Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
  • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
  • A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• sequence listing is submitted electronically via EFS- Web as an ASCII formatted sequence listing with a file named SeqList, modified on December 11, 2017 and having the size of 151 kilobyes (SEQ ID Nos:l-92), and is filed concurrently with the specification.
• SeqList modified on December 11, 2017 and having the size of 151 kilobyes (SEQ ID Nos:l-92), and is filed concurrently with the specification.
• sequence listing contained in the ACSII formatted document is part of the specification, and is incorporated herein by reference in its entirety.
• the present invention relates generally to genetic control of plant damage caused by insect pests (e.g., coleopteran pests).
• insect pests e.g., coleopteran pests
• the present invention relates to identification of target coding and non- coding polynucleotides, and the use of recombinant DNA technologies for post-transcriptionally repressing or inhibiting expression of target coding and non-coding polynucleotides in the cells of an insect pest to provide a plant protective effect.
• MCR Mexican corn rootworm
• SCR southern corn rootworm
• D. balteataheConte D.
• Both WCR and NCR eggs are deposited in the soil during the summer.
• the insects remain in the egg stage throughout the winter.
• the eggs are oblong, white, and less than 0.004 inches in length.
• the larvae hatch in late May or early June, with the precise timing of egg hatching varying from year to year due to temperature differences and location.
• the newly hatched larvae are white worms that are less than 0.125 inches in length.
• Corn rootworms go through three larval instars. After feeding for several weeks, the larvae molt into the pupal stage. They pupate in the soil, and then emerge from the soil as adults in July and August.
• Adult rootworms are about 0.25 inches in length.
• Corn rootworm larvae complete development on corn and several other species of grasses. Larvae reared on yellow foxtail emerge later and have a smaller head capsule size as adults than larvae reared on corn. Ellsbury et al. (2005) Environ. Entomol. 34:627-34.
• WCR adults feed on corn silk, pollen, and kernels on exposed ear tips. Jf WCR adults emerge before corn reproductive tissues are present, they may feed on leaf tissue, thereby slowing plant growth and occasionally killing the host plant. However, the adults will quickly shift to preferred silks and pollen when they become available. NCR adults also feed on reproductive tissues of the corn plant, but in contrast rarely feed on corn leaves.
• rootworm damage in corn is caused by larval feeding. Newly hatched rootworms initially feed on fine corn root hairs and burrow into root tips. As the larvae grow larger, they feed on and burrow into primary roots. When corn rootworms are abundant, larval feeding often results in the pruning of roots all the way to the base of the corn stalk. Severe root injury interferes with the roots' ability to transport water and nutrients into the plant, reduces plant growth, and results in reduced grain production, thereby often drastically reducing overall yield. Severe root injury also often results in lodging of corn plants, which makes harvest more difficult and further decreases yield. Furthermore, feeding by adults on the corn reproductive tissues can result in pruning of silks at the ear tip. If this "silk clipping" is severe enough during pollen shed, pollination may be disrupted.
• Control of corn rootworms may be attempted by crop rotation, chemical insecticides, biopesticides (e.g., the spore-foirning gram-positive bacterium, Bacillus thuringiensis), transgenic plants that express Bt toxins, or a combination thereof
• Crop rotation suffers from the disadvantage of placing unwanted restrictions upon the use of farmland.
• oviposition of some rootworm species may occur in soybean fields, thereby mitigating the effectiveness of crop rotation practiced with corn and soybean.
• Chemical insecticides are the most heavily relied upon strategy for achieving corn rootworm control. Chemical insecticide use, though, is an imperfect corn rootworm control strategy; over $1 billion may be lost in the United States each year due to corn rootworm when the costs of the chemical insecticides are added to the costs of the rootworm damage that may occur despite the use of the insecticides. High populations of larvae, heavy rains, and improper application of the insecticide(s) may all result in inadequate corn rootworm control. Furthermore, the continual use of insecticides may select for insecticide-resistant rootworm strains, as well as raise significant environmental concerns due to the toxicity to non-target species.
• PB European pollen beetles
• PB European pollen beetles
• the primary pest species is Meligethes aeneus.
• pollen beetle control in oilseed rape relies mainly on pyrethroids which are expected to be phased out soon because of their environmental and regulatory profile.
