Compositions and methods for restoring susceptibility to pyrethroid insecticides in resistant populations

Abstract

Disclosed are double stranded RNA molecules that are toxic to aphids including those that are resistant to pyrethroid insecticides. In particular, interfering RNA molecules capable of interfering with a voltage-gated sodium channel subunit h1 (vgsc-h1) target gene and that are toxic to aphids are provided. Further, methods of making and using the interfering RNA, for example in transgenic plants or as the active ingredient in insecticidal compositions, to confer protection from aphid damage are disclosed.

Description

TITLE: COMPOSITIONS AND METHODS FOR RESTORINGSUSCEPTIBILITY TO PYRETHROID INSECTICIDES IN RESISTANTPOPULATIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional applications U.S. Serial No. 63 / 610,693, filed December 15, 2023, and U.S. Serial No. 63 / 717,613, filed November 7, 2024, which are incorporated herein by reference in their entireties.GOVERNMENT SUPPORT

[0002] This invention was made with government support under award number 58-5030-1-056 awarded by the United States Department of Agriculture, and by funds Congressionally appropriated to the United States Department of Agriculture, Agricultural Research Service, Com Insects & Crop Genetics Research Unit, CRIS Projects 5030-22000-018-00D and 5030- 22000-019-00D. The government has certain rights in the invention.SEQUENCE LISTING XML

[0003] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on December 10, 2024, is named P14536WOOO.xml and is 25,056 bytes in size.TECHNICAL FIELD

[0004] The present disclosure relates generally to the control of insect pests that cause damage to crop plants by their feeding activities, and more particularly to the control of soybean aphid by insecticidal compositions comprising interfering RNA molecules. The disclosure further relates to the compositions and to methods of using such compositions comprising the interfering RNA molecules.BACKGROUND

[0005] Soybean aphid. Aphis glycines, is an insect pest of cultivated soybean, Glycine max, that traditionally has been controlled in the United States using tactics that include foliar applications of pyrethroid insecticides. The efficacy of pyrethroids has diminished due to the accumulation ofresistant aphids in populations in the Upper Midwest. This compromises the ability7of farmers to use inexpensive pyrethroid insecticides in these areas and thereby reduces profit margins.SUMMARY

[0006] The need outlined above is met by the present disclosure which, in various embodiments, provides methods of controlling economically important aphids including those that are resistant to pyrethroid insecticides. The disclosure in part comprises a method of inhibiting expression of a voltage-gated sodium channel subunit hl (ygsc-hl) target gene in aphids and other insect pests. The method comprises introduction of an interfering RNA molecule comprising a doublestranded RNA (dsRNA) or its modified forms such as short hairpin RNA (shRNA) or small interfering RNA (siRNA) sequences, into an insect pest, wherein the dsRNA, shRNA, or siRNA enters the cells and inhibits expression of the target gene, and wherein inhibition of the target gene exerts a deleterious effect upon the insect pest. It is contemplated that the methods and compositions of the disclosure will be useful in limiting or eliminating insect pest infestations in or on any plant by providing one or more compositions comprising interfering RNA molecules of the disclosure in the diet of the insect pest or through absorption through the cuticle of the insect pest. The disclosure 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 insect pest related damage or loss to crop plants. Such delivery may be through production of the interfering RNA in a transgenic plant, for example soybean, or by topically applying a composition comprising the interfering RNA to a plant or plant seed, such as a soybean plant or soybean seed. Delivery may further be through contacting the insect pest with the interfering RNA, such as when the insect pest feeds on plant material comprising the interfering RNA, either because the plant material is expressing the interfering RNA through a transgenic approach, or because the plant material is coated with a composition comprising the interfering RNA. The interfering RNA may also be provided in an artificial diet which the insect pest then contacts by feeding. The interfering RNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of an mRNA transcribable from the target gene or a portion of a nucleotide sequence of a mRNA transcribable from the target gene of the insect pest and therefore inhibits expression of the target gene by endogenous degradation of the transcribed mRNA from the target gene, which causes cessation of feeding, growth, development, reproduction and eventually results in death of the insect pest.

[0007] The disclosure further provides insecticidal compositions for inhibiting the expression of a vgsc-hl target gene that comprises an interfering RNA molecule of the disclosure and an agriculturally acceptable carrier. In certain embodiments, inhibition of the expression of a vgsc- hl gene described here leads to cessation of feeding and growth and ultimately results in the death of the insect pest. In certain embodiments, the vgsc-hl target gene encodes a VGSC-H1 polypeptide comprising an isoleucine at position 928, a leucine at position 928, a methionine at position 935, or a phenylalanine at position 1024, wherein the position numbering corresponds to the position in SEQ ID NO 3. By targeting such mutations that confer resistance to insecticides, the interfering RNA molecules of the disclosure not only kill the insect pest but also restore susceptibility to the insecticide in surviving resistant individuals. In certain embodiments, the interfering RNA molecules of the disclosure are provided in combination with an insecticide (e.g., a pyrethroid insecticide) to confer maximal insect control capabilities.

[0008] The disclosure is further drawn to transgenic plants which produce one or more interfering RNA molecules of the disclosure that are protected from insect pests and to methods of using the plants alone or in combination with other insect control strategies (e.g., pyrethroid insecticides) to confer maximal insect control capabilities. Plants or plant parts producing one or more interfering RNA molecules of the disclosure or treated with a composition comprising one or more interfering RNA molecules of the disclosure are resistant to insect pest infestation. For example, economically important insect pests can be controlled by a plant that produces an interfering RNA molecule of the disclosure or by a plant or plant seed that is treated with a composition comprising an interfering RNA molecule of the disclosure.

[0009] The disclosure provides a method of controlling an insect pest comprising inhibiting expression or activity of a VGSC-H1 polypeptide in the insect pest, wherein the VGSC-H1 polypeptide comprises an isoleucine at position 928, a leucine at position 928, a methionine at position 935, or a phenylalanine at position 1024, wherein the position numbering corresponds to the position in SEQ ID NO 3.

[0010] The disclosure also provides a method of controlling an insect pest comprising contacting the insect pest with an interfering RNA molecule of the disclosure for inhibiting expression of a vgsc-hl gene in the insect pest, thereby controlling the insect pest.

[0011] The disclosure provides a method of reducing the level of a target RNA transcribable from a vgsc-hl gene described herein in an insect pest comprising contacting the insect pest with a composition comprising an interfering RNA molecule of the disclosure, wherein the interfering RNA molecule reduces the level of the target RNA in a cell of the insect pest.

[0012] The disclosure provides a method of conferring insect pest tolerance to a plant or part thereof, comprising introducing into the plant, or part thereof, an interfering RNA molecule or a nucleic acid construct of disclosure, thereby conferring to the plant or part thereof tolerance to the insect pest.

[0013] The disclosure provides a method of reducing damage to a plant fed upon by an insect pest, comprising introducing into cells of the plant an interfering RNA molecule or a nucleic acid construct of the disclosure, thereby reducing damage to the plant fed upon by an insect pest.

[0014] The disclosure provides a method of producing a transgenic plant cell having toxicity to an insect pest, comprising introducing into a plant cell an interfering RNA molecule or a nucleic acid construct of the disclosure, thereby producing the transgenic plant cell having toxicity to the insect pest compared to a control plant cell.

[0015] The disclosure provides a method of producing a transgenic plant having enhanced tolerance to insect pest feeding damage, comprising introducing into a plant an interfering RNA molecule or a nucleic acid construct of the disclosure, thereby producing a transgenic plant having enhanced tolerance to insect pest feeding damage compared to a control plant.

[0016] The disclosure provides a method of enhancing control of an insect pest population comprising providing a transgenic plant or transgenic seed of the disclosure and applying to the transgenic plant or the transgenic seed an interfering RNA molecule of the disclosure that is insecticidal to an insect pest, thereby enhancing control of the insect pest population.

[0017] The disclosure provides a method of providing a soybean grower with a means of controlling an insect pest population below an economic threshold in a soybean crop comprising (a) selling or providing to the grower transgenic soybean seed comprising an interfering RNA molecule or a nucleic acid construct of the disclosure; and (b) advertising to the grower that the transgenic soybean seed produces transgenic soybean plants capable of controlling an insect pest population.

[0018] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent based on the detailed description, which shows and describes illustrative embodiments of the disclosure. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.BRIEF DESCRIPTION OF THE FIGURES

[0019] The following drawings form part of the specification and are included to further demonstrate certain embodiments. In some instances, embodiments can be best understood byreferring to the accompanying figures in combination with the detailed description presented herein. The description and accompanying figures may highlight a certain specific example, or a certain embodiment. However, one skilled in the art will understand that portions of the example or embodiment may be used in combination with other examples or embodiments.

[0020] FIG. 1A-B shows the location and impact of Aphis glycines voltage-gated sodium channel subunit hl (vgsc-hl) mutations. FIG. 1A shows the position of four predicted amino acid changes in the vgsc-hl protein of resistant / I. glycines. FIG. IB show s the change in resistance to pyrethroid insecticide associated with different vgsc-hl protein changes in laboratory colonies relative to the Boone-2019-ISO line. Heterozygotes and homozygote genotypes giving rise to amino acid change in each line are indicated as 1 and 2, respectively, for each pyrethroid resistant laboratory strain of A. glycines, Nashua-2018-ISO, MN1-2017- ISO, Kanawha-2019-ISO and Darwin-2- 19-ISO.

