# Patent

## Title
Biological control of coleopteran pests

## Abstract

Disclosed are double stranded RNA molecules that are toxic to coleopteran insects. In particular, dsRNA molecules that capable of interfering with pest IAP genes and that are toxic to the target pest are provided. Further, methods of making and using the interfering RNA, for example in transgenic plants to confer protection from insect damage are disclosed.

## Background
RELATED APPLICATION INFORMATION
This application is a 371 of International Application No. PCT/US2013/046450, filed 19 Jun. 2013, which claims priority to U.S. Provisional Application No. 61/662,958, filed Jun. 22, 2012, the disclosures of which are incorporated by reference herein in their entireties.


STATEMENT REGARDING ELECTRONIC SUBMISSION OF A SEQUENCE LISTING
A Sequence Listing in ASCII test format, submitted under 37 C.F.R. 1.821, entitled “73414_ST25.txt”, 111 kilobytes in size, generated on Dec. 10, 2014 and filed via EFS-Web is provided in lieu of a paper copy. This sequence listing is hereby incorporated by reference into the specification for its disclosure.
FIELD OF THE INVENTION
The invention relates generally to the control of pests that cause damage to crop plants by their feeding activities, and more particularly to the control of coleopteran pests by compositions comprising interfering RNA molecules. The invention further relates to the compositions and to methods of using such compositions comprising the interfering RNA molecules.
BACKGROUND
Insect species in the genus Diabrotica (corn rootworms and cucumber beetles) are considered some of the most important pests to crop plants. For example, species of corn rootworm, including Diabrotica virgifera virgifera, the western corn rootworm (WCR); D. barberi, the northern corn rootworm (NCR), D. undecimpunctata howardi, the southern corn rootworm (SCR), and D. virgifera zeae, the Mexican corn rootworm (MCR), are the most destructive corn pests in North America causing an estimated loss of over $1 billion annually. The western corn rootworm has also invaded Europe and causes an estimated 0.5 billion euros in damage each year. Diabrotica speciosa (common names include, among others, leaf beetle, little Brazilian beetle, cucurbit beetle and chrysanthemum beetle) is an important pest of corn, soybean and peanuts, in South America.
Most of the damage in corn is caused by larval rootworm feeding. Newly hatched rootworm larvae 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, reduces plant growth, and results in reduced grain production. Severe root injury also may result in lodging of corn plants, making mechanical harvest more difficult or impossible. Corn rootworm adults feed primarily on corn silk, pollen, and kernels on exposed ear tips. If corn rootworm adults begin emerging before corn reproductive tissues are present, adults may feed on leaf tissue, scraping away the green surface tissue and leaving a window-pane appearance. Silk feeding by adults can result in pruning of silks at the ear tip, commonly called silk clipping. In field corn, beetle populations may reach a level high enough to cause severe silk clipping during pollen shed, which may interfere with pollination and reduce yield. Thus, unlike lepidopteran pests of corn in which only the larval stages cause damage, both the larval and adult stages of corn rootworm are capable of causing economic damage to corn.
Diabrotica insect pests are mainly controlled by intensive applications of chemical pesticides, which may be active against both larval and adult stages, through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects. Additional problems occur in areas of high insecticide use where populations of corn rootworm beetles have become resistant to certain insecticides. This has been partially alleviated by various resistance management practices, but there is an increasing need for alternative pest control agents.
Several native Cry proteins from Bacillus thuringiensis, or engineered Cry proteins, have been expressed in transgenic crop plants and exploited commercially to control certain lepidopteran and coleopteran insect pests. For example, starting in 2003, transgenic corn hybrids that control corn rootworm by expressing a Cry3Bb1, Cry34Ab1/Cry35Ab1 or modified Cry3A (mCry3A) protein have been available commercially in the US.
The seed industry, university researchers and the US Environmental Protection Agency have worked together to develop management plans to help mitigate the onset of insect resistance to transgenic plants expressing insecticidal proteins. They are based primarily on a high dose and refuge strategy. A high dose strategy is to use corn hybrids that express high enough levels of an insecticidal protein such as a Cry protein to kill even partially resistant insects. The underlying hypothesis is that killing partially resistant insects and preventing their mating greatly delays the development of resistance. The success of a high dose strategy depends in part on the specific activity of the insecticidal protein to the particular insect species and how much of that insecticidal protein can be expressed in the transgenic corn plant. The higher the specific activity of an insecticidal protein to a pest, the less amount of the insecticidal protein is required to be expressed in a transgenic plant to achieve a high dose strategy. For example, corn hybrids expressing the lepidopteran-active Cry protein, Cry1Ab, are considered high-dose against the primary target pest European corn borer (Ostrinia nubilalis). Because Cry1Ab is very toxic to European corn borer larvae with an LC50<10 ng/cm2 (i.e. high specific activity), levels of expression of Cry1Ab that are achievable in transgenic plants easily places such corn hybrids in a high dose category. However, unlike the lepidopteran-active products, current rootworm products are not considered high-dose. The proteins they express are not active against adults and have limited activity against late instar larvae. Therefore, the current transgenic rootworm products allow some rootworm larvae to survive and emerge as adults.
Thus, economic levels of silk clipping by corn rootworm adults may still occur even in portions of fields planted to a transgenic corn rootworm hybrid. For example, densities of western corn rootworm adults may exceed economic levels in portions of fields planted to transgenic corn rootworm hybrids due to immigration of beetles as well as direct emergence of adults from transgenic root systems. There have been many reports that confirm western corn rootworm adult emergence from certain corn transgenic rootworm hybrids (Crowder et al. 2005. J. Econ. Entomol. 8:961-975). A recent publication suggests that western corn rootworm adults will exhibit similar feeding behaviors when encountering some transgenic corn plants or non-transgenic corn plants in the field and that it is unlikely that certain insecticidal proteins in transgenic plants will have significant effects on adults that might impact resistance management.
Therefore, indentifying alternative insect control agents with new modes of action would be beneficial. Particularly useful would be new insect control agents that are toxic to multiple life stages of the target insect pest. Such insect control agents may include those that target genetic elements in the target insect pest.
Apoptosis is a physiological cell death process that is critical for the development and maintenance of healthy biological systems in many living organisms. The Inhibitor of Apoptosis (IAP) family of proteins was first discovered in baculoviruses that attack insects and have been shown to be involved in suppressing the insect host cell death (apoptosis) response to baculovirus infection (Crook et al. 1993. J. Virol. 67:2168-2174; Birnbaum et al. 1994. J. Virol. 68:2521-2528). Subsequently, IAP proteins were discovered in other organisms including the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fly Drosophila melanogaster, and several mammalian species. Native IAP acts as an endogenous inhibitor of caspases, the main executioners of apoptosis and IAP family proteins are characterized by an approximately 70 amino acid domain termed the baculoviral IAP repeat (BIR). Up to three tandem copies of the BIR domain can occur within the known IAP family proteins of viruses and animal species. Although membership in the IAP family of proteins requires both the presence of a BIR domain and the ability to suppress apoptosis, many of these BIR-containing proteins are untested with respect to apoptosis suppression. Moreover, as it is debatable as to whether yeast possess an apoptosis program (Zha et al. 1996. Mol Cell Biol. 16:6494-6508), because the presence of BIR-containing proteins in S. pombe and S. cerevisiae raises the possibility that BIR domains are not devoted exclusively to apoptosis suppression. Rather, they may function as protein interaction domains that may have evolved to suit a variety of purposes.
Therefore, it is not clear that all IAPs are part of life-critical pathways for any given organism, particularly in certain insect species including coleopteran pest species like Diabrotica spp. It is also uncertain that IAP proteins in a pest Diabrotica species can be targeted as a pest control strategy. Furthermore, it is even more uncertain that the expression of such IAP proteins can be modulated using interfering RNA molecules and that if such protein expression can be modulated, whether such modulation will result in toxicity to the target Diabrotica pest.
RNA interference (RNAi) occurs when an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments of about 19-24 nucleotides in length, called small interfering RNAs (siRNAs). The siRNAs then diffuse or are carried throughout the organism, including across cellular membranes, where they hybridize to mRNAs (or other RNAs) and cause hydrolysis of the RNA. Interfering RNAs are recognized by the RNA interference silencing complex (RISC) into which an effector strand (or “guide strand”) of the RNA is loaded. This guide strand acts as a template for the recognition and destruction of the duplex sequences. This process is repeated each time the siRNA hybridizes to its complementary-RNA target, effectively preventing those mRNAs from being translated, and thus “silencing” the expression of specific genes from which the mRNAs were transcribed. Most plant miRNAs show extensive base pairing to, and guide cleavage of their target mRNAs (Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol. 57, 19-53; Llave et al. (2002) Proc. Natl. Acad. Sci. USA 97, 13401-13406). In other instances, interfering RNAs may bind to target RNA molecules having imperfect complementarity, causing translational repression without mRNA degradation. The majority of the animal miRNAs studied so far appear to function in this manner.
There is an ongoing need for compositions and methods for using such compositions having insecticidal activity, for instance for use in crop protection or insect-mediated disease control. Novel compositions are required to overcome the problem of resistance to existing insecticides and/or to help mitigate the development of resistance to existing transgenic plant approaches. Ideally such compositions have a high toxicity and are effective when ingested orally by the target pest and have applicability for use against both the larval and adult stages of the pest insect. Thus any invention which provided compositions in which any of these properties was enhanced would represent a step forward in the art.
SUMMARY
The needs outlined above are met by the invention which, in various embodiments, provides new methods of controlling economically important insect pests. The invention in part comprises a method of inhibiting expression of one or more target genes and proteins in insect pests such as members of the genus Diabrotica. Specifically, the invention comprises methods of modulating expression of one or more inhibitor of apoptosis (IAP) genes in Diabrotica species such as Diabrotica virgifera virgifera (western corn rootworm), Diabrotica barberi (northern corn rootworm), Diabrotica undecimpunctata howardi (southern corn rootworm), Diabrotica virgifera zeae (Mexican corn rootworm), Diabrotica speciosa (chrysanthemum beetle), and related species, that causes cessation of feeding, growth, development and reproduction, and eventually results in the death of the insect. The method comprises introduction of double-stranded RNA (dsRNA) or its modified forms such as small interfering RNA (siRNA) sequences, into cells or into the extracellular environment, such as the midgut, within a pest insect body wherein the dsRNA or siRNA enters the cells and inhibits expression of at least one or more IAP genes and wherein inhibition of the one or more IAP genes exerts a deleterious effect upon the pest insect. It is specifically contemplated that the methods and compositions of the invention will be useful in limiting or eliminating pest insect infestation in or on any plant by providing one or more compositions comprising dsRNA or siRNA molecules in the diet of the pest. The invention also provides interfering RNA molecules that when delivered to an insect pest inhibits, through a toxic effect, the ability of the insect pest to survive, grow, feed and/or reproduce, or to limit pest related damage or loss to crop plants. Such delivery may be through production of the interfering RNA in a transgenic plant, for example corn, or by topically applying a composition comprising the interfering RNA to a plant or plant seed, such as a corn plant or corn seed. The interfering RNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a mRNA transcribable from a IAP gene or a portion of a nucleotide sequence of a mRNA transcribable from a IAP gene of the pest insect and therefore inhibits expression of the IAP gene, which causes cessation of feeding, growth, development, reproduction and eventually results in death of the pest insect. The invention is further drawn to nucleic acid constructs, nucleic acid molecules and recombinant vectors that comprise at least one strand of a dsRNA molecule of the invention. The invention also provides chimeric nucleic acid molecules comprising an antisense strand of a dsRNA of the invention operably associated with a plant microRNA precursor molecule. The invention also provides artificial plant microRNA precursors comprising an antisense strand of a dsRNA of the invention.
The invention further provides compositions comprising two or more of the dsRNA molecules of the invention wherein the two or more RNA molecules each comprise a different antisense strand, or comprising two or more nucleic acid constructs or nucleic acid molecules or artificial plant microRNA precursors of the invention.
The invention further provides insecticidal compositions for inhibiting the expression of a Diabrotica insect IAP gene that comprises a dsRNA of the invention and an agriculturally acceptable carrier. Inhibition of the expression of the Diabrotica IAP gene leads to cessation of feeding and growth and ultimately results in the death of the Diabrotica insect.
The invention is further drawn to transgenic plants which produce one or more interfering RNA molecules of the invention that are self-protected from insect feeding damage and to methods of using the plants alone or in combination with other insect control strategies to confer maximal insect control capabilities. Plants and/or plant parts producing one or more interfering RNA molecules of the invention or treated with a composition comprising one or more interfering RNA molecules of the invention are highly resistant to insect pest infestation. For example, economically important coleopteran pests in the genus Diabrotica can be controlled by a plant that produces an interfering RNA molecule of the invention or by a plant or plant seed that is treated with a composition comprising an interfering RNA molecule of the invention.
The invention also provides a method of controlling a Diabrotica insect comprising contacting the Diabrotica insect with a nucleic acid molecule that is or is capable of producing an interfering RNA of the invention for inhibiting expression of an IAP gene in the Diabrotica insect thereby controlling the Diabrotica insect.
In other aspects, the invention provides a method of reducing an adult Diabrotica insect population on a transgenic plant expressing a Cry protein, a hybrid Cry protein or modified Cry protein comprising expressing in the transgenic plant a nucleic acid molecule that is or is capable of producing an interfering RNA of the invention capable of inhibiting expression of an IAP gene in an adult Diabrotica insect thereby reducing the adult Diabrotica insect population.
In other aspects, the invention provides a method of reducing resistance development in a Diabrotica insect population to an interfering RNA of the invention, the method comprising expressing in a transgenic plant fed upon by the Diabrotica insect population an interfering RNA of the invention that is capable of inhibiting expression of an IAP gene in a larval and adult Diabrotica insect, thereby reducing resistance development in the Diabrotica insect population compared to a Diabrotica insect population exposed to an interfering RNA capable of inhibiting expression of an IAP gene in only the larval stage or adult stage of a Diabrotica insect.
In other aspects, the invention provides a method of reducing the level of a target RNA transcribable from a IAP gene in a Diabrotica insect comprising contacting the Diabrotica insect with a composition comprising a dsRNA molecule of the invention, wherein the dsRNA molecule reduces the level of the target RNA in a cell of the Diabrotica insect.
In still other aspects, the invention provides a method of conferring Diabrotica insect tolerance to a plant, or part thereof, comprising introducing into the plant, or part thereof, a dsRNA molecule, a nucleic acid construct, a chimeric nucleic acid molecule, an artificial plant microRNA precursor molecule and/or a composition of the invention, thereby conferring tolerance of the plant or part thereof to the Diabrotica insect.
In further aspects, the invention provides a method of reducing root damage to a plant fed upon by a Diabrotica insect, comprising introducing into cells of the plant a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, an artificial plant microRNA precursor molecule and/or a composition of the invention, thereby reducing root damage to the plant.
In other aspects, the invention provides a method of producing a transgenic plant cell having toxicity to a Diabrotica insect, comprising introducing into a plant cell a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, an artificial plant microRNA precursor molecule and/or a composition of the invention, thereby producing the transgenic plant cell having toxicity to the Diabrotica insect compared to a control plant cell.
In further aspects, the invention provides a method of producing a transgenic plant having enhanced tolerance to Diabrotica insect feeding damage, comprising introducing into a plant a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, an artificial plant microRNA precursor molecule and/or a composition of the invention, thereby producing a transgenic plant having enhanced tolerance to Diabrotica insect feeding damage compared to a control plant.
In other aspects, the invention provides a method of enhancing control of a Diabrotica insect population comprising providing a transgenic plant or transgenic seed of the invention and applying to the transgenic plant or the transgenic seed a chemical pesticide that is insecticidal to a Diabrotica insect, thereby enhancing control of the Diabrotica insect population.
In other aspects, the invention provides a method of providing a corn grower with a means of controlling a Diabrotica insect pest population in a corn crop comprising (a) selling or providing to the grower at least one bag of corn seed comprising transgenic corn seed that comprises a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, an artificial plant microRNA precursor molecule and/or a composition of the invention; and (b) advertising to the grower that the transgenic corn seed produces transgenic corn plants capable of controlling a Diabrotica insect pest.
These and other aspects of the invention are set forth in more detail in the description of the invention below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows portions of the wcrIAP1 mRNA targeted by the wcrIAP1-A, wcrIAP1-B and wcrIAP1-C dsRNA.
FIG. 2 is an agarose gel of PCR products showing that the identical IAP1 gene is detected in larval stages and adult stage of Diabrotica virgifera virgifera (western corn rootworm).
FIG. 3 is a map of an expression vector comprising a nucleic acid molecule that encodes a dsRNA of the invention.
FIG. 4 is an alignment of Diabrotica IAP coding sequences. A “.” below a base (A, T, G or C) indicates an identical base as in the reference sequence. Bases that are different from the reference sequence are indicated.
FIG. 5 is a drawing comparing the root mass from transgenic corn plants expressing nucleic acids encoding dsRNA of the invention with control plants.



BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID NO:1 is a nucleotide sequence of a western corn rootworm IAP1 cDNA (wcrIAP1) including the 5′ and 3′ untranslated regions (UTR5).
SEQ ID NO:2 is a nucleotide sequence of the coding region of wcrIAP1 (SEQ ID NO:1).
SEQ ID NO:3 is a nucleotide sequence of a mRNA transcribable from the wcrIAP1 gene.
SEQ ID NO:4 is an antisense sequence of the wcrIAP1 mRNA designated wcrIAP1*.
SEQ ID NO:5 is a sense strand of a dsRNA designated wcrIAP1-B.
SEQ ID NO:6 is an antisense strand of wcrIAP1-8 designated wcrIAP1-8*.
SEQ ID NO:7 is a sense strand of a dsRNA designated wcrIAP1-A.
SEQ ID NO:8 is an antisense strand of wcrIAP1-A designated wcrIAP1-A*.
SEQ ID NO:9 is a sense strand of a dsRNA designated wcrIAP1-C.
SEQ ID NO:10 is an antisense strand of wcrIAP1-C designated wcrIAP1-C*.
SEQ ID NO:11 is a nucleotide sequence from a western corn rootworm IAP2 coding region (wcrIAP2).
SEQ ID NO:12 is a nucleotide sequence of a mRNA transcribable from wcrIAP2.
SEQ ID NO:13 is an antisense sequence of the wcrIAP2 mRNA designated wcrIAP2*.
SEQ ID NO:14 is a nucleotide sequence of a southern corn rootworm IAP1 coding region (scrIAP1).
SEQ ID NO:15 is a nucleotide sequence of a mRNA transcribable from scrIAP1 (SEQ ID NO:14).
SEQ ID NO:16 is an antisense sequence of the scrIAP1 mRNA designated scrIAP1*.
SEQ ID NO:17 is a nucleotide sequence of a northern corn rootworm IAP1 coding region (ncrIAP1)
SEQ ID NO:18 is a nucleotide sequence of a mRNA transcribable from ncrIAP1.
SEQ ID NO:19 is an antisense sequence of the ncrIAP1 mRNA designated ncrIAP1*.
SEQ ID NO:20 is the amino acid sequence encoded by SEQ ID NO:2.
SEQ ID NO:21 is the amino acid sequence encoded by SEQ ID NO:11.
SEQ ID NO:22 is the amino acid sequence encoded by SEQ ID NO:14
SEQ ID NO:23 is the amino acid sequence encoded by SEQ ID NO:17.
SEQ ID NO:24 is an amino acid sequence of a Drosophila melanogaster IAP protein (dmIAP).
SEQ ID NOs:25-28 are examples of 19-mer portions of wcrIAP1 mRNA (SEQ ID NO:3) targetable by siRNA.
SEQ ID NOs:29-32 are examples of 19-mer portions of wcrIAP2 mRNA (SEQ ID NO:12) targetable by siRNA.
SEQ ID NOs:33-36 are examples of 19-mer portions of scrIAP1 mRNA (SEQ ID NO:15) targetable by siRNA.
SEQ ID NOs:37-40 are examples of 19-mer portions of ncrIAP1 mRNA (SEQ ID NO:18) targetable by siRNA.
SEQ ID NOs:41-44 are examples of wcrIAP1* anti-sense 19-mer sequences.
SEQ ID NOs:45-48 are examples of wcrIAP2* anti-sense 19-mer sequences.
SEQ ID NOs:49-52 are examples of scrIAP1* anti-sense 19-mer sequences.
SEQ ID NOs:53-56 are examples of ncrIAP1* anti-sense 19-mer sequences.
SEQ ID NOs:57-62 are primers useful in the invention.
SEQ ID NO:63 is a nucleotide sequence coding for a hairpin RNA (hpRNA) designated hpwcrIAP1-C/wcrIAP1-C*.
SEQ ID NO:64 is a nucleotide sequence coding for a hairpin RNA (hpRNA) designated hpwcrIAP1-Ca/wcrIAP1-Ca*.
SEQ ID NO:65 is a nucleotide sequence of the pRNA21534 expression vector.
SEQ ID NO:66 is a nucleotide sequence of the pRNA21536 expression vector.
SEQ ID NO:67 is a nucleotide sequence of the pRNA21537 expression vector.
DETAILED DESCRIPTION
The following is a detailed description of the invention provided to aid those skilled in the art in practicing the invention. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments of the invention will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
For clarity, certain terms used in the specification are defined and presented as follows:
As used herein, “a,” “an” or “the” can mean one or more than one. For example, a cell can mean a single cell or a multiplicity of cells.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
Further, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.
As used herein, the transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
As used herein, “dsRNA” or “RNAi” refers to a polyribonucleotide structure formed either by a single self-complementary RNA strand or at least by two complementary RNA strands. The degree of complementary, in other words the % identity, need not necessarily be 100%. Rather, it must be sufficient to allow the formation of a double-stranded structure under the conditions employed.
Preferably, the % identity of a polyribonucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) using the default settings, wherein the query sequence is at least about 21 to about 23 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least about 21 nucleotides. In another embodiment, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. In a further embodiment, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. In yet another embodiment, the query sequence corresponds to the full length of the target RNA, for example mRNA, and the GAP analysis aligns the two sequences over the full length of the target RNA.
Conveniently, the dsRNA can be produced from a single open reading frame in a recombinant host cell, wherein the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. Alternatively, the sense strand and antisense strand can be made without an open reading frame to ensure that no protein will be made in the transgenic host cell. The two strands can also be expressed separately as two transcripts, one encoding the sense strand and one encoding the antisense strand.
RNA duplex formation can be initiated either inside or outside the cell. The dsRNA can be partially or fully double-stranded. The RNA can be enzymatically or chemically synthesized, either in vitro or in vivo.
The dsRNA need not be full length relative to either the primary transcription product or fully processed RNA. Generally, higher identity can be used to compensate for the use of a shorter sequence. Furthermore, the dsRNA can comprise single stranded regions as well, e.g., the dsRNA can be partially or fully double stranded. The double stranded region of the dsRNA can have a length of at least about 18 to about 25 base pairs, optionally a sequence of about 18 to about 50 base pairs, optionally a sequence of about 50 to about 100 base pairs, optionally a sequence of about 100 to about 200 base pairs, optionally a sequence of about 200 to about 500, and optionally a sequence of about 500 to about 1000 or more base pairs, up to molecule that is double stranded for its full length, corresponding in size to a full length target RNA molecule.
The dsRNA can contain known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiralmethyl phosphonates and 2-O-methyl ribonucleotides.
As used herein, the term “specifically reduce the level of a target RNA and/or the production of a target protein encoded by the RNA”, and variations thereof, refers to the sequence of a portion of one strand of the dsRNA being sufficiently identical to the target RNA such that the presence of the dsRNA in a cell reduces the steady state level and/or the production of said RNA. In many instances, the target RNA will be mRNA, and the presence of the dsRNA in a cell producing the mRNA will result in a reduction in the production of said protein. Preferably, this accumulation or production is reduced at least 10%, more preferably at least 50%, even more preferably at least 75%, yet even more preferably at least 95% and most preferably 100%, when compared to a wild-type cell.
The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as, but not limited to, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), and other immunoassays.
MicroRNAs (miRNAs) are non-protein coding RNAs, generally of between about 18 to about 25 nucleotides in length (commonly about 20-24 nucleotides in length in plants). These miRNAs direct cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways (Bartel, Cell, 116:281-297 (2004); Zhang et al. Dev. Biol. 289:3-16 (2006)). As such, miRNAs have been shown to be involved in different aspects of plant growth and development as well as in signal transduction and protein degradation. In addition, small endogenous mRNAs including miRNAs may also be involved in biotic stress responses such as pathogen attack. Since the first miRNAs were discovered in plants (Reinhart et al. Genes Dev. 16:1616-1626 (2002), Park et al. Curr. Biol. 12:1484-1495 (2002)) many hundreds have been identified. Furthermore, many plant miRNAs have been shown to be highly conserved across very divergent taxa. (Floyd et al. Nature 428:485-486 (2004); Zhang et al. Plant J. 46:243-259 (2006)). Many microRNA genes (MIR genes) have been identified and made publicly available in a database (miRBase; microrna.sanger.ac.uk/sequences). miRNAs are also described in U.S. Patent Publications 2005/0120415 and 2005/144669A1, the entire contents of which are incorporated by reference herein.
Genes encoding miRNAs yield primary miRNAs (termed a “pri-miRNA”) of 70 to 300 bp in length that can form imperfect stem-loop structures. A single pri-miRNA may contain from one to several miRNA precursors. In animals, pri-miRNAs are processed in the nucleus into shorter hairpin RNAs of about 65 nt (pre-miRNAs) by the RNaselll enzyme Drosha and its cofactor DGCR8/Pasha. The pre-miRNA is then exported to the cytoplasm, where it is further processed by another RNaselll enzyme, Dicer, releasing a miRNA/miRNA* duplex of about 22 nt in size. In contrast to animals, in plants, the processing of pri-miRNAs into mature miRNAs occurs entirely in the nucleus using a single RNaselll enzyme, DCL1 (Dicer-like 1). (Zhu. Proc. Natl. Acad. Sci. 105:9851-9852 (2008)). Many reviews on microRNA biogenesis and function are available, for example, see, Bartel Cell 116:281-297 (2004), Murchison et al. Curr. Opin. Cell Biol. 16:223-229 (2004), Dugas et al. Curr. Opin. Plant Biol. 7:512-520 (2004) and Kim Nature Rev. Mol. Cell Biol. 6:376-385 (2005).
The term “plant microRNA precursor molecule” as used herein describes a small ( ̃70-300 nt) non-coding RNA sequence that is processed by plant enzymes to yield a  ̃19-24 nucleotide product known as a mature microRNA sequence. The mature sequences have regulatory roles through complementarity to messenger RNA. The term “artificial plant microRNA precursor molecule” describes the non-coding miRNA precursor sequence prior to processing that is employed as a backbone sequence for the delivery of a siRNA molecule via substitution of the endogenous native miRNA/miRNA* duplex of the miRNA precursor molecule with that or a non-native, heterologous miRNA (amiRNA/amiRNA*; e.g. siRNA/siRNA*) that is then processed into the mature miRNA sequence with the siRNA sequence.
In the context of the invention, the term “toxic” used to describe a dsRNA of the invention means that the dsRNA molecules of the invention and combinations of such dsRNA molecules function as orally active insect control agents that have a negative effect on an insect. When a composition of the invention is delivered to the insect, the result is typically death of the insect, or the insect does not feed upon the source that makes the composition available to the insect. Such a composition may be a transgenic plant expressing the dsRNA of the invention.
A “coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.
As used herein, “complementary” polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A.” It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.
The terms “complementary” or “complementarity,” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single-stranded molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
As used herein, the terms “substantially complementary” or “partially complementary mean that two nucleic acid sequences are complementary at least about 50%, 60%, 70%, 80% or 90% of their nucleotides. In some embodiments, the two nucleic acid sequences can be complementary at least at 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their nucleotides. The terms “substantially complementary” and “partially complementary” can also mean that two nucleic acid sequences can hybridize under high stringency conditions and such conditions are well known in the art
To “control” or “controlling” insects means to inhibit, through a toxic effect, the ability of one or more insect pests to survive, grow, feed, and/or reproduce, or to limit insect-related damage or loss in crop plants. To “control” insects may or may not mean killing the insects, although it preferably means killing the insects.
To “deliver” or “delivering” a composition or dsRNA means that the composition or dsRNA comes in contact with an insect, resulting in a toxic effect and control of the insect. The composition or dsRNA can be delivered in many recognized ways, e.g., orally by ingestion by the insect via transgenic plant expression, formulated composition(s), sprayable composition(s), a bait matrix, or any other art-recognized RNA delivery system.
“Diabrotica” is a genus of beetles commonly referred to as “corn rootworms” or “cucumber beetles.” There are many species of Diabrotica that are pests of crop plants, including without limitation, Diabrotica barberi (northern corn rootworm; NCR), D. virgifera virgifera (western corn rootworm; WCR), D. undecimpunctata howardii (southern corn rootworm; SCR) and D. virgifera zeae (Mexican corn rootworm; MCR). In the context of the invention, the term “corn rootworm” or “cucumber beetle” is interchangeable with the term “Diabrotica.”
A “Diabrotica life stage” or “corn rootworm life stage” means the egg, larval, pupal or adult developmental form of a Diabrotica species.
“Effective insect-controlling amount” means that concentration of dsRNA that inhibits, through a toxic effect, the ability of insects to survive, grow, feed and/or reproduce, or to limit insect-related damage or loss in crop plants. “Effective insect-controlling amount” may or may not mean a concentration that kills the insects, although it preferably means that it kills the insects.
