Bacillus Thuringiensis Δ-endotoxin Research Articles

Transgenic japonica rice expressing the cry1C gene is resistant to striped stem borers in Northeast China

Rice production and quality are seriously affected by the lepidopteran pest, striped stem borer (SSB), in Northeast China. In this study, a synthetic cry1C gene encoding Bacillus thuringiensis (Bt) δ-endotoxin, which is toxic to lepidopteran pest, was transformed into a japonica rice variety (Jigeng 88) in Northeast China by Agrobacterium-mediated transformation. Through molecular detection and the Basta resistance germination assay, a total of 16 single-copy homozygous transgenic lines were obtained from 126 independent transformants expressing cry1C. Finally, four cry1C-transgenic lines (JL16, JL23, JL41, and JL42) were selected by evaluation of the Cry1C protein level, insect-resistance and agronomic traits. The cry1C-transgenic lines had higher resistance to SSB and higher yield compared with non-transgenic (NT) control plants. T-DNA flanking sequence analysis of the transgenic line JL42 showed that the cry1C gene was inserted into the intergenic region of chromosome 11, indicating that its insertion may not interfere with the genes near insertion site. In summary, this study developed four cry1C-transgenic japonica rice lines with high insect resistance and high yield. They can be used as insect-resistant germplasm materials to overcome the problem of rice yield reduction caused by SSB and reduce the use of pesticides in Northeast China.

Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice

DNA test is broadly used in diagnosis of crop disease and identification of genetic modified organism (GMO) in agriculture. However, rapid, low-cost, user-friendly, and field-deployable DNA test method is still limit. Recently, the RNA programmable nuclease of CRISPR/Cas is engineered as a new nucleic acid detection platform, but their application in plant remains to explore. In this study, we evaluated the Cas12a-based DNA detection for crop disease diagnosis and GMO test. A total of 14 crRNAs were designed to target two Magnaporthe oryzae genes and a synthetic Cry1C gene which encodes Bacillus thuringiensis δ-endotoxin and has been used to develop transgenic rice cultivar (Bt-rice) in China. Using a fluorescent reporter, targeted genes were easily detected by LbCas12a after recombinase polymerase amplification (RPA) for all crRNAs, despite that the signal strength varied 2–3-folds between different crRNAs. We further combined the filter paper-based DNA extraction and lateral flow assay (LFA) with RPA-Cas12a for DNA detection. This optimized Cas12a diagnostics method is carried at body temperature and does not require extra instrument except filter paper and LFA strip. Our data show that rice blast pathogen and Bt-rice were efficiently identified from leaf disc samples using this optimized DNA test method with highly active crRNAs. Moreover, LbCas12a exhibited variable nuclease activities on different targets; therefore, highly active crRNA is critical for successful DNA test using Cas12a and LFA. Owing to its simplicity, efficiency, and low-cost, DNA test using CRISPR/Cas12a would be easily applied in field for crop disease diagnosis and GMO administration.

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

Toxin stability improvement and toxicity increase against dipteran and lepidopteran larvae of Bacillus thuringiensis crystal protein Cry2Aa.

Bacillus thuringiensis δ-endotoxins are the most widely used biopesticides for controlling economically important crop pests and disease vectors. Improving their efficacy is of great benefit. Here, an improvement in Cry2Aa δ-endotoxin toxicity was attempted via a cry gene over expression system using P20 from B. thuringiensis israelensis. The coexpression of Cry2Aa with P20 resulted in a seven fold increase in its production yield in B. thuringiensis. Generated crystals proved to be significantly more toxic (505.207 µg g-1 , 1.99 mg L-1 and 1.49 mg L-1 ) than the P20-lacking control (720.78 µg g-1 , 705.69 mg L-1 and 508.51 mg L-1 ) against Ephestia kuehniella, Aedes aegypti and Culex pipiens larvae respectively. In vitro, processing experiments revealed a P20-mediated protection of Cry2Aa against degradation under larval gut conditions. Thus, P20 could promote the maintenance of a tightly packaged conformation of Cry2Aa toxins in the larval midgut upon correct activation and binding to its membrane receptors. Based on their resistance against excessive proteolysis, Cry2Aa δ-endotoxins, produced in the presence of P20, could be considered as a successful control agent for E. kuehniella and an effective alternative for mosquito control, implying its possible exploitation in pest management programmes. © 2016 Society of Chemical Industry.

