Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Chemical pesticides have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Therefore, synthetic chemical pesticides are being increasingly scrutinized, and correctly so, for their potential toxic environmental consequences. Synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result of runoff, and destroy non-target life forms. Synthetic chemical control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They may also provide health hazards to applicants, especially if the proper application techniques are not followed. Regulatory agencies around the world are restricting and/or banning the uses of many pesticides and particularly the synthetic chemical pesticides which are persistent in the environment and enter the food chain. Examples of widely used synthetic chemical pesticides include the organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb, and dieldrin; the organophosphates, e.g., chlorpyrifos, parathion, malathion, and diazinon; and carbamates. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests.
Because of the problems associated with the use of synthetic chemical pesticides, there exists a clear need to limit the use of these agents and a need to identify alternative control agents. The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides, could reduce the levels of toxic chemicals in the environment.
A biological pesticidal agent that is being used with increasing popularity is the soil microbe Bacillus thuringiensis (B.t.). The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium. Most strains of B.t. do not exhibit pesticidal activity. Some B.t. strains produce, and can be characterized by, parasporal crystalline protein inclusions. These xe2x80x9cxcex4-endotoxins,xe2x80x9d which typically have specific pesticidal activity, are different from exotoxins, which have a non-specific host range. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and are specific in their toxic activity.
Preparations of the spores and crystals of B. thuringietisis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD- 1 produces a crystalline xcex4-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
The cloning and expression of a B.t. crystal protein gene in Escherichia coli was described in the published literature more than 15 years ago (Schnepf, H. E., H. R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897.). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. Recombinant DNA-based B.t. products have been produced and approved for use.
Commercial use of B.t. pesticides was originally restricted to a narrow range of lepidopteran (caterpillar) pests. More recently, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] xe2x80x9cCellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,xe2x80x9d in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255).
New subspecies of B.t. have now been identified, and genes responsible for active xcex4-endotoxin proteins have been isolated and sequenced (Hxc3x6fte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hxc3x6fte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were cryI (Lepidoptera-specific), cryII (Lepidoptera- and Diptera-specific), cryIII (Coleoptera-specific), and cryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). For example, the designations CryV and CryVI have been proposed for two new groups of nematode-active toxins.
The 1989 nomenclature and classification scheme of Hxc3x6fte and Whiteley was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. The number of sequenced Bacillus thuringiensis crystal protein genes currently stands at more than 50. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmore et al. [1996 ] Society for Invertebrate Pathology, 29th Annual Meeting, IIIrd International Colloquium on Bacillus thiuringiensis, University of Cordoba, Cordoba, Spain, Sep. 1-6, 1996, abstract). The mnemonic xe2x80x9ccryxe2x80x9d has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, although a number have been reclassified.
With the use of genetic engineering techniques, new approaches for delivering B.t. toxins to agricultural environments are under development, including the use of plants genetically engineered with B.t. toxin genes for insect resistance and the use of stabilized, microbial cells as delivery vehicles of B.t. toxins (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Various improvements have been achieved by modifying B.t. toxins and/or their genes. For example, U.S. Pat. Nos. 5,380,831 and 5,567,862 relate to the production of synthetic insecticidal crystal protein genes having improved expression in plants.
Obstacles to the successful agricultural use of B.t. toxins include the development of resistance to B.t. toxins by insects. In addition, certain insects can be refractory to the effects of B.t. The latter includes insects such as boll weevil and black cutworm as well as adult insects of most species which heretofore have demonstrated no apparent significant sensitivity to B.t. xcex4-endotoxins.
Thus, resistance management strategies in B.t. plant technology have become of great interest, and there remains a great need for new toxin genes. For example, WO 97/40162 (published PCT application) discloses 15 kDa and 45 kDa coleopteran-active proteins obtainable from B.t. isolates PS80JJ1 and PS149B1.
As a result of extensive research and resource investment, patents continue to issue for new B.t. isolates, toxins, and genes, and for new uses of B.t. isolates. See Feitelson et al., supra, for a review. U.S. Pat. No. 5,589,382 discloses B.t. isolate PS80JJ1 as having activity against nematodes. U.S. Pat. No. 5,632,987 discloses B.t. isolate PS80JJ1 as having activity against corn rootworm. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
There remains a great need for new toxin genes that can be successfully expressed at adequate levels in plants in a manner that will result in the effective control of insects and other pests.