“Expression cassette” as used herein means a nucleic acid sequence capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the nucleic acid sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleic acid sequence. The expression cassette comprising the nucleic acid sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleic acid sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.
A “gene” is a defined region that is located within a genome and that, besides the aforementioned coding sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5′ and 3′ untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
As used herein, the term “grower” means a person or entity that is engaged in agriculture, raising living organisms, such as crop plants, for example corn, for food or raw materials.
A “heterologous” nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.
A “homologous” nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.
“Insecticidal” is defined as a toxic biological activity capable of controlling insects, preferably by killing them.
An “isolated” nucleic acid molecule or nucleotide sequence or nucleic acid construct or dsRNA molecule or protein of the invention is generally exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or nucleotide sequence or nucleic acid construct or dsRNA molecule or protein may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell such as a transgenic plant.
In the context of the invention, a number in front of the suffix “mer” indicates a specified number of subunits. When applied to RNA or DNA, this specifies the number of bases in the molecule. For example, a 19 nucleotide subsequence of an mRNA having the sequence ACUGGUCGCGUUGCAUGCU is a “19-mer.”
A “plant” is any plant at any stage of development, particularly a seed plant.
A “plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
“Plant cell culture” means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
“Plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
A “plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
“Plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
A corn rootworm “transcriptome” is a collection of all the ribonucleic acid (RNA) transcripts in a corn rootworm cell.
“Transformation” is a process for introducing heterologous nucleic acid into a host cell or organism. In particular, “transformation” means the stable integration of a DNA molecule into the genome of an organism of interest.
“Transformed/transgenic/recombinant” refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed”, “non-transgenic”, or “non-recombinant” host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
The nomenclature used herein for DNA or RNA bases and amino acids is as set forth in 37 C.F.R. §1.822.
The invention is based on the unexpected discovery that double stranded RNA (dsRNA) or small interfering RNAs (siRNA) designed to target a mRNA transcribable from an IAP gene of a Diabrotica insect are toxic to the Diabrotica insect pest and can be used to control Diabrotica infestation of a plant and impart to a transgenic plant tolerance to a Diabrotica infestation. Thus, in some embodiments, the invention provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein a nucleotide sequence of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a Diabrotica IAP gene that comprises a IAP coding sequence having at least 90% identity, or at least 91% identity, or at least 92% identity, or at least 93% identity, or at least 94% identity, or at least 95% identity, or least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to SEQ ID NO:2, and wherein the dsRNA molecule is toxic to a Diabrotica insect. In some embodiments the Diabrotica IAP gene is from a Diabrotica insect selected from the group consisting of Diabrotica barberi (northern corn rootworm), D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), D. virgifera zeae (Mexican corn rootworm), D. beniensis, D. cristata, D. curvipustulata, D. dissimilis, D. elegantula, D. emorsitans, D. graminea, D. hispanolae, D. lemniscata, D. linsleyi, D. milleri, D. nummularis, D. occlusa, D. porracea, D. scutellata, D. tibialis, D. trifasciata and D. viridula. In further embodiments, the Diabrotica insect is D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi (southern corn rootworm) or D. barberi (northern corn rootworm). In some embodiments, the IAP coding sequence comprises SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:17.
In some embodiments, the dsRNA molecule can comprise, consist essentially of or consist of from at least 18 to about 25 consecutive nucleotides (e.g. 18, 19, 20, 21, 22, 23, 24 or 25) to at least about 400 consecutive nucleotides. In some embodiments the dsRNA molecule can comprise, consist essentially of or consist of about 500, or about 50 or about 543 consecutive nucleotides. Additional nucleotides can be added at the 3′ end, the 5′ end or both the 3′ and 5′ ends to facilitate manipulation of the dsRNA molecule but that do not materially affect the basic characteristics or function of the dsRNA molecule in RNA interference (RNAi).
In some embodiments, the portion of the mRNA polynucleotide transcribable from a Diabrotica IAP gene that the antisense strand is complementary to comprises at least 18 consecutive nucleotides of SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18. In other embodiments, the portion of mRNA comprises, consists essentially of or consists of at least from 19, 20 or 21 consecutive nucleotides to at least 400 consecutive nucleotides of SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18. In other embodiments, the portion of mRNA comprises, consists essentially of or consists of at least about 500, or at least about 507 or at least about 543 consecutive nucleotides of SEQ ID NO:3.
In other embodiments, the portion of the mRNA polynucleotide that is complementary to the antisense strand of a dsRNA of the invention comprises any 19-mer subsequence of SEQ ID NO:3 (wcrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to 1604 of SEQ ID NO:3. In other words, the portion of the mRNA that is targeted comprises any of the 1604 19 consecutive nucleotide subsequences (i.e. 19-mer) of SEQ ID NO:3, for example, bases 1-19 (5′-GAGUAUCGAGUGAGAAAUC-3′) (SEQ ID NO:25), bases 2-20 (5′-AGUAUCGAGUGAGAAAUCG-3′) (SEQ ID NO:26), bases 3-21 (5′-GUAUCGAGUGAGAAAUCGU-3′) (SEQ ID NO:27) and so forth to bases 1604-1622 (5′-AUAUAGUAAUUUAUAAUAU-3′) (SEQ ID NO:28).
In other embodiments, the portion of the mRNA polynucleotide that is complementary to the antisense strand of a dsRNA of the invention comprises any 19-mer subsequence of SEQ ID NO:12 (wcrIAP2) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1444 of SEQ ID NO:12. In other words, the portion of the mRNA that is targeted comprises any of the 1444 19 consecutive nucleotide subsequences (i.e. 19-mers) of SEQ ID NO: 12, for example bases 1-19 (5′-UCAGAAUGGAAUUAUGGCG-3′) (SEQ ID NO:29), bases 2-20 (5′-CAGAAUGGAAUUAUGGCGA-3′) (SEQ ID NO:30), bases 3-21 (5′-AGAAUGGAAUUAUGGCGAU-3′) (SEQ ID NO:31) and so forth to bases 1444-1462 (5′-AUUAAAAUAAUUGUUUCCU-3′) (SEQ ID NO:32).
In other embodiments, the portion of the mRNA polynucleotide that is complementary to the antisense strand of a dsRNA of the invention comprises any 19-mer subsequence of SEQ ID NO:15 (scrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1018 of SEQ ID NO:15. In other words, the portion of the mRNA that is targeted comprises any of the 1015 19 consecutive nucleotide subsequences (i.e. 19-mer) of SEQ ID NO:15, for example, bases 1-19 (5′-AUGGCAGGUAGUCAAUCAA-3′) (SEQ ID NO:33), bases 2-20 (5′-UGGCAGGUAGUCAAUCAAA-3′) (SEQ ID NO:34), bases 3-21 (5′-GGCAGGUAGUCAAUCAAAU-3′) (SEQ ID NO:35) and so forth to bases 1015-1033 (5′-GUCAGAGCGUUUCCUAUCA-3′) (SEQ ID NO:36).
In still other embodiments, the portion of the mRNA polynucleotide that is complementary to the antisense strand of a dsRNA of the invention comprises any 19-mer subsequence of SEQ ID NO:18 (ncrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1020 of SEQ ID NO:18. In other words, the portion of the mRNA that is targeted comprises any of the 1020 19 consecutive nucleotide subsequences (i.e. 19-mer) of SEQ ID NO:18, for example, bases 1-19 (5′-AGUCAAUCAAAUUACAUUC-3′) (SEQ ID NO:37), bases 2-20 (5′-GUCAAUCAAAUUACAUUCA-3′) (SEQ ID NO:38), bases 3-21 (5′-UCAAUCAAAUUACAUUCAA-3′) (SEQ ID NO:38) and so forth to bases 1020-1038 (5′-AGUCAGAGCGUUUCCUAUC-3′) (SEQ ID NO:40).
In still other embodiments, the portion of the mRNA that is complementary to the antisense sequence of a dsRNA of the invention consists essentially of SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9. In other embodiments, the portion of the mRNA polynucleotide consists essentially of SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18.
In further embodiments of the dsRNA molecule of this invention, the nucleotide sequence of the antisense strand can consist essentially of the nucleotide sequence of any 19-mer subsequence of SEQ ID NO:4 (wcrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to 1603 of SEQ ID NO:4. In other words, the antisense strand consists essentially of any of the 1604 19 consecutive nucleotide subsequences (i.e. 19-mer) of SEQ ID NO:4, for example, bases 1-19 (5′-GAGUAUCGAGUGAGAAAUC-3′) (SEQ ID NO:41), bases 2-20 (5′-AGUAUCGAGUGAGAAAUCG-3′) (SEQ ID NO:42), bases 3-21 (5′-GUAUCGAGUGAGAAAUCGU-3′) (SEQ ID NO:43) and so forth to bases 1604-1622 (5′-AUAUAGUAAUUUAUAAUAU-3′) (SEQ ID NO:44).
In other embodiments, the nucleotide sequence of the antisense strand can consist essentially of the nucleotide sequence of any 19-mer subsequence of SEQ ID NO:13 (wcrIAP2*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1444 of SEQ ID NO:13. In other words, the antisense strand consists essentially of the 1444 19 consecutive nucleotide subsequences (i.e. 19-mer) of SEQ ID NO:13, for example, bases 1-19 (5′-CGCCAUAAUUCCAUUCUGA-3′) (SEQ ID NO:45), bases 2-20 (5′-UCGCCAUAAUUCCAUUCUG-3′) (SEQ ID NO:46), bases 3-21 (5′-AUCGCCAUAAUUCCAUUCU-3′) (SEQ ID NO:47) and so forth to bases 1444-1462 (5′-AGGAAACAAUUAUUUUAAU-3′) (SEQ ID NO:48).
In other embodiments, the nucleotide sequence of the antisense strand can consist essentially of the nucleotide sequence of any 19-mer subsequence of SEQ ID NO:16 (scrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1018 of SEQ ID NO:16. In other words, the antisense strand consists essentially of the nucleotide sequence of any of the 1015 19 consecutive nucleotide subsequences (i.e. 19-mer) of SEQ ID NO:16, for example, bases 1-19 (5′-UUGAUUGACUACCUGCCAU-3′) (SEQ ID NO:49), bases 2-20 (5′-UUUGAUUGACUACCUGCCA-3′) (SEQ ID NO:50), bases 3-21 (5′-AUUUGAUUGACUACCUGCC-3′) (SEQ ID NO:51) and so forth to bases 1015-1033 (5′-UGAUAGGAAACGCUCUGAC-3′) (SEQ ID NO:52).
In other embodiments, the nucleotide sequence of the antisense strand can consist essentially of the nucleotide sequence of any 19-mer subsequence of SEQ ID NO:19 (ncrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1020 of SEQ ID NO:19. In other words, the antisense strand consists essentially of the nucleotide sequence of any of the 1020 19 consecutive nucleotide subsequences (i.e. 19-mer) of SEQ ID NO:19, for example, bases 1-19 (5′-GAAUGUAAUUUGAUUGACU-3′) (SEQ ID NO:53), bases 2-20 (5′-UGAAUGUAAUUUGAUUGAC-3′) (SEQ ID NO:54), bases 3-21 (5′-UUGAAUGUAAUUUGAUUGA-3′) (SEQ ID NO:55) and so forth to bases 1015-1033 (5′-GAUAGGAAACGCUCUGACU-3′) (SEQ ID NO:56).
In still other embodiments, the nucleotide sequence of the antisense strand of a dsRNA of the invention that is complementary or substantially complementary to a portion of a mRNA polynucleotide transcribable from a Diabrotica insect IAP gene comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10. In other embodiments, the antisense strand comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18. It is to be understood that any of the 19-mer sequences of SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:16 or SEQ ID NO:19 can have one nucleotide at either the 3′ or 5′ end deleted or can have up to 6 nucleotides added at the 3′ end, the 5′ end or both, in any combination to achieve an antisense strand consisting essentially of the 19-mer nucleotide sequence of any of SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:16 or SEQ ID NO:19, as it would be understood that the deletion of the one nucleotide or the addition of up to the six nucleotides do not materially affect the basic characteristics or function of the double stranded RNA molecule of the invention. Such additional nucleotides can be nucleotides that extend the complementarity of the antisense strand along the target sequence and/or such nucleotides can be nucleotides that facilitate manipulation of the RNA molecule or a nucleic acid molecule encoding the RNA molecule, as would be known to one of ordinary skill in the art. For example, a TT overhang at the 3; end may be present, which is used to stabilize the siRNA duplex and does not affect the specificity of the siRNA.