Development of lepidopteran pest-resistant transgenic japonica rice harboring a synthetic cry2A* gene

A synthetic cry2A* gene encoding Bacillus thuringiensis (Bt) δ-endotoxin that resistance to lepidopteran pest was transformed into japonica rice variety Jijing 88, which is the most widely cultivated variety in Jilin Province, Northeast China, by Agrobacterium-mediated transformation. A total of 106 independent transformants overexpressing cry2A* gene driven by ubiquitin (Ubi) promoter was produced. Three single-copy homozygous transgenic lines were finally selected based on the results of PCR analysis, segregation ratio of Basta resistance, and Southern hybridization analyses. RT-PCR and enzyme linked immune sorbent assay (ELISA) revealed that cry2A* transcripts and protein were highly expressed in these lines. The high level of Cry2A* protein expression resulted in high resistance to rice striped stem borer as evidenced by insect feeding bioassays. Our results demonstrate that cry2A* transgenic japonica rice confers resistance to the rice striped stem borer in the laboratory conditions.

Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.

Development of Insect-Resistant Hybrid Rice by Introgressing the Bt Gene from Bt Rice Huahui 1 into II-32A/B, a Widely Used Cytogenic Male Sterile System

Huahui 1 is an elite transgenic male sterile restorer line of wild rice abortive-type that expresses a Bacillus thuringiensis (Bt) δ-endotoxin and provides effective and economic control of lepidopteran insects. To exploit Huahui 1 to develop a new Bt rice, the insertion site of the Bt gene was determined by thermal asymmetric interlaced PCR (TAIL-PCR). Bt was located in the promoter region of LOC.Os10g10360, approximately 5.35 Mb from the telomere of the short arm of chromosome 10. For the first time, a Bt cytoplasmic male sterile (CMS) system was developed by introgressing Bt from Huahui 1. The recipient CMS system used consisted of Indonesia paddy rice-type II-32B (maintainer line) and II-32A (male sterile line). Marker-assisted selection was used to increase selection efficiency in the backcrossing program. In BC5F1, the Bt plant 85015-8 was selected for further analyses, as it had the highest SSR marker homozygosity. In addition, the linkage drag of the foreign Bt gene in 85015-8 was minimized to 8.01-11.46 Mb. The foreign Bt gene was then delivered from 85015-8 into II-32A. The resultant Bt II-32A and Bt II-32B lines were both resistant to lepidopteran in field trials, and agronomic traits were not disturbed. The maintainability of II-32B, and the male sterility and general combining ability of II-32A, were not affected by the Bt introgression. This study demonstrates a simple and fast approach to develop Bt hybrid rice.

Production of marker-free transgenic Jatropha curcas expressing hybrid Bacillus thuringiensis δ-endotoxin Cry1Ab/1Ac for resistance to larvae of tortrix moth (Archips micaceanus).

BackgroundThe potential biofuel plant Jatropha curcas L. is affected by larvae of Archips micaceanus (Walker), a moth of the family Tortricidae. The hybrid Bacillus thuringiensis (Bt) δ-endotoxin protein Cry1Ab/1Ac confers resistance to lepidopteran insects in transgenic rice.ResultsHere, we report the production of a marker-free transgenic line of J. curcas (L10) expressing Cry1Ab/1Ac using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L10 carries a single copy of marker-free T-DNA that contains the Cry1Ab/1Ac gene under the control of a maize phosphoenolpyruvate carboxylase gene promoter (P Pepc :Cry1Ab/1Ac:T Nos ). The P Pepc :Cry1Ab/1Ac:T Nos gene was highly expressed in leaves of L10 plants. Insecticidal bioassays using leaf explants of L10 resulted in 80-100% mortality of larvae of A. micaceanus at 4 days after infestation.ConclusionThe results demonstrate that the hybrid Bt δ-endotoxin protein Cry1Ab/1Ac expressed in Jatropha curcas displays strong insecticidal activity to A. micaceanus. The marker-free transgenic J. curcas line L10 can be used for breeding of insect resistance to A. micaceanus.

Increased Bacillus thuringiensis δ-endotoxin Cry3Aa toxicity against longhorned beetle by fusing to peptide specifically binding to beetle Cx-cellulase

Background Bacillus thuringiensis (Bt) Cry toxins have specific toxicity to susceptible insects. They are being used in transgenic plants or spray to control insect pests in agriculture [1, 2, 3]. Cry3A toxins are used extensively for biological control of coleopteran larvae [4, 5]. A Bt886-Cry3Aa gene that exhibited a high activity against Coleoptera insects isolated Our laboratory. Insect bioassay performed on Anoplophora glabripennis Motsch and Apriona germari Hope showed that the mortality of larvae fed with the product of this gene was over 60% [6]. However, both transgenic poplar with native Cry3Aa and withmodified-Cry3Aa by using poplar-prefered codons did little effects on longhorned beetles probably due to its low expression level in poplar.A peptide (LPPNPTK) named PCx that specifically bind to cellulase from midgut of longhorned beetle larvae was screened out from a phage display library previously in our laboratory[7].

Metabolic profiling of transgenic rice progeny using gas chromatography–mass spectrometry: the effects of gene insertion, tissue culture and breeding

The Bacillus thuringiensis δ-endotoxin and cowpea trypsin inhibitor genes have been introduced into the rice genome to improve its pest resistance via Agrobacterium-mediated transformation. A gas chromatography-mass spectrometry (GC–MS) based metabolic profiling method was employed to determine the unpredictable metabolic changes resulting from the gene insertion and tissue culture separately. Descendants of the same transformant were obtained from different breeding programs, including both the transgenic and null-segregant progeny. The comparison of the transgenic and respective null-segregant plants enabled the evaluation of variations caused by transgenes; also the null-segregant plants were compared with the wild-type control to identify the influence of tissue culture. Based on the GC–MS metabolic profiles, the principal component analysis and significant differences determined by Student’s t-test suggested that there were more metabolic changes from the tissue culture than those from the insertion of the transgenes. By comparing different breeding programs, it was clear that the progeny which was developed after several generations of backcross with the non-transformed rice as the recurrent parent, displayed fewer metabolic differences from the non-transformed parent. A GC–MS based metabolic profiling study confirmed that backcrossing can help to reduce unwanted variations that occur during transformation processes.

Developmental and photosynthetic regulation of δ-endotoxin reveals that engineered sugarcane conferring resistance to ‘dead heart’ contains no toxins in cane juice

The phosphoenolpyruvate (PEP) carboxylase is regulated at the levels of transcription and post-translation in C4 plants in light and abundantly accumulates in leaf mesophyll cells. We report here developmental and photosynthetic regulation of stably accumulated Bacillus thuringiensis δ-endotoxin under the control of PEP-C promoter in transgenic sugarcane. In young leaves of plants, the transprotein is accumulated to 39% of the levels in mature leaves (135 ng mg(-1)), and is induced with the cell development, from base to tip. Nevertheless, these levels are decreased up to 99.98% in non-photosynthetic cells as cane matures, from top to bottom, suggesting the photosynthesis regulation of δ-endotoxin in cane cells. Further, transgenic plants are highly resistant to 'dead heart'. In these studies, Scirpophaga nivela larvae causing 'dead heart' were killed within one hour of release to the transgenic plants. Therefore, this report may be regarded as the first report that provides a better strategy for developing transgenic sugarcane lines with absolute protection against invading larvae and no toxin residues in cane juice.

Binding of Bacillus thuringiensis Cry1A toxins with brush border membrane vesicles of maize stem borer ( Chilo partellus Swinhoe)

Maize stem borer ( Chilo partellus) is a major insect pest of maize and sorghum in Asia and Africa. Bacillus thuringiensis ( Bt) δ-endotoxins have been found effective against C. partellus, both in diet-overlay assay and in transgenic plants. Gene stacking as one of the resistance management strategies in Bt maize requires an understanding of receptor sharing and binding affinity of δ-endotoxins. In the present study, binding affinity of three fluorescein isothiocyanate labeled Cry1A toxins showed high correlation with the toxicity of respective δ-endotoxins. Competitive binding studies showed that Cry1Ab toxins share some of the binding sites with Cry1Aa and Cry1Ac with low affinity and that Cry1Ab may have additional binding sites that are unavailable to the other two toxins tested.

Histo- and cyto-pathological changes induced by Bacillus thuringiensis δ-endotoxin in the midgut epithelium of silkworm larvae

Histo- and cyto-pathological changes induced by Bacillus thuringiensis δ-endotoxin in the midgut epithelium of silkworm larvae

Production of marker-free transgenic rice expressing tissue-specific Bt gene

The hybrid Bacillus thuringiensis (Bt) δ-endotoxin gene Cry1Ab/Ac was used to develop a transgenic Bt rice (Oryza sativa L.) targeting lepidopteran insects of rice. Here, we show the production of a marker-free and tissue-specific expressing transgenic Bt rice line L24 using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L24 carries a single copy of marker-free T-DNA that contains the Cry1Ab/Ac gene driven by a maize phosphoenolpyruvate carboxylase (PEPC) gene promoter. The marker-free T-DNA was integrated into the 3' untranslated region of rice gene Os01g0154500 on the short arm of chromosome 1. Compared to the constitutive and non-specific expression of the P (Actin1):Cry1Ab/Ac:T (Nos) gene in the control Bt rice line T51-1, the P ( Pepc ):Cry1Ab/Ac:T (Nos ) gene was detected only in the leaf and stem tissues of L24. More importantly, compared to high levels of CRY1Ab/Ac proteins accumulated in T51-1 seeds, the CRY1Ab/Ac proteins were not detectable in L24 seeds by Western blot analysis. As demonstrated by insect bioassay, L24 provided similar level of resistance to rice leaffolder (Cnaphalocrocis medinalis) as T51-1. The marker-free transgenic line L24 can be used directly in rice breeding for insect resistance to lepidopteran insects where absence of Bt toxin protein in the seed is highly desirable.

