곤충병원선충인 Steinernema longicaudum에 공생하는 Xenorhabdus ehlersii KSY 세균은 나방류에 대한 높은 병원력을 발휘한다. 본 연구에서 이 세균의 병원력이 아이코사노이드 생합성을 억제하여 기주 곤충의 면역 저하를 유발한다는 것을 확인하였다. 그러나 이 세균의 병원력은 혈강 주입에 의해 야기된다. 섭식을 통해 이 세균을 혈강으로 전달하기 위해 곤충의 중장벽을 파괴하여 병원력을 발휘하는 Bacillus thuringiensis (Bt)와 혼합하여 처리하였다. 배추좀나방(Plutella xylostella) 유충에 대해서 X. ehlersii 세균 배양액의 혼합 처리는 Bt 살충력을 현격하게 증가시켰다. 이러한 살충효과는 또 다른 나비목 해충인 콩명나방에 대해서도 확인되었다. 제형화를 위해 X. ehlersii 세균 배양액을 동결건조하여 Bt 수 화제와 혼합하였다. 이를 기반으로 간이 포장실험을 수행하였다. Bt 단독으로 처리한 결과 약 80%의 방제 효과를 보인 반면 X. ehlersii 혼합제는 95% 이상의 방제효과를 나타냈다. 본 연구는 곤충병원세균 X. ehlersii가 새로운 해충 방제제로 개발될 가능성을 제시하고 있다.

Honeybees are inevitably threatened by various pathogens including Sacbrood virus (SBV) and Galleria mellonella. Recently, RNA interference (RNAi) has been suggested as a promising strategy for suppression of honey bee viruses. Also, Bacillus thuringiensis (Bt) has been widely applied for the control of lepidopteran pests such as G. mellonella. In this study, it was intended to develop dsRNA production platform using Bt. For this, the pHT1K-SBV vp1 vector which transcribes sense and anti-sense SBV vp1 gene under control of Cyt1Aa sporulation-dependent promoter was introduced into Bt strain NT0423 expressing Cry1-types toxins. SBV replication was suppressed in the worker A. cerana ingested dsRNA produced from the Bt transformant. Crystal proteins from the Bt transformant showed high level of insecticidal activity against 4th instar larvae of G. mellonella.

The insecticidal activity of Bacillus thuringiensis subsp. israelensis (Bti) is due to synergistic interactions among its four major proteins (Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa), while the activity of Lysinibacillus sphaericus (Ls) is due to a binary toxin (Bin) consisting of a toxin domain, BinA, and a midgut receptor-binding domain, BinB. Although used commercially for almost three decades, reports of mosquito resistance to Bti have been rare. However, levels of resistance greater than 10,000-fold to Bin have been reported where Ls has been used intensively for mosquito control. Cyt1Aa is a lipophilic protein, and in previous studies we showed it delays the evolution of resistance to the Cry proteins of Bti, and can overcome high levels of resistance Bin. In a previous study, we fused Cyt1Aa to BinA, using the lipophile as a broad-spectrum binding domain and showed that the Cyt1Aa-BinA chimera was remarkably toxic to five major vector species of mosquitoes, Anopheles gambiae, An. stephensi, An. quadrimaculatus, Bin-sensitive and Bin-resistant strains of Culex quinquifasciatus, and Aedes aegypti, the latter not normally sensitive to Ls. However, toxicity against Aedes aegypti was not as high as against other mosquito species. Here we show that introducing another highly mosquitocidal protein, Cry11B from B. thuringiensis subsp. jegathesan, enhances the chimera’s toxicity against Ae. aegypti significantly but not against Cx. quinquefasciatus.

RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pest whereby double-stranded RNA triggers a potent and specific inhibition of its homologous mRNA. Since small dsRNAs are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. In this study, Bacillus thuringiensis (Bt) based dsRNA production platform was established under control of sporulation-dependent promoter and vp1 gene of Sacbrood virus (SBV) was introduced. The dsRNA against vp1 gene produced from the Bt suppressed the replication of SBV. In addition, the dsRNA was assembled into inulin coated-nanoparticle to increase stability of dsRNA in environment.

RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pests whereby double-strandedRNA triggers a potent and specific inhibition of its homologous mRNA. Since small double-stranded RNAs are requiredfor various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-qualitydsRNA. Bacillus thuringiensis produces much insecticidal proteins with expression of their encoding genes being drivenby sporulation-dependent promoters. To develop dsRNA mass-production platform utilizing Bt, the pHT1K-EGFP plasmidvector which has cyt1Aa sporulation-dependent promoter was constructed. The transcriptional level of target gene (EGFP)is higher 113 times than Bt reference gene (ssPE). It was applied to protect honeybee from Sacbrood virus, so targetgene was replaced to SBV-vp1. By ingestion of Bt-derived dsRNA to honeybee shows positive effect on SBV suppression.

RNA interference (RNAi) is an universal gene-knockdown mechanism in eukaryotic organisms including insects. RNAi has been considered as an alternative strategy to control agricultural pests whereby double-stranded RNA triggers a potent and specific inhibition of its homologous mRNA. Bacillus thuringiensis (Bt) is a spore-forming bacterium that produces a copious amount of crystal proteins δ-endotoxins under the control of sporulation-dependent promoter. In order to develop dsRNA mass-production platform utilizing recombinant Bt, the pHT1K-EGFP which expresses dsRNA against EGFP under the control of Cyt1-Aa sporulation-dependent promoter was constructed and the expression level of transgene (EGFP) was confirmed by qPCR analysis. These results suggested that Bt’s potential of becoming a new platform in dsRNA production.

Insecticidal crystal toxins from the bacterium Bacillus thuringiensis (Bt) kill insects via a complex mode of action resulting in the creation of cytolytic pores in the membrane of midgut epithelial cells. These toxins are expressed in transgenic cotton and maize which have been adopted worldwide to control lepidopteran pests while reducing dependence on chemical insecticides. However, insect resistance to Bt toxins is increasing in certain key pest species. Beginning with Heliothis virescens, genetic studies in Bt-resistant Lepidoptera and Coleoptera have found mutations in ABC transporters. Cry1A, Cry1C, Cry2A, and Cry3B toxins each appear to target a different member of the ABC superfamily. These studies confirm the essential role of ABC proteins in Bt toxin mode of action. It is proposed that ABC proteins assist in the insertion of the toxin into the midgut epithelial membrane, a crucial step for which the mechanism has not been known in detail. Properties of ABC transporters suggest strategies to increase efficacy of Bt toxins and to delay the evolution of Bt toxin resistance in target insect pests.

Bacillus thuringiensis (Bt) produces a variety of insecticidal crystal proteins and widely used as one of the most successful biological control agents. Recently, studies that introduce cry genes into crops to create pest resistance have made much progress, and the total area of land planted with Bt crops has increased substantially. In this study, pest resistance of 8 transgenic Bt rice events with a synthetic cry1Ac gene linked to rice rbcS-tp sequence were assessed under laboratory conditions. Bioassays were performed against Cnaphalocrocis medinalis, which is a significant pest of rice in Asia. C. medinalis larvae were shown to be susceptible to all eight events, even though there were differences between the causes of death. The results differed between developmental stages of the larvae, despite the fact that all 8 events led to high mortalities. These results may be a significant foundation for the evaluation of improved transgenic Bt rice.

작은뿌리파리(Bradysia agrestis)는 많은 어린 작물의 뿌리를 직접 가해하여 생육을 억제하거나 고사시키는 심각한 피해를 일으키고 있는 해충이다. 대부분 이러한 피해를 막기 위하여 살충제에 의존하고 있지만 살충제는 저항성 개체의 출현, 인축에 대한 독성, 환경오염 등의 문제점이 있다. 반면 대표적인 곤충병원성 세균인 Bacillus thuringiensis는 인축 및 식물에 무해하며 내독소단백질을 형성하여 곤충에 대해 강력한 독성을 가진 것으로 알려져 있다. 따라서 본 연구는 곤충병원성 미생물을 이용한 친환경적 방제 방법을 탐색하고자 B. thuringiensis 균주를 대상으로 생물검정을 실시하였다. 파리목 유충에 대해 살충활성을 보이는 것으로 알려진 spherical type의 내독소단백질을 가진 16균주를 선발하여 생물검정을 실시한 결과, 높은 방제효과를 나타내며 속효성을 지닌 균주가 확인되었다. 방제효과를 나타낸 균주는 농도별 살충활성을 확인하였으며 SDS-PAGE를 통하여 단백질패턴을 분석하였다.

