Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from Bacillus thuringiensis

A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.     

Crystal structure of a Vip3B family insecticidal protein reveals a new fold and a unique tetrameric assembly

Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full‐length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening—over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate‐binding domains, lectins, or from other insecticidal proteins.     

Effective control of yellow stem borer and rice leaf folder in transgenic rice indica varieties Basmati 370 and M 7 using the novel δ-endotoxin cry2A Bacillus thuringiensis gene

Transgenic rice indica varieties Basmati 370 and M 7 expressing the novel cry2A (Bt) insecticidal gene were generated by particle bombardment. Molecular and biochemical analyses in R0 and R1 populations confirmed stable integration and expression of this novel Bt transgene. We estimated that the gene product was expressed up to 5% of total leaf protein. Insect feeding bioassays demonstrated that the Cry2A protein was effective against the yellow stem borer and the rice leaf folder, two major rice pests in the Indian Subcontinent. This is the first report of the control of major rice pests using this specific Bt gene. The cry2A gene can now be used in combination with other insecticidal genes for pyramiding resistance against insect pests. This will delay, or perhaps in combination with integrated pest management practices, prevent evolution of insect populations resistant to single insecticidal genes.     

苏云金杆菌营养期杀虫蛋白的研究

营养期杀虫蛋白 (vegetativeinsecticidalproteins ,VIPs)是苏云金杆菌 (Bacillusthuringiensis,Bt)在对数生长中期分泌的一类新型杀虫毒蛋白。VIPs主要分为VIP1、VIP2和VIP3三种。VIP1和VIP2构成二元毒素 ,对鞘翅目叶甲科的昆虫具有杀虫特异性 ;而VIP3对鳞翅目昆虫具有较广谱的杀虫活性。VIP1和VIP2的杀虫作用机理还不清楚 ;VIP3通过诱发细胞凋亡 ,最终导致昆虫死亡 ,这种作用机理与Bt杀虫晶体蛋白的作用机理完全不同 ,这为筛选新的杀虫活性物质提供了新的思路。vip基因现已被应用于转基因杀虫植物的构建 ,得到高效抗虫的多价转基因玉米。此外 ,VIPs嵌合蛋白的构建、vip及其融合基因导入其它许多宿主微生物等方面的研究也具有诱人的潜在应用前景。     

Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a cry1ia-type toxin from a Brazilian Bacillus thuringiensis strain

Different isolates of the soil bacterium Bacillus thuringiensis produce multiple crystal (Cry) proteins toxic to a variety of insects, nematodes and protozoans. These insecticidal Cry toxins are known to be active against specific insect orders, being harmless to mammals, birds, amphibians, and reptiles. Due to these characteristics, genes encoding several Cry toxins have been engineered in order to be expressed by a variety of crop plants to control insectpests. The cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, are the major economically devastating pests of cotton crop in Brazil, causing severe losses, mainly due to their endophytic habit, which results in damages to the cotton boll and floral bud structures. A cry1Ia-type gene, designated cry1Ia12, was isolated and cloned from the Bt S811 strain. Nucleotide sequencing of the cry1Ia12 gene revealed an open reading frame of 2160 bp, encoding a protein of 719 amino acid residues in length, with a predicted molecular mass of 81 kDa. The amino acid sequence of Cry1Ia12 is 99% identical to the known Cry1Ia proteins and differs from them only in one or two amino acid residues positioned along the three domains involved in the insecticidal activity of the toxin. The recombinant Cry1Ia12 protein, corresponding to the cry1Ia12 gene expressed in Escherichia coli cells, showed moderate toxicity towards first instar larvae of both cotton boll weevil and fall armyworm. The highest concentration of the recombinant Cry1Ia12 tested to achieve the maximum toxicities against cotton boll weevil larvae and fall armyworm larvae were 230 microg/mL and 5 microg/mL, respectively. The herein demonstrated insecticidal activity of the recombinant Cry1Ia12 toxin against cotton boll weevil and fall armyworm larvae opens promising perspectives for the genetic engineering of cotton crop resistant to both these devastating pests in Brazil.     

Specific primers for the detection of vip3A insecticidal gene within a Bacillus thuringiensis collection

A PCR-based method was developed for the rapid detection of vip3A gene encoding a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Specific primer combinations (three primers for the normal strand and two primers for the complementary strand) were capable of generating diagnostic fragments that successfully predicted the presence of the gene encoding the Vip3A insecticidal toxin in various B. thuringiensis strains. Specificity of amplicons generated by primer pairs was confirmed by restriction endonuclease digestion and DNA sequence analysis. The evaluation of B . thuringiensis strains for biological activity against insect pests of rice is thus aided by the grouping of strains based on their potential insecticidal spectrum.     

