops such as cotton, brinjal, and corn. For regulatory compliance, there is a need for a sensitive and reliable detection method, which can distinguish between approved and nonapproved genetically modified (GM) events and quantify GM contents as well. A quantitative immunopolymerase chain reaction (IPCR) method has been developed for the detection and quantification of Vip protein in GM crops. The developed assay displayed a detection limit of 1 ng/mL (1 ppb) and linear quantification range between 10 and 1000 ng/mL of Vip-S protein. The sensitivity of the assay was found to be 10 times higher than an analogous enzyme-linked immunosorbent assay for Vip-S protein. The results suggest that IPCR has the potential to become a standard method to quantify GM proteins.

Increase of the Bacillus thuringiensis Secreted Toxicity Against Lepidopteron Larvae by Homologous Expression of the vip3LB Gene During Sporulation Stage

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against several agricultural insect pests. The fact that the expression of vip3 genes occurs only during the vegetative growth phase of Bacillus thuringiensis is a limiting factor in term of production level. Therefore, extending the synthesis of the Vip proteins to the sporulation phase is a good alternative to reach high levels of toxin synthesis. In this study, we have demonstrated that the maximal production of the secreted Vip3LB (also called Vip3Aa16) during the growth of the wild-type strain B. thuringiensis BUPM 95 is reached at the end of the vegetative growth phase, and that the protein remains relatively stable in the culture supernatant during the late sporulation stages. The vip3LB gene was cloned and expressed under the control of the sporulation dependant promoters BtI and BtII in B. thuringiensis BUPM 106 (Vip3(-)) and BUPM 95 (Vip3(+)) strains. The examination of the culture supernatants during the sporulation phase evidenced the synthesis of Vip3LB and its toxicity against the second-instars larvae of the Lepidopteron insect Spodoptera littoralis for the recombinant BUPM 106. Moreover, there was an increase of the Vip3LB synthesis level and an enhancement of the oral toxicity for the recombinant BUPM 95 resulting from the expression of the vip3LB gene during both the vegetative and sporulation phases and the relative stability of the Vip3LB protein.

The trends and future of biotechnology crops for insect pest control

Biotech crops, including those that are genetically modified (GM) with Bacillus thuringiensis (Bt) endotoxins for insect resistance, have been cultivated commercially and adopted in steadily increasing numbers of countries over the past 14 years. This review discusses the current status of insect resistant transgenic crops and the often raised concern that its resilience is limited and that its efficacy will be compromised by insect resistance. We consider this trait as it is currently deployed in fields across the world as well as potential candidates that are at various stages of development along the pathway between the laboratory and deregulation. Future trends and prospects for biotechnological applications to mediate crop protection against insects are also considered. These include strategies employing stacked genes, modified Bt toxins, vegetative insecticidal proteins, lectins, endogenous resistance mechanisms as well as novel approaches. In addition, the benefits and risks associated with the adoption of GM insect resistant crops, especially for developing countries and resource-poor smallholder farmers are also discussed. Key words : Bacillus thuringiensis (Bt), endotoxins, Cry proteins, transgenic crops, insect resistance

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin

BUPM95 is a Bacillus thuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth ( E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22 kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62 kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65 kDa compared to the 55 and 100 kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.

Complete Genome Sequence of Bacillus thuringiensis subsp. chinensis Strain CT-43

Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.