[0021] FIG. 2 shows the region of sequence in the Aphis glycines voltage-gated sodium channel subunit hl (ygsc-hl) encoding nucleotide substitutions within the double-stranded RNA (dsRNA) probe (SEQ ID NOs: 4-8). Nucleotide sequence of vgsc-hl susceptible allele from Boone-2019-ISO (vgsc-hl _S) and resistant alleles from field-derived A. glycines strains SBA- Darwin-2019-ISO (vgsc-hl _D), -Kanawha-2019-ISO (vgsc-hl JQ, -MN1-2017-ISO (vgsc- hl _M), and -Nashua-2018-ISO (vgsc-hl _N) lines corresponding to positions 2711 to 3195 of gene model AG6007485-PA. Substitution mutations are highlighted grey, and sequence overlay ed with the amino acid (aa) translation from position 905 to 1065 of protein model AG6007485-PA (SEQ ID NO: 9). Double underlined regions indicate locations of sequence corresponding to the forward primer, 5’-GTT CAT TTC GTT TGC TTC GAG T-3' (SEQ ID NO: 10), and reverse complementary to the reverse primer, 5’-GAA TCG GCC AAT ACG TTC AAA-3’ (SEQ ID NO: 11), each appended at their 5’ end with the T7 RNA polymerase promoter sequence, 5’-TAA TAC GAC TCA CTA TAG GGA GA-3’ (SEQ ID NO: 12). Altered triplet codons leading to amino acid changes are underlined: ATG -> ATA = M -> E ATG -> TTG = M -> L; TTG -> ATG = L -> M; CTT -> TTT = L -> F. Nucleic acids altered compared to the susceptible allele vgsc-hl _S are highlighted grey.

[0022] FIG. 3A-C shows relative quantitative PCR demonstrating reductions in vgsc-hl mRNA transcripts after applying the dsvgsc-hlmdsRNA probe.

[0023] FIG. 4 shows mortality at 24-hours post dsvgsc-hl^" dsRNA applications to A. glycines from MN1-2017-ISO, demonstrating synergistic effect of the dsRNA with the pyrethroid insecticide Warrior II.

[0024] FIG. 5A-B shows positions in the nucleic acid sequence of the Aphis glycines voltage gated sodium channel subunit (vgsc-hl) coding sequence (CDS) from gene model AG6007485-RA predicted from the A. glycines genome sequence assembly, Ag_Btl_v6.0, that was assembled from a pyrethroid susceptible strain of A. glycines. For all sequences, the start ATG and stop TAG codons highlighted grey, and cumulative positions of the mutations from dsRNA probes described in FIG. 2 are in bold. Location of the dsRNA probe at nucleic acid positions 2711 to 3195 of gene model AG6007485-PA is underlined. FIG. 5A shows the vgsc- hl CDS from a pyrethroid susceptible allele with nucleotide positions 2782 (A), 2782 (T), 2803 (G), in 3070 (C) in bold (SEQ ID NO: 1). FIG. 5B shows a consensus vgsc-hl CDS representing all nucleic acids among alleles defined from pyrethroid resistant glycines genotypes, with nucleotide positions 2782 (T), 2782 (A), 2803 (A), in 3070 (T) in bold, and location of the dsRNA probe at nucleic acid positions 2711 to 3195 of gene model AG6007485- PA is underlined. (SEQ ID NO: 2).

[0025] FIG. 6 shows the amino acid sequence of the Aphis glycines voltage gated sodium channel subunit hl(ygsc-hl) protein model AG6007485-PA predicted from the susceptible allele encoded in the A. glycines genome sequence assembly, Ag_Btl_v6.0 (SEQ ID NO:3). Sequence in this VGSC-H1 protein corresponding to amino acid positions changed and encoded by pyrethroid resistant A. glycines alleles, methionine (M). leucine (L) and leucine (L) at positions 928, 935, and 1024 respectively, are in bold. Per FIG. 2, the amino acid changes at positions 928, 935, and 1024 correspond to M -> I or M -> L, L -> M, and L -> F, respectively.

[0026] FIG. 7A-B shows the results of dsRNA treatments applied via spray on soybean plants infested with aphids resistant to lambda-cyhalothrin with the LI 014F mutation heterozygous in the vgsc-hl gene. FIG. 7A shows the average efficacy of the treatments, calculated across 2, 5, and 7 days after spray (DAS). Letters indicate significant differences between treatments. FIG. 7B shows the efficacy at 2, 5, and 7 days after spray (DAS) for each treatment.BRIEF DESCRIPTION OF THE SEQUENCES

[0027] SEQ ID NO: 1 is e Aphis glycines vgsc-hl CDS from gene model AG6007485-RA; pyrethroid susceptible allele.

[0028] SEQ ID NO: 2 is the Aphis glycines vgsc-hl CDS from gene model AG6007485-RA; consensus nucleotides from pyrethroid resistant alleles.

[0029] VGSC-H1 protein sequence from gene model AG6007485-PA representative of those encoded by pyrethroid susceptible alleles.

[0030] SEQ ID NO: 4 is a dsRNA probe designed to silence transcripts from the susceptible allele vgsc-hlBoone.

[0031] SEQ ID NO: 5 is a dsRNA probe designed to silence transcripts from the resistance allele L1014F vgsc-hl^s]im

[0032] SEQ ID NO: 6 is a dsRNA probe designed to silence transcripts from the resistance allele M918I + L1014F vgsc-hlMm.

[0033] SEQ ID NO: 7 is a dsRNA probe designed to silence transcripts from the resistance allele L1014F vgsc- / i / K:i,,a"li;i.

[0034] SEQ ID NO: 8 is a dsRNA probe designed to silence transcripts from the resistance allele M918L + L925M vgsc-ha™ia.

[0035] SEQ ID NO: 9 is the amino acid translation from position 905 to 1065 of protein model AG6007485-PA.

[0036] SEQ ID NO: 10 is a forward primer.

[0037] SEQ ID NO: 11 is a reverse primer.

[0038] SEQ ID NO: 12 is the T7 RNA polymerase promoter sequence.

[0039] SEQ ID NO: 13 is the Musca domestica VGSC protein sequence (GenBank accession AAB47604.1).DETAILED DESCRIPTION

[0040] RNA interference (RNAi) is a natural mechanism of gene silencing found in most eukaryote organisms, including insects. The RNAi pathway regulates gene expression by switching off genes via production of double-stranded RNA (dsRNA). The induction of RNA interference by intracellular dsRNA) was first discovered in the nematode Caenorhabditis elegans as an endogenous viral defense mechanism and has been developed into a reverse genetic tool for inducing the degradation of mRNA transcripts in a sequence-specific manner. Interfering RNAs of a particular sequence can be synthetically manufactured and used to silence a specific gene or set of genes in insects, thus blocking its expression. The ensuing process interrupts protein synthesis by either destroying the corresponding messenger RNA molecules (mRNA), interfering with their ability to be translated into proteins, or modifying the DNA so that it cannot be transcribed.

[0041] An insecticide product based on RNAi has been commercialized for the control of the Colorado potato beetle (Rodriques et al., 2021; USEPA 2023, and more are expected to hit the market within the next few years. RNAi-based insecticides offer new alternatives to causespecific toxic effects on selected arthropod pests. The technique is part of an emerging suite of genetic engineering technologies applied to agriculture, which already includes the insertion of transgenes into commodity crops and, potentially, CRISPR-based genome editing. RNA interference is a naturally occurring process that regulates gene expression in eucaryote organisms by silencing selected genes. This process can be triggered by using synthetic RNAi molecules, thus killing a pest by turning off genes that are essential for its survival. Genesilencing insecticide formulations largely consist of dsRNA, siRNA or shRNA molecules combined with additional compounds that enhance their efficacy, such as nanoparticles that increase stability in the environment or assist their penetration into plant and insect cells.

[0042] Gene-silencing RNAi insecticides are inherently sequence-specific and can be designed to control insect pests in a highly specific manner by turning off genes that are essential for their survival. Besides the so-called lethal genes (which would directly kill the pest if disrupted), sequences related to immunity, oviposition and olfactory systems may also be targeted, thus preventing insects from identifying crops and / or laying their eggs therein. However, target organisms must ingest intact and biologically active dsRNA molecules to trigger an RNAi response. This can be achieved either by genetically engineering crops to produce dsRNA in planta, or externally applying dsRNA molecules in open-air agricultural settings.

[0043] After being applied to a crop, RNAi insecticides can reach targeted pests via direct contact, ingestion followed by digestion, and potentially by inhalation. Alternatively, interfering RNAs can be taken up into plants (either by leaf absorption or root uptake), transported throughout its tissues and transferred to pests that feed on it. Once within the pest’s gut, the ingested dsRNA is taken up by the cells and processed into smaller, active fragments (20-25 bp) by the endonuclease Dicer. These short interfering RNAs (siRNA) are then integrated into the RNA-induced silencing complex (RISC) and bind to a specific mRNA, as they share a sequence that is matching or similar to each other. Finally, the targeted mRNA is cleaved and destroyed, blocking protein synthesis and resulting in interference of gene expression (i.e., gene-silencing). Interfering RNAs have been demonstrated to provide targeted effects on single species as well as on specific alleles at a genetic locus.

[0044] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherw ise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosurewithout undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.

[0045] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms "a," "an" and "the" can include plural referents unless the content clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicate otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.

[0046] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various embodiments of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1 ! , and 4%. This applies regardless of the breadth of the range.

[0047] As used herein, the phrase “biological sample” refers to either intact or non-intact (e.g., milled seed or plant tissue, chopped plant tissue, lyophilized tissue) plant tissue. It may also be an extract comprising intact or non-intact seed or plant tissue. The biological sample can comprise flour, meal, flakes, syrup, oil, starch, and cereals manufactured in whole or in part to contain crop plant by-products. In certain embodiments, the biological sample is “non- regenerable” (i.e., incapable of being regenerated into a plant or plant part).

[0048] 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 ifthey are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.

[0049] 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 certain 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.

[0050] 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. A composition that controls a target insect has insecticidal activity' against the target insect.

[0051] 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 toxicant delivery system.

[0052] “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.

[0053] As used herein, “expression cassette” 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 signal sequences. 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 withrespect 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. for example, 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.

[0054] A “gene’' is a defined region that is located within a genome and that, besides the 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.

[0055] 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 soybean, for food, feed or raw materials.

[0056] 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.