水稻中cry1Ah1基因密码子优化方案的比较

cry1Ah1基因是本实验室克隆的具有自主知识产权的模式基因,对鳞翅目害虫水稻二化螟等具有高毒力,具有较好的应用前景。为提高cry1Ah1基因在水稻中的表达量,探讨密码子使用频率对基因表达的影响,依据水稻密码子使用频率设计5种不同的优化方案,提高GC含量并去除剪切信号等不稳定因素后合成cry1Ah1基因杀虫活性区域。优化后的基因在大肠杆菌Rosetta(DE3)中正常表达了65 kDa蛋白,表达蛋白对2龄小菜蛾和水稻二化螟初孵幼虫都具有良好的杀虫活性。优化的基因转化水稻日本晴后,PCR阳性率达到87%以上,实时荧光定量RT-PCR和ELISA分析表明全部采用最高频率密码子的优化方案效果最好,Cry1Ah蛋白平均表达量占可溶性蛋白的0.104%。     

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua

AIMS: The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520. METHODS AND RESULTS: A vegetative insecticidal protein gene vip3Aa7, under the control of its native promoter and cry3A promoter, was subcloned into B. thuringiensis acrystalliferous BMB171 to generate BMB8901 and BMBvip respectively. It was found that the amount of Vip3Aa7 protein produced by BMBvip was 3.2-fold more than that produced by BMB8901. Therefore, the vip3Aa7 gene under the control of cry3A promoter was transformed into strain YBT1520. The toxicity of the resulting strain BMB218V against Spodoptera exigua was 10-fold more than that of YBT1520, and that the toxicity of BMB218V against Helicoverpa armigera retained the same level as that of strain YBT1520. CONCLUSIONS: Strain YBT1520 obtained high toxicity against S. exigua after it was transformed and expressed the foreign vip3Aa7 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial B. thuringiensis strain YBT1520 has high toxicity against H. armigera and Plutella xylostella, but almost no activity against S. exigua, which is a major crop pest in China. This work provides a new strategy for widening the activity spectrum of B. thuringiensis against agriculture pests.     

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.     

Novel chlorantraniliprole derivatives as potential insecticides and probe to chlorantraniliprole binding site on ryanodine receptor

The lepidopteran pests such as diamondback moth are the regularly harmful pests of crops in the world, which brings enormous losses in crop production. Chlorantraniliprole is an anthranilic diamide insecticide registered for the control of lepidopteran pests with high insecticidal activity, however with uncertain binding site action target of chlorantraniliprole on ryanodine receptor, a series of new chlorantraniliprole derivatives were synthesized and the insecticidal activities of these compounds against diamondback moth were evaluated with chlorantraniliprole and indoxacarb as control. All compounds except 8h, 8p and 8t exhibited varying degree of activities against diamondback moth. Especially, compounds 8c, 8i, 8k and 8l displayed good insecticidal activities against diamondback moth and the activities are even better than that of indoxacarb during 72 h period. The K_i values of all synthesized compounds were calculated through autodocking program respectively. The relationship between calculation value of molecular docking and results of insecticidal activities indicated that the proposed specific receptor, the membrane-spanning domain protein of diamondback moth ryanodine receptor in our study might have chlorantraniliprole binding sites.     

Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1

Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD) and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi.     

Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis

The potential of insecticidal Vip3Aa toxin peptide of Bacillus thuringiensis (Bt) as a resource for development of lepidopteran insect resistant transgenic crop plants has not yet been fully fathomed. The single mode of protection offered by the insecticidal Vip3Aa toxin against a broad spectrum of lepidopteran insect pests that invade crop field as secondary insect pests, carry definitive significance. However, lack of diversity amongst insecticidal Vip3A toxin towards toxicity for lepidopteran insects is often considered as disadvantage. In order to bring in improvement at this front, search for diversity and protein engineering of the toxin molecule for creation of diversity require to be undertaken in future. In that context, identification of the bioactive core component of Vip3BR toxin peptide of Bt an analogue of Vip3Aa toxin has been accomplished. The core component was found to contain enhanced potency of the insecticidal property 2–3 folds more than the native toxin against four major crop pests.     

Structure-function relationships affecting the insecticidal and miticidal activity of sugar esters

