L'invention concerne des sequences de polynucleotides isolees codant les proteines ET37, TIC8... more L'invention concerne des sequences de polynucleotides isolees codant les proteines ET37, TIC810 et TIC812 issues de Bacillus thuringiensis et des sequences de nucleotides a utiliser dans l'expression de TIC809, ET37, TIC810 et TIC812 et des fusions de diverses combinaisons efficaces sur le plan insecticides de ces proteines, telles que TIC 127, chez des vegetaux. L'invention concerne egalement des procedes de preparation et d'utilisation des sequences de polynucleotides et des proteines dans le developpement de cellules de vegetaux transgeniques et des vegetaux transgeniques presentant une resistance amelioree contre les insectes suivants: (1) des insectes coleopteres, notamment la chrysomele occidentale des racines du mais (Dibrotica virgifera), la chrysomele maculee du concombre (Dibrotica undecempunctata), la chrysomele des racines du mais (Diabrotica barbed), la chrysomele des racines du mais mexicaine (Diabrotica virgifera zeae), la chrysomele des racines du mai...
L'invention porte sur des isolats du Bacillus thuringiensis agissant contre les insectes nuis... more L'invention porte sur des isolats du Bacillus thuringiensis agissant contre les insectes nuisibles du type lepidopteres, et sur certaines proteines en cristaux toxiques pour les lepidopteres produites par les isolats du Bacillus thuringiensis.
Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RN... more Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI β; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRN...
Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 a... more Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal a...
Tn5401 is a class II transposable element derived from the gram-positive bacterium Bacillus thuri... more Tn5401 is a class II transposable element derived from the gram-positive bacterium Bacillus thuringiensis. The 4,837-bp transposon encodes a Tn3-like transposase (TnpA) and an integrase-like recombinase (TnpI) and is notable for its unusually long 53-bp terminal inverted repeats (TIRs). ThetnpA and tnpI genes are transcribed from a common promoter, designated PR, that is subject to negative regulation by TnpI. The TIRs of Tn5401 each contain a 38-bp sequence that can be aligned with the 38- to 40-bp TIR sequences of Tn3-like transposons and an adjacent 12-bp sequence that binds TnpI. This unique juxtaposition of TnpA and TnpI binding sites suggests that TnpI may regulate the binding or catalytic activity of TnpA. The results of the present study indicate that TnpI, in addition to functioning as a site-specific recombinase and as a transcriptional repressor, is required for TnpA binding to the TIRs of Tn5401.
Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity agai... more Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant incorporated protectants (PIP's) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors, demonstrate that receptor utilization of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together, should provide durability in the field against this economically important pest. IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently ex...
The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed... more The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed protein that is toxic to coleopteran insect larvae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the asporogenous B. thuringiensis subsp. morrisoni strain EG1351, which harbors the native cryIIIA-encoding 88-MDa plasmid, showed a 2.5-fold overproduction of the CryIIIA protein compared with that of an isogenic wild-type strain. Further studies showed that neither CryIIIA protein synthesis nor CryIIIA protein processing was affected in strain EG1351 during vegetative growth. In an attempt to characterize the EG1351 mutation by complementation of function, the hknA gene was identified and cloned from a B. thuringiensis cosmid library. Primer extension analysis of hknA mRNA in wild-type B. thuringiensis demonstrated that the hknA gene is transcribed during vegetative growth from a sigma A-like promoter. Multiple copies of either the hknA gene or the Bacillus subtili...
The Bacillus thuringiensis class II transposon Tn5401 encodes a recombinase protein, TnpI, that m... more The Bacillus thuringiensis class II transposon Tn5401 encodes a recombinase protein, TnpI, that mediates the resolution of cointegrate molecules generated as intermediates during Tn5401 transposition by the TnpA transposase. This recombination event requires a specific target site, or internal resolution site, at which TnpI binds and catalyzes the exchange of DNA strands. Gel mobility shift assays and DNase I footprinting analyses were used to localize the TnpI binding region to the sequence extending from nucleotides 637 to 747 of Tn5401. Deletions within this region blocked TnpI-mediated recombination in vivo. The 12-bp sequence ATGTCC RCTAAY, present in four copies within the TnpI binding region, is proposed to be the recognition sequence for TnpI binding. TnpI also binds to a single copy of this sequence located within the 53-bp terminal inverted repeats of Tn5401. The unique juxtaposition of recombinase and transposase binding sites at the terminal inverted repeats of Tn5401 su...
The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation... more The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.