[0057] A “homologous” nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0058] The term “insect” as used herein includes any organism now known or later identified that is classified in the animal kingdom, phylum Arthropoda, class Insecta, including but not limited to insects in the orders Coleoptera (beetles), Lepidoptera (moths, butterflies), Diptera (flies), Protura, Collembola (springtails), Diplura, Microcoryphia (jumping bristletails), Thysanura (bristletails, silverfish), Ephemeroptera (mayflies), Odonata (dragonflies, damselflies), Orthoptera (grasshoppers, crickets, katydids), Phasmatodea (walkingsticks), Grylloblattodea (rock crawlers). Mantophasmatodea, Dermaptera (earwigs), Plecoptera (stoneflies), Embioptera (web spinners), Zoraptera, Isoptera (termites), Mantodea (manti ds), Blattodea (cockroaches), Hemiptera (true bugs, cicadas, leafhoppers, aphids, scales), Thysanoptera (thrips), Psocoptera (book and bark lice), Phthiraptera (lice; including but not limited to suborders Amblycera. Ischnocera and Anoplura). Neuroptera (lacewings, owlflies, mantispids, antlions), Hymenoptera (bees, ants, wasps), Trichoptera (caddisflies), Siphonaptera(fleas), Mecoptera (scorpion flies), Strepsiptera (twisted-winged parasites), and any combination thereof.

[0059] “Insecticidal” is defined as a toxic biological activity' capable of controlling insects, preferably by killing them.

[0060] An “isolated” nucleic acid molecule or nucleotide sequence or nucleic acid construct or dsRNA molecule or protein of the disclosure 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 or host cell such as a transgenic plant or transgenic plant cell.

[0061] In the context of the disclosure, 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 RNA is a “19-mer”.

[0062] A “plant” is any plant at any stage of development, particularly a seed plant.

[0063] 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.

[0064] “Plant cell culture” means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embry o sacs, zy gotes and embry os at various stages of development.

[0065] “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.

[0066] 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.

[0067] “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 ty pe 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.

[0068] The term “primer” as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR.Typically, primers are oligonucleotides from 10 to 30 nucleotides in length, but longer sequences may be used. Primers may be provided in single or double-stranded form. Probes may be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process.

[0069] In the context of the disclosure, the term “toxic” used to describe a dsRNA of the disclosure means that the dsRNA molecules of the disclosure 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 disclosure 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. In certain embodiments, such a composition may be a transgenic plant expressing the dsRNA of the disclosure.

[0070] “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.

[0071] As used herein, the terms “transformed”, “transgenic”, and “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.

[0072] By “operably linked” or “operably associated,” it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term “operably linked” or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Therefore, a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For instance, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of the nucleotide sequence. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences functionto direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered “operably linked” to the nucleotide sequence.

[0073] A “promoter” is an untranslated DNA sequence upstream of a coding region that contains the binding site for RNA polymerase and initiates transcription of the DNA. A “promoter region” can also include other elements that act as regulators of gene expression. Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules.

[0074] “Regulatory elements” refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory’ elements may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. Regulatory elements present on a recombinant DNA construct that is introduced into a cell can be endogenous to the cell, or they can be heterologous with respect to the cell. The terms "regulatory element" and "regulatory sequence" are used interchangeably herein.

[0075] The terms “sequence similarity” or “sequence identity” of nucleotide or amino acid sequences mean a degree of identity or similarity of two or more sequences and may be determined conventionally by using known software or computer programs such as the Best-Fit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive. Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of identity or similarity between two sequences. Sequence comparison between two or more polynucleotides or polypeptides is generally performed by comparing portions of the tw o sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally from about 20 to 200 contiguous nucleotides. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit to determine the degree of DNA sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Similarly, when using a program such as BestFit to determine sequence identify, similarity or homology between tw o differentamino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.

[0076] The phrase “substantially identical,” in the context of two nucleic acids or two amino acid sequences, refers to two or more sequences or subsequences that have at least about 50% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, substantially identical sequences have at least about 60%, or at least about 70%, or at least about 80%, or even at least about 90% or 95% nucleotide or amino acid residue identity. In certain embodiments, substantial identity exists over a region of the sequences that is at least about 50 residues in length, or over a region of at least about 100 residues, or the sequences are substantially identical over at least about 150 residues. In certain embodiments, the sequences are substantially identical when they are identical over the entire length of the coding regions.

[0077] The term “homology” in the context of the disclosure refers to the level of similarity between nucleic acid or amino acid sequences in terms of nucleotide or amino acid identity or similarity, respectively, i.e., sequence similarity or identity. Homology, homologue, and homologous also refers to the concept of similar functional properties among different nucleic acids or proteins. Homologues include genes that are orthologous and paralogous. Homologues can be determined by using the coding sequence for a gene, disclosed herein or found in appropriate database (such as that atNCBI or others) in one or more of the following ways. For an amino acid sequence, the sequences should be compared using algorithms (for instance see section on “identity” and “substantial identity”). For nucleotide sequences the sequence of one DNA molecule can be compared to the sequence of a known or putative homologue in much the same way. Homologues are at least 20% identical, or at least 30% identical, or at least 40% identical, or at least 50% identical, or at least 60% identical, or at least 70% identical, or at least 80% identical, or at least 88% identical, or at least 90% identical, or at least 92% identical, or at least 95% identical, across any substantial region of the molecule (DNA. RNA, or protein molecule).

[0078] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology' algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0079] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). This algorithm involves first identilying high scoring sequence pairs (HSPs) by identifying short words of length W in the query' sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty' score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5. N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0080] In addition to calculating percent sequence identity', the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0. 1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0081] Another widely used and accepted computer program for performing sequence alignments is CLUSTALW vl.6 (Thompson, et al. Nuc. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between a 200 and a 400 amino acid protein, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches is divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.

[0082] Two nucleotide sequences can also be considered substantially identical when the two sequences hybridize to each other under stringent conditions. In representative embodiments, two nucleotide sequences considered to be substantially identical hybridize to each other under highly stringent conditions.

[0083] The terms "stringent conditions” or ‘'stringent hybridization conditions” include reference to conditions under which a polynucleotide will hybridize to its target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target polynucleotides can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity’ are detected (heterologous probing). Typically, stringent conditions will be those in which the salt concentration is less than approximately 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides). Stringent conditions also may be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (w / v; sodium dodecyl sulphate) at 37° C, and a wash in 1 x to 2xSSC (20xSSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55° C. Moderate stringency conditions detect sequences that share at least 80% sequence identity. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in 0.5x to 1 *SSC at 55 to 60° C. High stringency conditions detect sequences that share at least 90% sequence identity.Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in 0. 1 *SSC at 60 to 65° C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem, 138:267-284. 1984): Tm=81.5° C+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500 / L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary- target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C for each 1% of mismatching; thus, Tm, hybridization and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity- are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize hybridization and / or wash at 1, 2, 3, or 4° C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and / or wash at 6, 7, 8. 9, or 10° C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and / or wash at 11, 12, 13, 14, 15, or 20° C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C (aqueous solution) or 32° C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 ‘"Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., eds., Greene Publishing and Wiley- Interscience, New York (1995). Methods of stringent hybridization are known in the art which conditions can be calculated by means known in the art. This is disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.. Cold Spring Harbor Laboratory Press. 1989, Cold Spring Harbor, N.Y. and Current Protocols in Molecular Biology, Ausebel et al, eds., John Wiley and Sons, Inc., 2000. Methods of determining percent sequence identity are known in theart, an example of which is the GCG computer sequence analysis software (GCG, Inc, Madison Wis.).

[0084] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical (e.g., due to the degeneracy of the genetic code). A further indication that two nucleic acids or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.

[0085] The disclosure is based on the discovery that double stranded RNA (dsRNA), short hairpin RNA (shRNA), or small interfering RNAs (siRNA) designed to target a mRNA transcribable from the vgsc-hl genes described herein are toxic to the aphids and can be used to control an aphid infestation of a plant and impart to a transgenic plant tolerance to an aphid infestation. Thus, in certain embodiment, the disclosure 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 vgsc-hl gene described in the present disclosure, wherein the dsRNA molecule is toxic to an aphid.

[0086] As used herein, “dsRNA’’ 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. As used herein, the term “fully complementary” means that all the bases of the nucleotide sequence of the dsRNA are complementary to or ‘match’ the bases of the target nucleotide sequence. The term “at least partially complementary” means that there is less than a 100% match between the bases of the dsRNA and the bases of the target nucleotide sequence. The skilled person will understand that the dsRNA need only be at least partially complementary to the target nucleotide sequence in order to mediate down-regulation of expression of the target gene. It is known in the art that RNA sequences with insertions, deletions and mismatches relative to the target sequence can still be effective at RNAi. According to the current disclosure, it is preferred that the dsRNA and the target nucleotide sequence of the target gene share at least 80% or 85% sequence identity, preferably at least 90% or 95% sequence identity, or more preferably at least 97% or 98% sequence identity and still more preferably at least 99% sequenceidentity. Alternatively, the dsRNA may comprise 1, 2 or 3 mismatches as compared with the target nucleotide sequence over every length of 24 partially complementary nucleotides. It will be appreciated by the person skilled in the art that the degree of complementarity shared between the dsRNA and the target nucleotide sequence may vary depending on the target gene to be down-regulated or depending on the insect pest species in which gene expression is to be controlled.

[0087] The interfering RNA molecule may contain DNA bases, non-natural bases or non-natural backbone linkages or modifications of the sugar-phosphate backbone, for example to enhance stability during storage or enhance resistance to degradation by nucleases. Furthermore, the interfering RNA may be produced chemically or enzymatically by one skilled in the art through manual or automated reactions.