The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides new, plant-optimized polynucleotide sequences that encode pesticidal proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. Using the polynucleotide sequences described herein, the transformation of plants can be accomplished, using techniques known to those skilled in the art, in order to confer pest resistance upon said plants. In a preferred embodiment, the subject invention provides plant-optimized polynucleotide sequences which encode approximately 15 kDa and approximately 45 kDa pesticidal proteins.
SEQ ID NO. 1 is a polynucleotide sequence for a gene designated 80JJ1-15-PO5, which is optimized for expression in maize. This gene encodes an approximately 15 kDa protein. This gene and protein were disclosed in WO 97/40162.
SEQ ID NO. 2 is a novel polynucleotide sequence for a gene designated 80JJ1-15-PO7, which is optimized for expression in maize. This is an alternative gene that encodes an approximately 15 kDa protein.
SEQ ID NO. 3 is an amino acid sequence for a novel pesticidally active protein encoded by the gene designated 80JJ1-15-PO7.
SEQ ID NO. 4 is a polynucleotide sequence for a gene designated 80JJ1-45-PO, which is optimized for expression in maize. This gene encodes an approximately 45 kDa protein. This gene was disclosed in WO 97/40162.
SEQ ID NO. 5 is a novel polynucleotide sequence for a gene designated 149B1-15-PO, which is optimized for expression in Zea mays. This gene encodes an approximately 15 kDa protein obtainable from PS149B1 that is disclosed in WO 97/40162.
SEQ ID NO. 6 is a novel polynucleotide sequence for a gene designated 149B1-45-PO, which is optimized for expression in Zea mays. This gene encodes an approximately 45 kDa protein obtainable from PS149B1 that is disclosed in WO 97/40162.
The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides new, plant-optimized polynucleotide sequences that encode pesticidal proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimized expression in plants. Using the polynucleotide sequences described herein, the transformation of plants can be accomplished, using techniques known to those skilled in the art, in order to confer pest resistance upon said plants. In a preferred embodiment, the subject invention provides plant-optimized polynucleotide sequences which encode approximately 15 kDa and approximately 45 kDa pesticidal proteins.
Using techniques such as computer- or software-assisted sequence alignments, differences can be noted in the nucleotide sequence of the subject plant-optimized genes as compared to the wild-type genes or to previously known genes. Similarly, differences in the unique amino acid sequences of the subject invention can be noted as compared to wild-type toxins or to previously known toxins.
It should be apparent to a person skilled in this art that, given the sequences of the genes as set forth herein, the genes of the subject invention can be obtained through several means. In preferred embodiments, the subject genes may be constructed synthetically by using a gene synthesizer, for example. The specific genes exemplified herein can also be obtained by modifying, according to the teachings of the subject invention, certain wild-type genes (for example, by point-mutation techniques) from certain isolates deposited at a culture depository as discussed below.
Certain cultures discussed in this application have been deposited in the Agricultural Research Service Pat. Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA. The deposited strains listed below are disclosed in the patent references as discussed above in the section entitled xe2x80x9cBackground of the Invention.xe2x80x9d
It should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Genes and toxins. The subject invention includes, in preferred embodiments, polynucleotide sequences optimized for expression in plants, wherein said sequences are selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 6. SEQ ID NO. 2 encodes a preferred protein that is shown in SEQ ID NO. 3.
The polynucleotides of the subject invention can be used to form complete xe2x80x9cgenesxe2x80x9d to encode proteins or peptides in a desired host cell. For example, as the skilled artisan would readily recognize, SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 6 are shown without stop codons. SEQ ID NO. 2, SEQ ID NO. 5, and/or SEQ ID NO. 6 can be appropriately placed under the control of a promoter in a host of interest, as is readily known in the art.
As the skilled artisan would readily recognize, DNA can exist in a double-stranded form. In this arrangement, one strand is complementary to the other strand and vice versa. The xe2x80x9ccoding strandxe2x80x9d is often used in the art to refer to the strand having a series of codons (a codon is three nucleotides that can be read three-at-a-time to yield a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest. In order to express a protein in vivo, a strand of DNA is typically translated into a complementary strand of RNA which is used as the template for the protein. As DNA is replicated in a plant (for example) additional, complementary strands of DNA are produced. Thus, the subject invention includes the use of either the exemplified polynucleotides shown in the attached sequence listing or the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the specifically exemplified, novel DNA molecules are included in the subject invention.