A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulati... more A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulation-deficient variant of Bacillus thuringiensis subsp. morrisoni EG2158 following its insertion into a recombinant plasmid. Sequence analysis of the insert revealed a 4,837-bp transposon with two large open reading frames, in the same orientation, encoding proteins of 36 kDa (306 residues) and 116 kDa (1,005 residues) and 53-bp terminal inverted repeats. The deduced amino acid sequence for the 36-kDa protein shows 24% sequence identity with the TnpI recombinase of the B. thuringiensis transposon Tn4430, a member of the phage integrase family of site-specific recombinases. The deduced amino acid sequence for the 116-kDa protein shows 42% sequence identity with the transposase of Tn3 but only 28% identity with the TnpA transposase of Tn4430. Two small open reading frames of unknown function, designated orf1 (85 residues) and orf2 (74 residues), were also identified. Southern blot analysis i...
The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysano... more The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysanopteran insect pests in cotton was characterized by selecting and screening multiple pest and non-pest species, based on representation of ecological functional groups, taxonomic relatedness (e.g. relationship to species where activity was observed), and availability for effective testing. Seven invertebrate orders, comprising 12 families and 17 representative species were screened for susceptibility to Cry51Aa2.834_16 protein and/or the ability of the protein to protect against feeding damage in laboratory, controlled environments (e.g. greenhouse/growth chamber), and/or field studies when present in cotton plants. The screening results presented for Cry51Aa2.834_16 demonstrate selective and limited activity within three insect orders. Other than Orius insidiosus, no activity was observed for Cry51Aa2.834_16 against several groups of arthropods that perform key ecological roles in some a...
Lygus species of plant-feeding insects have emerged as economically important pests of cotton in ... more Lygus species of plant-feeding insects have emerged as economically important pests of cotton in the United States. These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton varieties resulting in economic losses and increased application of insecticide. Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity against Lygus spp. However, transgenic cotton plants expressing this protein did not exhibit effective protection from Lygus feeding damage. Here we employ various optimization strategies, informed in part by protein crystallography and modelling, to identify limited amino-acid substitutions in Cry51Aa2 that increase insecticidal activity towards Lygus spp. by >200-fold. Transgenic cotton expressing the variant protein, Cry51Aa2.834_16, reduce populations of Lygus spp. up to 30-fold in whole-plant caged field trials. One transgenic event, designated MON88702, has been selected for further development of cotton varieties tha...
L'invention concerne des sequences de polynucleotides isolees codant les proteines ET37, TIC8... more L'invention concerne des sequences de polynucleotides isolees codant les proteines ET37, TIC810 et TIC812 issues de Bacillus thuringiensis et des sequences de nucleotides a utiliser dans l'expression de TIC809, ET37, TIC810 et TIC812 et des fusions de diverses combinaisons efficaces sur le plan insecticides de ces proteines, telles que TIC 127, chez des vegetaux. L'invention concerne egalement des procedes de preparation et d'utilisation des sequences de polynucleotides et des proteines dans le developpement de cellules de vegetaux transgeniques et des vegetaux transgeniques presentant une resistance amelioree contre les insectes suivants: (1) des insectes coleopteres, notamment la chrysomele occidentale des racines du mais (Dibrotica virgifera), la chrysomele maculee du concombre (Dibrotica undecempunctata), la chrysomele des racines du mais (Diabrotica barbed), la chrysomele des racines du mais mexicaine (Diabrotica virgifera zeae), la chrysomele des racines du mai...
L'invention porte sur des isolats du Bacillus thuringiensis agissant contre les insectes nuis... more L'invention porte sur des isolats du Bacillus thuringiensis agissant contre les insectes nuisibles du type lepidopteres, et sur certaines proteines en cristaux toxiques pour les lepidopteres produites par les isolats du Bacillus thuringiensis.
Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RN... more Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI β; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRN...
Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 a... more Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal a...
Tn5401 is a class II transposable element derived from the gram-positive bacterium Bacillus thuri... more Tn5401 is a class II transposable element derived from the gram-positive bacterium Bacillus thuringiensis. The 4,837-bp transposon encodes a Tn3-like transposase (TnpA) and an integrase-like recombinase (TnpI) and is notable for its unusually long 53-bp terminal inverted repeats (TIRs). ThetnpA and tnpI genes are transcribed from a common promoter, designated PR, that is subject to negative regulation by TnpI. The TIRs of Tn5401 each contain a 38-bp sequence that can be aligned with the 38- to 40-bp TIR sequences of Tn3-like transposons and an adjacent 12-bp sequence that binds TnpI. This unique juxtaposition of TnpA and TnpI binding sites suggests that TnpI may regulate the binding or catalytic activity of TnpA. The results of the present study indicate that TnpI, in addition to functioning as a site-specific recombinase and as a transcriptional repressor, is required for TnpA binding to the TIRs of Tn5401.
Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity agai... more Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant incorporated protectants (PIP's) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors, demonstrate that receptor utilization of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together, should provide durability in the field against this economically important pest. IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently ex...
The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed... more The Bacillus thuringiensis CryIIIA insecticidal crystal protein (ICP) is a vegetatively expressed protein that is toxic to coleopteran insect larvae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the asporogenous B. thuringiensis subsp. morrisoni strain EG1351, which harbors the native cryIIIA-encoding 88-MDa plasmid, showed a 2.5-fold overproduction of the CryIIIA protein compared with that of an isogenic wild-type strain. Further studies showed that neither CryIIIA protein synthesis nor CryIIIA protein processing was affected in strain EG1351 during vegetative growth. In an attempt to characterize the EG1351 mutation by complementation of function, the hknA gene was identified and cloned from a B. thuringiensis cosmid library. Primer extension analysis of hknA mRNA in wild-type B. thuringiensis demonstrated that the hknA gene is transcribed during vegetative growth from a sigma A-like promoter. Multiple copies of either the hknA gene or the Bacillus subtili...
The Bacillus thuringiensis class II transposon Tn5401 encodes a recombinase protein, TnpI, that m... more The Bacillus thuringiensis class II transposon Tn5401 encodes a recombinase protein, TnpI, that mediates the resolution of cointegrate molecules generated as intermediates during Tn5401 transposition by the TnpA transposase. This recombination event requires a specific target site, or internal resolution site, at which TnpI binds and catalyzes the exchange of DNA strands. Gel mobility shift assays and DNase I footprinting analyses were used to localize the TnpI binding region to the sequence extending from nucleotides 637 to 747 of Tn5401. Deletions within this region blocked TnpI-mediated recombination in vivo. The 12-bp sequence ATGTCC RCTAAY, present in four copies within the TnpI binding region, is proposed to be the recognition sequence for TnpI binding. TnpI also binds to a single copy of this sequence located within the 53-bp terminal inverted repeats of Tn5401. The unique juxtaposition of recombinase and transposase binding sites at the terminal inverted repeats of Tn5401 su...
The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation... more The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.
A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulati... more A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulation-deficient variant of Bacillus thuringiensis subsp. morrisoni EG2158 following its insertion into a recombinant plasmid. Sequence analysis of the insert revealed a 4,837-bp transposon with two large open reading frames, in the same orientation, encoding proteins of 36 kDa (306 residues) and 116 kDa (1,005 residues) and 53-bp terminal inverted repeats. The deduced amino acid sequence for the 36-kDa protein shows 24% sequence identity with the TnpI recombinase of the B. thuringiensis transposon Tn4430, a member of the phage integrase family of site-specific recombinases. The deduced amino acid sequence for the 116-kDa protein shows 42% sequence identity with the transposase of Tn3 but only 28% identity with the TnpA transposase of Tn4430. Two small open reading frames of unknown function, designated orf1 (85 residues) and orf2 (74 residues), were also identified. Southern blot analysis i...
The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysano... more The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysanopteran insect pests in cotton was characterized by selecting and screening multiple pest and non-pest species, based on representation of ecological functional groups, taxonomic relatedness (e.g. relationship to species where activity was observed), and availability for effective testing. Seven invertebrate orders, comprising 12 families and 17 representative species were screened for susceptibility to Cry51Aa2.834_16 protein and/or the ability of the protein to protect against feeding damage in laboratory, controlled environments (e.g. greenhouse/growth chamber), and/or field studies when present in cotton plants. The screening results presented for Cry51Aa2.834_16 demonstrate selective and limited activity within three insect orders. Other than Orius insidiosus, no activity was observed for Cry51Aa2.834_16 against several groups of arthropods that perform key ecological roles in some a...
Lygus species of plant-feeding insects have emerged as economically important pests of cotton in ... more Lygus species of plant-feeding insects have emerged as economically important pests of cotton in the United States. These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton varieties resulting in economic losses and increased application of insecticide. Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity against Lygus spp. However, transgenic cotton plants expressing this protein did not exhibit effective protection from Lygus feeding damage. Here we employ various optimization strategies, informed in part by protein crystallography and modelling, to identify limited amino-acid substitutions in Cry51Aa2 that increase insecticidal activity towards Lygus spp. by >200-fold. Transgenic cotton expressing the variant protein, Cry51Aa2.834_16, reduce populations of Lygus spp. up to 30-fold in whole-plant caged field trials. One transgenic event, designated MON88702, has been selected for further development of cotton varieties tha...
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