This study tested a hypothesis that the bacterial immunosuppresants enhance BtI susceptibility of two mosquitoes, the forest mosquito (Aedes albopictus) and the house mosquito (Culex pipiens pallens). Three symbiotic bacteria Xenorhabdus nematophila (Xn), X. hominickii (Xh), and Photorhabdus temperata subsp. temperata (Ptt) were isolated from their symbiotic nematodes and cultured in nutrient broth to allow them to produce the secondary metabolites. BtI gave significant toxicities to A. albopictus and C. pipiens pallens larvae: 50% of lethal concentration to be 2.9 × 105 spores/mL and 2.2 × 105 spores/mL at 16 h after treatment, respectively. Addition of each bacteria-cultured broth significantly enhanced BtI toxicity to the mosquito larvae by lowering LC50 values of BtI to A. albopictus larvae (1.5 × 105 to Xn, 1.7 × 105 to Xh, and 1.9 × 105 to Ptt, respectively) and to C. pipiens pallens larvae (1.2 × 105 to Xn, 1.3 × 105 to Xh, and 1.5 × 105 to Ptt, respectively). Based on these results, we developed a new mosquitocidal Bt formulation called ‘Dip-Kill’, which consisted of 80% Xn-cultured broth, 10% BtI (1010 spores/mL), and 10% preservative. Only 400 ppm of Dip-Kill showed 100% mortality to fourth instar larvae of A. albopictus and C. pipiens pallens 16 h after treatment.

A large number of transgenic crop varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins have been commercialized in 13 countries since 1996. Although the use of these insect-resistant Bt crops can increase crop quality and yields, concerns remain about the potential negative effects of such crops on ecosystems. Transgenic soybean containing cry1Ac gene have been developed to control Lepidopteran pests of soybean and we aimed to investigate whether this soybean could affect non-target arthropods, which play a major role in ecological functions in agricultural ecosystems. In the present study, we first measured the levels of Cry1Ac protein in Bt soybean at different growth stages of soybean and then we compared the community structure of arthropods occurred in fields of transgenic and wild-type soybean. The levels of Cry1Ac protein in transgenic soybean leaves ranged from 252.9 to 604.5 μg g-1 DW. Multivariate analyses (PerMANOVA and NMDS) showed that the composition of the non-target arthropod community was affected by sampling date but not by soybean genotype. These results suggest that transgenic soybean expressing Cry1Ac protein may not adversely affect such non-target arthropod communities.

Recently, Bacillus thuringiensis (Bt) cry genes encoding insecticidal Cry proteins have been widely applied for the construction of transgenic crops resistant to insect pests. This study aimed to construct novel mutant cry1Ac genes for genetically modified crops with enhanced insecticidal activities. Using multi-site directed mutagenesis, 34 mutant cry1Ac genes were synthesized and converted at 24 amino acid residues, located on domain I (8 residues) and domain II (16 residues). These mutant genes were expressed as a fusion protein with polyhedrin using the baculovirus expression system. The expressed proteins were occluded into polyhedra and activated stably to 65 kDa by trypsin. Among these, Mut-N04, N06, and N16 showed high levels of insecticidal activites against larvae of Plutella xylostella, Spodoptera exigua, and Ostrinia furnacalis. Mut-N16, which showed the highest insecticidal activity, is expected to be a desirable cry gene for introduction into transgenic crops. This study could provide useful means to construct mutant cry genes with improved insecticidal activities and expanded host spectrum for transgenic crops.

Bacillus thuringiensis (Bt) is a gram-positive and spore-forming bacterium that produces parasporal inclusions containing Cry and Cyt proteins during sporulation. These inclusions or proteins are highly toxic to lepidopteran, dipteran, coleopteran larvae, and nematodes. Because of their selectable specificity and safety to non-target organisms and environment, Bt is considered as a valuable and safe alternative to chemical pesticides for eradication of insect pests. Bt biopesticide products has been commercialized and used very widely and successfully in agriculture, forestry and medical care markets since 1930s. In Korea, the screening for isolation of new Bt strains such as Bt Nt0423 (Tobbagi) and Bt GB-413 (Solbichae) have been performed and their commercial products were launched in agricultural markets. In terms of public health, mosquito larvicidal products made by Bt subsp. israelensis have been registered by Korean ministry of food and drug safety and used since 1995. Recently, a new mosquitocidal serovar (H3a3b3d), Bt subsp. mogi was isolated and its molecular characteristics were analyzed. In our opinion, Bt biopesticides might be still an effective and safe tool for insect pest control in agriculture and public health. Key words: Bacillus thuringiensis, biopesticide, Bt subsp. israelensis, mosqui

During the last century, the overuse of synthetic chemical insecticides led to problems associated with natural environment such as harmful effects on nontarget organisms and development of resistance. Therefore, the application of insect pathogenic microorganisms including Bacillus thuringiensis (Bt), baculoviruses, and entomopathogenic fungi has been an environmentally favorable methods for insect control. However, their use has been limited by several factors such as narrow host spectrum and slow speed of insecticidal activities. A number of approaches have been taken to overcome these defects of microbial insecticides. A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of the NeuroBactrus, the Bt cry1-5 crystal protein gene was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but also activated by treatment with trypsin, resulting in an approximately 65-kDa active toxin. The NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Expression of the foreign proteins (Bt toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. These results suggested that the NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging. The cry gene from Bt, encoding the Cry protein, has been recently introduced into crops to generate transgenic plants that are resistant to pest insects. Through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of S. exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 34 mutant cry genes. These mutant cry genes were expressed, as a polyhedrin fusion form, using a baculovirus expression system. The expressed proteins were 95 kDa and SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.

Evolution of resistance to entomopathogenic bacterium, Bacillus thuringiensis (Bt), can potentially reduce the efficacy of insecticidal proteins from Bt to insect pests in fields. Bt resistance is involved in modification of the toxin binding to its specific midgut membrane receptors, such as cadherin, aminopeptidase N, alkaline phosphatase, and ABC transporters. The beet armyworm, Spodoptera exigua, is one of major lepidopteran insect pest in Korea and showed highly susceptible to Cry1Ca. We investigated the Cry1Ca toxicity with respect to its binding affinity to a Bt receptor, cadherin compared with Cry1Ac. RNA interference (RNAi) of a cadherin of S. exigua (SeCad1) significantly suppressed the Cry1Ca to the toxic level of Cry1Ac. Binding affinity of Cry1Ca to brush border membrane vesicle (BBMV) of S. exigua midgut was significantly lost after SeCad1 RNAi. Binding affinity of Cry1Ac to BBMV was much low compared to that of Cry1Ca and less sensitive to SeCad1 RNAi. Direct binding assay of Cry toxins to SeCad1 was assessed using a recombinant cadherin repeat 10-11 (rCR10-11) of SeCad1. The addition to rCR10-11 to Cry1Ca significantly enhanced the toxicity under SeCad1 RNAi. However, the synergistic effect of rCR10-11 on toxicity of Cry1Ac was not much significant with poor binding affinity of Cry1Ac compared to Cry1Ca. These results indicate that the differential toxicity of Cry toxins against S. exigua is caused by the different affinities to the Bt receptor, cadherin.

The cry gene from Bacillus thuringiensis (Bt), encoding the Cry protein, has been recently introduced into crops to generate transgenic plants that are resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 31 mutant cry genes. These mutant cry genes were expressed, as a polyhedrin fusion form, using a baculovirus expression system. The expressed proteins were 95 kDa and SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.

Crystals of proteinaceous insecticidal proteins, Cry proteins, produced by Bacill us thuringiensis (Bt) have been generally used used to control insect pests. In this st udy, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Plutella xylostella, Spodopt era exigua and Ostrinia furnacalis were identified. To construct novel cry genes wi th enhanced insecticidal activity, we randomly mutated these 24 amino acid sequen ces by in vitro muti site-directed mutagenesis, resulting in totally 34 mutant cry gen es. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded in to polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activit ies of these mutant Cry proteins against to larvae of P. xylostella, S. exigua, and O. furnacalis were assayed, they showed higher or similar insecticidal activity compar ed to those of Cry1Ac and Cry1C. Especially, among them Mutant-N16 showed th e highest insecticidal activity against to both of P. xylostella, S. exigua and Ostrinia furnacalis. Therefore, Mutant-N16 is estimated to have the potential for the efficac ious bioagent.

Six of cadherins have been selected from the P. xylostella genome 52 open reading frames are annotated as cadherin-like genes. Compared to other 5 cadherins of P. xylostella (PxCads), PxCad1 has the highest homology with other lepidopteran insect cadherins and PxCad1 was expressed in all developmental stages specially in gut tissue. Expression of PxCad1 was suppressed by feeding its specific double-stranded RNA (dsRNA, 150ng/larva) and treatment of dsPxCad1 significantly reduced susceptibility to Bt Cry1Ac toxin. To confirm the specific interaction between PxCad1 and Cry1Ac, a toxin-binding assay was performed using enzyme-linked immunosorbent assay (ELISA). The ELISA indicates that BBMV extracted from PxCad1-silenced P. xylostella have significantly lower binding activity to active form of Cry1Ac than control BBMV. Moreover, the analysis of the binding parameters showed that the toxin affinity (Kd) of the control BBMV extract (BBMV-dsCON) was 6.08 ± 0.84 nM, which was not much different to the affinity value (6.72 ± 0.81 nM) of the dsPxCad1 treatment. However, there was a remarkable difference in number of binding sites (Bmax), in which BBMV-dsCON extract had 1.61 ± 0.04, but the BBMV-dsPxCad1 extract had 0.88 ± 0.02. Taken together, these results are suggest that PxCad1 is a functional receptor for Cry1Ac toxicity against P. xylostella larva.

파밤나방에 활성있는 Bacillus thuringiensis subsp. kurstaki KB100균주에 protease inhibitor인 tannic acid을 혼합처리 했을 때 상승효과를 나타냈다. 본 연구에서는 tannins이 proteases의 활성을 떨어뜨려 나비목 유충의 생장을 억제한다 라는 가설을 토대로 실험을 진행하였다. B. thuringiensis균주와 중장액의 배양시간에 따른 독소의 분해정도를 확인하기 위해 SDS-PAGE를 수행하였다. KB100 toxin을 중장액으로 소화 시켜 시간대별로 단백질 밴드패턴을 확인 한 결과 60kDa의 활성독소가 시간이 지날수록 옅어지는 것을 확인한 반면 tannic acid를 첨가 했을 때는 60kDa의 살충활성 밴드가 계속해서 유지됨을 확인하였다. Trypsin에 의한 B. thuringiensis의 분해능을 SDS-PAGE로 분석한 결과 약 60kDa과 70kDa의 밴드를 나타냈고 tannic acid에 의해서 억제됨을 확인하였다. 파밤나방 중장액에 40mM tannic acid를 처리한 후 각각의 기질에 활성을 측정한 결과 trypsin의 기질인 BApNA, BPVApNA는 각각 62.2±0.3%, 54.5±1.1%의 활성을 나타냈다. 40mM tannic acid의 기질에 대한 단백질 분해 활성 억제정도를 비교한 결과 trypsin 각각의 기질에 대한 활성은 40mM tannic acid를 처리 했을 때 약 30∼40% 활성을 억제하는 것을 확인 하였다. Tannic acid는 serine 계열의 proteases 중에서 trypsin의 활성을 효과적으로 억제하는 것을 예상할 수 있었다. 이와 같은 결과를 토대로 파밤나방 중장 proteases중 trypsin의 서열을 밝히기 위해 5'RACE PCR법을 이용하여 실험을 수행하고 있다.

Bacillus thuringiensis (Bt) is a Gram-positive and soil-dwelling bacterium and well known for its ability to produce insecticidal parasporal crystalline protein inclusions, which have attracted worldwide interest for effective pest management. A diseased silkworm by Bt was first discovered by Japanese scientist Ishiwata Shigetane in 1901, and in 10 years, it was re-discovered in Germany by Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars. The first commercial product, Bt. kurstaki HD-1 was released to a market in France in 1938. Optimization of mass production for crystal production and cost down enabled the industrialization to be successful, and now many products, such as WP, EC, SC and tablets are used worldwide. In 1976 Robert A. Zakharyan found that plasmids in Bt are involved in the production of crystal proteins and endospores. Pore formation model and signal transduction model were revealed to explain the mode of action of Bt. Works on Bt resistance included a group of receptors of crystal proteins, such as cadherin, APN and ALP. In 1996, a Bt cry gene was integrated to cotton, which successfully reached markets. AtMT technology was used to generate Bt crops. Now the area planted worldwide to genetically engineered Bt crops increased to 66 million hectares. Refuge may be particularly important in slowing the spread of insects resistant to the Bt insecticides. Researchers are trying to increase the insecticidal efficacy of integrated Bt crystal proteins using recent biotechnology.