• pollen beetle resistance to existing chemical insecticides has been reported. Therefore, urgently needed are environmentally fiiendly pollen beetle control solutions with novel modes of action.
• pollen beetles overwinter as adults in the soil or under leaf litter.
• the adults emerge from hibernation and start feeding on flowers of weeds, and migrate onto flowering oilseed rape plants.
• the eggs are laid in oilseed rape flower buds.
• the larvae feed and develop in the buds and on the flowers. Late stage larvae find a pupation site in the soil.
• the second generation of adults emerge in July and August and feed on various flowering plants before finding sites for overwintering.
• RNA interference is a process utilizing endogenous cellular pathways, whereby an interfering RNA (iRNA) molecule (e.g., a dsRNA molecule) that is specific for all, or any portion of adequate size, of a target gene results in the degradation of the mRNA encoded thereby.
• iRNA interfering RNA
• RNAi has been used to perform gene "knockdown" in a number of species and experimental systems; for example, Caenorhabditiselegans, plants, insect embryos, and cells in tissue culture. See, e.g., ⁇ ke et al. (1998) Nature 391 :806- 11; Martinez et al. (2002) Cell 110:563-74; McManus and Sharp (2002) Nature Rev. Genetics 3 :737-47.
• RNAi accomplishes degradation of mRNA through an endogenous pathway including the DICER protein complex.
• DICER cleaves long dsRNA molecules into short fragments of approximately 20 nucleotides, termed small interfering RNA (siRNA).
• the siRNA is unwound into two single- stranded RNAs: the passenger strand and the guide strand.
• the passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC).
• RISC RNA-induced silencing complex
• Micro ribonucleic acids are structurally very similar molecules that are cleaved from precursor molecules containing a polynucleotide "loop" connecting the hybridized passenger and guide strands, and they may be similarly incorporated into RISC.
• Post- transcriptional gene silencing occurs when the guide strand binds specifically to a complementary mRNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout the organism despite initially limited concentrations of siRNA and/or miRNA in some eukaryotes such as plants, nematodes, and some insects.
• DICER genes In plants, several functional groups of DICER genes exist. The gene silencing effect of RNAi persists for days and, under experimental conditions, can lead to a decline in abundance of the targeted transcript of 90% or more, with consequent reduction in levels of the corresponding protein. In insects, there are at least two DICER genes, where DICERl facilitates miRN A- directed degradation by Argonautel . Lee et al. (2004) Cell 117 (1):69-81. DICER2 facilitates siRNA- directed degradation by Argonaute2.
• U.S. Patent 7,612,194 and U.S. Patent Publication Nos. 2007/0050860, 2010/0192265, and 2011/0154545 disclose a library of 9112 expressed sequence tag (EST) sequences isolated from D. v. virgiferaLeCo te pupae. It is suggested in U.S. Patent 7,612, 194 and U.S. Patent Publication No. 2007/0050860 to operably link to a promoter a nucleic acid molecule that is complementary to one of several particular partial sequences of D. v. virgifera vacuolar-type H + -ATPase (V-ATPase) disclosed therein for the expression of anti- sense RNA in plant cells.
• V-ATPase V-ATPase
• 2010/0192265 suggests operably linking a promoter to a nucleic acid molecule that is complementary to a particular partial sequence of aD. v. virgifera gene of unknown and undisclosed function (the partial sequence is stated to be 58% identical to C56C10.3 gene product in C. elegans) for the expression of anti- sense RNA in plant cells.
• U.S. Patent Publication No. 2011/0154545 suggests operably linking a promoter to a nucleic acid molecule that is complementary to two particular partial sequences of D. v. virgifera coatomer beta subunit genes for the expression of anti- sense RNAin plant cells. Further, U.S.
• Patent 7,943,819 discloses a library of 906 expressed sequence tag (EST) sequences isolated from / ) , v. virgif era LeConte larvae, pupae, and dissected midguts, and suggests operably linking a promoter to a nucleotide sequence that is complementary to a particular partial sequence of a D. v. virgifera charged multivesicular body protein 4b gene for the expression of double- stranded RNA in plant cells.