In some embodiments of this invention, the antisense strand of the double stranded RNA molecule can be fully complementary to the target RNA polynucleotide or the antisense strand can be substantially complementary or partially complementary to the target RNA polynucleotide. By substantially or partially complementary is meant that the antisense strand and the target RNA polynucleotide can be mismatched at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide pairings. Such mismatches can be introduced into the antisense strand sequence, e.g., near the 3′ end, to enhance processing of the double stranded RNA molecule by Dicer, to duplicate a pattern of mismatches in a siRNA molecule inserted into a chimeric nucleic acid molecule or artificial microRNA precursor molecule of this invention (see Examples section), and the like, as would be known to one of skill in the art. Such modification will weaken the base pairing at one end of the duplex and generate strand asymmetry, therefore enhancing the chance of the antisense strand, instead of the sense strand, being processed and silencing the intended gene (Geng and Ding “Double-mismatched siRNAs enhance selective gene silencing of a mutant ALS-causing Allele1” Acta Pharmacol. Sin. 29:211-216 (2008); Schwarz et al. “Asymmetry in the assembly of the RNAi enzyme complex” Cell 115:199-208 (2003)). Other such mismatches can be introduced into the antisense strand due to eliminating fortuitous open reading frames created in making dsRNA encoding expression cassettes. Such open reading frames are eliminated by making point mutations in the dsRNA encoding nucleotide sequence thus creating some mismatches in the dsRNA compared to the target gene. In one embodiment of this invention, such mismatches are introduced into a nucleotide sequence encoding the sense strand wcrIAP1-C(SEQ ID NO:9) resulting in a nucleotide sequence encoding a mutated sense strand designated wcrIAP1-Ca (nucleotides 1-500 of SEQ ID NO:64).
In some embodiments of this invention, the dsRNA molecule of the invention is a short hairpin RNA (shRNA) molecule. Expression of shRNA in cells is typically accomplished by delivery of plasmids or recombinant vectors, for example in transgenic plants such as transgenic corn.
The invention encompasses a nucleic acid molecule encoding at least one strand of a dsRNA molecule of the invention. The invention further encompasses a nucleic acid construct comprising at least one strand of a dsRNA molecule of the invention or comprising the nucleic acid molecule encoding the at least one strand of a dsRNA molecule of the invention. In one embodiment of the invention, the nucleic acid molecule encodes a short hairpin RNA. In another embodiment, the nucleic acid molecule that encodes the short hairpin RNA comprises SEQ ID NO:63 or SEQ ID NO:64. In another embodiment, the nucleic acid molecule is a recombinant vector. In yet another embodiment, the recombinant vector comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:67.
The invention further encompasses chimeric nucleic acid molecules comprising an antisense strand of a dsRNA of the invention operably linked with a plant microRNA precursor molecule. In some embodiments, the chimeric nucleic acid molecule comprises an antisense strand having the nucleotide sequence of any of the 19-mer subsequences of SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:16 or SEQ ID NO:19 operably linked with a plant microRNA precursor molecule. In some embodiments, the plant microRNA precursor molecule is a maize microRNA precursor.
In some embodiments, the invention encompasses an artificial plant microRNA precursor molecule comprising an antisense strand of a dsRNA molecule of the invention. In other embodiments, the artificial plant microRNA precursor molecule comprises an antisense strand having the nucleotide sequence of any of the 19-mer subsequences of SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:16 or SEQ ID NO:19. The use of artificial plant microRNAs to deliver a nucleotide sequence of interest (e.g an artificial miRNA; siRNA/siRNA*) into a plant is known in the art (see, e.g., Schwab et al. 2006. The Plant Cell 18:1121-1133 and Examples section herein). In the invention, the artificial microRNAs are chimeric or hybrid molecules, having a plant microRNA precursor backbone and an insect (i.e. animal) siRNA sequence inserted therein. As would be understood by one of ordinary skill in the art, it is typically desirable to maintain mismatches that normally occur in the plant microRNA precursor sequence in any nucleotide sequence that is substituted into the plant microRNA precursor backbone. In still other embodiments, the artificial plant microRNA precursor comprises portions of a corn microRNA precursor molecule. Any corn microRNA (miRNA) precursor is suitable for the compositions and methods of the invention. Nonlimiting examples include miR156, miR159, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR319, miR390, miR393, miR394, miR395, miR396, miR397, miR398, miR399, miR408, miR482, miR528, miR529, miR827, miR1432, as well as any other plant miRNA precursors now known or later identified.
In some embodiments, the invention encompasses nucleic acid constructs, nucleic acid molecules or recombinant vectors comprising at least one strand of a dsRNA molecule of the invention, or comprising a chimeric nucleic acid molecule of the invention, or comprising an artificial plant microRNA of the invention. In some embodiments the nucleic acid construct comprises a nucleic acid molecule of the invention. In other embodiments, the nucleic acid construct is a recombinant expression vector.
In some embodiments, the invention encompasses compositions comprising two or more dsRNA molecules of the invention wherein the two or more RNA molecules each comprise a different antisense strand. In some embodiments the two or more dsRNA molecules are present on the same nucleic acid construct, on different nucleic acid constructs or any combination thereof. In other embodiments, the composition comprises an RNA molecule comprising an antisense strand consisting essentially of the nucleotide sequence of SEQ ID NO:6 and an RNA molecule comprising an antisense strand consisting essentially of the nucleotide sequence of SEQ ID NO:8 and/or an RNA molecule comprising an antisense strand consisting essentially of the nucleotide sequence of SEQ ID NO:10. In other embodiments, the composition comprises two or more nucleic acid constructs, two or more nucleic acid molecules, two or more chimeric nucleic acid molecules, two or more artificial plant microRNA precursors of the invention, wherein the two or more nucleic acid constructs, two or more nucleic acid molecules, two or more chimeric nucleic acid molecules, or two or more artificial plant microRNA precursors, each comprise a different antisense strand.
In some embodiments, the invention encompasses an insecticidal composition for inhibiting the expression of a Diabrotica insect IAP gene, comprising a dsRNA or siRNA of the invention and an agriculturally acceptable carrier. In some embodiments, the acceptable agricultural carrier is a transgenic organism expressing a dsRNA or siRNA of the invention. In some embodiments the transgenic organism may be a transgenic plant expressing the dsRNA or siRNA of the invention that when fed upon by a target pest causes the target pest to stop feeding, growing or reproducing or causing death of the target pest. In other embodiments, the transgenic plant is a transgenic corn plant and the target pest is a Diabrotica insect pest. In still other embodiments, the Diabrotica insect pest is selected from the group consisting of Diabrotica barberi (northern corn rootworm), D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), D. virgifera zeae (Mexican corn rootworm).
In other embodiments, the transgenic organism is selected from, but not limited to, the group consisting of: yeast, fungi, algae, bacteria, virus or an arthropod expressing the dsRNA or siRNA of the invention. In some embodiments, the transgenic organism is a virus, for example an insect baculovirus that expresses a dsRNA or siRNA of the invention upon infection of an insect host. Such a baculovirus is likely more virulent against the target insect than the wildtype untransformed baculovirus. In other embodiments the transgenic organism is a transgenic bacterium that is applied to an environment where a target pest occurs or is known to have occurred. In some embodiments, non-pathogenic symbiotic bacteria, which are able to live and replicate within plant tissues, so-called endophytes, or non-pathogenic symbiotic bacteria, which are capable of colonizing the phyllosphere or the rhizosphere, so-called epiphytes, are used. Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium are also possible hosts for expression of the inventive dsRNA or siRNA for the same purpose.
In some embodiments, an acceptable agricultural carrier is a formulation useful for applying the composition comprising the dsRNA or siRNA to a plant or seed. In some embodiments, the dsRNA or siRNA molecules are stabilized against degradation because of their double stranded nature and the introduction of Dnase/Rnase inhibitors. For example, the dsRNA or siRNA can be stabilized by including thymidine or uridine nucleotide 3′ overhangs. The dsRNA or siRNA contained in the compositions of the invention can be chemically synthesized at industrial scale in large amounts. Methods available would be through chemical synthesis or through the use of a biological agent.
In other embodiments the formulation comprises a transfection promoting agent. In other embodiments, the transfection promoting agent is a lipid-containing compound. In further embodiments, the lipid-containing compound is selected from the group consisting of; Lipofectamine, Cellfectin, DMRIE-C, DOTAP and Lipofectin. In another embodiment, the lipid-containing compound is a Tris cationic lipid.
In some embodiments, the formulation further comprises a nucleic acid condensing agent. The nucleic acid condensing agent can be any such compound known in the art. Examples of nucleic acid condensing agents include, but are not limited to, spermidine (N-[3-aminopropyl]-1,4-butanediamine), protamine sulphate, poly-lysine as well as other positively charged peptides. In some embodiments, the nucleic acid condensing agent is spermidine or protamine sulfate.
In still further embodiments, the formulation further comprises buffered sucrose or phosphate buffered saline.
In some embodiments, the invention encompasses transgenic plants, or parts thereof, comprising a dsRNA molecule, a nucleic acid construct, a chimeric nucleic acid molecule, a artificial plant microRNA precursor molecule and/or a composition of the invention, wherein the transgenic plant has enhanced resistance to a Diabrotica insect as compared to a control plant. In other embodiments, the transgenic plant, or part thereof, is a transgenic corn plant, or part thereof. The invention further encompasses transgenic seed of the transgenic plants of the inventions, wherein the transgenic seed comprises a dsRNA molecule, a nucleic acid construct, a chimeric nucleic acid molecule, a artificial plant microRNA precursor molecule and/or a composition of the invention. In some embodiments the transgenic seed is a transgenic corn seed.
Transgenic plants expressing dsRNA or siRNA of the invention are tolerant or resistant to attack by target insect pests. When the insect starts feeding on such a transgenic plant, it also ingests the expressed dsRNA or siRNA. This will deter the insect from further biting into the plant tissue or may even harm or kill the insect. A nucleic acid sequence encoding a dsRNA or siRNA of the invention is inserted into an expression cassette, which is then preferably stably integrated in the genome of the plant. Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, corn, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees. In further embodiments, the transgenic plant is a transgenic corn plant.
Expression of the dsRNA or siRNA in transgenic plants is driven by promoters that function in plants. The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the insect target species. Thus, expression of the interfering RNAs of this invention in leaves, in stalks or stems, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings is contemplated. In many cases, however, protection against more than one type of insect pest is sought, and thus expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the dsRNA or siRNA in the desired cell.
Promoters useful with the invention include, but are not limited to, those that drive expression of a nucleotide sequence constitutively, those that drive expression when induced, and those that drive expression in a tissue- or developmentally-specific manner. These various types of promoters are known in the art.
Examples of constitutive promoters include, but are not limited to, cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as U.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), figwort mosaic virus (fmv) promoter (Govindarajulu et al. 2008. Mol Plant Microbe Interact 21:1027-35) and the ubiquitin promoter (Ubi). The constitutive promoter derived from ubiquitin accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632), and arabidopsis (Norris et al. 1993. Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926.
In some embodiments, tissue specific/tissue preferred promoters can be used. Tissue specific or preferred expression patterns include, but are not limited to, green tissue specific or preferred, root specific or preferred, stem specific or preferred, and flower specific or preferred. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. In one embodiment, a promoter useful with the invention is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Non-limiting examples of tissue-specific promoters include those associated with genes encoding the seed storage proteins (such as β-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378). Tissue-specific or tissue-preferential promoters useful for the expression of the nucleotide sequences of the invention in plants, particularly maize, include but are not limited to those that direct expression in roots or particular cells in roots, pith, leaf or pollen. Such promoters are disclosed, for example without limitation, in WO 93/07278, herein incorporated by reference in its entirety. Other non-limiting examples of tissue specific or tissue preferred promoters useful with the invention the cotton rubisco promoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Pat. No. 5,604,121; the root specific promoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452 269 to Ciba-Geigy); the stem specific promoter described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene; and the cestrum yellow leaf curling virus promoter disclosed in WO 01/73087, all incorporated herein by reference.