[0088] The disclosure provides an interfering RNA molecule comprising at least one dsRNA, where the dsRNA is a region of double-stranded RNA comprising annealed at least partially complementary strands. In certain embodiments, one strand of the dsRNA comprises a sequence of at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85. at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 contiguous nucleotides which is at least partially complementary to a target nucleotide sequence within a vgsc-hl target gene. In certain embodiments, the interfering RNA molecule (i) has at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity', at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity, to at least a 19, at least a 20. at least a 21, at least a 22, at least a 23, at least a 24. at least a 25. at least a 26, at least a 27, at least a 28, at least a 29, at least a 30, at least a 35, at least a 40, at least a 45, at least a 50, at least a 55, at least a 60, at least a 65, at least a 70, at least a 75, at least a 80, at least a 85, at least a 90, at least a 95, at least a 100, at least a 110, at least a 120, at least a 130, at least a 140, at least a 150. at least a 160, at least a 170. at least a 180, at least a 190, at least a 200, at least a 210, at least a 220, at least a 230, at least a 240, at least a 250, at least a 260, at least a 270, at least a 280, at least a 290, or at least a 300 contiguous nucleotide fragment of SEQ ID NO: 1 or2, or the complement thereof; (ii) comprises at least a 19, at least a 20, at least a 21, at least a 22, at least a 23, at least a 24, at least a 25, at least a 26, at least a 27, at least a 28, at least a 29, at least a 30, at least a 35, at least a 40, at least a 45, at least a 50, at least a 55, at least a 60, at least a 65, at least a 70, at least a 75, at least a 80. at least a 85, at least a 90, at least a 95, at least a 100, at least a 110. at least a 120, at least a 130, at least a 140, at least a 150, at least a 160. at least a 170, at least a 180, at least a 190, at least a 200, at least a 210, at least a 220, at least a 230, at least a 240, at least a 250, at least a 260, at least a 270, at least a 280, at least a 290, or at least a 300 contiguous nucleotide fragment of SEQ ID NO: 1 or 2, or the complement thereof; (iii) comprises at least a 19, at least a 20, at least a 21. at least a 22, at least a 23, at least a 24, at least a 25, at least a 26, at least a 27, at least a 28, at least a 29, at least a 30, at least a 35, at least a 40, at least a 45, at least a 50, at least a 55, at least a 60, at least a 65, at least a 70, at least a 75, at least a 80, at least a 85, at least a 90, at least a 95, at least a 100, at least a 110, at least a 120, at least a 130, at least a 140, at least a 150, at least a 160, at least a 170. at least a 180, at least a 190, at least a 200, at least a 210, at least a 220, at least a 230, at least a 240, at least a 250, at least a 260, at least a 270, at least a 280, at least a 290, or at least a 300 contiguous nucleotide fragment of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, or the complement thereof; or (iv) can hybridize under stringent conditions to a polynucleotide selected from SEQ ID NO: 1 or 2. and the complements thereof.

[0089] In certain embodiments, the disclosure encompasses an interfering RNA molecule which comprises at least one dsRNA wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands. One strand of the dsRNA comprises a sequence of at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25. at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least170, at least 180, at least 190, at least 200. at least 210, at least 220. at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 contiguous nucleotides which is at least partially complementary to a target nucleotide sequence within a vgsc-hl target gene. In certain embodiments, the interfering RNA molecule (i) has at least 80% identity, at least 85% identity', at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity’, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identify, at least 96% identify, at least 97% identify', at least 98% identify, at least 99% identify, or 100% identify, to at least a 19, at least a20, at least a 21, at least a 22, at least a 23, at least a 24, at least a 25, at least a 26, at least a 27, at least a 28, at least a 29, at least a 30, at least a 35, at least a 40, at least a 45, at least a 50, at least a 55, at least a 60, at least a 65, at least a 70, at least a 75, at least a 80, at least a 85, at least a 90, at least a 95, at least a 100, at least a 110, at least a 120, at least a 130, at least a 140, at least a 150. at least a 160, at least a 170. at least a 180, at least a 190, at least a 200, at least a 210, at least a 220, at least a 230, at least a 240, at least a 250, at least a 260, at least a 270, at least a 280, at least a 290, or at least a 300 contiguous nucleotide fragment of SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof; (ii) comprises at least a 19, at least a 20, at least a 21, at least a 22, at least a 23, at least a 24. at least a 25, at least a 26, at least a 27, at least a 28. at least a 29, at least a 30, at least a 35, at least a 40, at least a 45, at least a 50, at least a 55, at least a 60, at least a 65, at least a 70, at least a 75, at least a 80, at least a 85, at least a 90, at least a 95, at least a 100, at least a 110, at least a 120, at least a 130, at least a 140, at least a 150, at least a 160, at least a 170, at least a 180, at least a 190, at least a 200, at least a 210, at least a 220. at least a 230, at least a 240, at least a 250, at least a 260, at least a 270, at least a 280, at least a 290, or at least a 300 contiguous nucleotide fragment of SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof; (iii) comprises at least a 19, at least a 20, at least a 21, at least a 22, at least a 23, at least a 24, at least a 25, at least a 26. at least a 27, at least a 28, at least a 29, at least a 30, at least a 35, at least a 40, at least a 45, at least a 50. at least a 55, at least a 60, at least a 65, at least a 70, at least a 75, at least a 80, at least a 85, at least a 90, at least a 95, at least a 100, at least a 110, at least a 120, at least a 130, at least a 140, at least a 150, at least a 160, at least a 170, at least a 180, at least a 190, at least a 200, at least a 210, at least a 220, at least a 230, at least a 240. at least a 250, at least a 260, at least a 270, at least a 280, at least a 290, or at least a 300 contiguous nucleotide fragment of a nucleotide sequence encoding an amino acid sequence encoded by SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof; or (iv) can hybridize under stringent conditions to a polynucleotide selected from SEQ ID NO: 4, 5, 6, 7, or 8, and the complements thereof.

[0090] It will be appreciated that the dsRNA may comprise or consist of a region of doublestranded RNA comprising annealed complementary strands, one strand of which, the sense strand, comprises a sequence of nucleotides at least partially complementary to a target nucleotide sequence within the target gene.

[0091] In certain embodiments, the interfering RNA molecule comprises a dsRNA that 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, 25, 26, 27, 28, or 29) to at least about 300 consecutivenucleotides. 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).

[0092] In certain embodiments, the interfering RNA molecule comprises a dsRNA which comprises an antisense strand that is complementary to at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140. at least 150, at least 160. at least 170, at least 180. at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2. In certain embodiments, the portion of dsRNA comprises, consists essentially of or consists of at least from 19, 20, or 21 consecutive nucleotides to at least 19, at least 20, at least 21, at least 22. at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220. at least 230, at least 240. at least 250, at least 260. at least 270, at least 280, at least 290, or at least 300 consecutive nucleotides of SEQ ID NO: 1 or 2, or the complement thereof.

[0093] In certain embodiments, the interfering RNA molecule comprises a dsRNA which comprises an antisense strand that is complementary to at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160. at least 170, at least 180. at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260. at least 270, at least 280, at least 290, or at least 300 consecutive nucleotides of SEQ ID NO: 4, 5, 6, 7, or 8. In certain embodiments, the portion of dsRNA comprises, consists essentially of or consists of at least from 19, 20, or 21 consecutive nucleotides to at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29. at least 30. at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 consecutive nucleotides of SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof.

[0094] In certain embodiments, the antisense strand of the double stranded RNA of the interfering 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. The dsRNA of the interfering RNA molecule may comprise a dsRNA which is a region of double-stranded RNA comprising substantially complementary annealed strands, or which is a region of double-stranded RNA comprising fully complementary annealed strands. 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 disclosure, 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 “Doublemismatched siRNAs enhance selective gene silencing of a mutant ALS-causing Allelel” Acta Pharmacol. Sin. 29:211-216 (2008); Schwarz et al. “Asymmetry in the assembly of the RNAi enzyme complex" Cell 115: 199-208 (2003)).

[0095] In certain embodiments, the interfering RNA comprises a dsRNA which comprises 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 soybean.

[0096] The interfering RNA molecules of the disclosure may comprise one dsRNA or multiple dsRNAs, wherein each dsRNA comprises or consists of a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene and that functions upon uptake by an insect pest species to down-regulate expression of said target gene. Concatemeric RNA constructs of this type are described in W02006 / 046148 as incorporated herein by reference. In the context of the present disclosure, the term ’multiple’ means at least two, at least three, at least four, etc and up to at least 10, 15, 20 or at least 30. In oneembodiment the interfering RNA comprises multiple copies of a single dsRNA i.e. repeats of a dsRNA that binds to a particular target nucleotide sequence within a specific target gene. In another embodiment, the dsRNAs within the interfering RNA comprise or consist of different sequences of nucleotides complementary’ to different target nucleotide sequences. It should be clear that combinations of multiple copies of the same dsRNA combined with dsRNAs binding to different target nucleotide sequences are within the scope of the current disclosure.

[0097] The dsRNAs may be arranged as one contiguous region of the interfering RNA or may be separated by the presence of linker sequences. The linker sequence may comprise a short random nucleotide sequence that is not complementary to any target nucleotide sequences or target genes. In one embodiment, the linker is a conditionally self-cleaving RNA sequence, preferably a pH-sensitive linker or a hydrophobic-sensitive linker. In one embodiment, the linker comprises a sequence of nucleotides equivalent to an intronic sequence. Linker sequences of the current disclosure may range in length from about 1 base pair to about 10000 base pairs, provided that the linker does not impair the ability7of the interfering RNA to down-regulate the expression of target gene(s).

[0098] In addition to the dsRNA(s) and any linker sequences, the interfering RNA of the disclosure may comprise at least one additional polynucleotide sequence. In different embodiments of the disclosure, the additional sequence is chosen from (i) a sequence capable of protecting the interfering RNA against RNA processing, (ii) a sequence affecting the stability of the interfering RNA, (iii) a sequence allowing protein binding, for example to facilitate uptake of the interfering RNA by cells of the insect pest species, (iv) a sequence facilitating large-scale production of the interfering RNA, (v) a sequence which is an aptamer that binds to a receptor or to a molecule on the surface of the insect pest cells to facilitate uptake, or (v) a sequence that catalyzes processing of the interfering RNA within the insect pest cells and thereby enhances the efficacy of the interfering RNA. Structures for enhancing the stability of RNA molecules are well known in the art and are described further in W02006 / 046148 as incorporated herein by reference.

[0099] In certain embodiments, the interfering RNA molecule comprises at least two dsRNAs, wherein each dsRNA comprises a sequence of nucleotides which is at least partially complementary' to a target nucleotide sequence within the target gene. In certain embodiments, each of the dsRNAs comprise a different sequence of nucleotides which is complementary to a different target nucleotide sequence within the target gene. In certain embodiments, each of thedsRNAs comprise a different sequence of nucleotides which is complementary to a target nucleotide sequence within two different target genes.

[0100] The disclosure encompasses a nucleic acid construct comprising an interfering RNA of the disclosure. The disclosure further encompasses a nucleic acid molecule encoding at least one interfering molecule of the disclosure. The disclosure further encompasses a nucleic acid construct comprising at least one interfering molecule of the disclosure or comprising a nucleic acid molecule encoding the at least one interfering molecule of the disclosure. The disclosure further encompasses a nucleic acid construct wherein the nucleic acid construct is an expression vector. The disclosure further encompasses a recombinant vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes an interfering RNA molecule of the disclosure. A regulatory sequence may refer to a promoter, enhancer, transcription factor binding site, insulator, silencer, or any other DNA element involved in the expression of a gene.

[0101] In certain embodiments, the disclosure encompasses a composition comprising one or more or two or more of the interfering RNA molecules of the disclosure. In certain embodiments, the interfering RNA molecules are present on the same nucleic acid construct, on different nucleic acid constructs, or any combination thereof. For example, one interfering RNA molecule of the disclosure may be present on a nucleic acid construct, and a second interfering RNA molecule of the disclosure may be present on the same nucleic acid construct or on a separate, second nucleic acid construct. The second interfering RNA molecule of the disclosure may be to the same target gene or to a different target gene.

[0102] The disclosure encompasses an insecticidal composition for inhibiting the expression of a vgsc-hl gene described herein, comprising an interfering RNA of the disclosure and an agriculturally acceptable carrier.

[0103] An '‘agriculturally acceptable carrier” includes adjuvants, mixers, enhancers, etc. beneficial for application of an active ingredient, such as an interfering RNA molecule of the disclosure. Suitable carriers should not be phytotoxic to valuable crops, particularly at the concentrations employed in applying the compositions in the presence of crops, and should not react chemically with the compounds of the active ingredient herein, namely an interfering RNA of the disclosure, or other composition ingredients. Such mixtures can be designed for application directly to crops, or can be concentrates or formulations which are normally diluted with additional carriers and adjuvants before application. They may include inert or active components and can be solids, such as, for example, dusts, granules, water dispersible granules, or wettable powders, or liquids, such as, for example, emulsifiable concentrates, solutions,emulsions or suspensions. Suitable agricultural carriers may include liquid carriers, for example water, toluene, xylene, petroleum naphtha, crop oil, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, but l acetate, propylene glycol monomethyl ether and diethylene glycol monomethyl ether, methanol, ethanol, isopropanol, amyl alcohol, ethylene glycol, propylene glycol, glycerine, and the like. Water is generally the carrier of choice for the dilution of concentrates. Suitable solid carriers may include talc, pyrophyllite clay, silica, attapulgus clay, kieselguhr, chalk, diatomaceous earth, lime, calcium carbonate, bentonire clay, Fuller's earth, cotton seed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour, lignin, and the like.

[0104] For the present disclosure, an agriculturally acceptable carrier may also include non- pathogenic, attenuated strains of microorganisms, which carry the insect control agent, namely an interfering RNA molecule of the disclosure. In this case, the microorganisms carrying the interfering RNA may also be referred to as insect control agents. The microorganisms may be engineered to express a nucleotide sequence of a target gene to produce interfering RNA molecules comprising RNA sequences homologous or complementary to RNA sequences typically found within the cells of an insect. Exposure of the insects to the microorganisms result in ingestion of the microorganisms and down-regulation of expression of target genes mediated directly or indirectly by the interfering RNA molecules or fragments or derivatives thereof.

[0105] In certain embodiments, the interfering RNA molecules may be encapsulated in a synthetic matrix such as a polymer and applied to the surface of a host such as a plant. Ingestion of the host cells by an insect permits delivery of the insect control agents to the insect and results in down-regulation of a target gene in the host.

[0106] In certain embodiments, the interfering RNA molecule of the disclosure may be encapsulated in a nanoparticle. As used herein, the term “nanoparticle” refers to any material having dimensions in the 1-1,000 nm range. A nanoparticle can have at least one cross-sectional dimension of less than about 500 nm, less than about 250 nm. less than about 100 nm, less than about 75 nm. less than about 50 nm. less than about 25 nm. or less than about 10 nm. A variety of nanoparticles can be used. Examples of nanoparticles include, but are not limited to, lipid- based nanoparticles, protein-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots (e.g., carbon dots), polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerene, graphene, star polycations, nanotubes (e.g, carbon nanotubes), among others. 1

[0107] In certain embodiments, the nanoparticles include chitosan as a component. Generally, chitosans are a family of cationic, binary hetero-polysaccharides composed of (1^4)-linked 2- acetamido-2-deoxy-P-D-glucose (GlcNAc, A-unit) and 2-amino-2-deoxy-P-D-glucose, (GlcN; D-unit). The chitosan has a positive charge, stemming from the de-acetylated amino group ( — NH3+). Chitosan, chitosan derivatives, or salts (e.g., nitrate, phosphate, sulphate, hydrochloride, glutamate, lactate or acetate salts) of chitosan may be used and are included within the meaning of the term “chitosan”. As used herein, the term “chitosan derivatives” is intended to include ester, ether, or other derivatives formed by bonding of acyl and / or alkyl groups with — OH groups, but not the NH2 groups, of chitosan. Examples are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Modified chitosans, particularly those conjugated to polyethylene glycol, are also considered “chitosan derivatives.” Many chitosans and their salts and derivatives are commercially available (e.g., SigmaAldrich). Methods of preparing chitosans and their derivatives and salts are also known, such as boiling chitin in concentrated alkali (50% w / v) for several hours. This produces chitosan wherein 70%-75% of the N-acetyl groups have been removed. Chitosans may be obtained from any source know n to those of ordinary skill in the art. For example, chitosans may be obtained from commercial sources. Chitosans may be obtained from chitin, the second most abundant biopolymer in nature. Chitosan is prepared by N- deacetylation of chitin. Chitosan is commercially available in a wide variety of molecular weight (e g., 10-1000 kDa) and usually has a degree of deacetylation ranging betw een 70%-90%. In certain embodiments, the chitosan (or chitosan derivative or salt) has a molecular weight of about 4,000 Dalton or more, in the range about 25,000 to about 2,000,000 Dalton, or about 50,000 to about 300,000 Dalton. Chitosans of different molecular weights can be prepared by enzymatic degradation of high molecular weight chitosan using chitosanase or by the addition of nitrous acid. Some methods of producing chitosan involve recovery from microbial biomass, such as the methods taught by U.S. Pat. No. 4,806,474 and U.S. Patent Application No. 2005 / 0042735. herein incorporated by reference. Additional information regarding chitosan and chitosan derivatives can be found in U.S. Patent App. Pub. Nos. 2007 / 0167400, 2007 / 0116767, 2007 / 0311468, 2006 / 0277632, 2006 / 0189573, 2006 / 0094666, 2005 / 0245482, 2005 / 0226938, 2004 / 0247632, and 2003 / 0129730, each of which is herein specifically incorporated by reference.

[0108] A composition of the disclosure, for example a composition comprising an interfering RNA molecule of the disclosure and an agriculturally acceptable carrier, may be used in conventional agricultural methods. For example, the compositions of the disclosure may bemixed with water and / or fertilizers and may be applied preemergence and / or postemergence to a desired locus by any means, such as airplane spray tanks, irrigation equipment, direct injection spray equipment, knapsack spray tanks, cattle dipping vats, farm equipment used in ground spraying (e.g., boom sprayers, hand sprayers), and the like. The desired locus may be soil, plants, and the like.

[0109] A composition of the disclosure may be applied to a seed or plant propagule in any physiological state, at any time between harvest of the seed and sowing of the seed; during or after sowing; and / or after sprouting. It is preferred that the seed or plant propagule be in a sufficiently durable state that it incurs no or minimal damage, including physical damage or biological damage, during the treatment process. A formulation may be applied to the seeds or plant propagules using conventional coating techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters.

[0110] In certain embodiments, the acceptable agricultural carrier is a transgenic organism expressing an interfering RNA of the disclosure. In certain embodiments, the transgenic organism may be a transgenic plant expressing the interfering RNA of the disclosure that when fed upon by an aphid causes the target aphid to stop feeding, growing or reproducing or causing death of the aphid. In certain embodiments, the transgenic plant is a transgenic soybean plant and the aphid is Aphis glycines.

[0111] In certain embodiments, the transgenic organism is selected from, but not limited to, yeast, fungi, algae, bacteria, virus or an arthropod expressing the interfering RNA molecule of the disclosure. In certain embodiments, the transgenic organism is a virus, for example an insect baculovirus that expresses an interfering RNA molecule of the disclosure upon infection of an insect host. Such a baculovirus is likely more virulent against the target insect than the wildtype untransformed baculovirus. In certain 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 certain 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 interfering RNA molecules of the disclosure for the same purpose.

[0112] In certain embodiments, an acceptable agricultural carrier is a formulation useful for applying the composition comprising the interfering RNA molecule to a plant or seed. In certain embodiments, the interfering RNA molecules are stabilized against degradation because of their double stranded nature and the introduction of Dnase / Rnase inhibitors. For example, dsRNA, shRNA, or siRNA can be stabilized by including thymidine or uridine nucleotide 3' overhangs. The dsRNA, shRNA, or siRNA contained in the compositions of the disclosure 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.

[0113] In certain embodiments, the formulation comprises a transfection promoting agent. In certain embodiments, the transfection promoting agent is a lipid-containing compound. In certain embodiments, the lipid-containing compound is selected from Lipofectamine, Cellfectin, DMRIE-C, DOTAP and Lipofectin. In certain embodiments, the lipid-containing compound is a Tris cationic lipid.

[0114] In certain 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]-l,4-butanediamine). protamine sulphate, poly -lysine as well as other positively charged peptides. In certain embodiments, the nucleic acid condensing agent is spermidine or protamine sulfate. In certain embodiments, the formulation further comprises buffered sucrose or phosphate buffered saline.

[0115] Transgenic plants expressing an interfering RNA of the disclosure are tolerant or resistant to attack by aphids. When the aphid starts feeding on such a transgenic plant, it also ingests the expressed dsRNA, shRNA, or siRNA. This may deter the aphid from further feeding or may even harm or kill the insect. In certain embodiments, a nucleic acid sequence encoding a dsRNA, shRNA, or siRNA of the disclosure is inserted into an expression cassette, which is then preferably stably integrated in the genome of the plant. The nucleic acid sequences of the expression cassette introduced into the genome of the plant are heterologous to the plant and non-naturally occurring. Plants transformed in accordance with the present disclosure may be monocots or dicots and include, but are not limited to, com, 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 certain embodiments, the transgenic plant is a transgenic soybean plant.

[0116] Expression of the interfering RNA molecule in transgenic plants is driven by regulatory’ sequences comprising 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 target species. Thus, expression of the interfering RNAs of this disclosure in leaves, or stems, in pods, 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, shRNA, or siRNA in the desired cell.

[0117] Promoters useful with the disclosure 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.

[0118] In certain embodiments, tissue-specific / tissue-preferred promoters can be used. Tissuespecific or tissue-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. In addition, promoters functional in plastids can be used. In certain embodiments of the disclosure, inducible promoters can be used. In further aspects, the nucleotide sequences of the disclosure can be operably associated with a promoter that is wound inducible or inducible by pest or pathogen infection (e.g., an insect pest)

[0119] In certain embodiments of the present disclosure, 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 certain 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 certain 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 disclosure can be selected for use as a minimal promoter.

[0120] In certain embodiments, a recombinant nucleic acid molecule of the disclosure 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 disclosure), wherein the nucleotide sequence is operably associated with at least a control sequence (e.g.. a promoter). Thus, certain embodiments of the disclosure provide expression cassettes designed to express nucleotides sequences encoding the dsRNAs, shRNAs, or siRNAs of the disclosure. In this manner, for example, one or more plant promoters operably associated with one or more nucleotide sequences of the disclosure are provided in expression cassettes for expression in a soybean plant, plant part and / or plant cell.

[0121] 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 comprises a native promoter driving its native gene, however it has been obtained in a recombinant form useful for heterologous expression. Such usage of an expression cassette makes it so it is not naturally occurring in the cell into which it has been introduced.

[0122] 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.

[0123] An expression cassette of the disclosure 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.

[0124] Examples of selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptll, 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 b*n 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 disclosure.

[0125] An expression cassette of the disclosure 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 conventionalmethodology, 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 single transgene can comprise multiple expression cassettes, such that multiple expression cassettes are introduced into the genome of a transformed cell at a single genomic location. Alternatively, 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 disclosure, 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., Infl Patent Application Publication Nos. WO 99 / 25821; WO 99 / 25854; WO 99 / 25840; WO 99 / 25855 and WO 99 / 25853.

[0126] 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.

[0127] Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carry ing at least one T- DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construct 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 Agrobacteri um- 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 of the disclosure may also comprise other selectable marker genes, for example, phosphomannose isomerase (pmi), which provides forpositive 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 acety ltransferase (pat), which provides tolerance to the herbicide phosphinotricin (glufosinate). The choice of selectable marker is not, however, critical to the disclosure.

[0128] In certain embodiments, a nucleic acid sequence of the disclosure 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 rpsl2 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-cletoxifying 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 disclosure. 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 totalsoluble plant protein. In a preferred embodiment, a nucleic acid sequence of the present disclosure 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 disclosure are obtained, and are preferentially capable of high expression of the nucleic acid sequence.

[0129] Transgenic plants or seed comprising an interfering RNA of the disclosure 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 disclosure are active against the same target insect, for example an aphid, the combination is useful (i) in a method for further enhancing activity of the composition of the disclosure against the target insect, and (ii) in a method for preventing development of resistance to the composition of the disclosure by providing yet another mechanism of action against the target insect. Thus, the disclosure provides a method of enhancing control of an aphid population comprising providing a transgenic plant or seed of the disclosure and applying to the plant or the seed an insecticide or insecticidal seed coating to a transgenic plant or seed of the disclosure. 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® (carbofuran), LANATE® (methomyl, metomil, mesomile), SEVIN® (carbaryl), TALSTAR® (bifenthrin), FORCE® (tefluthrin), AMMO® (cypermethrin). CYMBUSH® (cypermethrin). DELTA GOLD® (deltamethrin), KARATE® (lambda- cyhalothrin), AMBUSH® (permethrin), POUNCE® (permethrin), BRIGADE® (bifenthrin), CAPTURE® (bifenthrin), PROSHIELD® (tefluthrin), WARRIOR® (lambda-cyhalothrin), DURSBAN® (chlorphyrifos), FORTRESS® (chlorethoxyfos), MOCAP® (ethoprop), THIMET® (phorate), AASTAR® (phorate, flucythinate). RAMPART® (phorate).COUNTER® (terbufos), CYGON® (dimethoate), Dicapthon, REGENT® (fipronil),CRUISER® (thiamethoxam), GAUCHO® (imidacloprid), PRESCRIBE® (imidacloprid), PONCHO® (clothianidin) and AZTEC® (cyfluthrin, tebupirimphos).

[0130] The compositions of the disclosure can also be combined with other biological control agents to enhance control of insect or aphid populations. Thus, the disclosure provides a method of enhancing control of an insect population or an aphid population by providing a transgenic plant that produces an interfering RNA of the disclosure and further comprises a polynucleotide that encodes a second insecticidal agent. The second insecticidal agent may be an insecticidal protein derived from Bacillus thuringiensis . A B. thuringiensis insecticidal protein can be any of a number of insecticidal proteins including but not limited to a Cry 1 protein, a Cry 3 protein, a Cry 7 protein, a Cry8 protein, a Cryl 1 protein, a Cry 22 protein, a Cry 23 protein, a Cry 36 protein, a Cry37 protein, a Cry34 protein together with a Ciy35 protein, a binary insecticidal protein CryET33 and CiyTT34. a binary insecticidal protein TIC 100 and TIC 101, a binary insecticidal protein PS149B1, a VIP, a TIC900 or related protein, a TIC901, TIC1201, TIC407, TIC417, a modified Cry 3 A protein, or hybrid proteins or chimeras made from any of the preceding insecticidal proteins. In certain embodiments, the B. thuringiensis insecticidal protein is selected from the group consisting of Cry3Bbl, Cry34Abl together with Cry35Abl, mCry3A and eCry 3.1 Ab.

[0131] In certain embodiments, the transgenic plant may produce an interfering RNA of the disclosure and a second insecticidal agent which is derived from sources other than B. thuringiensis . The second insecticidal agent can be an agent selected from the group comprising a patatin, a protease, a protease inhibitor, a chitinase, a urease, an alpha-amylase inhibitor, a pore-forming protein, a lectin, an engineered antibody or antibody fragment, a Bacillus cereus insecticidal protein, aXenorhabdus spp. (such as A nematophila or V bovienii) insecticidal protein, a Photorhabdus spp. (such as P. luminescens or P. asymobiotica) insecticidal protein, a Brevibacillus later osporous insecticidal protein, a Lysinibacillus sphearicus insecticidal protein, a Chromobacterium spp. insecticidal protein, a Yersinia entomophaga insecticidal protein, a Paenibacillus popiliae insecticidal protein, a Clostridium spp. (such as C. bifermentans) insecticidal protein, and a lignin. In certain embodiments, the second agent may be at least one insecticidal protein derived from an insecticidal toxin complex (Tc) from Photorhabdus , Xenorhabus, Serratia, or Yersinia. In certain embodiments, the insecticidal protein may be an ADP-ribosyltransferase derived from an insecticidal bacterium, such as Photorhabdus spp. In certain embodiments, the insecticidal protein may be a VIP protein, such as VIP1 or VIP2 from B. cereus. In certain embodiments, the insecticidal protein may be a binary toxin derived frominsecticidal bacteria, such as ISP1A and ISP2A from B. laterosporous or BinA and BinB from L. sphaericus . In certain embodiments, the insecticidal protein may be engineered or may be a hybrid or chimera of any of the preceding insecticidal proteins.

[0132] 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 disclosure, which has activity against hemipteran insects, the treated plant or coated transgenic seed controls both lepidopteran and hemipteran insect pests.

[0133] In further embodiments, the disclosure encompasses a biological sample from a transgenic plant, seed, or parts thereof, of the disclosure, wherein the sample comprises a nucleic acid that is or encodes at least one strand of a dsRNA of the disclosure. In certain embodiments, the disclosure encompasses a commodity product derived from a transgenic plant, seed, or parts thereof, of the disclosure. In certain embodiments, the commodity product is selected from the group consisting of whole or processed seeds, beans, grains, hulls, meals, 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 certain embodiments, the biological sample or commodity product is toxic to insects. In certain embodiments, the biological sample or commodity product is toxic to aphids. In certain embodiments, the transgenic plant is a transgenic soybean plant.

[0134] The following numbered embodiments also form part of the present disclosure:

[0135] 1. An interfering ribonucleic acid (RNA) molecule comprising a region of doublestranded RNA (dsRNA), wherein a strand of the dsRNA comprises a sequence of at least 19 contiguous nucleotides that is at least partially complementary to a target nucleotide sequence within an aphid voltage-gated sodium channel subunit hl (ygsc-hl) target gene.

[0136] 2. The interfering RNA molecule of embodiment 1, wherein the interfering RNA molecule comprises a nucleotide sequence having at least 80%. at least 90%. at least 95%. or at least 99% sequence identity to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 1 or 2, or the complement thereof.

[0137] 3. The interfering RNA molecule of embodiment 1 or embodiment 2, wherein the interfering RNA molecule comprises at least a 19 contiguous nucleotide fragment of SEQ ID NO: 1 or 2, or the complement thereof.

[0138] 4. The interfering RNA molecule of any one of embodiments 1-3, wherein the interfering RNA molecule comprises a T at a position corresponding to position 2782 of SEQ ID NO: 1 or 2, an A at a position corresponding to position 2784 of SEQ ID NO: 1 or 2, an A at a position corresponding to position 2803 of SEQ ID NO: 1 or 2, or a T at a position corresponding to position 3070 of SEQ ID NO: 1 or 2.

[0139] 5. The interfering RNA molecule of any one of embodiments 1-4, wherein the interfering RNA molecule comprises at least a 19 contiguous nucleotide fragment of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, or the complement thereof.

[0140] 6. The interfering RNA molecule of any one of embodiments 1-5. wherein the interfering RNA molecule can hybridize under stringent conditions to the polynucleotide set forth in SEQ ID NO: 1 or 2, or the complement thereof.

[0141] 7. The interfering RNA molecule of any one of embodiments 1 -6, wherein the interfering RNA molecule comprises a nucleotide sequence having at least 80%. at least 90%. at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof.

[0142] 8. The interfering RNA molecule of any one of embodiments 1-7, wherein the interfering RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof.

[0143] 9. The interfering RNA molecule of any one of embodiments 1-8. wherein the interfering RNA molecule has insecticidal activity on an aphid.

[0144] 10. A nucleic acid construct encoding the interfering RNA molecule of any one of embodiments 1-9.

[0145] 11. A vector comprising the nucleic acid construct of embodiment 10 operably linked to a regulatory sequence.

[0146] 12. An insecticidal composition comprising the interfering RNA molecule of any one of embodiments 1 -9 and an agriculturally acceptable carrier.

[0147] 13. The insecticidal composition of embodiment 12. further comprising a pyrethroid insecticide.

[0148] 14. The insecticidal composition of embodiment 12 or embodiment 13, further comprising a nanoparticle.

[0149] 15. The insecticidal composition of embodiment 14, wherein the nanoparticle comprises chitosan.

[0150] 16. A transgenic plant, or part thereof, comprising the interfering RNA molecule of any one of embodiments 1-9, the nucleic acid construct of embodiment 10, or the vector or embodiment 11.

[0151] 17. The transgenic plant, or part thereof, of embodiment 16. wherein the transgenic plant, or part thereof, is a soybean plant or part thereof.

[0152] 18. A seed or an asexual propagate of the transgenic plant of embodiment 14 or embodiment 17.

[0153] 19. A biological sample from the transgenic plant, or part thereof, of embodiment 16 or embodiment 17.

[0154] 20. A commodity plant product derived from the transgenic plant, or part thereof, of embodiment 16 or embodiment 17.

[0155] 21. A method of controlling an insect pest, the method comprising contacting the insect pest with the interfering RNA molecule of any one of embodiments 1-9, wherein expression of a vgsc-hl target gene in the insect pest is inhibited.

[0156] 22. The method of embodiment 21, wherein contacting comprises planting a transgenic seed capable of producing a transgenic plant that expresses interfering RNA molecule, and wherein the insect pest feeds on the transgenic plant, or part thereof.

[0157] 23. The method of embodiment 21 or embodiment 22, wherein contacting comprises applying an insecticidal composition comprising the interfering RNA molecule to a seed or plant, or part thereof, and wherein the insect pest feeds on the seed, the plant, or a part thereof.

[0158] 24. The method of any one of embodiments 21-23, wherein the insecticidal composition further comprises a pyrethroid insecticide.

[0159] 25. The method of embodiment 23 or embodiment 24, wherein the insecticidal composition further comprises a nanoparticle.

[0160] 26. The method of embodiment 25, wherein the nanoparticle comprises chitosan.

[0161] 27. The method of any one of embodiments 21-26, wherein the insect pest is resistant to a pyrethroid insecticide.

[0162] 28. The method of any one of embodiments 21-27, wherein the insect pest is an aphid.

[0163] 29. A method of controlling an insect pest, the method comprising: inhibiting expression or activity of a VGSC-H1 polypeptide in the insect pest, wherein the VGSC-H1 polypeptide comprises an isoleucine at position 928, a leucine at position 928, a methionine at position 935, or a phenylalanine at position 1024, wherein the position numbering corresponds to the position in SEQ ID NO 3.

[0164] 30. The method of embodiment 29, wherein the VGSC-H1 polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3.

[0165] 31. The method of embodiment 29 or embodiment 30, wherein the gene encoding the VGSC-H1 polypeptide comprises a nucleotide sequence having at least 80%. at least 90%. at least 95%, or at least 99% sequence identity to SEQ ID NO: 1 or 2.

[0166] 32. The method of any one of embodiments 29-31, further comprising contacting the insect pest with a pyrethroid insecticide.

[0167] 33. The method of any one of embodiments 29-32, wherein the insect pest is an aphid.

[0168] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure 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.

[0169] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

[0170] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1: RNA interference (RNAi) mediated knockdown of alleles of the voltage-gated sodium channel gene that confer pyrethroid insecticide resistance to enhance efficacy and restore susceptibility in resistant soybean aphid populations

[0171] RNAi-mediated silencing of resistance genes could be used as a synergistic means to increase the susceptibility of insect pests to chemical insecticides. This approach could be applied to enhance control of resistant strains or field populations of agriculturally important insect pests, such as soybean aphids.

[0172] The invasive species A. glycines is a major pest of soybean (Glycine max) in the United States, with potential to spread into other countries, such as Brazil. The damage caused by this insect can reduce soybean yield by < 40 % when left unmanaged. Soybean aphids have traditionally been controlled using foliar applications of pyrethroids, a class of insecticides that accounts for 15 % of the global market. Pyrethroids bind to the voltage-gated sodium channel (vgsc) protein, subsequently altering the function of the pore channel, causing repetitiveneurological impulses and killing the insect by paralysis. Wide exposure to these insecticides has led to an increasing occurrence of pyrethroid-resistant populations in aphids and other insect pests.

[0173] Pyrethroid resistance is conferred by two mechanisms: increased expression and activity of detoxification enzymes (e.g., cytochrome P450 monooxygenases (P450) and glutathione transferases (GST)), or amino acid substitutions that alter the target site of the vgsc gene. Multiple non-synonymous nucleic acid mutations have been reported in the voltage gated sodium channel subunit hl (vgsc-hl) of aphids. Specifically , A. glycines genotypes with one or more nucleotide mutations causing amino acid sequence changes, M928I, M928L, L935M and L1024F, which are associated with increased resistance to pyrethroid insecticides (FIG. 1A). The numbered positions of these amino acid changes were adjusted with respect to the homologous positions of the VGSC protein for the house fly, Musca domestica (SEQ ID NO: 13; GenBank accession AAB47604.1), as done by convention in the published literature. The amino acid sequence changes M928I, M928L, L935M, and L1024F in the glycines VGSC protein (SEQ ID NO: 3) correspond to M918I, M918L, L925M, and L1014F, respectively, in the VGSC Musca domestica protein (SEQ ID NO: 13). Field-evolved resistance in A. glycines populations are becoming increasingly frequent throughout northcentral USA, potentially jeopardizing the long-term efficacy of pyrethroids as a control tool for this pest and highlighting the need to develop alternative strategies within an insecticide resistance management (IRM) plan to prevent this outcome.

[0174] One such strategy could make use of exogenously applied RNAi, combining it with pyrethroid insecticides in the same spray and, potentially, as a single formulation product. By spraying dsRNA molecules targeting mutated vgsc genes, pyrethroid resistance in soybean aphids can be reversed, increasing control efficacy of the insecticides themselves and improving the long-term “life” and durability of the pyrethroid molecules. Moreover, this spraying approach may lead to a limited time exposure of non-target organisms, compared to a constitutive expression of dsRNA on plants (i.e., insertion of transgenes into crops that encode for interfering RNAs). Due to demonstrated sequence specificity of interfering RNAs, those interfering RNAs that target mutated forms of insect genes and alleles that enhance insecticide resistance are likely to have no or fewer unintended consequences (e.g., gene silencing effects) on non-target species, thus avoiding off-target adverse activity. Major RNAi-related concerns, such as how it impacts different non-target organisms exposed, could be overcome with this approach.

[0175] A dsRNA was developed to target vgsc-hl transcripts in A. glycines, and the efficacy was validated through subsequent significant reductions in corresponding levels of transcript and level of phenoty pic pyrethroid resistance in treated aphids. Poly merase chain reaction (PCR) primers were designed to anneal nucleic acid locations flanking the A. glycines vgsc-hl allele positions encoding M9181. M918L, L925M. and L1014F mutations (FIG. 2), that when PCR amplified from complementary DNA (cDNA) template generated from A. glycines in the pyrethroid resistant colonies (FIG. IB) provided the subsequent template for synthesis of dsRNAs. These synthesized dsRNAs were shown to reduce vgsc-hl transcript levels more effectively among resistant compared to susceptible^, glycines and cause significantly greater mortality among resistant A. glycines when applied in conjunction with a pyrethroid insecticide compared to the pyrethroid alone. Specifically, experiments show that a dsRNA synthesized from cDNA template derived from the Kanawha-2019-ISO colony, dsvgsc-h 1^"' . that is homozygous for the mutation causing the L1014F mutation more effectively cause reductions in vgsc-hl transcripts among resistant compared to susceptible aphids, as demonstrated by relative quantitative real time PCR (FIG. 3).