• EST expressed sequence tag
• Patent 7,943,819 provides no suggestion to use any particular sequence of the more than nine hundred sequences listed therein for RNA interference, other than the particular partial sequence of a charged multivesicular body protein 4b gene. Furthermore, U.S. Patent 7,943,819 provides no guidance as to which other of the over nine hundred sequences provided would be lethal, or even otherwise useful, in species of corn rootworm when used as dsR A or siRNA.
• U.S. Patent Application Publication No. U.S. 2013/040173 and PCT Application Publication No. WO 2013/169923 describe the use of a sequence derived from a Diabrotica virgif era Snf7 gene for R A interference in maize. (Also disclosed in Bolognesi et al. (2012) PLOS ONE 7(10): e47534. doi:10.1371/journal.pone.0047534).
• dsRNA double- stranded RNAs
• nucleic acid molecules e.g., target genes, DNAs, dsR As, siRNAs, miRNAs, shRNAs, and hpRNAs
• methods of use thereof for the control of insect pests, including, for example, coleopteran pests, such asD. v. virgiferaLeConte (western corn rootworm, "WCR”); D. barberi Smith and Lawrence (northern corn rootworm, 'NCR”); D. u. howardi Barber (southern corn rootworm, "SCR”); D. v. zeae Krysan and Smith (Mexican corn rootworm, "MCR”); D. balteata LeConte; D.
• coleopteran pests such asD. v. virgiferaLeConte (western corn rootworm, "WCR”); D. barberi Smith and Lawrence (northern corn rootworm, 'NCR”); D. u. howardi Barber (
• exemplary nucleic acid molecules are disclosed that may be homologous to at least a portion of one or more native nucleic acids in an insect pest.
• the native nucleic acid sequence may be a target gene, the product of which may be, for example and without limitation: involved in a metabolic process; or involved in larval development.
• post- transcriptional inhibition of the expression of a target gene by a nucleic acid molecule comprising a polynucleotide homologous thereto may be lethal to an insect pest or result in reduced growth and/or development of an insect pest.
• pre- mRNA processing fact or 8 (prp8) or a vpShomolog may be selected as a target gene for post-transcriptional silencing.
• a target gene useful for post- transcriptional inhibition is aprp8 gene selected from the group consisting oiDiabrotica prp8 (e.g., SEQ ID NO:l (prp8-l) and SEQ ID NO:3 (prp8-2)) and Meligethes prpS (e.g., SEQ ID NO:5).
• oiDiabrotica prp8 e.g., SEQ ID NO:l (prp8-l) and SEQ ID NO:3 (prp8-2)
• Meligethes prpS e.g., SEQ ID NO:5
• An isolated nucleic acid molecule comprising the polynucleotide of SEQ ID NO :1; the complement of SEQ ID NO:l; SEQ ID NO:3; the complement of SEQ ID NO:3; SEQ ID NO:5; the complement of SEQ ID NO:5; and/or fragments of any of the foregoing (e.g., SEQ ID NOs:7-12 and the complements thereof) is therefore disclosed herein.
• nucleic acid molecules comprising a polynucleotide that encodes a polypeptide that is at least about 85% identical to an amino acid sequence within a target gene product (for example, the product of a prp8 gene).
• a nucleic acid molecule may comprise a polynucleotide encoding a polypeptide that is at least 85% identical to Diabrotica PRP8 (e.g., SEQ ID NO:2 and SEQ ID NO:4); MeligethesPKPS (e.g., SEQ ID NO:6); and/or an amino acid sequence within a product of a prp8 gene.
• nucleic acid molecules comprising a polynucleotide that is the reverse complement of a polynucleotide that encodes a polypeptide at least 85% identical to an amino acid sequence within a target gene product.
• cDNA polynucleotides that may be used for the production of iRNA (e.g., dsR A, siR A, shRNA, miR A, and hpR A) molecules that are complementary to all or part of an insect pest target gene, for example, an prp8 gene.
• dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be produced in vitro or in vivo by a genetically- modified organism, such as a plant or bacterium.
• cDNA molecules are disclosed that may be used to produce iRNA molecules that are complementary to all or part of a prp8 gene (e.g., SEQ ID NCvl, SEQ ID N03, and SEQ ID NO:5).