Additional examples of tissue-specific/tissue preferred promoters include, but are not limited to, the root hair-specific cis-elements (RHEs) (Kim et al. The Plant Cell 18:2958-2970 (2006)), the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Pat. No. 5,459,252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), corn light harvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), corn heat shock protein promoter (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J. 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, “Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), bean glycine rich protein 1 promoter (Keller et al. (1989) Genes Dev. 3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol. 12:579-589), R gene complex-associated promoters (Chandler et al. (1989) Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al. (1991) EMBO J. 10:2605-2612). In some particular embodiments, the nucleotide sequences of the invention are operably associated with a root-preferred promoter. Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as the seed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) Science 270:1986-1988).
In addition, promoters functional in plastids can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5′ UTR and other promoters disclosed in U.S. Pat. No. 7,579,516. Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).
In some embodiments of the invention, inducible promoters can be used. Thus, for example, chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences of the invention via promoters that are chemically regulated enables the polypeptides of the invention to be synthesized only when the crop plants are treated with the inducing chemicals. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
Chemical inducible promoters are known in the art and include, but are not limited to, the maize Int-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid (e.g., the PR1a system), steroid steroid-responsive promoters (see, e.g., the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156, Lac repressor system promoters, copper-inducible system promoters, salicylate-inducible system promoters (e.g., the PR1a system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. 11:605-612), and ecdysone-inducible system promoters.
Other non-limiting examples of inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoid glycosyl-transferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol. 29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; and Quigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are the benzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) and alcohol-inducible (Intl Patent Application Publication Nos. WO 97/06269 and WO 97/06268) systems and glutathione S-transferase promoters. Likewise, one can use any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically inducible promoters useful for directing the expression of the nucleotide sequences of this invention in plants are disclosed in U.S. Pat. No. 5,614,395 herein incorporated by reference in its entirety. Chemical induction of gene expression is also detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. In some embodiments, a promoter for chemical induction can be the tobacco PR-1a promoter.
In further aspects, the nucleotide sequences of the invention can be operably associated with a promoter that is wound inducible or inducible by pest or pathogen infection (e.g., a nematode plant pest). Numerous promoters have been described which are expressed at wound sites and/or at the sites of pest attack (e.g., insect/nematode feeding) or phytopathogen infection. Ideally, such a promoter should be active only locally at or adjacent to the sites of attack, and in this way expression of the nucleotide sequences of the invention will be focused in the cells that are being invaded. Such promoters include, but are not limited to, those described by Stanford et al., Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), Warner et al. Plant J. 3:191-201 (1993), U.S. Pat. No. 5,750,386, U.S. Pat. No. 5,955,646, U.S. Pat. No. 6,262,344, U.S. Pat. No. 6,395,963, U.S. Pat. No. 6,703,541, U.S. Pat. No. 7,078,589, U.S. Pat. No. 7,196,247, U.S. Pat. No. 7,223,901, and U.S. Patent Application Publication 2010043102.
In some embodiments of the present invention, a “minimal promoter” or “basal promoter” is used. A minimal promoter is capable of recruiting and binding RNA polymerase II complex and its accessory proteins to permit transcriptional initiation and elongation. In some embodiments, a minimal promoter is constructed to comprise only the nucleotides/nucleotide sequences from a selected promoter that are required for binding of the transcription factors and transcription of a nucleotide sequence of interest that is operably associated with the minimal promoter including but not limited to TATA box sequences. In other embodiments, the minimal promoter lacks cis sequences that recruit and bind transcription factors that modulate (e.g., enhance, repress, confer tissue specificity, confer inducibility or repressibility) transcription. A minimal promoter is generally placed upstream (i.e., 5′) of a nucleotide sequence to be expressed. Thus, nucleotides/nucleotide sequences from any promoter useable with the present invention can be selected for use as a minimal promoter.
In some embodiments, a recombinant nucleic acid molecule of the invention can be an “expression cassette.” As used herein, “expression cassette” means a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the nucleotide sequences of the invention), wherein the nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter). Thus, some embodiments of the invention provide expression cassettes designed to express nucleotides sequences encoding the dsRNAs or siRNAs of the invention. In this manner, for example, one or more plant promoters operably associated with one or more nucleotide sequences of the invention are provided in expression cassettes for expression in a corn plant, plant part and/or plant cell.
An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in plants. A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the plant host, or any combination thereof). Appropriate transcriptional terminators include, but are not limited to, the CAMV 35S terminator, the tml terminator, the nopaline synthase terminator and/or the pea rbcs E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a coding sequence's native transcription terminator can be used.
An expression cassette of the invention also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part and/or plant cell. As used herein, “selectable marker” means a nucleotide sequence that when expressed imparts a distinct phenotype to the plant, plant part and/or plant cell expressing the marker and thus allows such transformed plants, plant parts and/or plant cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., the R-locus trait). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.
Examples of selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptII, which confers resistance to kanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); a nucleotide sequence encoding bar, which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); a nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyl tryptophan; and/or a nucleotide sequence encoding hph that confers resistance to hygromycin. One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette of the invention.
Additional selectable markers include, but are not limited to, a nucleotide sequence encoding β-glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac,” pp. 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes a catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding aequorin, which may be employed in calcium-sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette of the invention.
An expression cassette of the invention also can include polynucleotides that encode other desired traits. Such desired traits can be other polynucleotides which confer insect resistance, or which confer nematode resistance, or other agriculturally desirable traits. Such polynucleotides can be stacked with any combination of nucleotide sequences to create plants, plant parts or plant cells having the desired phenotype. Stacked combinations can be created by any method including, but not limited to, cross breeding plants by any conventional methodology, or by genetic transformation. If stacked by genetically transforming the plants, nucleotide sequences encoding additional desired traits 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 additional nucleotide sequences can be introduced simultaneously in a co-transformation protocol with a nucleotide sequence, nucleic acid molecule, nucleic acid construct, and/or other composition of the invention, provided by any combination of expression cassettes. For example, if two nucleotide sequences will be introduced, they can be incorporated in separate cassettes (trans) or can be incorporated on the same cassette (cis). Expression of the nucleotide sequences can be driven by the same promoter or by different promoters. It is further recognized that nucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, e.g., Intl Patent Application Publication Nos. WO 99/25821; WO 99/25854; WO 99/25840; WO 99/25855 and WO 99/25853.
Thus, an expression cassette can include a coding sequence for one or more polypeptides for agronomic traits that primarily are of benefit to a seed company, grower or grain processor. A polypeptide of interest can be any polypeptide encoded by a polynucleotide sequence of interest. Non-limiting examples of polypeptides of interest that are suitable for production in plants include those resulting in agronomically important traits such as herbicide resistance (also sometimes referred to as “herbicide tolerance”), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, and/or fungal resistance. See, e.g., U.S. Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431.
Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4:1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) undertaken with a selectable marker that may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta). Plant transformation vectors comprising expression cassettes that encode a short hairpin RNA of the invention may also comprise genes, for example, phosphomannose isomerase (pmi), which provides for positive selection of the transgenic plants as disclosed in U.S. Pat. Nos. 5,767,378 and 5,994,629, herein incorporated by reference, or phosphinotricin acetyltransferase (pat), which provides tolerance to the herbicide phosphinotricin (glufosinate). The choice of selectable marker is not, however, critical to the invention. In one embodiment, the vector suitable for plant transformation comprises, consists essentially of or consists of a nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:67.
In other embodiments, a nucleic acid sequence of the invention is directly transformed into the plastid genome. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. In a preferred embodiment, a nucleic acid sequence of the present invention is inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleic acid sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleic acid sequence.
Transgenic plants or seed comprising a dsRNA or siRNA of the invention can also be treated with an insecticide or insecticidal seed coating as described in U.S. Pat. Nos. 5,849,320 and 5,876,739, herein incorporated by reference. Where both the insecticide or insecticidal seed coating and the transgenic plant or seed of the invention are active against the same target insect, for example a Diabrotica target pest, the combination is useful (i) in a method for further enhancing activity of the composition of the invention against the target insect, and (ii) in a method for preventing development of resistance to the composition of the invention by providing yet another mechanism of action against the target insect. Thus, the invention provides a method of enhancing control of a Diabrotica insect population comprising providing a transgenic plant or seed of the invention and applying to the plant or the seed an insecticide or insecticidal seed coating to a transgenic plant or seed of the invention. Examples of such insecticides and/or insecticidal seed coatings include, without limitation, a carbamate, a pyrethroid, an organophosphate, a friprole, a neonicotinoid, an organochloride, a nereistoxin, or a combination thereof. In another embodiment, the insecticide or insecticidal seed coating are selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chlorethoxyfos, dimethoate, ethoprophos, malathion, methyl-parathion, phorate, terbufos, tebupirimiphos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, bensultap, and a combination thereof. Commercial products containing such insecticides and insecticidal seed coatings include, without limitation, Furadan®, Lanate®, Sevin®, Talstar®, Force®, Ammo®, Cymbush®, Delta Gold®, Karate®, Ambush®, Pounce®, Brigade®, Capture®, ProShield®, Warrior®, Dursban®, Fortress®, Mocap®, Thimet®, AAstar®, Rampart®, Counter®, Cygon®, Dicap®, Regent®, Cruiser®, Gaucho®, Prescribe®, Poncho® and Aztec®.
The compositions of the invention can also be combined with other biological control agents to enhance control of Diabrotica insect populations. Thus, the invention provides a method of enhancing control of a Diabrotica insect population by providing a transgenic plant that produces a dsRNA of the invention and further comprises a polynucleotide that encodes a pesticidal agent selected from the group consisting of a patatin, a protease, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein or protein complex, a Photorhabdus insecticidal protein or protein complex, a Bacillus laterosporus insecticidal protein or protein complex, and a Bacillus sphaericus insecticidal protein. In some embodiments, the Bacillus thuringiensis insecticidal protein is selected from the group consisting of a Cry1 protein, a Cry3 protein, a Cry 7 protein, a Cry8 protein, a Cry 23 protein, a Cry 36 protein, a Cry37 protein, a Cry34 protein together with a Cry35 protein, a modified Cry3A protein, and hybrid proteins made therefrom. In other embodiments, the Bacillus thuringiensis insecticidal protein is selected from the group consisting of Cry3Bb1, Cry34Ab1 together with Cry35Ab1, mCry3A and eCry3.1Ab. In another embodiment, the transgenic plant and transgenic seed is a corn plant or corn seed. In another embodiment, the transgenic corn plant is provided by crossing a first transgenic corn plant comprising a dsRNA of the invention with a transgenic corn plant comprising a transgenic event selected from the group consisting of MIR604, Event 5307, DAS51922-7, MON863 and MON88017.
Even where the insecticide or insecticidal seed coating is active against a different insect, the insecticide or insecticidal seed coating is useful to expand the range of insect control, for example by adding an insecticide or insecticidal seed coating that has activity against lepidopteran insects to the transgenic plant or seed of the invention, which has activity against coleopteran insects, the treated plant or coated transgenic seed controls both lepidopteran and coleopteran insect pests.
In further embodiments, the invention encompasses a biological sample from a transgenic plant, seed, or parts thereof, of the invention, wherein the sample comprises a nucleic acid that is or encodes at least one strand of a dsRNA of the invention. In other embodiments, the invention encompasses a commodity product derived from a transgenic plant, seed, or parts thereof, of the invention. In some embodiments, the commodity product is selected from the group consisting of whole or processed seeds, beans, grains, kernels, hulls, meals, grits, flours, sugars, sugars, starches, protein concentrates, protein isolates, waxes, oils, extracts, juices, concentrates, liquids, syrups, feed, silage, fiber, paper or other food or product produced from plants. In other embodiments, the biological sample or commodity product is toxic to insects. In other embodiments, the transgenic plant is transgenic corn plant.
The invention further encompasses a method of controlling a Diabrotica insect comprising contacting the Diabrotica insect with a nucleic acid molecule that is or is capable of producing an interfering RNA of the invention for inhibiting expression of an IAP gene in the Diabrotica insect thereby controlling the Diabrotica insect. In some embodiments, the IAP gene comprises a IAP coding sequence having from at least about 90% identity to at least about 99% identity to SEQ ID NO:2. In some embodiments the IAP coding sequence is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:14 and SEQ ID NO:17. In other embodiments, the interfering RNA is complementary to a portion of a mRNA polynucleotide transcribable from the IAP gene. In other embodiments, the portion of the mRNA polynucleotide comprises from 18, 19, 20 or 21 consecutive nucleotides to at least about 400 consecutive nucleotides of SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18. In some embodiments, the portion of the mRNA polynucleotide consists essentially of (a) any 19-mer subsequence of SEQ ID NO:3 (wcrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to 1603 of SEQ ID NO:3; or (b) any 19-mer subsequence of SEQ ID NO:12 (wcrIAP2) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1444 of SEQ ID NO:12; or (c) any 19-mer subsequence of SEQ ID NO:15 (scrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1018 of SEQ ID NO:15; or (d) any 19-mer subsequence of SEQ ID NO:18 (ncrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1020 of SEQ ID NO:18. In some embodiments, the portion of the mRNA polynucleotide consists essentially of the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18.
In some embodiments of the method of controlling a Diabrotica pest, the nucleotide sequence of the interfering RNA consists essentially of (a) any 19-mer subsequence of SEQ ID NO:4 (wcrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to 1603 of SEQ ID NO:4; or (b) any 19-mer subsequence of SEQ ID NO:13 (wcrIAP2*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1444 of SEQ ID NO:13; or (c) any 19-mer subsequence of SEQ ID NO:16 (scrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1018 of SEQ ID NO:16; or (d) any 19-mer subsequence of SEQ ID NO:19 (ncrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1020 of SEQ ID NO:19. In yet further embodiments, the nucleotide sequence of the interfering RNA consists essentially of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, or SEQ ID NO:19.
In some embodiments of the method of controlling Diabrotica, the Diabrotica insect is selected from the group consisting of D. barberi (northern corn rootworm), D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle) and D. virgifera zeae (Mexican corn rootworm).
In other embodiments of the method of controlling the Diabrotica insect, the contacting comprises (a) planting a transgenic seed capable of producing a transgenic plant that expresses the nucleic acid molecule, wherein the Diabrotica insect feeds on the transgenic plant, or part thereof; or (b) applying a composition comprising the nucleic acid molecule to a seed or plant, or part thereof, wherein the Diabrotica insect feeds on the seed, the plant, or a part thereof. In some embodiments, the transgenic seed and the transgenic plant is a corn seed or a corn plant. In other embodiments the seed or plant is a corn seed or a corn plant.
The invention also encompasses a method of reducing an adult Diabrotica insect population on a transgenic plant expressing a Cry protein, a hybrid Cry protein or modified Cry protein comprising expressing in the transgenic plant a nucleic acid molecule that is or is capable of producing an interfering RNA capable of inhibiting expression of an IAP gene in an adult Diabrotica insect thereby reducing the adult Diabrotica insect population.
In some embodiments, the invention encompasses a method of reducing the level of a target mRNA transcribable from a IAP gene in a Diabrotica insect comprising feeding to the Diabrotica insect a composition comprising the dsRNA molecule of the invention, wherein the dsRNA molecule reduces the level of the target mRNA in a cell of the Diabrotica insect. In other embodiments, production of a IAP protein encoded by the target mRNA is reduced. In other embodiments, the IAP protein is an IAP1 or IAP2 protein. In other embodiments, the IAP protein comprises an amino acid having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO:20. In other embodiments the IAP protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In other embodiments, the dsRNA is contacted with a Diabrotica insect through a transgenic organism expressing the dsRNA. In other embodiments, the transgenic organism is a transgenic plant, a transgenic bacterium or a transgenic endophyte. In other embodiments, the dsRNA is contacted with a Diabrotica insect by topically applying a dsRNA in an acceptable agricultural carrier to a plant or plant part on which the Diabrotica insects feeds. In some embodiments, the dsRNA that reduces the level of a target mRNA transcribable from a IAP gene in a Diabrotica insect is lethal to the Diabrotica insect. In other embodiments, the Diabrotica insect is selected from the group consisting of D. barberi (northern corn rootworm), D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle) and D. virgifera zeae (Mexican corn rootworm).
In some embodiments, the invention encompasses a method of conferring Diabrotica insect tolerance to a plant, or part thereof, comprising introducing into the plant, or part thereof, a dsRNA molecule, a nucleic acid construct, a chimeric nucleic acid molecule, a artificial plant microRNA precursor molecule and/or a composition of the invention, thereby conferring tolerance of the plant or part thereof to the Diabrotica insect. In one embodiment the nucleic acid construct or the chimeric nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:63 or SEQ ID NO:64. In another embodiment, the chimeric nucleic acid molecule is a vector comprising a nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:67.
In other embodiments, the invention encompasses a method of reducing root damage to a plant fed upon by a Diabrotica insect, comprising introducing into cells of the plant a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, a artificial plant microRNA precursor molecule and/or a composition of any of the respective preceding claims, thereby reducing root damage to the plant. In one embodiment the nucleic acid construct or the chimeric nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:63 or SEQ ID NO:64. In another embodiment, the chimeric nucleic acid molecule is a vector comprising a nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:67.
In still other embodiments, the invention encompasses a method of producing a transgenic plant cell having toxicity to a Diabrotica insect, comprising introducing into a plant cell a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, a artificial plant microRNA precursor molecule and/or a composition of the invention, thereby producing the transgenic plant cell having toxicity to the Diabrotica insect compared to a control plant cell. In some embodiments, the invention encompasses a plurality of transgenic plant cells produced by this method. In other embodiments, the plurality of transgenic plant cells is grown under conditions which include natural sunlight. In one embodiment the nucleic acid construct or the chimeric nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:63 or SEQ ID NO:64. In another embodiment, the chimeric nucleic acid molecule is a vector comprising a nucleotide sequence of SEQ ID NO:65 or SEQ ID NO:67.
In some embodiments, the invention encompasses a method of producing a transgenic plant having enhanced tolerance to Diabrotica insect feeding damage, comprising introducing into a plant a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, an artificial plant microRNA precursor molecule and/or a composition of the invention, thereby producing a transgenic plant having enhanced tolerance to Diabrotica insect feeding damage compared to a control plant. In other embodiments, the introducing step is performed by transforming a plant cell and producing the transgenic plant from the transformed plant cell. In still other embodiments, the introducing step is performed by breeding two plants together.
In some embodiments, the invention encompasses a method of providing a corn grower with a means of controlling a Diabrotica insect pest population in a corn crop comprising (a) selling or providing to the grower at least one bag of corn seed comprising transgenic corn seed that comprises a dsRNA, a nucleic acid molecule, a nucleic acid construct, a chimeric nucleic acid molecule, an artificial plant microRNA precursor molecule and/or a composition of the invention; and (b) advertising to the grower that the transgenic corn seed produces transgenic corn plants that control a Diabrotica pest population.
EXAMPLES
The invention will be further described by reference to the following detailed examples. These examples are provided for the purposes of illustration only, and are not intended to be limiting unless otherwise specified.
Example 1
Identification of IAP Genes in Diabrotica 
This example describes the cloning and sequencing of inhibitor of apoptosis (iap) genes and coding sequences from Diabrotica insects.
Diabrotica virgifera virgifera (Western Corn Rootworm; WCR)
A whole-body neonate WCR transcriptome was sequenced by pyrosequencing on a 454 platform (454 Life Sciences, Branford, Conn.) essentially according to the manufacturer's instructions. The resulting reads (i.e., short fragments of nucleic acid sequence) were trimmed and assembled using a MIRA assembler (See, for example, Chevreux et al. 2004. Genome Res. 14:1147-1159, incorporated herein by reference). The resulting contigs were used to make a BLAST database (Attschul et al. 1990. J. Mol. Biol. 215:403-410) using formatdb (NCBI, Bethesda, Md.).
To identify assembled contigs with similarity to a known insect iap gene, a Drosophila melanogaster IAP1 (dmIAP1) amino acid sequence (Genbank Accession No. NP730097; SEQ ID NO:24) was used as a query against the WCR transcriptome BLAST database using tblastn (protein query vs. translated nucleotide database). An expect value cutoff of 1e-05 was used. Fifteen WCR sequences were identified and used in an assembly using Contig Express (Vector NTI version 11, Invitrogen, Carlsbad, Calif.) with default parameters. Three contiguous sequences were assembled and the resulting sequences were compared to the Genbank NR database by blastx. A first contig (contg1) was assembled using five 454 contigs sequences and was found to be similar to dmIAP1. The assembly of contig 1 was edited manually to remove low quality base calls and to resolve discrepancies between 454 contigs. The final sequence (wcrIAP1; SEQ ID NO:1) was confirmed by sequencing with the primers shown in Table 1 using standard Sanger sequencing methods. The wcrIAP1 gene is 1622 nucleotides in length and comprises a 145 bp 5′ UTR, 1050 bp coding region (position 146 to 1195), and a 427 bp 3′ UTR. The encoded WCR-IAP1 amino acid sequence is shown in SEQ ID NO:20.







