Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-12-28
2004-04-27
Prouty, Rebecca E. (Department: 1652)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S295000, C530S350000, C424S093461, C514S012200, C435S252300, C435S419000, C536S023710
Reexamination Certificate
active
06727409
ABSTRACT:
This invention relates to four novel strains of
Bacillus thuringiensis
(the “BTS02617A strain”, the “BTS02618A strain”, the “BTS02654B strain” and the “BTS02652E strain”), each of which produces crystallized proteins (the “BTS02617A crystal proteins”, the “BTS02618A crystal proteins”, the “BTS02654B crystal proteins” and the “BTS02652E crystal proteins”, respectively) which are packaged in crystals (the “BTS02617A crystals”, the “BTS02618A crystals”, the “BTS02654B crystals” and the “BTS02652E crystals”, respectively) during sporulation. The BTS02617A, BTS02618A, BTS02654B and BTS02652E strains were deposited under the provisions of the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms—Collection Laboratorium voor Microbiologie Belgium (“BCCM-LMG”), R.U.G., K. Ledeganckstraat 35, B-9000 Gent.
This invention also relates to an insecticide composition that is active against Lepidoptera and that comprises the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, as such, or preferably the BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals, crystal proteins or the active component(s) thereof as an active ingredient.
This invention further relates to a gene (the “bTS02618A gene”), which is present in the genome of the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains and which encodes an insecticidal protein (the “BTS02618A protoxin”) that is found in the BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals. The BTS02618A protoxin is the protein that is produced by the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains before being packaged into their respective BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals.
This invention still further relates to a toxin (the “BTS02618A toxin”) which can be obtained (e.g., by trypsin digestion) from the BTS02618A protoxin. The BTS02618A toxin is an insecticidally active protein which can be liberated from the BTS02617A crystals, the BTS02618A crystals, the BTS02654B crystals, and the BTS02652E crystals, which are produced by the BTS02617A strain, the BTS02618A strain, the BTS02654B strain and the BTS02652E strain, respectively. This toxin and its protoxin have a high activity against a wide range of lepidopteran insects, particularly against Noctuidae, especially against Spodoptera and Agrotis spp., but also against other important lepidopteran insects such as Pyralidae, particularly the European corn borer,
Ostrinia nubilalis
, and Yponomeutidae such as
Plutella xylostella
. This new characteristic of the BTS02618A protoxin and toxin (“(pro)toxin”), i.e., the combination of activity against different economically important Lepidopteran insect families such as Noctuidae, Yponomeutidae and Pyralidae, makes this (pro)toxin an ideally suited compound for combatting a wide range of insect pests by contacting these insects with the (pro)toxin, e.g., by spraying or by expressing the bTS02618A gene in plant-associated bacteria or in plants. The BTS02618A toxin is believed to represent the smallest portion of the BTS02618A protoxin which is insecticidally effective against Lepidoptera.
This invention yet further relates to a chimeric gene that can be used to transform a plant cell and that contains the following operably linked DNA fragments:
1) a part of the bTS02618A gene (the “insecticidally effective bTS02618A gene part”) encoding an insecticidally effective portion of the BTS02618A protoxin, preferably a truncated part of the bTS02618A gene (the “truncated bTS02618A gene”) encoding just the BTS02618A toxin;
2) a promoter suitable for transcription of the insecticidally effective bTS02618A gene part in a plant cell; and
3) suitable 3′ end transcript formation and polyadenylation signals for expressing the insecticidally effective bTS02618A gene part in a plant cell.
This chimeric gene is hereinafter generally referred to as the “bTS02618A chimeric gene”.
This invention also relates to:
1) a cell (the “transformed plant cell”) of a plant, such as corn or cotton, the genome of which is transformed with the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene; and
2) a plant (the “transformed plant”) which is regenerated from the transformed plant cell or is produced from the so-regenerated plant and their seeds, the genome of which contains the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene, and which is resistant to Lepidoptera.
This invention still further relates to
1) a microbial organism, such as
B. thuringiesis
or Pseudomonas spp., the genome of which is transformed with all or part of the bTS02618A gene; and
2) a microbial spore, containing a genome which is transformed with all or parts of the bTS02618A gene.
BACKGROUND OF THE INVENTION
B. thuringiesis
(“Bt”) is a Gram-positive bacterium which produces endogenous crystals upon sporulation. The crystals are composed of proteins which are specifically toxic against insect larvae. These crystal proteins and corresponding genes have been classified based on their structure and insecticidal spectrum (Höfte and Whiteley, 1989). The four major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), and Diptera-specific (cryIV) genes.
The fact that conventional submerged fermentation techniques can be used to produce Bt spores on a large scale makes Bt bacteria commercially attractive as a source of insecticidal compositions.
Gene fragments from some Bt strains, encoding insecticidal proteins, have heretofore been identified and integrated into plant genomes in order to render the plants insect-resistant. However, obtaining expression of such Bt gene fragments in plants is not a straightforward process. In order to achieve optimal expression of an insecticidal protein in plant cells, it has been found necessary to engineer each Bt gene fragment in a specific way so that it encodes a part of a Bt protoxin that retains substantial toxicity against its target insects (European patent application (“EPA”) 86/300,291.1 and 88/402,115.5; U.S. patent application Ser. No. 821,582, filed Jan. 22, 1986).
SUMMARY OF THE INVENTION
In accordance with this invention, four novel Bt strains, i.e., the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains, are provided. The BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals and crystal proteins, the BTS02618A protoxin and toxin produced by the strains during sporulation, and insecticidally effective portions of the BTS02618A protoxin, as well as equivalents of these crystals, crystal proteins, protoxin, toxin and insecticidally effective protoxin portions, each possess insecticidal activity and can therefore be formulated into insecticidal compositions against Lepidoptera in general, and particularly against Noctuidae, such as Agrotis spp. (cutworms such as
Agrotis ipsilon
), Mamestra spp. (e.g., the cabbage moth,
Mamestra brassica
) and Spodoptera spp. (armyworms, such as
Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis
and
Spodoptera litura
), against Pyralidae (e.g., the European corn borer,
Ostrinia nubilalis
) and Yponomeutidae (such as
Plutella xylostella
) which are major pests of various economically important crops, such as corn, cotton and many vegetables such as Brassicas.
Also in accordance with this invention, a plant cell genome is transformed with the insecticidally effective bTS02618A gene part, preferably the truncated bTS02618A gene, or an equivalent thereof such as a modified, synthetic bTS02618A gene. It is preferred that this transformation be carried out with the bTS02618A chimeric gene. The resulting transformed plant cell can be used to produce transformed plants, seeds of transformed plants and plant cell cultures consisting essentially of the transformed cells. The transformed cells in some or all of the tissues of the transformed plants: 1) contain the insecticidally effective bTS02618A gene part as a stable insert in their genome, and 2) express the insecticidally effective bTS02618A gene part by producing an insecticidally effective po
Jansens Stefan
Lambert Bart
Peferoen Marnix
Van Audenhove Katrien
Bayer BioScience N.V.
Prouty Rebecca E.
LandOfFree
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