# Patent

## Title
Synthetic insecticidal proteins active against corn rootworm

## Abstract

Traditionally, the primary method for impacting insect pest populations is the application of broad-spectrum chemical insecticides. However, there is increasing concern about the environmental hazards associated with the production and use of synthetic chemical pesticides. Thus, there is substantial interest in developing alternative pesticides, including biological control of insect pests of agricultural significance using a microbial agent or another species of insect. The present invention provides compositions and methods for such biological control. Modified Cry3 pesticidal polypeptides, polynucleotides encoding such polypeptides, and methods of use are disclosed. The modified polynucleotides provided herein can be used to transform organisms and cells of hosts comprising plant, insects, and microorganisms. The expression of modified polypeptides can provide the host with improved insecticidal activity against one or more insect pathogens.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/441,816, filed Feb. 11, 2011, which is hereby incorporated herein in its entirety by reference.


REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB
The official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of 414541 SEQLIST.txt, a creation date of Feb. 8, 2012 and a size of 167 kilobytes. The sequence listing filed via EFS-Web is part of the specification and is incorporated herein by reference as if set forth in its entirety.
FIELD OF THE INVENTION
The present invention relates to the fields of plant molecular biology and plant pest control. More particularly, the present invention relates to nucleic acids obtained from Bacillus thuringiensis Cry3-family genes that encode δ-endotoxins characterized by pesticidal activity against insect pests. Compositions and methods of the invention utilize disclosed nucleic acids, and their encoded modified pesticidal polypeptides, to control pests.
BACKGROUND OF THE INVENTION
Insect pests are a major factor in the loss of agricultural crops. The western corn rootworm, Diabrotica virgifera virgifera LeConte, is one of the most devastating corn rootworm species in North America, especially in the midwestern corn-growing areas. A related species, the Northern corn rootworm, D. barberi Smith and Lawrence, co-inhabits in much of the range, and is fairly similar in biology to western corn rootworm. A third corn rootworm species, the Southern corn rootworm, D. undecimpunctata howardi, causes significant economic damage in other regions.
Corn rootworm larvae can destroy significant percentages of corn if left untreated. In the United States, it is presently estimated that 30 million acres (120,000 km2) of corn (out of 80 million grown) are infested with corn rootworms, and that the area is expected to grow over the next 20 years. The United States Department of Agriculture estimates that corn rootworms cause $1 billion in lost revenue each year, which includes $800 million in yield loss and $200 million in cost of treatment for corn growers.
Most of the damage in corn is caused by larval feeding. Newly hatched rootworms locate corn roots in the soil and initially begin feeding on the fine root hairs and burrow into root tips of the corn plant. As larvae grow larger, they feed on and tunnel into primary roots. When rootworms are abundant, larval feeding and deterioration of injured roots by root rot pathogens can result in roots being pruned to the base of the stalk. Severe root injury interferes with the roots' ability to transport water and nutrients into the plant reducing plant growth and resulting in reduced grain production. Severe root injury also may result in lodging of corn plants, making harvest more difficult. Silk feeding by adults can result in pruning of silks at the ear tip, commonly called silk clipping. In field corn, beetle populations are occasionally high enough to cause severe silk clipping during pollen shed, which may interfere with pollination.
Corn rootworms of the genus Diabrotica (Coleoptera: Chrysomelidae) are among the most important insect pest of agricultural crops in the United States. For example, the Southern corn rootworm (SCRW), Diabrotica undecimpunctata howardi Barber is an economically important pest of corn, cucurbits and peanuts. SCRW Diabrotica undecimpunctata howardi Barber, or the spotted cucumber beetle, is widely distributed in North America, occurring in most areas east of the Rocky Mountains, in southern Canada, and in Mexico. It is most abundant and destructive in the southern United States. This insect is multivoltine and overwinters as adults in the southern parts of its range (Branson & Krysan (1981) Environmental Entomology 10:826-831). Southern corn rootworms infest the roots of many grass crops and weeds, as well as those of peanuts, alfalfa, and occasionally cucurbits. Annually, 20 to 25 million acres of corn are treated with soil insecticides to protect the crop from corn rootworm larval feeding damage (Fuller et al. (1997) J Econ Entomol 90:1332-1340). Soil insecticides applied for the corn rootworm represent one of the major uses of insecticide in the United States. Costs associated with insecticides applied to control larval damage to corn roots and adult damage to corn silks, along with crop losses can approach $1 billion annually (Metcalf (1986) In M. Kogan [ed.], Ecological Theory and Integrated Pest Management Practice. John Wiley & Sons, New York).
Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria, or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards. Biopesticides also provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecticides. Biopesticides are often less expensive to produce and, thus, improve economic yield for a wide variety of crops.
Microbial insecticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control. Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a broad range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera, and others. Bacillus thuringiensis and Bacillus papilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus (Harwook, ed., ((1989) Bacillus (Plenum Press), 306) and B. cereus (International Application Publication No. WO 96/10083). Pesticidal activity appears to be concentrated in parasporal crystalline protein inclusions, although pesticidal proteins have also been isolated from the vegetative growth stage of Bacillus. Several genes encoding these pesticidal proteins have been isolated and characterized (see, for example, U.S. Pat. Nos. 5,366,892 and 5,840,868).
Recently, agricultural scientists have developed crop plants with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of B. thuringiensis. These proteins are known as δ-endotoxins or Cry toxins (see, e.g., Aronson (2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998) Microbiol Mol Biol Rev. 62(3):775-806). These genetically engineered crops are now widely used in American agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods. In addition, potatoes genetically engineered to contain pesticidal Cry toxins have been sold to the American farmer. While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants provide resistance to only a narrow range of the economically important insect pests.
Accordingly, there remains a need for new Bt toxins with a broader range of insecticidal activity against insect pests including, for example, toxins which are active against a greater variety of insects from the order Coleoptera. In addition, there remains a need for biopesticides having activity against a variety of insect pests and for biopesticides which have improved insecticidal activity.
BRIEF SUMMARY OF THE INVENTION
Compositions and methods for protecting a plant from a plant pest are provided. The compositions include novel nucleotide and amino acid sequences for Cry3 pesticidal polypeptides. The presently disclosed polypeptides display enhanced pesticidal activity against plant pests. Polynucleotides comprising nucleotide sequences that encode the pesticidal polypeptides of the invention are further provided. In some embodiments, modified Cry polypeptides of the invention display an improved pesticidal activity relative to the Cry3 native polypeptide (SEQ ID NO:6).