TABLE 1







Primers used to sequence wcrIAP1











Sequence 


Primer Name
Sequence (5′→3′)
Identifier





WCR_IAP_FP01
AGAGCATTAAACAGAGGCCTTC
SEQ ID NO: 57


 


WCR_IAP_RP01
TGATAGGAACGCTCTGACTGTG
SEQ ID NO: 58


 


WCR_IAP_FP02
ATGGCAGTAGTTCAATCAAATT
SEQ ID NO: 59



ACATT



 


WCR_IAP_RP02
GCCAACCCTGGAATGTTGC
SEQ ID NO: 60


 


WCR_IAP_FP03
TTGTGGTGGGGGATTAAAAG
SEQ ID NO: 61


 


WCR_IAP_RP03
CATTTGCTAAACCAAAGGGC
SEQ ID NO: 62









A second contig (contig2) was assembled using three 454 contigs and was also manually edited as with contig1. The final assembled sequence is 1462 bp in length and designated wcrIAP2 (SEQ ID NO:11), which contains an incomplete coding sequence at the 5′ end. The last 31 bp of wcrIAP2 belongs to the 3′ untranslated region (UTR). The encoded WCR-IAP2 amino acid sequence is shown in SEQ ID NO:21.
Diabrotica undecimpunctata howardi (Southern Corn Rootworm; SCR)
Commercially-available SCR eggs were purchased (Crop Characteristics, Inc, Farmington, Minn.) and incubated at approximately 30° C. and ambient relative humidity. Newly emerged neonate SCR were collected (approximately 100-200) and total RNA was extracted with a PicoPureTM RNA Isolation Kit (Life Technologies, Carlsbad, Calif.) essentially according to the manufacturer's instructions. RNA concentration was measured by spectrophotometry and purity was assessed by absorbance ratios A260/280 and A260/230.
SCR total RNA was reverse transcribed to cDNA using an anchored oligo (dT)17 primer (Sigma Aldrich, St. Louis, Mo.) and Superscript III reverse transcriptase (Cat. No. 18080-051; Invitrogen, Carlsbad, Calif.) essentially according to the manufacturers' instructions. The reaction was incubated at about 50° C. for 1 h, followed by about 70° C. for 15 min and then diluted with 80 μl ddH2O and stored at −20° C.
Primers to the wcrIAP1 gene were used to amplify an IAP gene from SCR. The reaction conditions for all amplifications were as follows: 95° C. for 5 min, followed by 35 cycles of 95° C. for 30 s, 50° C. for 30 s, 72° C. for 90 s, and then a final step of 72° C. for 2 min. The entire coding sequence of the SCR IAP gene was amplified with FP02: 5′-ATGGCAGTAGTTCAATCAAATTACATT-3′ (SEQ ID NO:59) and RP01: 5′-TGATAGGAACGCTCTGACTGTG-3′ (SEQ ID NO:58) and analyzed on a 1% agarose gel. The PCR product was purified using a MinElute PCR Purification Kit (Qiagen, Valencia, Calif.) essentially according to the manufacturer's instructions. The resulting PCR product was 1033 bp long. Using the 3 forward and 3 reverse primers shown in Table 5, the entire amplicon was sequenced using standard Sanger sequencing methods.
Individual sequences were assembled using ContigExpress (Vector NTI, Invitrogen) using default parameters. The resulting full-length scrIAP1 coding sequence is shown in SEQ ID NO:14. The encoded SCR-IAP1 amino acid sequence is shown in SEQ ID NO:22.
Diabrotica barberi (Northern Corn Rootworm; NCR)
NCR eggs were obtained from the insect rearing facility at the USDA ARS NCARL (Brookings, S. Dak.) and incubated at about 30° C. and ambient relative humidity. Newly emerged neonates were collected ( ̃20 total) and total RNA was extracted with a PicoPureTM RNA Isolation Kit (Life Technologies, Carlsbad, Calif.) essentially according to the manufacturer's instructions. RNA concentration was measured by spectrophotometry and purity was assessed by A260/280 and A260/230 ratios.
NCR total RNA was reverse transcribed to cDNA using an anchored oligo (dT)17 primer (Sigma-Aldrich, St. Louis, Mo.) and Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) according to the manufacturers' recommendations. The reaction was incubated at about 42° C. for 50 min, followed by 70° C. for 15 min. RNA was removed by treatment with 2 μl RNase H (Invitrogen) and incubate at about 37° C. for 20 min followed by 65° C. for 20 min, and then diluted with 80 μl ddH2O and stored at −20° C.
Primers to the wcrIAP1 gene described above (Table 1) were used to amplify a NCR IAP gene. The reaction conditions for all amplifications were as follows: 95° C. for 5 min, followed by 35 cycles of 95° C. for 30 s, 50° C. for 30 s, 72° C. for 90 s, and then a final step of 72° C. for 10 min. The entire coding sequence of the NCR IAP gene was amplified with primer FP02 (SEQ ID NO:64) and primer RP01 (SEQ ID NO:63) and analyzed on a 1% agarose gel. The PCR product was purified using a Qiagen (Valencia, Calif.) MinElute kit essentially according to the manufacturer's instructions. The PCR product was 1038 bp long. Using the 3 forward and 3 reverse primers shown in Table 1, the entire amplicon was sequenced using standard Sanger sequencing methods.
Individual sequences are assembled using ContigExpress (Vector NTI, Invitrogen) using default parameters. The assembly was manually edited to produce a final contiguous sequence, designated ncrIAP1 (SEQ ID NO:17) that comprises 1038 bp of the coding sequence. The encoded NCR-IAP1 amino acid sequence is shown in SEQ ID NO:23.
The wcrIAP1, scrIAP1 and ncrIAP1 coding sequences were aligned using Vector NTI AdvanceTM 11.1.0 (Invitrogen). The scrIAP1 (SEQ ID NO:14) and the ncrIAP1 (SEQ ID NO:17) coding sequences have 95% identity and 97% identity, respectively, with the wcrIAP1 coding sequence (SEQ ID NO:2). The scrIAP1 coding sequence has 94% identity with the ncrIAP1 coding sequence. An alignment of the three coding sequences is shown in FIG. 4 wherein the wcrIAP1 coding sequence (SEQ ID NO:2) was used as the reference sequence.
The WCR-IAP1, SCR-IAP1 and NCR-IAP1 amino acid sequences were aligned using Vector NTI AdvanceTM 11.1.0 (Invitrogen). The SCR-IAP1 (SEQ ID NO:22) and the NCR-IAP1 (SEQ ID NO:23) amino acid sequences have 95% identity and 97% identity, respectively, with the WCR-IAP1 amino acid sequence (SEQ ID NO:20). The SCR-IAP1 amino acid sequence has 94% identity with the NCR-IAP1 amino acid sequence. An alignment of the three amino sequences is shown in FIG. 5 wherein the WCR-IAP1 amino acid sequence (SEQ ID NO:20) was used as the reference sequence. The WCR-IAP1 and the WCR-IAP2 amino acid sequences have 28% identity.
Example 2
Detection of IAP1 in Different Life Stages
This example describes the detection of wcrIAP1 gene in different life stages of Diabrotica virgifera (western corn rootworm).
Total RNA was isolated from larvae and adults of western corn rootworm using standard RNA extraction methods. Reverse transcription of mRNA to cDNA was carried out as described above. IAP1-specific PCR primers from those listed in Table 1 above were used in PCR reactions with the larval and adult DNA to detect the presence of IAP1. The results shown in FIG. 2 demonstrate that adult western corn rootworm have the same or substantially the same IAP1 as the larval stages indicating that wcrIAP1 in the adult stage can be targeted with the same or similar dsRNA as that used to target IAP1 in the larval stages. As a consequence, expression of an IAP dsRNA in both above and below ground tissues of a corn plant may affect both larvae and adults and provide increased protection over technologies that target only larval stages.
Example 3
Construction of Interfering RNA Molecules
This example describes the construction of interfering RNA molecules designed to target mRNA transcribable from Diabrotica IAP1 genes.
Constructing wcrIAP1 dsRNA
Double stranded RNA (dsRNA) molecules were designed to hybridize to at least three different targets on the wcrIAP1 mRNA (SEQ ID NO:3). wcrIAP1-B (sense strand, SEQ ID NO:5)/wcrIAP-B* (antisense strand, SEQ ID NO:6), wcrIAP1-A (sense strand, SEQ ID NO:7)/wcrIAP1-A* (antisense strand, SEQ ID NO:8) and wcrIAP1-C (sense strand, SEQ ID NO:9)/wcrIAP-C* (antisense strand, SEQ ID NO:10) target base pairs 693-1192, 146-689 and 473-972 of SEQ ID NO:3, respectively. Synthesis of dsRNA was performed using a AmpliScribeTM T7-FlashTM Transcription Kit (Epicentre, Madison, Wis.) essentially according to the manufacturer's instructions. Briefly, 1 μg of template DNA with opposing T7 promoters was used in each 20 μl reaction. Each sample was treated with DNase and precipitated with an equal volume of 5 M ammonium acetate and then washed with 500 μl ice-cold 70% ethanol. The dsRNA was checked for integrity on a 1% agarose gel.
Constructing scrIAP1 dsRNA
Double stranded RNA (dsRNA) molecules were designed to hybridize to the entire southern corn rootworm IAP1 mRNA (SEQ ID NO:15). Synthesis of dsRNA was performed using a AmpliScribeTM T7-FlashTM Transcription Kit (Epicentre, Madison, Wis.) essentially according to the manufacturer's instructions. Briefly, 1 μg of template DNA with opposing T7 promoters was used in each 20 μl reaction. Each sample was treated with DNase and precipitated with an equal volume of 5 M ammonium acetate and then washed with 500 μl ice-cold 70% ethanol. The dsRNA was checked for integrity on a 1% agarose gel. Sequence of the sense strand and antisense strand is shown in SEQ ID NOs: 15 and 16, respectively.
Example 4
Bioassay of Interfering RNA Molecules
This example describes testing dsRNAs of the invention for biological activity against Diabrotica insects.
The dsRNA molecules described above were tested for toxicity against several Diabrotica species in laboratory bioassays. Bioassays were performed using an RNA-treated artificial diet method. Briefly, molten artificial diet, modified from the diet of Marrone et al. 1985. J. Econ. Entomol. 78:290-293, was poured into each well of 24-well plates and allowed to solidify. dsRNA molecules were diluted to appropriate concentration so that 60 μl of solution was added to the surface of the diet in each well, with a final overlay concentration of 100 ng dsRNA/cm2. One Diabrotica larva was added to each well and each 24-well plate was maintained at approximately 28° C. and 16:8 light:dark photoperiod. Mortality was recorded at 7 d post-infestation. dsRNA designed to target green florescent protein (GFP) was used in all bioassays as a negative control. The bioassay was repeated three times.
Double stranded RNAs designed to target different portions of Diabrotica virgifera (western corn rootworm) IAP1 mRNA were tested against western corn rootworm larvae. The results, shown in Table 2, demonstrate that a dsRNA molecule designed to target a portion of a mRNA transcribable from a Diabrotica insect IAP gene is highly toxic to Diabrotica virgifera (western corn rootworm). Both wcrIAP1-A/wcrIAP1-A* (SEQ ID NO:7/SEQ ID NO:8) and wcrIAP1-8/wcrIAP1-8* (SEQ ID NO:5/SEQ ID NO:6) produced a mean of about 90% mortality after 7d.







TABLE 2







Activity of dsRNA against Diabrotica virgifera (western corn rootworm).










