Vacuolar Targeting of Cry1Ac and its Effects on Expression and Stability in Tobacco.

Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes.Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes

Protein engineering of \u3b4-endotoxins of Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a valuable environment-friendly biopesticide, which occupies 90% of the world biopesticide market. Its insecticidal properties are attributed to the presence of \u3b4-endotoxins which are synthesized during the sporulation phase of the bacterium. \u3b4-endotoxin or crystal toxin is a multi-domain protein molecule comprising of three distinct domains. Domain I is made of seven \u3b1-helices, domain II comprises three antiparallel \u3b2 sheets, which are folded into loops and domain III is made of a \u3b2 sandwich of two antiparallel \u3b2 strands. Molecular studies on the structure and functional properties of different \u3b4-endotoxins revealed that the domain I by virtue of its membrane spanning hydrophobic and amphipathic \u3b1-helices is capable of forming pores in the cell membranes of the larval midgut. Domain II being hyper variable in nature determines the insecticidal specificity of a toxin and domain III is involved in varied functions like structural stability, ion channel gating, binding to Brush Border Membrane Vesicles and insecticidal specificity. Recent studies on toxin aggregation and interaction revealed that the three domains interact closely to bring about the insecticidal activity of Bt. In this review we describe the protein engineering studies conducted on different \u3b4-endotoxins which led to an understanding of their molecular mode of action and construction of novel toxins with enhanced insecticidal activity and specificity

Protein engineering of δ-endotoxins of Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a valuable environment-friendly biopesticide, which occupies 90% of the world biopesticide market. Its insecticidal properties are attributed to the presence of d-endotoxins which are synthesized during the sporulation phase of the bacterium. d-endotoxin or crystal toxin is a multi-domain protein molecule comprising of three distinct domains. Domain I is made of seven a-helices, domain II comprises three antiparallel b sheets, which are folded into loops and domain III is made of a b sandwich of two antiparallel b strands. Molecular studies on the structure and functional properties of different d-endotoxins revealed that the domain I by virtue of its membrane spanning hydrophobic and amphipathic a-helices is capable of forming pores in the cell membranes of the larval midgut. Domain II being hyper variable in nature determines the insecticidal specificity of a toxin and domain III is involved in varied functions like structural stability, ion channel gating, binding to Brush Border Membrane Vesicles and insecticidal specificity. Recent studies on toxin aggregation and interaction revealed that the three domains interact closely to bring about the insecticidal activity of Bt. In this review we describe the protein engineering studies conducted on different d-endotoxins which led to an understanding of their molecular mode of action and construction of novel toxins with enhanced insecticidal activity and specificity

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Not AvailableCastor is an industrially valued non-edible oilseed crop. Susceptibility of the crop to foliage feeders like Achaea janata (semilooper) and Spodoptera litura accounts for 30–50% of yield losses. Owing to a lack of reliable sources of resistance to these lepidopteran pests, attempts were made to develop transgenic events expressing the Bacillus thuringiensis (Bt) Cry1Aa gene. Transformation of decotyledonated embryo axes through Agrobacterium tumefaciens, particle gun bombardment and in planta methods resulted in transformation frequencies of 2.4%, 1.1% and 2.1%, respectively. The presence and integration of the cry1Aa gene in the T0 plants was confirmed by polymerase chain reaction (PCR) and Southern hybridization analysis. Based on segregation for a Mendelian ratio of 3:1, eight events (AMT-894, AMT-899, AK1304-PB-1, AK1304-PB-4, AK1304-PB-785, AK1304-PB-830, AK1304-PB-837 and DTS-43) were advanced. ELISA analysis detected protein from 0.16 to 2.76 ng/mg fresh leaf tissue across events and in different generations. In laboratory insect bioassays, the mortality of S. litura and A. janata ranged from 20 to 80% in different transgenic events and the weight reduction of surviving larvae over the control larvae after 8 days of feeding was 28.4–87.2% in the case of S. litura and 27.9–78.1% for A. janata. In field bioassays, the event AMT-894 was most promising with 43% of plants showing less than 25% leaf damage. As part of the regulatory requirement to check the toxicity of the transgenic events to beneficial insects, larval bioassays against Samia cynthia ricini (eri silkworm) using three transgenic events (AK1304-PB-1, AK1304-PB-4 and AMT-894) showed a 20.2–78.5% reduction in weight.Not Availabl

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Not AvailableCastor is an industrially valued non-edible oilseed crop. Susceptibility of the crop to foliage feeders like Achaea janata (semilooper) and Spodoptera litura accounts for 30-50% of yield losses. Owing to a lack of reliable sources of resistance to these lepidopteran pests, attempts were made to develop transgenic events expressing the Bacillus thuringiensis (Bt) cry1Aa gene. confirmed by polymerase chain reaction (PCR) and Southern hybridization analysis. Based on segregation for a Mendelian ratio of 3:1, eight events (AMT-894, AMT-899, AK1304-PB-1, AK1304-PB-4, AK1304-PB-785, AK1304-PB-830, AK1304-PB-837 and DTS-43) were advanced. ELISA analysis detected protein from 0.16-2.76 ng/mg fresh leaf tissue across events and in different generations. In laboratory insect bioassays, the mortality of S. litura and A. janata ranged from 20-80% in different transgenic events and the weight reduction of surviving larvae over the control larvae after 8 days of feeding was 28.4-87.2% in the case of S. litura and 27.9-78.1% for A. janata. In field bioassays, the event AMT-894 was most promising with 43% 34 of plants showing less than 25% leaf damage. As part of the regulatory requirement to check the toxicity of the transgenic events to beneficial insects, larval bioassays against Samia cynthia ricini (eri silkworm) using three transgenic events (AK1304-PB-1, AK1304-PB-4 and AMT- 894) showed a 20.2 to 78.5% reduction in weightNot Availabl

Delineating the glycoproteome of elongating cotton fiber cells

The data presented here delineates the glycoproteome component in the elongating cotton fiber cells attained using complementary proteomic approaches followed by protein and N-linked glycosylation site identification (Kumar et al., 2013) [1]. Utilizing species specific protein sequence databases in proteomic approaches often leads to additional information that may not be obtained using cross-species databases. In this context we have reanalyzed our glycoproteome dataset with the Gossypium arboreum, Gossypium raimondii (version 2.0) and Gossypium hirsutum protein databases that has led to the identification of 21 N-linked glycosylation sites and 18 unique glycoproteins that were not reported in our previous study. The 1D PAGE and solution based glycoprotein identification data is publicly available at the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) [2] using the dataset identifier PXD000178 and the 2D PAGE based protein identification and glycopeptide approach based N-linked glycosylation site identification data is available at the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) [2] using the dataset identifier PXD002849

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Not AvailableWeed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T 0, T 1 and T 2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1 % of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.Not Availabl