Compositions of the invention include nucleic acid molecules encoding sequences for modified Cry3 pesticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include the polypeptide sequences. The nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions also comprise transformed bacteria, plants, plant cells, plant tissues, and seeds.
In particular, modified Cry3 proteins are provided having improved activity. Using in silico protein sequence design and DNA shuffling, polypeptides were identified having improved insecticidal activities against coleopteran pests. Methods are provided for producing the modified polypeptides of the invention, and for using such polypeptides for controlling or killing pests of interest, particularly Coleopteran pests.
The compositions and methods of the invention are useful for the production of organisms with pesticide resistance, specifically bacteria and plants. These organisms and compositions derived from them are desirable for agricultural purposes. The compositions of the invention are also useful for generating altered or improved pesticidal proteins.
The following embodiments are encompassed by the present invention:
1. A modified Cry3 pesticidal polypeptide, said polypeptide having one or more amino acid substitutions which confer greater solubility to said polypeptide in a solution having a pH of 5 to 9.
2. The modified Cry3 pesticidal polypeptide of embodiment 1, wherein said one or more substitutions are at a position selected from positions corresponding to positions 12, 63, 97, 106, 117, 119, 140, 152, 158, 186, 206, 221, 222, 230, 232, 258, 292, 294, 340, 346, 384, 468, 472, 491, 496, 503, 557, 584, 589, 593, and 610 of SEQ ID NO:6.
3. A modified Cry3 pesticidal polypeptide, said polypeptide comprising at least one amino acid substitution wherein said substitution makes the polypeptide less basic than the wild type Cry3 polypeptide.
4. The modified Cry3 pesticidal polypeptide of embodiment 3, wherein said substitution comprises replacing a basic amino acid with a neutral or acidic amino acid, replacing a neutral amino acid with an acidic amino acid, or a combination thereof.
5. The modified Cry3 pesticidal polypeptide of embodiment 3, wherein said substitution is at a position selected from positions corresponding to positions 12, 63, 97, 106, 117, 119, 140, 152, 158, 186, 206, 221, 222, 230, 232, 258, 292, 294, 340, 346, 384, 468, 472, 491, 496, 503, 557, 584, 589, 593, and 610 of SEQ ID NO:6.
6. The modified Cry3 pesticidal polypeptide of embodiment 4, wherein said basic amino acid residue comprises a histidine, lysine, or arginine residue and said acidic and/or neutral amino acid residue comprises a glutamic acid or aspartic acid residue.
7. The modified Cry3 pesticidal polypeptide of embodiment 4, wherein said basic amino acid residue comprises a histidine, lysine, or arginine residue and said acidic and/or neutral amino acid residue comprises a serine, threonine, leucine, or valine residue.
8. The modified Cry3 pesticidal polypeptide of any one of embodiments 1-7, wherein said polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, 2, 3, 8, 11, or 14.
9. The modified Cry3 pesticidal polypeptide of embodiment 8, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 8, 11, or 14.
10. The modified Cry3 pesticidal polypeptide of any one of embodiments 1-8, wherein said polypeptide further comprises at least one amino acid substitution corresponding to position 152 or 158 of SEQ ID NO: 6, wherein said polypeptide exhibits resistance to protease digestion.
11. A fragment or variant of the modified Cry3 pesticidal polypeptide of any one of embodiments 1-10, wherein said fragment or variant retains pesticidal activity.
12. A polynucleotide having a nucleotide sequence encoding the modified Cry3 pesticidal polypeptide of any one of embodiments 1-10.
13. A DNA construct comprising the polynucleotide of embodiment 12.
14. The DNA construct of embodiment 13, wherein said polynucleotide is operably linked to a promoter that drives expression in a microorganism or a plant.
15. The DNA construct of embodiment 14, wherein said promoter drives expression in a plant.
16. The DNA construct of embodiment 14, wherein said promoter is a root-preferred promoter.
17. A plant comprising the polynucleotide of embodiment 12.
18. A plant comprising the DNA construct of any one of embodiments 13-16.
19. The plant of embodiment 17 or 18, wherein said plant is a monocotyledonous plant.
20. The plant of embodiment 17 or 18, wherein said plant is a dicotyledonous plant.
21. The plant of embodiment 19, wherein said monocotyledonous plant is selected from the group consisting of maize, sugarcane, wheat, rice, barley, sorghum, and rye.
22. The plant of embodiment 21, wherein said monocotyledonous plant is maize.
23. A transgenic plant expressing the polypeptide of any one of embodiments 1-10.
24. The plant of embodiment 23, wherein said plant is a monocotyledonous plant.
25. The plant of embodiment 23, wherein said plant is a dicotyledonous plant.
26. The plant of embodiment 24, wherein said monocotyledonous plant is selected from the group consisting of maize, sugarcane, wheat, rice, barley, sorghum, and rye.
27. The plant of embodiment 26, wherein said monocotyledonous plant is maize.
28. A transgenic seed produced by the plant of any one of embodiments 17-27.
29. A method for producing a plant having improved pesticidal activity, said method comprising introducing into said plant a polynucleotide having a nucleotide sequence that encodes a modified Cry3 pesticidal polypeptide, said polypeptide having at least one amino acid substitution, wherein said substitution comprises replacing a basic amino acid residue with an acidic or neutral amino acid, wherein said polynucleotide sequence is operably linked to a promoter sequence that drives expression in a plant cell, and wherein said polypeptide has improved pesticidal activity relative to a wild-type Cry3 polypeptide.
30. The method of embodiment 29, wherein said modified Cry3 pesticidal polypeptide has at least one amino acid substitution at a position selected from the group consisting of positions corresponding to 12, 63, 97, 106, 117, 119, 140, 152, 158, 186, 206, 221, 222, 230, 232, 258, 292, 294, 340, 346, 384, 468, 472, 491, 496, 503, 557, 584, 589, 593, and 610 of SEQ ID NO: 6.
31. The method of embodiment 29, wherein said modified Cry3 pesticidal polypeptide comprises an amino acid substitution that replaces one or more histidine, lysine, or arginine residues with a glutamic acid or aspartic acid residue.
32. The method of embodiment 29, wherein said modified Cry3 pesticidal polypeptide comprises an amino acid substitution that replaces one or more histidine, lysine, or arginine residues with a serine, threonine, leucine, or valine residue.
33. The method of any one of embodiments 29-32, wherein said modified Cry3 pesticidal polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, 2, 3, 8, 11, or 14.
34. The method of embodiment 33, wherein said modified Cry3 pesticidal polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 8, 11, or 14.
35. The method of any one of embodiments 29-33 wherein said modified Cry3 pesticidal polypeptide comprises at least one amino acid substitution corresponding to positions 152 or 158 of SEQ ID NO: 6 that renders the polypeptide resistant to protease digestion.
36. The method of embodiment 31 or 32, wherein said modified Cry3 pesticidal polypeptide comprises a fragment or variant of SEQ ID NO: 1, 2, 3, 8, 11, or 14, wherein said fragment or variant retains insecticidal activity.
37. A method for controlling an insect pest in an area of cultivation comprising planting the area with the transgenic seed of embodiment 28.
38. A microorganism comprising the polynucleotide of embodiment 12 or the DNA construct of embodiment 13 or 14.
39. A transgenic microorganism that expresses the modified Cry3 pesticidal polypeptide of any one of embodiments 1-10.
40. A pesticidal composition comprising at least one microorganism of embodiment 38 or 39.
41. A method for controlling an insect pest in an area of cultivation comprising applying an effective amount of the pesticidal composition of embodiment 40 to an environment of said insect pest.
42. The method of embodiment 41, wherein said insect pest is Coleopteran.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 presents Cry3 pesticidal sequences of the invention with replaced residues and their positions relative to SEQ ID NO: 6 highlighted.