Sample Size
% WCR Mortality














dsRNA Treatment
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Mean

















wcrIAP1-A/
30
48
48
77
96
94
89


wcrIAP1-A*


wcrIAP1-B/
30
48
24
87
92
88
89


wcrIAP1-B*


GFP
30
48
24
3
22
20
15









The wcrIAP1-A/wcrIAP1-A* dsRNA was also tested against Diabrotica undecimpunctata (southern corn rootworm) and produced 100% mortality in this species compared to 12% mortality in a GFP control. The results of this bioassay demonstrates that a dsRNA designed to target one Diabrotica's IAP1 mRNA is toxic to a different Diabrotica species.
Example 5
Interaction Between Interfering RNA Molecules and Insecticidal Proteins
This example describes the synergistic interaction, with regard to insecticidal activity, between a dsRNA that targets mRNA transcribable from Diabrotica IAP genes and insecticidal proteins with activity against Diabrotica insects.
The effects of the insecticidal proteins mCry3A or eCry3.1Ab on the sensitive pest species Diabrotica virgifera (western corn rootworm; WCR) are investigated in the presence and absence of wcrIAP1-A/wcrIAP1-A*, wcrIAP1-B/wcrIAP1-B*, wcrIAP1-C/wcrIAP1-C*, scrIAP1/scrIAP1* and/or ncrIAP1/ncrIAP1* dsRNA. First instar larvae are used to conduct the WCR diet incorporation bioassay. WCR percent mortality is assessed at approximately 4-7 days after infestation. Approximately two concentrations of mCry3A and eCry3.1Ab giving intermediate level of response are chosen from a dose-response curve to conduct the interaction bioassay. The results indicate a higher WCR percent mortality when wcrIAP1-A/wcrIAP1-A*, wcrIAP1-B/wcrIAP1-B*, wcrIAP1-C/wcrIAP1-C*, scrIAP1/scrIAP1* and/or ncrIAP1/ncrIAP1* dsRNA is present, indicating potential elevated efficacy between dsRNA of the invention and an insecticidal protein when used in combination based upon WCR bioassay. Similar results are observed when mCry3A or eCry3.1Ab is tested in the presence of wcrIAP1-A/wcrIAP1-A*, wcrIAP1-B/wcrIAP1-B*, wcrIAP1-C/wcrIAP1-C*, scrIAP1/scrIAP1* and/or ncrIAP1/ncrIAP1* dsRNA against Diabrotica barberi (northern corn rootworm).
Example 6
Activity of dsRNA Expressed in a Plant Cells
This example describes the expression of dsRNA molecules in corn cells.
Vector Construction.
Expression vectors for transforming plant cells generally comprised two expression cassettes, a first expression cassette comprising a Ubi1 promoter operably linked to a nucleotide sequence designed to produce a hairpin RNA (hpRNA) which was operably linked to a Ubi361 terminator; and a second expression cassette comprising a Ubi1 promoter operably linked to a pmi (phosphomannose isomerase) selected marker coding sequence, which was operably linked to a Ubi1 terminator. The nucleotide sequence in the first expression cassette designed to form a hairpin RNA comprised a nucleotide sequence coding for a sense RNA strand fused to an intron spacer, which functions to form a loop sequence, fused to a nucleotide sequence coding for an antisense RNA strand, an example of which is shown in FIG. 3, and having the general formula, sense strand-intron spacer-antisense strand.
Three such expression vectors were constructed to test in plants, 1) pRNA21537—in which the hpRNA encoding nucleotide sequence was designated hpwcrIAP1-C/wcrIAP1-C* SEQ ID NO:63), designed to target a corn rootworm IAP gene; 2) pRNA21534—in which the hpRNA encoding nucleotide sequence was designated hpwcrIAP1-Cα/wcrIAP1-Cα* (SEQ ID NO:64), designed to target a corn rootworm IAP gene; and 3) pRNA21536—in which the hpRNA encoding nucleotide sequence was designated hpNC/NC*, which functioned as a negative control having no known homology to corn rootworm or to corn plant DNA. The vectors also contained selectable markers for selection in bacteria.
To ensure that no unintended proteins were produced in transgenic plants transformed with a vector of the invention, all fortuitous open reading frames in the pRNA21534 vector were disrupted by making single nucleotide point mutations in the hpRNA encoding nucleotide sequence. Eight such point mutations were made. Therefore, the hpRNA encoding sequences in pRNA21537 and pRNA21534 are identical except for the eight point mutations in pRNA21534.
Agrobacterium Preparation.
The resulting vectors containing the hairpin RNA encoding expression cassette described above can first be tested in a corn cell culture system such as black Mexican sweet (BMS) corn cells or calli. The vectors are transformed into Agrobacterium tumefaciens using standard molecular biology techniques known to those skilled in the art. To prepare the Agrobacteria for transformation, cells are suspended in BMS culture media and the density is adjusted to an OD600 of  ̃0.45. Alternatively, the Agrobacteria are cultured in liquid YPC media at 28° C. and 220 rpm overnight. Approximately, four hours prior to BMS transformation, acetosyringone is added to a concentration of 100 μM and the cell density adjusted to an OD600 of  ̃0.45.
BMS Cell Preparation.
Approximately 3 to 4 d prior to plant cell transformation, the BMS cells are split into new cell culture media by adding 5 ml of BMS cells to 45 ml AW5 media in 125 ml flasks and culture at 25° C., shaking at 125 rpm and no light. Next approximately 1.4 ml of BMS cells culture is pelleted by centrifugation at 2300×g for 5 min at room temperature. The cells are washed once with AW5 media and the cells are pelleted again under the same conditions. The cells are heat shocked for 5 min at 45° C.
BMS Cell Transformation.
Approximately 1 ml of Agrobacterium suspension is added to heat shocked BMS cells. The tube is shaken at 80 rpm for 15 min, briefly centrifuged and the Agrobacterium media is removed. The BMS cells are spread onto M11/02K media plates with sterile filter paper or nylon netting and cultured at room temperature for 3 d without light. The cells are then transferred to media plates containing the antibiotic timentin and cultured at room temperature for 7 d without light to eliminate Agrobacterium. Cells are transferred to media plates containing mannose for selection and cultured at room temperature for 2 to 3 wk without light. The cells are finally transferred to fresh selectable media plates or liquid media and allowed to grow for another 2 wks.
Example 7
Insecticidal Activity of Corn Cells Expressing IAP dsRNA
This example describes testing transformed corn cells expressing IAP dsRNA for bioactivity against Diabrotica. 
Assays for insect toxicity are performed by feeding transformed black Mexican sweet calli described in Example 6 to western corn rootworm larvae (Diabrotica virgifera). Alternatively, an extract containing the interfering RNA is prepared from transformed maize cells and this extract is incorporated into an appropriate insect diet which is fed to Diabrotica larvae. Rootworm larvae feeding on transformed calli or extracts of such calli are negatively impacted from the effects of the dsRNA compared to larvae feeding on non-transformed calli or extracts from non-transformed calli.
Example 8
Insecticidal Activity of Transgenic Corn Plants Expressing dsRNA
The vectors described in Example 6 were transformed into maize plants. Agrobacterium transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. For this example, all media constituents were essentially as described in Negrotto et al., supra. However, various media constituents known in the art may be substituted.
Briefly, Agrobacterium strain LBA4404 (pSB1) containing a plant transformation plasmid was grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl (5 g/L), 15 g/I agar, pH 6.8) solid medium for 2-4 days at 28° C. Approximately 0.8×109 Agrobacterium were suspended in LS-inf media supplemented with 100 μM As (Negrotto et al., supra). Bacteria were pre-induced in this medium for 30-60 minutes.
Immature embryos from a suitable genotype were excised from 8-12 day old ears into liquid LS-inf+100 μM As. Embryos were rinsed once with fresh infection medium. Agrobacterium solution was then added and embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate were transferred to LSDc medium supplemented with cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l) and cultured in the dark for 28° C. for 10 days.
Immature embryos, producing embryogenic callus were transferred to LSD1M0.55 medium. The cultures were selected on this medium for about 6 weeks with a subculture step at about 3 weeks. Surviving calli was transferred to Reg1 medium supplemented with mannose. Following culturing in the light (16 hour light/8 hour dark regiment), green tissues were then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and grown in the light. After about 2-3 weeks, plants were tested for the presence of the pmi gene and hpRNA encoding sequences by qRT-PCR. Positive plants from the PCR assay were transferred to the greenhouse and subsequently tested for resistance to at least Diabrotica virgifera (western corn rootworm) using a root excision assay and/or a whole plant assay.
Root Excision Assay
Roots were excised from transgenic plants comprising the constructs described in Example 6. The excised root was placed on moistened sterile germination paper in small snap-cap petri dishes. Ten corn rootworm first-instar larvae were added to each dish. Data were collected at 48, 72 and 96 hours post-infestation. Roots were scored by counting the number of entry wounds caused by CRW and root scaring severity on a scale from 0-10, with 0 being “no scaring” and 10 being “severe scaring.” A scaring score of 0 to about 6 indicated that an hpRNA-IAP plant was positive compared to a negative control hpRNA plant.
Whole Plant Assay
Corn plants growing in 3′′ pots were infested with at least  ̃30-75 neonate corn rootworm larvae per plant. For each assay, 3 plants were used as uninfested controls, which typically were a heterozygous plant that performed well in the root excision bioassay, a homozygous plant not tested in the root excision bioassay and negative control plant. These plants acted as controls for growth conditions during the course of the assay. Data were collected 10-14 days after infestation. Evaluations were primarily subjective measures comparing infested test plants to those of uninfested and infested control plants. One key visual evaluation that was made was whether the plants showed signs of lodging, a condition indicative of severe damage caused by extensive corn rootworm feeding on the root system.
Bioassay Results
Although bioassay results did not correlate 100% with the levels of hpRNA-encoding nucleic acids in the transgenic plants, results from the root-excision assay indicated that roots from plants transformed with both the pRNA21534 and pRNA21537 vectors were active against Diabrotica, corn rootworm. Positive roots from plants transformed with either vector caused CRW mortality and had the lowest number of entry wounds at about 1.3/root compared to negative plants which had as many as to 8.3 wounds/root. In general, many roots from pRNA21534 and pRNA21537 transformed plants had lower scaring severity compared to controls indicating that the dsRNA-treated larvae were negatively impacted by targeting the IAP gene.
Results of the whole plant assay indicated that the level of corn rootworm infestation was high enough to cause severe lodging in many of the negative control plants, whereas only one positive plant transformed with either of the pRNA21534 and pRNA21537 vectors was lodged during the same time period. When plants were removed from the soil at the end of the whole-plant bioassay period to inspect the roots for damage and the root systems compared, the root systems on plants transformed with pRNA21534 and pRNA21537 were generally far more extensive than the control root mass (See FIG. 5 for example), indicating that the expression of a dsRNA targeted at the corn rootworm IAP gene was sufficient to protect the plant from economically significant damage.
Although not all events transformed with the vectors encoding hpRNA targeted at the corn rootworm IAP gene provided good corn rootworm control, the results observed in many of the events clearly showed that expression of nucleic acids encoding such dsRNA that target a corn rootworm IAP gene in transgenic corn is a viable way to control corn rootworm and protect the plant from corn rootworm feeding damage.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof of the description will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art that this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

## Claims

1. A double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein at least 20 consecutive nucleotides of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a Diabrotica insect IAP gene wherein the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 3, and wherein the dsRNA molecule is toxic to a Diabrotica insect.

2. The dsRNA molecule of claim 1, wherein the IAP coding sequence comprises SEQ ID NO:2.

3. The dsRNA molecule of claim 1, wherein the portion of the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO:3.

4. The dsRNA molecule of claim 3, wherein the portion of the mRNA polynucleotide comprises from 20 or 21 consecutive nucleotides to at least 400 consecutive nucleotides of SEQ ID NO:3.

5. The dsRNA of claim 4, wherein the portion of the mRNA polynucleotide consists essentially of any 20-mer subsequence of SEQ ID NO:3 (wcrIAP1) consisting of N to N+19 nucleotides, wherein N is nucleotide 1 to 1602 of SEQ ID NO:3.

6. The dsRNA molecule of claim 4, wherein the portion of the mRNA polynucleotide consists essentially of the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

7. The dsRNA molecule of claim 1, wherein the nucleotide sequence of the antisense strand consists essentially of (a) any 20-mer subsequence of SEQ ID NO:4 (wcrIAP1*) consisting of N to N+19 nucleotides, wherein N is nucleotide 1 to 1602 of SEQ ID NO:4.

8. The dsRNA molecule of claim 6, wherein the nucleotide sequence of the antisense strand consists essentially of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.

9. The dsRNA molecule of claim 1, wherein the nucleotide sequence of the sense strand is substantially complementary to the nucleotide sequence of the antisense strand.

10. The dsRNA molecule of claim 1, wherein the nucleotide sequence of the sense strand is fully complementary to the nucleotide sequence of the antisense strand.

11. The dsRNA molecule of claim 1, wherein the double stranded RNA molecule is a short hairpin RNA (shRNA) molecule.

12. The dsRNA molecule of claim 1, wherein the Diabrotica insect is selected from the group consisting of D. barberi (northern corn rootworm), D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle) and D. virgifera zeae (Mexican corn rootworm).

13. A nucleic acid molecule encoding the dsRNA molecule of claim 1.

14. A recombinant vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes the dsRNA of claim 1.

15. A composition comprising two or more of the dsRNA molecules of claim 1 wherein the two or more RNA molecules each comprise a different antisense strand.

16. The composition of claim 15, comprising an RNA molecule comprising an antisense strand consisting essentially of the nucleotide sequence of SEQ ID NO:6 and an RNA molecule comprising an antisense strand consisting essentially of the nucleotide sequence of SEQ ID NO:8 and/or an RNA molecule comprising an antisense strand consisting essentially of the nucleotide sequence of SEQ ID NO:10.

17. A composition comprising two or more of the nucleic acid molecules of claim 13, wherein the two or more nucleic acid molecules each encode a different antisense strand.