Certain DNA sequences of the subject invention have been specifically exemplified herein. These sequences are exemplary of the subject invention. It should be readily apparent that the subject invention includes not only the genes and sequences specifically exemplified herein but also equivalents and variants thereof (such as mutants, fusions, chimerics, truncations, fragments, and smaller genes) that exhibit the same or similar characteristics relating to expressing toxins in plants, as compared to those specifically disclosed herein. As used herein, xe2x80x9cvariantsxe2x80x9d and xe2x80x9cequivalentsxe2x80x9d refer to sequences which have nucleotide (or amino acid) substitutions, deletions (internal and/or terminal), additions, or insertions which do not materially affect the expression of the subject genes, and the resultant pesticidal activity, in plants. Fragments of polynucleotide proteins retaining pesticidal activity, and xe2x80x9cpesticidal portionsxe2x80x9d of full-length proteins, are also included in this definition.
Genes can be modified, and variations of genes may be readily constructed, using standard techniques. For example, techniques for making point mutations are well known in the art. In addition, commercially available exonucleases or endonucleases can be used according to standard procedures, and enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Useful genes can also be obtained using a variety of restriction enzymes.
It should be noted that equivalent genes will encode toxins that have high amino acid identity or homology with the toxins encoded by the subject genes. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table I provides a listing of examples of amino acids belonging to each class.
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the ability of plants to express the subject DNA sequences or from the biological activity of the toxin.
As used herein, reference to xe2x80x9cisolatedxe2x80x9d polynucleotides and/or xe2x80x9cpurifiedxe2x80x9d toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature and would include their use in plants. Thus, reference to xe2x80x9cisolatedxe2x80x9d and/or xe2x80x9cpurifiedxe2x80x9d signifies the involvement of the xe2x80x9chand of manxe2x80x9d as described herein.
Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In some embodiments of the subject invention, transformed microbial hosts can be used in preliminary steps for preparing precursors, for example, that will eventually be used to transform, in preferred embodiments, plant cells and plants so that they express the toxins encoded by the genes of the subject invention. Microbes transformed and used in this manner are within the scope of the subject invention. Recombinant microbes may be, for example, a B.t., E. coli, or Pseudomnonas. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.
Thus, in preferred embodiments, expression of a gene of this invention results, directly or indirectly, in the intracellular production and maintenance of the protein of interest. When transformed plants are ingested by the pest, the pests will ingest the toxin. The result is a control of the pest.
The B.t. toxin gene can be introduced via a suitable vector into a host, preferably a plant host. There are many crops of interest, such as corn, wheat, rice, cotton, soybeans, and sunflowers. The genes of the subject invention arc particularly well suited for providing stable maintenance and expression, in the transformed plant, of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
Thus, the subject invention includes recombinant hosts comprising a polynucleotide sequence optimized for expression in a plant, wherein said sequence is selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 5, and SEQ ID NO. 6. The recombinant host can be, for example, a plant cell. Entire plants comprising the subject polynucleotides are also within the scope of the subject invention. In particularly preferred embodiments, a plant can be made resistant to corn rootworm damage by being transformed to express a polynucleotide, such as SEQ ID NO. 2, that encodes an approximately 15 kDa protein, together with a second polynucleotide, such as SEQ ID NO. 4, which encodes a 45 kDa protein. Likewise, SEQ ID NO. 5 and SEQ ID NO. 6 can be used together, under one promoter or separate promoters, such as the ubiquitin promoter. For that matter, the polynucleotide of SEQ ID NO. 2 and SEQ ID NO. 6 can be used together, or SEQ ID NO. 4 and SEQ ID NO. 5 can be used, for example.
While the subject invention provides specific embodiments of synthetic genes, other genes that are functionally equivalent to the genes exemplified herein can also be used to transform hosts, preferably plant hosts. Additional guidance for the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.
All of the publications and patent references referred to or cited herein are hereby incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification.