Secreted Insecticidal Protein and Gene Compositions from Bacillus Thuringiensis and Uses Therefor

Abstract

The present invention relates to the isolation and characterization of nucleotide sequences encoding novel insecticidal proteins secreted into the extracellular space from Bacillus thuringiensis and related strains. The proteins are isolated from culture supernatants of Bacillus thuringiensis and related strains and display insecticidal activity against lepidopteran insects including European corn borer (ECB), tobacco budworm (TBW) and diamondback moth (DBM). Insecticidal proteins encoded by nucleotide sequences that hybridize under stringent conditions to the isolated and characterized nucleotide sequences are disclosed. Methods are disclosed for making and using transgenic cells and plants comprising the novel nucleotide sequence of the invention.

Description

BACKGROUND OF INVENTION[0001]The present invention relates to a new family of genes encoding lepidopteran-toxic proteins and insecticidal fragments thereof. In particular, the present invention is directed to exemplary proteins designated herein as TIC900, TIC402, TIC403, TIC404, TIC961, TIC962, TIC963, TIC965 and TIC966, and insecticidal fragments thereof, each encoded by exemplary nucleotide coding sequences designated herein respectively as tic900, tic402, tic403, tic404, tic434, tic961, tic962, tic963, tic965, and tic966, as well as to nucleotide sequence homologs that (1) encode insecticidal proteins and (2) hybridize to the tic900, tic402, tic403, tic404, tic434, tic961, tic962, tic963, tic965, and tic966 coding sequences under stringent hybridization conditions. The present invention also relates to host cells transformed with one or more nucleotide sequences of the present invention or transformed with variants of the nucleotide sequences set forth herein, genes related by i...

AI Technical Summary

Benefits of technology

[0024]A particular advantage of the present invention comprises an improvement in insect resistance management (RM). The ability to combine two or more insecticidal agents, each toxic to the same insect pest species, into a single composition, and each agent exhibiting a mode of action different from the other insecticidal agents with which it is combined, present a means for more effectively controlling a particular insect pest species by substantially reducing the likelihood that resistance to the insecticidal composition will develop in a population. The TIC900 protein an insecticidal fragment thereof, or any homolog thereof, of the present invention can be combined with any number of known insecticidal agents to achieve the level of resistance management in a particular composition, preferably by expression of the combination of insecticidal agents in plants. In particular TIC900 or related insecticidal protein compositions can be combined with a Cry1 or Cry2 amino acid sequence or a variant thereof to achieve control of various lepidopteran plant pest species, or with other appropriate Cry proteins, and with various insecticidal compositions derived from Xenorhabdus and Photorhabdus bacterium species that have been shown to exhibit insecticidal bioactivity directed to lepidopteran plant pest species. Preferably the in planta use of these compositions would be directed to enhanced expression of the proteins in the parts of the plant that exhibit the greatest vulnerability to lepidopteran insect predation. For protection of maize species against European corn borer (ECB), it would be preferable to achieve the highest levels of expression in the leaves and stems of the plant. For tobacco species susceptible to budworm, it would be preferable to achieve the highest levels of expression in the sprouting parts of the plant, i.e., within the bud systems of the plant. For protection of a cruciferous vegetable species against diamondback moth (DBM), it would be preferable to achieve the highest levels of expression in the leaves and stems of the plant.

[0025]The insecticidal proteins of the present invention can also be combined with insecticidal and / or fungicidal toxins expressed in planta to achieve a recombinant plant that exhibits multiple levels of resistance to infestation by pests that are not beneficial to plants. For example, a protein of the present invention can be expressed along with a protein that exhibits coleopteran insect control, and / or along with a protein or other agent that exhibits antifungal activity, to achieve a recombinant transgenic plant that exhibits improved resistance to lepidopteran insect pests, coleopteran insect pests, and fungal pests. Other permutations of levels of resistance are known to those of skill in the art, such as means for resistance to piercing and sucking insect infestation, and nematode infestation, etc. The insecticidal proteins of the present invention can also be combined with one or more nucleotide sequences expressed as one or more dsRNA's for use in suppression of one or more genes (1) in the target pest as a means for achieving a plant that exhibits multiple layers of resistance to infestation by a particular pest, (2) in the plant as a means for achieving desired plant traits, or (3) in various combinations to achieve the desired properties of (1) or (2) collectively.