Synthetic sugar esters are a relatively new class of insecticidal compounds that are produced by reacting sugars with fatty acids. The objective of this research was to determine how systematic alterations in sugar or fatty acid components of sugar ester compounds influenced their insecticidal properties. Sucrose octanoate, sorbitol octanoate, sorbitol decanoate, sorbitol caproate, xylitol octanoate, xylitol decanoate and xylitol dodecanoate were synthesized and evaluated against a range of arthropod pests. Dosage-mortality studies were conducted on pear psylla (Cacopsylla pyricola Foerster) on pear, tobacco aphid (Myzus nicotianae) Blackman and tobacco hornworm (Manduca sexta [Johannson]) on tobacco, and twospotted spider mite (Tetranychus urticae Koch) on apple in laboratory bioassays. These sugar esters were compared with insecticidal soap (M-Pede, Dow AgroSciences L.L.C., San Diego, CA), to determine how toxicologically similar these materials were against the arthropod pests. Substitutions in either the sugar or fatty acid component led to significant changes in the physical properties and insecticidal activity of these compounds. The sugar esters varied in their solubility in water and in emulsion stability, yet, droplet spread upon pear leaves occurred at low concentrations of 80-160 ppm and was strongly correlated with psylla mortalities (R2 = 0.73). Sequentially altering the sugar or fatty acid components from lower to higher numbers of carbon chains, or whether the sugar was a monosaccharide or disaccharide did not follow a predictable relationship to insecticidal activity. Intuitively, changing the hydrophile from sorbitol (C6) to xylitol (C5) would require a decrease in lipophile chain length to maintain hydrophilic-lipophilic balance (HLB) relationships, yet an increase in lipophile chain length was unexpectedly needed for increasing insecticidal activity. Thus, the HLB of these materials did not correlate with pear psylla mortalities. Initial insect bioassays and dosage-mortality data found significant differences among sugar ester compounds' toxicity to the range of arthropod species. Sucrose octanoate high in monoester content had the highest activity against the range of arthropod pests at low concentrations of 1200-2400 ppm. No single chemical structure for the xylitol or sorbitol esters were optimally effective against the range of arthropods we tested and sorbitol octanoate and xylitol decanoate had the highest insecticidal activity of this group. All of the sugar ester materials produced high T. urticae mortalities on apple at very low concentrations of 400 ppm. Overall, most of the sugar esters that were examined had superior insecticidal activity compared with insecticidal soap. Sugar ester chemistry offers a unique opportunity to design an insecticide or miticide specific to certain arthropod pests which would be valuable in crop integrated pest management (IPM) programs. Sucrose esters are currently used as additives in the food industry which makes them especially attractive as safe and effective insecticides.     

Functional GNA expressed in Escherichia coli with high efficiency and its effect on Ceratovacuna lanigera Zehntner

The mannose-specific GNA (Galanthus nivalis agglutinin, snowdrop lectin) are the resistant proteins with many bioactivities. Snowdrop lectin is different with plant organs and development periods in lectin species, content, and bioactivities. It is an effective and cheap way to obtain much active GNA through overexpression of GNA gene in Escherichia coli. Constructs encoding mature GNA fused with an N-terminal pelB signal sequence protein (PelB) were expressed in E. coli with high efficiency. Recombinant protein productivity was higher than values published before. The insecticidal activity of purified recombinant proteins was assayed on feeding sugarcane wooly aphid (Ceratovacuna lanigera Zehntner), as well as spraying on sugarcane plants infected by aphids. The insecticidal activity was found to be comparable to native GNA. Oral delivery has obvious positive implications for crop protection against insect pests since peptides can be present in, or sprayed on, plant tissues susceptible to damage. A highly efficient expression of functional recombinant GNA would decrease the cost of GNA and promote its wide use, especially to give crop protection in the field.     

Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis

Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.     

一种新的Bt杀虫基因HJC原核表达蛋白活性的鉴定

HJC基因是由2个Bt基因(cry1Ab和vip3)经过人工融合而成,具有更广谱的杀虫活性,可延缓害虫产生交互抗性的时间。将已构建好的携带HJC基因的重组质粒pET28a-HJC转化到大肠杆菌BL21中诱导表达。该HJC融合蛋白主要以包涵体形式存在,变性条件下使用镍亲和层析柱对其进行纯化,并经尿素梯度透析复性后,进行免疫反应活性及美国白蛾杀虫活性测定。Western blot结果显示,该原核表达蛋白与转HJC基因水稻中的HJC蛋白有相同的免疫反应性,对美国白蛾也有一定的杀虫活性,可以替代植物外源蛋白进行转HJC基因产品的食用安全性评价。     

Characteristics of insecticidal substances from the entomopathogenic fungus Metarhizium pinghaense 15R against cotton aphid in Korea

Aphids (Hemiptera: Aphididae) are agricultural pests for a wide range of economically important crops worldwide. Entomopathogenic fungi have been studied and developed as a biological agents to control hard to control insect pests like the aphid. In particular, secondary metabolites produced by entomopathogenic fungi are being studied as insecticidal substances. This study evaluated the insecticidal activity and characteristics of insecticidal substances in culture filtrate against aphids to confirm the insecticidal substances produced by Metarhizium pinghaense 15R. After treating cotton aphids with the culture filtrate, strong insecticidal activity (100 % insect mortality) was observed on the third day of treatment, and it was confirmed that insecticidal substances in the culture filtrate had high thermal stability. In addition, the physicochemical properties of insecticidal substances in the culture filtrate were evaluated, and it was determined that the insecticidal substances were hydrophobic, with small molecules of 3 kDa or less. Based on these results, it was suspected that the insecticidal substances in the culture filtrate were destruxins (DTXs), which are representative secondary metabolites of Metarhizium known to have insecticidal activity. Through HPLC (High Performance Liquid Chromatography) analysis, the presence of DTX A and B was found in all active samples, and it was confirmed that the insecticidal substances of M. pinghaense 15R were DTXs. This study provides primary data for developing biochemical crop protection agents using these insecticidal substances, and we suggest the potential of using insecticidal substances of entomopathogenic fungi as biochemical pesticides in Korea.     

Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.     

The Role of Magnesium, Pyrophosphate, and Their Complexes as Substrates and Activators of the Vacuolar H+-Pumping Inorganic Pyrophosphatase (Studies Using Ligand Protection from Covalent Inhibitors)

Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.