DETAILED DESCRIPTION OF THE INVENTION
The invention is drawn to compositions and methods for impacting insect pests, particularly plant pests. Compositions and methods directed to increasing the toxicity of a Cry3 endotoxin are provided. Compositions of the invention include nucleotide and amino acid sequences for novel modified pesticidal polypeptides (e.g., proteins). Modifications to the nucleotide and amino acid sequences include the replacement of basic amino acid residues with acidic or neutral amino acid residues. For example, any one of the basic amino acids (arginine, histidine, and lysine) can be replaced with an acidic amino acid (e.g., aspartic acid and glutamic acid) or a neutral amino acid (e.g., serine, threonine, leucine, and valine).
The Cry3 polypeptides of the invention can be designed by any one of in silico protein design, shuffling of DNA sequences, and directed mutagenesis to change a basic amino acid residue to a neutral or acidic residue. After any modification, the resulting polypeptide is tested for insecticidal activity. Polypeptides having the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 8, 11, or 14, or variants or fragments thereof are provided. Polynucleotides comprising nucleotide sequences encoding the amino acid sequences set forth in SEQ ID NO: 1, 2, 3, 8, 11, or 14, or variants or fragments thereof are further provided, including but not limited to SEQ ID NOs: 7, 9, 10, 12, 13, and 15, and variants and fragments thereof.
Naturally occurring Cry3Aa protein is insoluble in a low salt buffer at and around neutral pH. These low salt and neutral pH conditions mimic the environment of the corn rootworm (CRW) gut. Accordingly, the activity of Cry3Aa against western corn rootworm (WCRW) and southern corn rootworm (SCRW) is very low. Some amino acid substitutions of the invention confer greater solubility to the modified Cry3Aa polypeptide, relative to the native Cry3Aa polypeptide, in a solution having a pH of about pH 5 to about pH 9. Amino acid substitutions of the invention include those that make the Cry3 polypeptide less basic. Thus, the amino acid substitutions include replacing basic amino acids with neutral or acidic amino acids, as well as the substitution of neutral amino acids to acidic amino acids. These amino acid substitutions can be made to any Cry3 polypeptide, including native (e.g., naturally occurring), mutated, or shuffled sequences. These changes increase the solubility of the Cry3 polypeptide in the rootworm gut. Nucleic acid molecules and nucleotide sequences of the invention can be used to transform any organism to produce the encoded modified Cry3 pesticidal polypeptides. Methods are provided that involve the use of such transformed organisms to impact or control plant pests.
Accordingly, the modified Cry3 polypeptides of the invention comprise Cry3 polypeptides that have been modified to make the polypeptide less basic. That is, the modified Cry3 polypeptides of the invention comprise substitutions that replace a basic amino acid with a neutral amino acid, or that replace a neutral amino acid with an acidic amino acid. The substitutions are made to replace a basic amino acid with a less basic (e.g., neutral or acidic) amino acid in a wild type (e.g., naturally occurring) Cry3 sequence, particularly, SEQ ID NO:6.
The novel pesticidal polypeptides and nucleotide sequences of the invention can be generated by recombinant engineering, such as through DNA shuffling followed by site-directed mutagenesis. These shuffled polypeptides display increased solubility as well as increased insecticidal activities against WCRW and SCRW that are significantly higher than naturally occurring Cry proteins such as Cry3Aa and Cry8Bb.
In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
As used herein, the term “recombinant engineering” or “engineered” connotes the utilization of recombinant DNA technology to introduce (e.g., engineer) a change in the protein structure based on an understanding of the protein's mechanism of action and a consideration of the amino acids being introduced, deleted, or substituted.
“DNA shuffling” as used herein refers to a method for the in vitro recombination of homologous genes, where the genes to be recombined are randomly fragmented by DNaseI. Methods are well known in the art (e.g., Joern (2003) Methods in Molecular Biology 231:85-89). The resulting fragments of the desired size are then purified from an agarose gel and reassembled using numerous cycles of denaturation, annealing, and extension by a polymerase. Recombination occurs when fragments from the different parental DNA strands anneal at a region of high sequence identity. Following this reassembly, full-length chimeras generated through multiple rounds of PCR amplification are cloned into suitable expression cassettes.
As used herein, “in silico protein sequence design” refers to use of computational molecular design and protein sequence analysis strategies for modifying proteins. These approaches employ analysis of the three-dimensional structure of a protein to guide the selection of appropriate amino acid sequences for creating desired properties or functions in the protein of interest. Various academic software packages have been developed for in silico protein design and can be used with the invention, including but not limited to 3D-JIGSAW, CPHModel, GeneSilico, I-TASSER, SWISS-MODEL, Selvita Protein Modeling Platform, Abalone, GOR, and Phobius.
The term “Cry3” or “Cry3 family” is used herein to refer to the nucleotide or amino acid sequences of the present invention, which share a high degree of sequence identity or similarity to previously described sequences categorized as Cry3 and/or CryIII. In some embodiments, the sequences of the invention share a high degree of sequence identity or similarity with Cry3Aa1 (SEQ ID NO: 6 or 16), Cry3Aa2 (SEQ ID NO: 17), Cry3Aa3 (SEQ ID NO: 18), Cry3Aa4 (SEQ ID NO: 19), Cry3Aa5 (SEQ ID NO: 20), Cry3Aa6 (SEQ ID NO: 21), Cry3Aa7 (SEQ ID NO: 22), Cry3Aa8 (SEQ ID NO: 23), Cry3Aa9 (SEQ ID NO: 24), Cry3Aa10 (SEQ ID NO: 25), Cry3Aa11 (SEQ ID NO: 26), Cry3Aa12 (SEQ ID NO: 27), Cry3Ba1 (SEQ ID NO: 28), Cry3Ba2 (SEQ ID NO: 29), Cry3Bb1 (SEQ ID NO: 30), Cry3Bb2 (SEQ ID NO: 31), Cry3Bb3 (SEQ ID NO: 32), or Cry3Ca1 (SEQ ID NO: 33). In some of these embodiments, the sequences of the invention share a high degree of sequence identity or similarity with a Cry3Aa protein.
The sequences of the invention are modified from naturally occurring (i.e., found in nature) Cry3 sequences in that they have at least one amino acid substitution and are referred to herein as “modified Cry3” sequences (nucleotide or polypeptide). In some embodiments, the modified Cry3 sequences are modified Cry3Aa sequences.
The modified Cry3 sequences of the invention display high solubility at a neutral pH in low salt solutions. Thus, the sequences of the invention are highly soluble in the gut of the CRW and in solutions having a pH of about pH 5 to about pH 9. In one embodiment, exposed basic amino acid residues are replaced with more acidic amino acid residues providing a negative charge. Likewise, basic amino acids can be replaced with neutral amino acids and neutral amino acids can be replaced with acidic amino acids. By “exposed basic amino acid residues” is intended those amino acids on the surface of the crystal structure of the toxin that are histidine, lysine, or arginine. Thus, the modified Cry3 sequences have one or more substitutions of the amino acids at positions 12, 63, 97, 106, 117, 119, 140, 152, 158, 186, 206, 221, 222, 230, 232, 258, 292, 294, 340, 346, 384, 468, 472, 491, 496, 503, 557, 584, 589, 593, and 610. These positions are relative to the wild type Cry3Aa1 polypeptide set forth in SEQ ID NO:6.
At least one modification can be made at the positions indicated above. Such modifications include the substitution of a basic amino acid for a more acidic or neutral amino acid residue. Such acidic amino acids include aspartic acid and glutamic acid. Acidic amino acids are polar and negatively charged at physiological pH. Examples of such acidic amino acid substitutions can include, without limitation, K63E, N97D, K152E, R158E, K222S, K384E, and K496E.
In another aspect, the present invention provides one or more modifications made to Cry3Aa such that solubility of the protein in the corn rootworm (CRW) gut environment is increased (e.g., enhanced). Native Cry3Aa protein, which does not exhibit insecticidal activity to CRW, is highly insoluble at the pH 5 to pH 9 range in the absence of high salt (Li et al. (1991) Nature 353: 815-821). Modified Cry3Aa sequences that demonstrate high CRW activity (see variants listed as IP3-H in Example 1) exhibited high solubility around pH 7 (i.e., pH 5 to pH 9) which is similar to the CRW gut pH.
It is recognized that one or more of the amino acids in a Cry3 polypeptide can be modified. In all instances, changes or modifications can be made at various sites, and the resulting toxin protein tested for activity. Thus, polypeptides encoded by nucleotide sequences comprising mutations will comprise at least one amino acid change or addition relative to the native (i.e., naturally occurring) or background sequence, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 620, 630, 640, or up to 652 amino acid changes.
In another embodiment, at least one of the protease sensitive residues at positions corresponding to positions 152 and 158 of the naturally occurrring protein (e.g., SEQ ID NO: 6) can be modified to render the protein resistant to protease digestion, particularly resistant against trypsin. In one embodiment, the modification includes at least one of the following substitutions: K152E and R158E corresponding to SEQ ID NO: 6. These residues are located at the loop between Alpha 3 and 4. At least one of the residues can be mutated. The mutation(s) render the protein resistant to trypsin digestion in the corn rootworm (CRW) gut. In this manner, changes can be made at this site and the protein tested for digestibility. Any changes that decrease digestibility in the gut at this site are encompassed herein. Other modifications can include at least one of the following substitutions: K63E, N97D, K152E, R158E, K222S, K384E, and K496E corresponding to SEQ ID NO: 6. As used herein, the terms “proteolytic site” or “cleavage site” refer to an amino acid sequence which confers sensitivity to a class of proteases or a particular protease such that a polypeptide containing the amino acid sequence is digested by the class of proteases or particular protease. A proteolytic site is said to be “sensitive” to the protease(s) that recognize that site. It is appreciated in the art that the efficiency of digestion will vary, and that a decrease in efficiency of digestion can lead to an increase in stability or longevity of the polypeptide in an insect gut. Thus, a proteolytic site may confer sensitivity to more than one protease or class of proteases, but the efficiency of digestion at that site by various proteases may vary. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, and elastase sites.
In some embodiments, the presently disclosed pesticidal polypeptides or variants or fragments thereof display improved pesticidal activity when compared to the parent polypeptide (e.g., SEQ ID NO: 6, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33) from which it was derived. As used herein, the terms “pesticidal activity” and “insecticidal activity” are used synonymously to refer to activity of an organism or a substance (such as, for example, a protein) that can be measured by, but is not limited to, pest mortality, pest weight loss, pest repellency, and other behavioral and physical changes of a pest after feeding and exposure for an appropriate length of time. In this manner, pesticidal activity impacts at least one measurable parameter of pest fitness. As used herein, “pest” means an organism that interferes with or is harmful to plant development and/or growth. One of skill in the art understands that not all substances or mixtures thereof are equally effective against all pests. Of particular interest herein are pesticidal polypeptides that act as insecticides and thus have biological activity against insect pests. Thus, an organism or substance having pesticidal activity adversely impacts at least one measurable parameter of pest fitness. For example, “pesticidal proteins” are proteins that display pesticidal activity by themselves or in combination with other proteins.
Assays for assessing pesticidal activity are well known in the art. See, e.g., U.S. Pat. Nos. 6,570,005 and 6,339,144. See also Brooke et al. (2001) Bull. Entomol. Res. 91:265-272; Chen et al. (2007) Proc. Natl. Acad. Sci. USA 104:13901-13906; Crespo et al. (2008) Appl. Environ. Microb. 74:130-135; Khambay et al. (2003) Pest Manag. Sci. 59:174-182; Liu & Dean (2006) Protein Eng. Des. Sel. 19:107-111; Marron et al. (1985) J. Econ. Entomol. 78:290-293; Robertson et al., Pesticide Bioassays with Arthropods (2nd ed., CRC Press 2007); Scott & McKibben (1976) J. Econ. Entomol. 71:343-344; Stickman (1985) Bull. Environ. Contam. Toxicol. 35:133-142; and Verma et al. (1982) Water Res. 16 525-529; as well as U.S. Pat. No. 6,268,181. Examples of insect bioassays include, but are not limited to, pest mortality, pest weight loss, pest repellency, pest attraction, and other behavioral and physical changes of the pest after feeding and exposure to a pesticide or pesticidal polypeptide for an appropriate length of time. General methods include addition of the pesticide, pesticidal polypeptide or an organism having the pesticidal polypeptide to the diet source in an enclosed container. See, e.g., U.S. Pat. Nos. 6,339,144 and 6,570,005. Pesticidal activity can be measured by, but is not limited to, changes in mortality, weight loss, attraction, repellency and other behavioral and physical changes after feeding and exposure for an appropriate length of time.
The preferred developmental stage for testing for pesticidal activity is larvae or immature forms of these above-mentioned insect pests. The insects may be reared in total darkness at from about 20° C. to about 30° C. and from about 30% to about 70% relative humidity. Bioassays may be performed as described in Czapla and Lang (1990) J. Econ. Entomol. 83(6):2480-2485. Methods of rearing insect larvae and performing bioassays are well known to one of ordinary skill in the art.
In some embodiments of the invention, the pesticidal gene encodes a Bacillus thuringiensis (Bt) toxin or a variant thereof, particularly a homologue of a known Cry toxin. “Bt” or “Bacillus thuringiensis” toxin is intended to mean the broader class of toxins found in various strains of Bacillus thuringiensis, which includes such toxins as, for example, the vegetative insecticidal proteins and the δ-endotoxins. See, for example, Crickmore et al. (1998) Microbiol. Molec. Biol. Rev. 62:807-813; and Crickmore et al. (2004) Bacillus Thuringiensis Toxin Nomenclature at lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt, both of which are herein incorporated by reference in their entirety. The vegetative insecticidal proteins (for example, members of the VIP1, VIP2, or VIP3 classes) are secreted insecticidal proteins that undergo proteolytic processing by midgut insect fluids. They have pesticidal activity against a broad spectrum of Lepidopteran insects. See, for example, U.S. Pat. No. 5,877,012, herein incorporated by reference in its entirety. The Bt δ-endotoxins are toxic to larvae of a number of insect pests, including members of the Lepidoptera, Diptera, and Coleoptera orders. These insect toxins include, but are not limited to, the Cry toxins, including, for example, Cry1, Cry3, Cry5, Cry8, and Cry9. Of particular interest are pesticidal genes that are homologous to known Cry3 genes. In some instances, polypeptide δ-endotoxins of the invention and the nucleotide sequences encoding them will share a high degree of sequence identity or similarity to wild-type (i.e., naturally occurring) Cry3 sequences (e.g., SEQ ID NO: 6, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33). In some of these embodiments, the pesticidal polypeptides of the invention and the nucleotide sequences encoding them will share a high degree of sequence identity or similarity to wild-type Cry3Aa sequences.
The Cry endotoxins initially are produced in an inactive protoxin form, which are proteolytically converted into an active endotoxin through the action of proteases in an insect's gut. Once active, the endotoxin binds to the gut epithelium and forms cation-selective channels that cause cell lysis and subsequent death. See, Carroll et al. (1997) J. Invertebr. Pathol. 70:41-49; Oppert (1999) Arch. Insect Biochem. Phys. 42:1-12; and Rukmini et al. (2000) Biochimie 82:109-116.
Bt Cry proteins have five conserved sequence domains, and three conserved structural domains (see, e.g., de Maagd et al. (2001) Trends Genetics 17:193-199). The most amino-terminal conserved structural domain (Domain I) consists of seven alpha helices, with a central hydrophobic helix-α5 encircled by six other amphipathic helices, and is involved in membrane insertion and pore formation. The second conserved structural domain (Domain II) consists of three antiparallel beta-sheets implicated in cell binding, and the most carboxy-terminal conserved structural domain (Domain III) consists of a beta-sandwich. Exposed regions in domains II and III are involved in receptor recognition and binding, and are therefore considered determinants of toxin specificity. The location and properties of these domains are known to those of skill in the art. See, for example, Grochulski et al. (1995) J Mol Biol 254:447-464; Morse, Yamamoto, and Stroud (2001) Structure 9:409-417; Li et al. (1991) Nature 353:815-821; Galitsky et al. (2001) Acta Cryst D57:1101-1109; Boonserm et al. (2006) J Bacteriol 188:3391-3401; Boonserm et al. (2005) J Mol Biol 348:363-382; and Guo et al. (2009) J Struct Biol 168:259-266.
A polypeptide having “improved pesticidal activity” or “improved pesticidal activity” can refer to a polypeptide exhibiting an increase in activity against a single plant pest or activity against a wider spectrum of plant pests as compared to a reference polypeptide (e.g., naturally occurring Cry3 polypeptide). In some embodiments, the presently disclosed pesticidal polypeptides or variants or fragments thereof display improved pesticidal activity when compared to a naturally occurring Cry 3 polypeptide (e.g., SEQ ID NO: 6, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33). In certain embodiments, the presently disclosed pesticidal polypeptide exhibits a 2-fold to 100-fold greater activity against at least one susceptible insect pest than a naturally occurring polypeptide (e.g., SEQ ID NO: 6, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33), including but not limited to, about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, and 100-fold. A finding of improved or enhanced pesticidal activity requires a demonstration of an increase of pesticidal activity of at least 10%, against the pest target, or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, or 300% or greater increase of pesticidal activity relative to the pesticidal activity of a naturally occurring polypeptide as determined against the same pest.
In certain embodiments, a presently disclosed pesticidal polypeptide or variant or fragment thereof exhibits greater pesticidal activity against a Coleopteran pest when compared to a naturally occurring Cry3 polypeptide. In some of these embodiments, the modified Cry3 polypeptide has greater pesticidal activity against a rootworm, including but not limited to a Southern Corn Rootworm or a Western Corn Rootworm, when compared to a naturally occurring Cry3 polypeptide.
Compositions of the invention include nucleic acids, and fragments and variants thereof that encode pesticidal polypeptides. In particular, the present invention provides for modified nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:1, 2, 3, 8, 11, or 14. Further provided are modified polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The invention encompasses isolated or substantially purified polynucleotide or protein compositions. An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.
Fragments and variants of the nucleotide and amino acid sequences and the polypeptides encoded thereby are also encompassed by the present invention. As used herein the term “fragment” refers to a portion of a nucleotide sequence of a polynucleotide or a portion of an amino acid sequence of a polypeptide of the invention. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native or corresponding full-length protein and hence possess the relevant biological activity such as pesticidal activity.
Nucleic acids that are fragments of a modified Cry3 family nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 1,000, 1,200, 1,400, 1,600, 1,800, or 1900 nucleotides, or up to the number of nucleotides present in the modified Cry3 family nucleotide sequences disclosed herein. In particular embodiments, the nucleic acids of the invention disclose fragments derived from (e.g., produced from) a nucleic acid of the invention, wherein the fragment encodes a truncated modified Cry3 family endotoxin characterized by pesticidal activity. The truncated polypeptide encoded by the polynucleotide fragments of the invention are characterized by pesticidal activity that is either equivalent to, or improved, relative to the activity of the corresponding full-length polypeptide encoded by the nucleic acid from which the fragment is derived. In some embodiments, nucleic acid fragments of the invention are truncated at the 3′ end of the native or corresponding full-length coding sequence. Nucleic acid fragments may also be truncated at both the 5′ and 3′ end of the native or corresponding full-length coding sequence.
Furthermore, it is understood that the invention also encompasses polypeptides that are fragments of the exemplary pesticidal proteins of the invention and having lengths of at least about 100, about 200, about 300, about 400, about 500, about 600, about 620, about 630, about 640, about 641, about 642, about 643, or about 644 contiguous amino acids of a pesticidal polypeptide of the invention and retain pesticidal activity.
The term “variant” is used herein to refer to a substantially similar sequence. Variant nucleotide sequences include nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a pesticidal protein of the invention. Generally, variants of a particular nucleotide sequence of the invention will have at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters. A variant of a nucleotide sequence of the invention may differ from that sequence by as few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 nucleotide.
Variants of a particular nucleotide sequence of the invention (i.e., an exemplary nucleotide sequence) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant nucleotide sequence and the polypeptide encoded by the reference nucleotide sequence. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs described elsewhere herein using default parameters. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, or at least about 98%, about 99% or more sequence identity. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
As used herein, the term “variant protein” encompasses polypeptides that are derived from the polypeptides of the invention by: deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Accordingly, the term “variant protein” encompasses biologically active fragments that comprise a sufficient number of contiguous amino acid residues to retain the biological activity of the polypeptides of the invention, i.e., to have pesticidal activity.
The modified Cry3 family proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the pesticidal proteins can be prepared by introducing mutations into a synthetic nucleic acid (e.g., DNA molecule). Methods for mutagenesis and nucleic acid alterations are well known in the art. For example, designed changes can be introduced using an oligonucleotide-mediated site-directed mutagenesis technique. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and the references cited therein.
The nucleotide sequences of the invention find direct use in methods for impacting pests, particularly insect pests such as pests of the order Coleoptera.
The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and, (d) “percentage of sequence identity.”
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wis. Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See the National Center for Biotechnology Information website on the world wide web at www.ncbi.hlm.nih.gov. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. The term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wis. Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wis. Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
(c) As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, an amino acid residue of a modified Cry3 polypeptide at the position corresponding to a particular amino acid residue of a naturally occurring Cry3 (e.g., SEQ ID NO: 6) refers to the amino acid residue within the modified Cry3 polypeptide that appears opposite the amino acid residue at a particular position in the naturally occurring Cry3 sequence when the modified Cry3 sequence is aligned with the naturally occurring Cry3 sequence (e.g., SEQ ID NO: 6) for maximum homology using an alignment program, such as one known in the art or one described herein.
The modified Cry3 family sequences of the invention are provided in expression cassettes for expression in the organism of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to a modified Cry3 family sequence of the invention. The term “operably linked” as used herein refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
Such an expression cassette is provided with a plurality of restriction sites for insertion of the modified Cry3 family sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The expression cassette will include in the 5′ to 3′ direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a modified Cry3 family DNA sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign or heterologous to the host organism and/or to the modified Cry3 family sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term “foreign” as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter is “foreign” or “heterologous” to the Cry3 family sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked modified Cry3 family sequence of the invention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the Cry3 family sequence of interest, the plant host, or any combination thereof).
Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term “host cell” as used herein refers to a cell which contains a vector and supports the replication and/or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a maize host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.
In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible, or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the present invention in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28: 425-449; Duan et al. (1996) Nature Biotechnology 14: 494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215: 200-208); systemin (McGurl et al. (1992) Science 225: 1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2): 141-150); and the like, herein incorporated by reference.
Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the present invention. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819, herein incorporated by reference.
Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).
Tissue-preferred promoters can be utilized to target enhanced pesticidal protein expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.
Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213; and Meijer et al. (1991) Plant Mol. Biol. 16:807-820); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol. 7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15:127-136); bromoxynil (Stalker et al. (1988) Science 242:419-423); glyphosate (Shaw et al. (1986) Science 233:478-481; and U.S. application Ser. Nos. 10/004,357; and 10/427,692); phosphinothricin (DeBlock et al. (1987) EMBO J. 6:2513-2518). See generally, Yarranton (1992) Curr. Opin. Biotech. 3: 506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); and Gill et al. (1988) Nature 334: 721-724. Such disclosures are herein incorporated by reference.
The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
The nucleic acids and nucleotide sequences of the invention may be used to transform any organism to produce the encoded pesticidal proteins. Methods are provided that involve the use of such transformed organisms to impact or control plant pests. The nucleotide sequences of the invention may also be used to transform organelles such as chloroplasts (McBride et al. (1995) Biotechnology 13: 362-365; and Kota et al. (1999) Proc. Natl. Acad. Sci. USA 96: 1840-1845). The invention therefore provides plants and microorganisms transformed with these novel polynucleotides, and methods involving the use of such nucleic acids, pesticidal compositions, transformed organisms, and products thereof in impacting insect pests.
As used herein, the term “impacting insect pests” refers to effecting changes in insect feeding, growth, and/or behavior at any stage of development, including but not limited to: killing the insect; retarding growth; preventing reproductive capability; antifeedant activity; and the like.
Of particular interest herein are insect pests. As used herein, “insect pest” means an organism in the phylum Arthropoda that interferes with or is harmful to plant development and/or growth, and more specifically means an organism in the class Insecta. The class Insecta can be divided into two groups historically treated as subclasses: (1) wingless insects, known as Apterygota; and (2) winged insects, known as Pterygota. Examples of insect pests include, but are not limited to, insects in the orders Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera, Mallophaga, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera and Thysanura, particularly Coleoptera and Lepidoptera. While technically not insects, arthropods such as arachnids, especially in the order Acari, are included in “insect pest.” Insect pests include economically important agronomic, forest, greenhouse, nursery, ornamentals, food and fiber, public and animal health, domestic and commercial structure, household, and stored product pests.
Of particular interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae, and Curculionidae including, but not limited to: Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (Diaprepes root weevil); Hypera punctata Fabricius (clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevil); M. hemipterus sericeus Olivier (silky cane weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug); S. livis Vaurie (sugarcane weevil); Rhabdoscelus obscurus Boisduval (New Guinea sugarcane weevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae including, but not limited to: Chaetocnema ectypa Horn (desert corn flea beetle); C. pulicaria Melsheimer (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberi Smith & Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); D. virgifera virgifera LeConte (western corn rootworm); Leptinotarsa decemlineata Say (Colorado potato beetle); Oulema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family Coccinellidae including, but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle); chafers and other beetles from the family Scarabaeidae including, but not limited to: Antitrogus parvulus Britton (Childers cane grub); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Dermolepida albohirtum Waterhouse (Greyback cane beetle); Euetheola humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French's cane grub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub); P. latifrons LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp. including M. communis Gyllenhal (wireworm); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae such as, but not limited to, Migdolus fryanus Westwood (longhorn beetle); and beetles from the Buprestidae family including, but not limited to, Aphanisticus cochinchinae seminulum Obenberger (leaf-mining buprestid beetle).
The invention also encompasses transformed or transgenic plants comprising at least one nucleotide sequence of the invention. In some embodiments, the plant is stably transformed with a nucleotide construct comprising at least one nucleotide sequence of the invention operably linked to a promoter that drives expression in a plant cell. As used herein, the terms “transformed plant” and “transgenic plant” refer to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome of a transgenic or transformed plant such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
It is to be understood that as used herein the term “transgenic” includes any cell, cell line, callus, tissue, plant part, or plant the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
“Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6: 923-926); and Led transformation (WO 00/28058). For potato transformation see Tu et al. (1998) Plant Molecular Biology 37: 829-838 and Chong et al. (2000) Transgenic Research 9: 71-78. Additional transformation procedures can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988) Bio/Technology 6: 923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91: 440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
In specific embodiments, the modified Cry3 family sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the modified Cry3 family protein or variants and fragments thereof directly into the plant or the introduction of the modified Cry3 family transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107: 775-784, all of which are herein incorporated by reference. Alternatively, the modified Cry3 family polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).
Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the invention can be contained in transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.
The nucleotide sequences of the invention may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired pesticidal protein. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a pesticidal protein of the invention, may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the present invention. Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules are known in the art. See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; and 5,316,931; herein incorporated by reference.
As used herein, the term plant also includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.
The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the present invention include crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.
Turfgrasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewings fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).
Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
In certain embodiments the nucleic acid sequences of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the polynucleotides of the present invention may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as other Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al., (1986) Gene 48:109), pentin (described in U.S. Pat. No. 5,981,722) and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,049); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122) and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which are herein incorporated by reference.
The invention further provides a method of increasing insect target range by using a pesticidal protein of the invention in combination with at least one other or “second” pesticidal protein. Any pesticidal protein known in the art can be employed in the methods of the present invention. Such pesticidal proteins include, but are not limited to Bt δ-endotoxins, protease inhibitors, α-amylases, and peroxidases.
The polynucleotides of the present invention can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene and GAT gene as disclosed in U.S. application Ser. Nos. 10/004,357; and 10/427,692); and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988)J. Bacteriol. 170: 5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides providing agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or TopCross® methodology, or genetic transformation. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.
Compositions of the invention find use in impacting insect pests, and protecting plants, seeds, and plant products in a variety of ways. For example, the compositions can be used in a method that involves placing an effective amount of the pesticidal composition in the environment of the pest by a procedure selected from the group consisting of spraying, dusting, broadcasting, or seed coating.
Before plant propagation material (fruit, tuber, bulb, corm, grains, seed), but especially seed, is sold as a commercial product, it is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these preparations, if desired together with further carriers, surfactants, or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal, or animal pests. In order to treat the seed, the protectant coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, e.g., treatment directed at the buds or the fruit.
The plant seed of the invention comprising a nucleotide sequence encoding a pesticidal protein of the invention may be treated with a seed protectant coating comprising a seed treatment compound, such as, for example, captan, carboxin, thiram, methalaxyl, pirimiphos-methyl, and others that are commonly used in seed treatment. In one embodiment within the scope of the invention, a seed protectant coating comprising a pesticidal composition of the invention is used alone or in combination with one of the seed protectant coatings customarily used in seed treatment.
It is recognized that the genes encoding the pesticidal proteins can be used to transform insect pathogenic organisms. Such organisms include baculoviruses, fungi, protozoa, bacteria, and nematodes.
The invention further encompasses a microorganism that is transformed with at least one nucleic acid of the invention, with an expression cassette comprising the nucleic acid, or with a vector comprising the expression cassette. In some embodiments, the microorganism is one that multiplies on plants. An embodiment of the invention relates to an encapsulated pesticidal protein which comprises a transformed microorganism capable of expressing at least one pesticidal protein of the invention.
A gene encoding a pesticidal protein of the invention may be introduced via a suitable vector into a microbial host, and said host applied to the environment, or to plants or animals. The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
Microorganism hosts that are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest may be selected. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the pesticidal protein, and desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinlandir and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.
A number of ways are available for introducing a gene expressing the pesticidal protein into the microorganism host under conditions that allow for stable maintenance and expression of the gene. For example, expression cassettes can be constructed which include the nucleotide constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the nucleotide constructs, and a nucleotide sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system that is functional in the host, whereby integration or stable maintenance will occur.
Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter “Sambrook II”; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y.; and the references cited therein.
Suitable host cells, where the pesticidal protein-containing cells will be treated to prolong the activity of the pesticidal proteins in the cell when the treated cell is applied to the environment of the target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells that do not produce substances toxic to higher organisms, such as mammals. However, organisms that produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and gram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes of pesticidal protein production include ease of introducing the pesticidal protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp., Erwinia spp., and Flavobacterium spp., and other such organisms, including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Escherichia coli, Bacillus spp., Bacillus thuringiensis, Bacillus subtilis, Bacillus cereus, and the like.
Genes encoding the pesticidal proteins of the invention can be introduced into microorganisms that multiply on plants (epiphytes) to deliver pesticidal proteins to potential target pests. Epiphytes, for example, can be gram-positive or gram-negative bacteria.
Root-colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a Bacillus cereus strain that colonizes roots can be isolated from roots of a plant (see, for example, Handelsman et al. (1991) Appl. Environ. Microbiol. 56:713-718). Genes encoding the pesticidal proteins of the invention can be introduced into a root-colonizing Bacillus cereus by standard methods known in the art.
Genes encoding pesticidal proteins can be introduced, for example, into the root-colonizing Bacillus by means of electrotransformation. Specifically, genes encoding the pesticidal proteins can be cloned into a shuttle vector, for example, pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218. The shuttle vector pHT3101 containing the coding sequence for the particular pesticidal protein gene can, for example, be transformed into the root-colonizing Bacillus by means of electroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218).
Expression systems can be designed so that pesticidal proteins are secreted outside the cytoplasm of gram-negative bacteria, such as E. coli, for example. Advantages of having pesticidal proteins secreted are: (1) avoidance of potential cytotoxic effects of the pesticidal protein expressed; and (2) improvement in the efficiency of purification of the pesticidal protein, including, but not limited to, increased efficiency in the recovery and purification of the protein per volume cell broth and decreased time and/or costs of recovery and purification per unit protein.
Pesticidal proteins can be made to be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal end of the pesticidal protein. Signal peptides recognized by E. coli can be found in proteins already known to be secreted in E. coli, for example the OmpA protein (Ghrayeb et al. (1984) EMBO J, 3:2437-2442). OmpA is a major protein of the E. coli outer membrane, and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide (Duffaud et al. (1987) Meth. Enzymol. 153: 492).
Pesticidal proteins of the invention can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bacillus thuringiensis strains have been used as insecticidal sprays. In the case of a pesticidal protein(s) that is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the pesticidal protein(s) into the growth medium during the fermentation process. The pesticidal proteins are retained within the cell, and the cells are then processed to yield the encapsulated pesticidal proteins. Any suitable microorganism can be used for this purpose. Pseudomonas has been used to express Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide (Gaertner et al. (1993), in: Advanced Engineered Pesticides, ed. Kim).
Alternatively, the pesticidal proteins are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticidal proteins may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.
In the present invention, a transformed microorganism (which includes whole organisms, cells, spore(s), pesticidal protein(s), pesticidal component(s), pest-impacting component(s), mutant(s), living or dead cells and cell components, including mixtures of living and dead cells and cell components, and including broken cells and cell components) or an isolated pesticidal protein can be formulated with an acceptable carrier into a pesticidal composition(s) that is, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, and an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymer substances.
The invention provides pesticidal compositions comprising a transformed microorganism of the invention. In such embodiments, the transformed microorganism is generally present in the pesticidal composition in a pesticidally effective amount, together with a suitable carrier. The invention also encompasses pesticidal compositions comprising an isolated protein of the invention, alone or in combination with a transformed organism of the invention and/or an encapsulated pesticidal protein of the invention, in an insecticidally effective amount, together with a suitable carrier.
Such compositions disclosed above may be obtained by the addition of a surface-active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent or fertilizers, micronutrient donors, or other preparations that influence plant growth. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaracides, plant growth regulators, harvest aids, and fertilizers, can be combined with carriers, surfactants or adjuvants customarily employed in the art of formulation or other components to facilitate product handling and application for particular target pests. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. For example, the compositions of the present invention may be applied to grain in preparation for or during storage in a grain bin or silo, etc. The compositions of the present invention may be applied simultaneously or in succession with other compounds. Methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention that contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention include, but are not limited to, foliar application, seed coating, and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
Suitable surface-active agents include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; a carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g., the sulfonated condensation product of oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g., the sodium sulfonate of dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate or oleate; or oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.
Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.
The compositions of the present invention can be in a suitable form for direct application or as a concentrate of primary composition that requires dilution with a suitable quantity of water or other diluant before application. The pesticidal concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition contains 1 to 98% of a solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of a surfactant. These compositions will be administered at the labeled rate for the commercial product, for example, about 0.01 lb-5.0 lb. per acre when in dry form and at about 0.01 pts.-10 pts. per acre when in liquid form.
In a further embodiment, the compositions, as well as the transformed microorganisms and pesticidal proteins, of the invention can be treated prior to formulation to prolong the pesticidal activity when applied to the environment of a target pest as long as the pretreatment is not deleterious to the pesticidal activity. Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously affect the properties of the composition(s). Examples of chemical reagents include but are not limited to halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride; alcohols, such as isopropanol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixative (see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freeman and Co.).