• a means for inhibiting expression of a/vpS gene in a Diabrotica pest is a double- stranded RNA molecule, wherein one strand of the molecule consists of a polyribonucleotide selected from the group consisting of SEQ ID NOs:86-90; and the complements thereof
• Functional equivalents of means for inhibiting expression of a prp8 gene in a Diabrotica pest include double- stranded RNA molecules comprising a polyribonucleotide that is substantially homologous to all or part of a Diabrotica prp8 gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:7- 11.
• a means for providing /vpS-mediated Diabrotica pest protection to a plant is a DNA molecule comprising a polynucleotide encoding a means for inhibiting expression of a prp8 gene in a Diabrotica pest operably linked to a promoter, wherein the DNA molecule is capable of being integrated into the genome of a plant.
• a means for inhibiting expression of a/vpS gene in aMeligethes pest is a double- stranded RNA molecule, wherein one strand of the molecule consists of the polyribonucleotide of SEQ ID NO:92; and the complements thereof
• Functional equivalents of means for inhibiting expression of aprp8 gene in a Meligethes pest include double- stranded R A molecules comprising a polyribonucleotide that is substantially homologous to all or part of a Meligethes prp8 gene comprising SEQ ID NO: 12.
• a means for providing mediated Meligethes pest protection to a plant is a DNA molecule comprising a polynucleotide encoding a means for inhibiting expression ofa/vpSgene in & Meligethes pest operably linked to a promoter, wherein the DNA molecule is capable of being integrated into the genome of a plant
• an insect pest e.g., a coleopteran pest
• methods for controlling a population of an insect pest comprising providing to an insect pest ⁇ e.g., a coleopteran pest) an iRNA (e.g., dsR A, siRNA, shRNA, miRNA, and hpRNA) molecule that functions upon being taken up by the pest to inhibit a biological function within the pest.
• an iRNA e.g., dsR A, siRNA, shRNA, miRNA, and hpRNA
• methods for controlling a population of a coleopteran pest comprises providing to the coleopteran pest an iR A molecule that comprises all or part of a polynucleotide selected from the group consisting of: SEQ ID NO:84; the complement of SEQ ID NO:84; SEQ ID NO:85; the complement of SEQ ID NO:85; SEQ ID NO:86; the complement of SEQ ID NO:86; SEQ IDNO:87; the complement of SEQ ID NO:87; SEQ ID NO:88; the complement of SEQ ID NO:88; SEQ ID NO:89; the complement of SEQ ID NO:89; SEQ ID NO:90; the complement of SEQ ID NO:90; SEQ ID NO:91; the complement of SEQ ID NO:91; SEQ IDNO:92; the complement of SEQ ID NO :92; a polynucleotide that hybridizes to a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising of a
• an iRNA that functions upon being taken up by an insect pest to inhibit a biological function within the pest is transcribed from a DNA comprising all or part of a polynucleotide selected from the group consisting of SEQ ID NO : 1 ; the complement of SEQ ID NO : 1 ; SEQ ID NO :3 ; the complement of SEQ ID NO:3; SEQ ID NO:5; the complement of SEQ ID NO:5; a native coding polynucleotide of a Diabrotica organism ⁇ e.g., WCR) comprising all or part of any of SEQ ID NOs:7- 11; the complement of a native coding polynucleotide of a Diabrotica organism comprising all or part of any of SEQ ID NOs:7- l 1; a native coding polynucleotide of a Meligethes organism (e.g., PB) comprising all or part of SEQ ID NO: 12; and the complement of a native coding
• dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be provided to an insect pest in a diet-based assay, or in genetically- modified plant cells expressing the dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs.
• the dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be ingested by the pest.
• RNAi ingestion of dsRNAs, siRNA, shRNAs, miRNAs, and/or hpRNAs of the invention may then result in RNAi in the pest, which in turn may result in silencing of a gene essential for viability of the pest and leading ultimately to mortality.
• a coleopteran pest controlled by use of nucleic acid molecules of the invention may be WCR, NCR, SCR, and/or PB.
• FIG. 1 includes a depiction of a strategy used to generate dsRNA from a single transcription template with a single pair of primers.
• FIG. 2 includes a depiction of a strategy used to generate dsRNA from two transcription templates.
• nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R ⁇ 1.822.
• the nucleic acid and amino acid sequences listed define molecules (i.e., polynucleotides and polypeptides, respectively) having the nucleotide and amino acid monomers arranged in the manner described.
• the nucleic acid and amino acid sequences listed also each define a genus of polynucleotides or polypeptides that comprise the nucleotide and amino acid monomers arranged in the manner described.
• nucleotide sequence including a coding sequence also describes the genus of polynucleotides encoding the same polypeptide as a polynucleotide consisting of the reference sequence. It will further be understood that an amino acid sequence describes the genus of polynucleotide ORFs encoding that polypeptide.
• nucleic acid sequence Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
• the complement and reverse complement of a primary nucleic acid sequence are necessarily disclosed by the primary sequence, the complementary sequence and reverse complementary sequence of a nucleic acid sequence are included by any reference to the nucleic acid sequence, unless it is explicitly stated to be otherwise (or it is clear to be otherwise from the context in which the sequence appears).
• RNA sequence is included by any reference to the DNA sequence encoding it.
• SEQ ID NO:l shows a contig containing an exemplary WCR prp8 DNA, referred to herein in some places as WC prp8 or WC prp8-l :
• SEQ ID NO:2 shows the amino acid sequence of aPRP8 polypeptide encoded by an exemplary WCR prp8 DNA, referred to herein in some places as WCR PRP8 or WCRPRP8-1:
• SEQ ID NO :3 shows a contig comprising a further exemplary WCR prp8 DNA, referred to herein in some places as WCR prp8-2:
• SEQ ID NO:6 shows the amino acid sequence of a PB PRP8 polypeptide encoded by an exemplary PB prp8 DNA:
• SEQ ID NO:7 shows a further exemplary WCRvpS DNA, referred to herein in some places as WCKprp8-l grommet (region 1), which is used in some examples for the production of a dsRNA:
• SEQ ID NO:8 shows a further exemplary WCRvpSDNA, referred to herein in some places as WCR prp8-2 gr (region 1), which is used in some examples for the production of a dsRNA:
• SEQ ID NO:l 1 shows a further exemplary WC prp8 DNA, referred to herein in some places as WCR prp8-3 v2 (version 2), which is used in some examples for the production of a dsRNA:
• SEQ ID NO:12 shows a further exemplary PB prp8 DNA, referred to herein in some places as PB prp8 gr (region 1), which is used in some examples for the production of a dsRNA:
• AAATGGAAGACCGCCGAAGAAGTAGCCGCTCTAATTCGTTCGCTTCCCGTAG AAGAACAACCCAAACAAATC ATC GT TAC CAGAAAAGGAAT GTT GGAT CCT CT TGAAG T GCATC TT CTT GATT TCC CC AACAT CGT TATCAAAGGATC TGAAC TACAACT GCC TT T CCAGGCT TGCCT CAAGATAGAAAAGTT CGGCGAT TTGAT TTT GAAAGCT ACCGAAC CTCAGATGGTTCTGTTCAATCTTTACGACGATTGGTTGAAGACCATTTCGTCTTACA CCGCCTTCTCCAGGTTGATT TTGAT ATTAAGGGCATTACACGTCAATACAGAAAGAA C TAAGG TT ATT CT TAAAC CC GACAAAAC CACAATT ACC GAGCC GC AT C
• SEQ ID NO:13 shows a nucleotide sequence of T7 phage promoter.
• SEQ ID NO:14 shows a fragment of an exemplary YFP coding region.
• SEQ ID NOs:15-26 show primers used to amplify portions of exemplary prp8 sequences comprising WCKprp8-l regl, WCR prp8-2 gr, WCR prp8-3 gr, WCR prp8-3 vl, WCR prp8-3 v2, and PB prp8 gr used in some examples for dsRNA production.
• SEQ ID NO:27 shows an exemplary YFP gene.
• SEQ ID NO :28 shows a DNA sequence of annexin region 1.
• SEQ ID NO:29 shows a DNA sequence of annexin region 2.
• SEQ ID NO :30 shows a DNA sequence of beta spectrin 2 region 1.