[0026]Chimeric proteins consisting of all or a part of one or more proteins of the present invention fused to other proteins that are useful in plant protection from infestation or otherwise are contemplated herein. For example, domains of the proteins of the present invention have been found to exhibit a low level of similarity to other Bt toxins, such as Cry3Aa toxin domain I, Cry1Ca toxin domain II, and Cry1Ja toxin domain III (in particular, Domains I, II, and III of the toxin portion of the TIC900 protein, respectively). The proteins of the present invention can be fused to the protoxin domains of any of the Cry1 proteins known in the art, resulting in crystal toxin protein formation when expressed in Bt or other Bacillus strains of bacteria. Furthermore, the domains identified herein within the amino acid sequence of the proteins of the present invention can be exchanged with other similar domains from insecticidal Bt toxin proteins to achieve improved insecticidal activity and / or host ranges that have not previously been observed with Cry1 toxin domain exchanges (Malvar et al. U.S. Pat. No. 6,017,534; Galizzi et al, PCT / EP90 / 0114, WO 91 / 01087).

[0027]Another embodiment comprises an isolated polynucleotide that encodes a Bacillus thuringiensis insecticidal toxin or insecticidal fragment thereof, active against an insect pest, wherein the toxin or insecticidal fragment has a molecular weight between approximately 65,000 Daltons and approximately 70,000 Daltons. In addition, the nucleotide sequence encoding the toxin, or the complement thereof, hybridizes under specific or stringent hybridization conditions to SEQ ID NO:3. The toxin preferably exhibits biological activity in controlling or killing a lepidopteran insect pest, preferably European corn borer (ECB), tobacco budworm (TBW) and / or diamondback moth (DBM). In one embodiment the nucleotide sequence encoding the toxin is optimized for expression in plants, yet encodes substantially the toxin or an insecticidal fragment thereof, i.e., encodes the same or substantially the same amino acid sequence as present in the native amino acid sequence.

[0028]Another embodiment of the present invention provides for host cells transformed to contain a polynucleotide encoding an insecticidal protein of the present invention or an insecticidal fragment thereof. Preferably the nucleotide sequences of the present invention are modified to improve expression of the proteins of the present invention in a preferred host cell. The host cell of the present invention is selected from the group consisting of a bacterial cell, a fungal cell, and a plant cell. Expression in a plant cell can comprise expression to achieve accumulation of the insecticidal protein in the cytoplasm, or can result in the insecticidal protein being accumulated into a subcellular organelle such as a plastid, chloroplast, or mitochondria. Alternatively the insecticidal protein of the present invention or insecticidal fragments thereof could be localized to the protein secretion machinery of the particular host cell and result in an accumulation of the protein product outside of the cell and into the extracellular spaces surrounding the cell.

[0029]An additional embodiment of the present invention provides a method for controlling infestation of a plant by a lepidopteran insect species. Preferably a pesticidal amount of an insecticidal protein of the present invention or insecticidal fragment thereof is provided for consumption by the insect pest in the diet of the insect. The diet can consist of a plant part that the insect normally feeds upon, such as a plant tissue or plant cell. The insecticidal protein or insecticidal fragment thereof can be provided in a composition that is applied to the surface of the plant tissue, plant part, or plant cell or more preferably can be produced by the protein synthesis machinery of the cell and, as described above, accumulated within the plant cell or secreted outside of the plant cell, so long as the amount of the protein toxin provided is an insecticidal amount sufficient to inhibit the insect pest from further feeding, or to inhibit the further growth and development of the insect pest, or to cause mortality to the insect pest. The insecticidal toxin or fragment thereof is derived from a nucleotide sequence that is encoded in Bacillus thuringiensis by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence substantially complementary to SEQ ID NO:3.

Problems solved by technology

Almost all field crops, plants, and commercial farming areas are susceptible to attack by one or more insect pests.

Particularly problematic are Coleopteran and Lepidoptern pests.

Likewise, pests such as armyworm, sod webworm, and tropical sod webworm often attack turf grasses.

These B. thuringiensis crystal proteins are often highly toxic to specific insects.

Commercial B. thuringiensis insecticide formulations typically contain dried sporulated B. thuringiensis fermentation cultures whose crystal proteins are toxic to various insect species.

However, three secreted non-crystal proteins of B. thuringiensis designated Vip1, Vip2 and Vip3 have been reported to be toxic to coleopteran or lepidopteran insects (Estruch et al., 1996; U.S. Pat. No. 5,866,326; WO94 / 21795; WO96 / 10083).