In other embodiments of the invention, it may be advantageous to treat the modified Cry3 family polypeptides with a protease, for example trypsin, to activate the protein prior to application of a pesticidal protein composition of the invention to the environment of the target pest. Methods for the activation of protoxin by a serine protease are well known in the art. See, for example, Cooksey (1968) Biochem. J. 6:445-454 and Carroll and Ellar (1989) Biochem. J. 261:99-105, the teachings of which are herein incorporated by reference. For example, a suitable activation protocol includes, but is not limited to, combining a polypeptide to be activated, for example a purified modified Cry3 polypeptide (e.g., having the amino acid sequence set forth in SEQ ID NO:2), and trypsin at a 1/100 weight ratio of Cry3 protein/trypsin in 20 nM NaHCO3, pH 8 and digesting the sample at 36° C. for 3 hours.
The compositions (including the transformed microorganisms and pesticidal proteins of the invention) can be applied to the environment of an insect pest, a plant, plant seed, plant part, or an area of cultivation by, for example, spraying, atomizing, dusting, scattering, coating or pouring, introducing into or on the soil, introducing into irrigation water, by seed treatment or general application or dusting at the time when the pest has begun to appear or before the appearance of pests as a protective measure. For example, the pesticidal protein and/or transformed microorganisms of the invention may be mixed with grain to protect the grain during storage. It is generally important to obtain good control of pests in the early stages of plant growth, as this is the time when the plant can be most severely damaged. The compositions of the invention can conveniently contain another insecticide if this is thought necessary. In an embodiment of the invention, the composition is applied directly to the soil, at a time of planting, in granular form of a composition of a carrier and dead cells of a Bacillus strain or transformed microorganism of the invention. Another embodiment is a granular form of a composition comprising an agrochemical such as, for example, an herbicide, an insecticide, a fertilizer, an inert carrier, and dead cells of a Bacillus strain or transformed microorganism of the invention.
The pesticidal composition can be applied to an area of cultivation before or after planting. The area of cultivation can comprise the insect pest or the environmental conditions of the area of cultivation can be conducive to the insect pest (e.g., preferred air temperature, season, soil temperature for growth of the insect pest). As used herein, an “area of cultivation” comprises any region in which one desires to grow a plant. Such areas of cultivations include, but are not limited to, a field in which a plant is cultivated (such as a crop field, a sod field, a tree field, a managed forest, a field for culturing fruits and vegetables, etc), a greenhouse, a growth chamber, etc.
In some embodiments of the invention, the polynucleotide encodes an antibody that binds to an epitope of the modified polypeptide of interest (i.e., modified Cry3 family proteins) when expressed in at least one organism from the group consisting or plants and microorganism. Such antibodies can be to assay for the expression levels of polypeptides of interest and/or protease activity.
In another embodiment, the binding of the antibody results in increased turnover of the antibody-Cry3 protein complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald (2003) Nature Biotech. 21:35-36, incorporated herein by reference.
By “epitope” is intended the part of an antigenic molecule to which an antibody is produced and to which the antibody will bind. Epitopes can comprise linear amino acid residues (i.e., residues within the epitope are arranged sequentially one after another in a linear fashion), nonlinear amino acid residues (referred to herein as “nonlinear epitopes”; these epitopes are not arranged sequentially), or both linear and nonlinear amino acid residues. Typically epitopes are short amino acid sequences, e.g. about five amino acids in length. Systematic techniques for identifying epitopes are known in the art and are described, for example, in U.S. Pat. No. 4,708,871. Briefly, a set of overlapping oligopeptides derived from the antigen may be synthesized and bound to a solid phase array of pins, with a unique oligopeptide on each pin. The array of pins may comprise a 96-well microtiter plate, permitting one to assay all 96 oligopeptides simultaneously, e.g., for binding to a biomarker-specific monoclonal antibody. Alternatively, phage display peptide library kits (New England BioLabs) are currently commercially available for epitope mapping. Using these methods, the binding affinity for every possible subset of consecutive amino acids may be determined in order to identify the epitope that a given antibody binds. Epitopes may also be identified by inference when epitope length peptide sequences are used to immunize animals from which antibodies are obtained.
Antigen-binding fragments and variants of the antibodies disclosed herein are further provided. Such variants will retain the desired binding properties of the parent antibody. Methods for making antibody fragments and variants are generally available in the art. For example, amino acid sequence variants of an antibody described herein, can be prepared by mutations in the cloned DNA sequence encoding the antibody of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference.
Antibodies having the binding characteristics of an antibody of the invention are also provided. “Binding characteristics” or “binding specificity” when used in reference to an antibody connotes that the antibody recognizes the same or similar antigenic epitope as a comparison antibody. Examples of such antibodies include, for example, an antibody that competes with an antibody of the invention in a competitive binding assay. One of skill in the art could determine whether an antibody competitively interferes with another antibody using standard methods.
In constructing variants of the antibody polypeptide of interest, modifications are made such that variants continue to possess the desired activity, i.e., similar binding specificity to Cry3 proteins. Obviously, any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See EP Patent Application Publication No. 75,444.
Preferably, variants of an antibody have amino acid sequences that have at least 70% or 75% sequence identity, preferably at least 80% or 85% sequence identity, more preferably at least 90%, 91%, 92%, 93%, 94% or 95% sequence identity to the amino acid sequence for the reference antibody molecule, or to a shorter portion of the reference antibody molecule. More preferably, the molecules share at least 96%, 97%, 98% or 99% sequence identity. For purposes of the present invention, percent sequence identity is determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math. 2:482-489. A variant may, for example, differ from the reference antibody by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
Methods for making antibodies and for selecting appropriate antibodies are known in the art. See, for example, Celis, ed. (in press) Cell Biology & Laboratory Handbook, 3rd edition (Academic Press, New York), which is herein incorporated in its entirety by reference. In some embodiments, commercial antibodies directed to Cr proteins may be used to practice the invention.
The following examples are presented by way of illustration, not by way of limitation.
EXPERIMENTAL
Example 1
IP3-1 Sequence Design
In order to introduce the amino acid sequence diversity to Cry3Aa, a new Cry3-type protein called IP3-1 was designed in silico. In the design process for introducing certain diversities to Cry3Aa, modest changes such as 1→V, L→F, K→H, G→A were made which are expected to induce very small changes in the protein structure. Such structural changes may contribute to higher activity when combined with additional diversity introduced by shuffling.
In order to design IP3-1, all available naturally occurring Cry3 peptide sequences were aligned. The Cry3 sequence was scanned residue by residue to find specific residues that differ among Cry3 proteins. These diversified residues found on Cry3Aa were changed one by one to another residue found in other Cry3 proteins. Each mutation was evaluated structurally by computer to assure structural integrity of the polypeptide. The 3-dimensional conformation of the Cry3Aa derivative was predicted from its amino acid sequence using Homology modeling. Accelrys Discovery StudioTM v 2.0 was used to produce homology models of Cry3 derivatives. The Cry3Aa structure (Protein Data Bank ID 1DLC, Chain A; see Li et al. (1991) Nature, 353:815-821) determined by X-ray crystallography was used as reference. Accelrys Discovery Studio recruits MODELER (Sali and Blundell (1993) J. Mol. Biol. 234:779-815) to generate model protein structures using conjugate gradient and simulated annealing optimization procedures. The homology model thus built confirms the location within secondary structure element, solvent exposure and detailed orientation of the side-chains of the mutated residue. When significant issues with certain residues were identified during the structural evaluation, such mutations were discarded. After the cycles of mutation and structural evaluation, the final amino acid sequence of IP3-1 was obtained.
The IP3-1 amino acid sequence was back-translated to a nucleotide sequence using E. coli codon usage. The IP3-1 nucleotide and amino acid sequences are set forth in SEQ ID NOs: 4 and 5, respectively. The IP3-1 nucleotide sequence was synthesized and cloned in pMAL E. coli vector to express the IP3-1 protein as an MBP fusion. The pMAL vector was obtained from New England Biolabs and the synthesized DNA was cloned in the vector, and the MBP-IP3-1 fusion protein was purified according to the manufacturer's recommended method. The fusion protein was expressed in E. coli as a soluble protein. However, when the fusion was digested with Factor Xa at pH 8 to isolate the IP3-1 protein from MBP, the IP3-1 protein precipitated. The MBP-free IP3-1 was almost completely insoluble in a wide pH range (pH 5 to pH 9 as tested). The solubility and corn rootworm (CRW) insecticidal activity of this computer-designed IP3-1 appeared to be the same as reported characteristics of naturally occurring Cry3Aa (data not shown).
In order to increase the solubility of IP3-1, several mutations were attempted (FIG. 1). A number of mutants were made as described below, and their solubility and insecticidal activity were assayed. Among those, two sets of mutations, Set 1: K152E-R158E and Set 2: E221S-K232S showed some improved solubility in around pH7. In addition, these mutants showed remarkable activity against WCRW.
IP3-1 DNA was shuffled under mutational conditions as described by Stemmer (1994) Nature 370:389-391. Five thousand shuffled variants were selected and screened for insecticidal activity against WCRW. Among those, 3 shuffled variants showed high CRW activity (Table 1). These variants were called IP3-H1, IP3-H4 and IP3-H7 (FIG. 1). Sequencing these variants, the following mutations were found over IP3-1:
IP3-H1: I340V, K384E, Q472L, F589L
IP3-H4: K63R, N97D, Q119H, F584L, I(M)593V
IP3-H7: I12T, K63E, Q232H, K496E, Y557H, S610T
As demonstrated in FIG. 1, each shuffled variant contained at least one mutation that converted a basic amino acid residue to a neutral or acidic residue. IP3-H variants were active against WCRW, SCRW and NCRW, while wild type Cry3Aa and IP3-1 showed low or no activity. From these shuffled CRW active variants, two sets of mutations described above were combined to produce new sequences as follows:

Addition of K152E-R158E mutations produced: H1→H2, H4→H5, and H7→H8

Further addition of E221S-K232S mutations produced: H2→H3, H5→H6, and H8→H9

Purified protein was analyzed by SDS-PAGE to determine the accurate concentration. The protein concentration was adjusted to 2.0 mg/ml and mixed into the insect diet in 96-well plates at 37° C. The diet was made with low temperature melting agarose allowing thorough mixing with IP3 proteins at 37° C. After the diet was solidified at the room temperature, insects (neonate CRW larvae) were infested in each well. The plates were sealed with air permeable plastic film and incubated at 28° C. for 4 days. Insect responses to the IP3 proteins were scored based on feeding inhibition and mortality. EC50 was calculated by Probit analysis and presented in Table 1.







TABLE 1







Insecticidal Activity of IP3 Variant Polypeptides












WCRW

SCRW












EC50 ppm

EC50 ppm
















IP3-H1
24
IP3-H1
177



IP3-H2
19
IP3-H2
68



IP3-H3
15
IP3-H3
74



IP3-H4
26
IP3-H4
117



IP3-H5
14
IP3-H5
104



IP3-H6
11
IP3-H6
156



IP3-H7
63
IP3-H7
97



IP3-H8
12
IP3-H8
38



IP3-H9
7
IP3-H9
39



IP3-1
500~1000
IP3-1
No Activity










Example 2
Generation of IP3 Variant Plant Transformation Constructs
The protein sequences of IP3 variants, H2 (SEQ ID NO: 8), H3 (SEQ ID NO: 1), H5 (SEQ ID NO: 11), H6 (SEQ ID NO: 2), H8 (SEQ ID NO: 14) and H9 (SEQ ID NO: 3) were backtranslated and optimized for expression in maize. The resulting sequences H2 (SEQ ID: 7), H3 (SEQ ID: 9), H5 (SEQ ID: 10), H6 (SEQ ID NO: 12), H8 (SEQ ID NO: 13) and H9 (SEQ ID NO; 15) were synthesized by Genscript USA Corp (Piscataway, N.J.) with BamHI and SnaBI restriction enzyme sites at the 5′- and 3′ ends respectively. Each variant was subcloned as a BamHI-SnaBI fragment into a Gateway entry vector containing a plant expression cassette with the maize Ubiquitin1 promoter-5′UTR-intron combination (UBIZM PRO-UBIZM-5UTR-UBIZM INTRON) and the potato PIN II terminator sequence. The resulting plant expression cassette contains the following components operatively linked together in this order; UBIZM PRO-UBIZM-5UTR-UBIZM INTRON, the IP3-variant gene, and the PIN II terminator. The expression cassette is flanked by Gateway attL3 and attL4 recombination sites and this entry vector was used to transfer the expression cassette into an attR3 and attR4 containing binary destination transformation vector. The final plant transformation vector contains the IP3-variant expression cassette upstream of a cassette containing the maize Ubiquitin1 promoter-5′UTR-intron controlling expression of a PAT selectable marker gene with the 35S terminator sequence.
Example 3
Agrobacterium-Mediated Transformation of Maize and Regeneration of Transgenic Plants
For Agrobacterium-mediated transformation of maize transformation vectors containing the IP3 variants, the method of Zhao was employed (U.S. Pat. No. 5,981,840, and international patent publication WO98/32326; the contents of each are hereby incorporated by reference in its entirety). Briefly, immature embryos were isolated from maize and the embryos contacted with a suspension of Agrobacterium transformed with the IP3 variant plant transformation construct under conditions whereby the bacteria were capable of transferring the gene sequences of the invention to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step, the immature embryos were immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos were co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos were cultured on solid medium following the infection step. Following the co-cultivation period, an optional “resting” step was performed. In this resting step, the embryos were incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos were cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos were cultured on medium containing a selective agent and growing transformed callus was recovered (step 4: the selection step). The immature embryos were cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus was then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium were cultured on solid medium to regenerate the plants.
Example 4
Expression and Characterization of IP3 Variants in Transgenic Maize Root Tissue
Tissue samples from V4-V6 roots were placed into 1.2 mL tubes and extracted in 0.6 mL of chilled PBST (Phosphate Buffered Saline plus Tween-20). Following centrifugation, supernatants were removed, diluted, and analyzed by ELISA for IP3 variant expression. The Cry3A ELISA kit used for protein determination was obtained from EnviroLogix, Inc. (Portland, Me.). The ELISA method for the IP3 variant proteins utilized a sequential “sandwich” format to determine the concentration of the protein in sample extracts. Standards (analyzed in triplicate wells) and diluted sample extracts (analyzed in duplicate wells) were incubated in plates pre-coated with an antibody against CRY3A. Following incubation, unbound substances were washed from the plate. A different specific antibody for the respective selected protein, conjugated to the enzyme horseradish peroxidase (HRP), was added to the plate and incubated. Then, unbound substances were washed from the plate, leaving the bound protein “sandwiched” between the antibody coated on the plate and the antibody-HRP conjugate. Detection of the bound antibody-protein complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the optical density (OD) of each well was determined using a plate reader. An average of the results from duplicate wells was used to determine the concentration of the IP3 variant expressed in root tissue in parts per million (PPM) of total souble protein.
The results of this analysis are shown in Table 2 as an average of each IP3 variant accumulation level in 25 transgenic events. Accumulation was observed with all IP3 variants tested in root tissue. Differences in expression levels between variants can be attributed to multiple factors including the inherent stability and toxicity of each variant in plant tissue, how effective gene optimization was for expression, and differences in cross-reactivity to the CRY3A Ab used for ELISA detection.







TABLE 2







Expression of IP3 variants in transgenic maize events.










Transgene
Expression in root (ppm)







Control
—



IP3-H2
1571



IP3-H3
852



IP3-H5
6493



IP3-H6
10094



IP3-H8
9942



IP3-H9
9309










Example 5
Efficacy of Events Expressing IP3 Variants Against Corn Rootworm
Root damage caused by Western Corn Rootworm (WCRW) was investigated with events expressing the IP3 variants. Potted plants at approximately the V4 growth stage were manually infested with approximately 400 WCRW eggs applied into the soil. Root injury scores were taken when adult WCRW begin to emerge from negative control pots. Plants were removed from the pot and gently washed with pressurized water. The root damage was rated using the 0-3 node injury scale (CRWNIS) (Oleson et al. (2005) J. Econ. Entomol. 98(1):1-8) and means were calculated for each treatment. Mean root damage ratings from WCRW feeding on events expressing different IP3 variants are shown in Table 3.







TABLE 3







Efficacy of maize expressing individual IP3 variants against


WCRW larvae.











Variant ID
CRWNIS score*
SD















Negative control
2.0
0.30



IP3-H2
0.12
0.28



IP3-H3
0.26
0.51



IP3-H5
0.23
0.34



IP3-H6
0.44
0.6



IP3-H8
0.64
0.76



IP3-H9
0.38
0.58







SD: standard deviation



*Corn rootworm node injury score (CRWNIS) rating system

















Value
Description







0.00
No feeding damage (lowest rating that can be given)


1.00
One node (circle of roots), or the equivalent of an entire node,



eaten back to within approximately two inches of the stalk



(soil line on the 7th node)


2.00
Two complete nodes eaten


3.00
Three or more nodes eaten (highest rating that can be given)





Damage in between complete nodes eaten is noted as the percentage of the node missing, i.e. 1.50 = 11⁄2 nodes eaten, 0.25 = 1⁄4 of one node eaten, etc.






The results of the rootworm damage assay demonstrate that these IP3 variants provided significantly improved control against WCRW when expressed in transgenic maize. Negative control plants suffered a mean of 2 nodes of root injury compared to node injury scores of within the range of 0.12 to 0.64 for IP3 variant expressing transgenics.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a polypeptide” is understood to represent one or more polypeptides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein, the term “about,” when referring to a value is meant to encompass variations of, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the presently disclosed subject matter be limited to the specific values recited when defining a range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. Although any methods and materials similar herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

## Claims

1. A modified Cry3 pesticidal polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 8, 11, or 14.

2. A polynucleotide having a nucleotide sequence encoding the modified Cry3 pesticidal polypeptide of claim 1.

3. The polynucleotide of claim 2, wherein said nucleotide sequence is set forth in SEQ ID NO: 7, 9, 10, 12, 13, or 15.

4. An expression cassette comprising the polynucleotide of claim 2.

5. The expression cassette of claim 4, wherein said polynucleotide is operably linked to a promoter that drives expression in a microorganism or a plant.

6. A host cell comprising the polynucleotide of claim 2.

7. A plant comprising the polynucleotide of claim 2, wherein said polynucleotide is operably linked to a promoter active in said plant.

8. The plant of claim 7, wherein said plant is a monocotyledonous plant.

9. The plant of claim 7, wherein said plant is a dicotyledonous plant.

10. The plant of claim 8, wherein said monocotyledonous plant is selected from the group consisting of maize, sugarcane, wheat, rice, barley, sorghum, and rye.

11. The plant of claim 10, wherein said monocotyledonous plant is maize.

12. A transgenic seed produced by the plant of claim 7.

13. A method for producing a plant having improved pesticidal activity, said method comprising introducing into said plant a polynucleotide having a nucleotide sequence that encodes a modified Cry3 pesticidal polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 8, 11, or 14.

14. A pesticidal composition comprising the modified Cry3 pesticidal polypeptide of claim 1.

15. The pesticidal composition of claim 14, further comprising a carrier.

16. A method for controlling an insect pest in an area of cultivation comprising applying an effective amount of the pesticidal composition of claim 14 to an environment of said insect pest.

17. The method of claim 16, wherein said insect pest is a Coleopteran.

18. An expression cassette comprising the polynucleotide of claim 3.

19. The expression cassette of claim 18, wherein said polynucleotide is operably linked to a promoter that drives expression in a microorganism or a plant.

20. A host cell comprising the polynucleotide of claim 3.

21. A plant comprising the polynucleotide of claim 3, wherein said polynucleotide is operably linked to a promoter active in said plant.

22. The plant of claim 21, wherein said plant is a monocotyledonous plant.

23. The plant of claim 21, wherein said plant is a dicotyledonous plant.

24. The plant of claim 22, wherein said monocotyledonous plant is selected from the group consisting of maize, sugarcane, wheat, rice, barley, sorghum, and rye.

25. The plant of claim 24, wherein said monocotyledonous plant is maize.

26. A transgenic seed produced by the plant of claim 21 comprising said polynucleotide.