Including dissolved oxygen dynamics into the Bt δ-endotoxins production process model and its application to process control

This paper proposes a model to characterize the Dissolved Oxygen Dynamics (DO) for the Bacillus thuringiensis (Bt) δ-endotoxins production process. The objective of this work is to include this dynamics into a phenomenological model of the process in order to facilitate the biomass estimation from the knowledge of oxygen consumption; and for control purposes, by allowing the addition of a new control variable in order to favorably influence the bioprocess evolution. The mentioned DO model is based on first principles and parameter estimation and model verification are supported by real experimental data. Finally, a control strategy is designed based on this model with its corresponding asymptotic stability and robustness analysis.

Biomass estimation in batch biotechnological processes by Bayesian Gaussian process regression

This paper proposes a biomass concentration estimator for a batch biotechnological process based on Bayesian regression with Gaussian process. On the basis of experimental data, a two-stage bootstrap technique has been developed for the estimator design. In the first stage, the biomass data set was augmented with virtual filtered measurements, and in the second stage, the biomass estimator design was completed. The method provides information on the confidence level of the estimates, and the biomass estimator performances are illustrated for the Bacillus thuringiensis δ-endotoxins production process.

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis ( Bt) δ-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.

OPTIMIZATION OF A CULTURE MEDIUM FOR BIOMASS AND δ-ENDOTOXIN PRODUCTION BY A RECOMBINANT ESCHERICHIA COLI STRAIN

A recombinant strain of Escherichia coli harboring a plasmid containing the Bacillus thuringiensis δ-endotoxin synthesis gene, was tested for its efficacy to synthesize δ-endotoxin, in a complex medium containing sucrose and yeast extract. Also, the recombinant E. coli strain was tested for its efficacy against the 2nd instars of Spodoptera littoralis. The recombinant strain of E. coli showed a good activity against the 2nd instars of S. littoralis, the mortality was 70 % after 7 days at room temperature. A high cell biomass (8.8gL-1) and δ-endotoxin concentration (6.8 mg L-1), were obtained by the shake flask culture (100 ml medium/250 ml flask, at 200 rpm), of the recombinant E. coli in modified MR medium containing sucrose (20g/L), as carbon source and yeast extract as nitrogen source, in the presence of CaCO3, K2HPO4, MgSO4, FeSO4 and ZnSO4 as mineral salts. The best pH values for cell biomass production and endotoxin production were 7.0 and 7.5, respectively. The corresponding figures for the best temperature were 37oC and 30oC, respectively. The use of some byproducts such as black-strap molasses, corn-steep liquor and cheese whey, as an alternative for carbon and nitrogen sources of medium, were found to enhance the cell growth but showed no effect on endotoxin production.

Development of insect-resistant transgenic indica rice with a synthetic cry1C* gene

Stemborers and leaffolders are two groups of lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, a cry1C* gene encoding Bacillus thuringiensis (Bt) δ-endotoxin was synthesized by codon optimization as the first step towards gene stacking in our resistance management strategy of transgenic rice. Agrobacterium-mediated transformation of this gene into Minghui 63 (Oryza sativa L.), an elite indica CMS restorer line, produced 120 independently transformed plants, 19 of which had a single-copy transgene. Preliminary screening of T1 families of these 19 transformants in the field identified five lines showing a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Hybrids were produced by crossing these five lines with Zhenshan 97A, the male-sterile line for Shanyou 63, the most widely cultivated hybrid in China. These five lines and their hybrids were highly resistant to yellow stemborer (Tryporyza incertulas) as revealed by an insect bioassay. The content of Cry1C* protein varied considerably among the five lines as well as among the corresponding hybrids. T1c-19, a line showing the highest content of Cry1C* protein, and its hybrid were tested in the field for insect resistance and agronomic performance and found to be highly resistant to stemborers and leaffolders throughout the growth period, resulting in a significantly increased grain yield compared with the respective controls. These results indicate that T1c-19 can be used for production of insect-resistant hybrid rice and as a germplasm for gene stacking to produce rice plants with two toxins.

Bacillus thuringiensis δ-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids

We investigated the role of domain III of Bacillus thuringiensis δ-endotoxin Cry1Ac in determining toxicity against Heliothis virescens. Hybrid toxins, containing domain III of Cry1Ac with domains I and II of Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, and Cry1Fb, respectively, were created. In this way Cry1Ca, Cry1Fb, and to a lesser extent Cry1Ba were made considerably more toxic.