[0176] This is shown by comparison of vgsc-hl transcript levels for aphids from the susceptible Boone colony with no topically-applied dsvgsc-hl^m(untreated) compared to vgsc-hl levels 12- , 24-. 36- and 48-hours post dsvgsc-h 1^"' application (FIG. 3A), where levels are significantly reduced compared to untreated aphids at 36- and 48-hours. In contrast, vgsc-hl transcripts were silenced more quickly and to a greater degree among A. glycines from resistant colonies that encode the L1014F mutation in one or more vgsc-hl alleles. For the MN1-2017-ISO colony that is heterozygous for the substitution mutation causing the L1014F amino acid change, vgsc-hl transcript levels were significantly reduced at all time points post dsvgsc-h application compared to untreated counterparts (FIG. 3B). Similar effects were shown for aphids from the Kanawha-2019-ISO colony that are homozygous for the alleles causing the L1014F change (FIG. 3C), where no estimates were provided for the 48-hour post dsvgsc-hl^' application timepoint due to 100% mortality being reached. Overall, the results showed that ds gsc-hl^'" leads to the most rapid silencing of vgsc-hl transcripts of pyrethroid resistant A. glycines, and reduction is most rapid where alleles have greater sequence similarity to the dsvgsc-h lKwdsRNA probe. The latter is most poignantly demonstrated by results from Kanawha-2009-ISO, where mRNAs expressed from both alleles were silenced by dsvgsc-hl^m.The rapid degradation of resistant dsvgsc-hl alleles by dsvgsc-h lKi'nimpacts the survival of resistant aphids when applied alone, but acts synergistically with concurrent application of apyrethroid insecticide. The was shown in laboratory bioassay by the increased mortality among A. glycines treated with dsvgsc-h 7Kanand a pyrethroid insecticide compared to treatment with the insecticide or dsvgsc-hlmalone (FIG. 4). This experiment that exposed MN1 -2017-ISO colony aphids to treatments with different levels of the pyrethroid insecticide, WARRIOR II®, and dsvgsc-hIKimfor 24-hours. firstly confirmed that aphids in both untreated and exposures to pyrethroid levels encountered in the field (0.29 parts per million, ppm) had low mortality (< 4%; confirming high levels of resistance in the colony). Application of dsvgsc-hl^"' alone led to mortality among —1 / 3 of MN1-2017-ISO individuals, an 11-fold increase in mortality compared to 0.29 ppm WARRIOR II® alone. More importantly, exposure to 0.29 ppm WARRIOR II® and dsvgsc-h lv'mled to 19.3-fold greater mortality' compared to 0.29 ppm WARRIOR II® alone. This nearly 20-fold increase in efficacy in laboratory bioassays shows that RNAi-mediated knockdow n (silencing) of mRNA transcripts for resistance alleles of the A. glycines vgsc-hl locus 1) by themselves cause increased mortality of resistant individuals compared to susceptible aphids, and 2) this silencing leads to an even greater increase in mortality among resistant A. glycines when applied in conjunction with a pyrethroid insecticide.

[0177] This technology' that causes RNAi-based silencing of dsvgsc-hl alleles from pyrethroid resistant A. glycines and concomitant reductions levels of phenotypic resistance to pyrethroid insecticides can be used in insect control strategies. Increased mortality occurs when dsvgsc-hl dsRNAs were used alone, but since evidence demonstrated the effects on mortality are greater (synergized) among resistant aphids when applied along with an insecticide (FIG. 4) this technology may remediate instances of pyrethroid resistance. Thus, this RNAi-based insecticide would be useful to growers through the transient restoration of pyrethroid susceptibility in field populations of A. glycines in geographic areas where resistance is widespread. This synergistic insecticidal activity restores efficacy of pyrethroid insecticides in the laboratory and may similarly do so under field conditions to prolong their efficacy for A. glycines control.Example 2: Spray application on soybean plants infested with resistant aphids

[0178] In this example, dsRNA targeting mutations in the vgcs gene in soybean aphid was tested both naked and encapsulated with nanoparticles based on chitosan.

[0179] The treatments were applied via spray on soybean plants infested with aphids resistant to lambda-cyhalothrin, with the L1014F mutation heterozygous in the vgsc gene. 25 uninjured adult aphids were transferred to each plant (V2 growth stage), which were placed on the abaxial side of the leaves. After allowing 48 hours for the aphids to acclimate and move freely acrossthe plant, the initial population was recorded. The plants were then treated in a spray chamber calibrated to deliver 20 gallons per acre. The lambda-cyhalothrin treatment was applied at a concentration of 5 ppm, corresponding to 0.38 g a.i. / acre. The dsRNA treatment was synthesized to target all mutations present in the isolines (L1014F, M918I, M918L, and M925), with a concentration of 1 pg / jrl. corresponding to 75.71 g / acre. The average aphid population before spraying was 145 aphids per plant. Post-treatment population counts were collected at 2, 5, and 7 days after spray. The efficacy of the treatments was calculated using Henderson-Tilton's formula, based on the population fluctuation in the control.

[0180] A generalized linear model (GLM) was used for the statistical analysis. There was no interaction between the day of evaluation and the treatments. Therefore, FIG. 7A-B summarizes the average efficacy of the treatments across 2, 5, and 7 days after spray (DAS). The ability of the dsRNA probes to kill insecticide resistant aphids was improved when encapsulated in the nanoparticles.References

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[0295] Zhang, L., Hou, D., Chen, X., Li, D., Zhu, L., Zhang, Y., Li, J., Bian, Z., Liang, X., Cai, X., Yin, Y., Wang, C., Zhang, T , Zhu, D., Zhang. D., Xu, J., Chen. Q., Ba. Y., Liu, J., Wang, Q., ... Zhang, C. Y. (2012). Exogenous plant MIR168a specifically targets mammalian LDLRAP1 : evidence of cross-kingdom regulation by microRNA. Cell Research, 22(1), 107— 126. doi: 10. 1038 / cr.2011.158

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[0297] Zheng, Y , Hu, Y„ Yan, S„ Zhou, H„ Song, D„ Yin, M„ et al. (2019). A polymer / detergent formulation improves dsRNA penetration through the body wall and RNAi- induced mortality in the soybean aphid Aphis glycines. Pest Manag. Sci. 75, 1993-1999. doi: 10. 1002 / ps.5313

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Claims

What is claimed is:1 . An interfering ribonucleic acid (RNA) molecule comprising a region of double-stranded RNA (dsRNA), wherein a strand of the dsRNA comprises a sequence of at least 19 contiguous nucleotides that is at least partially complementary to a target nucleotide sequence within an aphid voltage-gated sodium channel subunit hl (vgsc-hl) target gene.

2. The interfering RNA molecule of claim 1, wherein the interfering RNA molecule comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 1 or 2, or the complement thereof.

3. The interfering RNA molecule of claim 1, wherein the interfering RNA molecule comprises at least a 19 contiguous nucleotide fragment of SEQ ID NO: 1 or 2, or the complement thereof.

4. The interfering RNA molecule of claim 1, wherein the interfering RNA molecule comprises a T at a position corresponding to position 2782 of SEQ ID NO: 1 or 2, an A at a position corresponding to position 2784 of SEQ ID NO: 1 or 2. an A at a position corresponding to position 2803 of SEQ ID NO: 1 or 2, or a T at a position corresponding to position 3070 of SEQ ID NO: 1 or 2.

5. The interfering RNA molecule of claim 1, wherein the interfering RNA molecule comprises at least a 19 contiguous nucleotide fragment of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, or the complement thereof.

6. The interfering RNA molecule of claim 1, wherein the interfering RNA molecule can hybridize under stringent conditions to the polynucleotide set forth in SEQ ID NO: 1 or 2, or the complement thereof.

7. The interfering RNA molecule of claim 1, wherein the interfering RNA molecule comprises a nucleotide sequence having at least 80%. at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof.

8. The interfering RNA molecule of claim 1, wherein the interfering RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 4, 5, 6, 7, or 8, or the complement thereof.

9. The interfering RNA molecule of claim 1. wherein the interfering RNA molecule has insecticidal activity on an aphid.

10. A nucleic acid construct encoding the interfering RNA molecule of any one of claims 1 - 9.11 A vector comprising the nucleic acid construct of claim 10 operably linked to a regulatory sequence.

12. An insecticidal composition comprising the interfering RNA molecule of any one of claims 1-9 and an agriculturally acceptable carrier.

13. The insecticidal composition of claim 12, further comprising a pyrethroid insecticide.

14. The insecticidal composition of claim 12, further comprising a nanoparticle.

15. The insecticidal composition of claim 14, wherein the nanoparticle comprises chitosan.

16. A transgenic plant, or part thereof, comprising the interfering RNA molecule of any one of claims 1 -9.

17. The transgenic plant, or part thereof, of claim 16, wherein the transgenic plant, or part thereof, is a soybean plant or part thereof.

18. A seed or an asexual propagate of the transgenic plant of claim 16.

19. A biological sample from the transgenic plant, or part thereof, of claim 16.

20. A commodity plant product derived from the transgenic plant, or part thereof, of claim 16.

21. A method of controlling an insect pest, the method comprising contacting the insect pest with the interfering RNA molecule of any one of claims 1-9, wherein expression of a vgsc-hl target gene in the insect pest is inhibited.

22. The method of claim 21, wherein contacting comprises planting a transgenic seed capable of producing a transgenic plant that expresses interfering RNA molecule, and wherein the insect pest feeds on the transgenic plant, or part thereof.

23. The method of claim 21, wherein contacting comprises applying an insecticidal composition comprising the interfering RNA molecule to a seed or plant, or part thereof, and wherein the insect pest feeds on the seed, the plant, or a part thereof.

24. The method of claim 23, wherein the insecticidal composition further comprises a pyrethroid insecticide.

25. The method of claim 23, wherein the insecticidal composition further comprises a nanoparticle.

26. The method of claim 25, wherein the nanoparticle comprises chitosan.

27. The method of claim 21, wherein the insect pest is resistant to a pyrethroid insecticide.

28. The method of claim 21 , wherein the insect pest is an aphid.

29. A method of controlling an insect pest, the method comprising: inhibiting expression or activity7of a VGSC-H1 polypeptide in the insect pest, wherein the VGSC-H1 polypeptide comprises an isoleucine at position 928, a leucine at position 928, a methionine at position 935, or a phenylalanine at position 1024, wherein the position numbering corresponds to the position in SEQ ID NO 3.

30. The method of claim 29, wherein the VGSC-H1 polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3.

31. The method of claim 29. wherein the gene encoding the VGSC-H1 polypeptide comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1 or 2.

32. The method of claim 29, further comprising contacting the insect pest with a pyrethroid insecticide.

33. The method of claim 29, wherein the insect pest is an aphid.

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