• SEQ ID NO :31 shows a DNA sequence of beta spectrin 2 region 2.
• SEQ ID NO 32 shows a DNA sequence oimtRP-L4 region 1.
• SEQ ID NO 3 shows a DNA sequence oimtEP-L4 region 2.
• SEQ ID NOs:34-61 show primers used to amplify gene regions of annexin, beta spectrin 2, mtRP-L4, and YFP for dsRNA synthesis.
• SEQ ID NO:62 shows a maize DNA sequence encoding a TTP41-like protein.
• SEQ ID NO:63 shows the nucleotide sequence of a T20VN primer oligonucleotide.
• SEQ ID NOs:64-68 show primers and probes used for dsRNA transcript expression analyses in maize.
• SEQ ID NO:69 shows a nucleotide sequence of a portion of a SpecR coding region used for binary vector backbone detection.
• SEQ ID NO:70 shows a nucleotide sequence of an AAD1 coding region used for genomic copy number analysis.
• SEQ ID NO:71 shows a DNA sequence of a maize invertase gene.
• SEQ ID NOs:72-80 show the nucleotide sequences of DNA oligonucleotides used for gene copy number determinations and binary vector backbone detection.
• SEQ ID NOs:81-83 show primers and probes used for dsRNA transcript maize expression analyses.
• SEQ ID NOs:84-92 show exemplary RNAs transcribed from nucleic acids comprising exemplary prp8 polynucleotides and fragments thereof
• RNA interference as a tool for insect pest management, using one of the most likely target pest species for transgenic plants that express dsRNA; the western corn rootworm.
• dsRNA RNA interference
• most genes proposed as targets for RNAi in rootworm larvae do not actually achieve their purpose.
• RNAi- mediated knockdown of prp8 in the exemplary insect pests, western corn rootworm and pollen beetle which is shown to have a lethal phenotype when, for example, iRNA molecules are delivered via ingested or injected prp8 dsRNA.
• the ability to deliver prp8 dsRNAby feeding to insects confers aRNAi effect that is very useful for insect (e.g., coleopteran) pest management.
• insect e.g., coleopteran
• the potential to affect multiple target sequences for example, in larval rootworms and pollen beetle, may increase opportunities to develop sustainable approaches to insect pest management involving RNAi technologies.
• RNAi-mediated control of an insect pest population are also provided.
• DNA plasmid vectors encoding an RNA molecule may be designed to suppress one or more target gene(s) essential for growth, survival, and/or development.
• the RNA molecule may be capable of foirning dsRNA molecules.
• methods are provided for post- transcriptional repression of expression or inhibition of a target gene via nucleic acid molecules that are complementary to a coding or non-coding sequence of the target gene in an insect pest.
• a pest may ingest one or more dsRNA, siRNA, shRNA, miRNA, and/or hpRNA molecules transcribed from all or a portion of a nucleic acid molecule that is complementary to a coding or non- coding sequence of a target gene, thereby providing a plant- protective effect.
• some embodiments involve sequence-specific inhibition of expression of target gene products, using dsRNA, siRNA, shRNA, miRNA and/or hpRNA that is complementary to coding and/or non-coding sequences of the target gene(s) to achieve at least partial control of an insect (e.g., coleopteran) pest.
• an insect e.g., coleopteran
• a set of isolated and purified nucleic acid molecules comprising a polynucleotide, for example, as set forth in one of SEQ ID NOs:l, 3, 5, and fragments thereof
• a stabilized dsRNA molecule may be expressed from these polynucleotides, fragments thereof or a gene comprising one or more of these polynucleotides, for the post- transcriptional silencing or inhibition of a target gene.
• isolated and purified nucleic acid molecules comprise all or part of any of SEQ ID NOs:l, 3, and 5 (e.g., SEQ ID NOs: 7- 12), and/or a complement thereof
• a recombinant host cell e.g., a plant cell
• a recombinant DNA encoding at least one iRNA (e.g., dsRNA) molecule(s).
• an encoded dsRNA molecule(s) may be provided when ingested by an insect (e.g., coleopteran) pest to post- transcriptionally silence or inhibit the expression of a target gene in the pest.
• the recombinant DNA may comprise, for example, any of SEQ ID NOs:l, 3, 5, and 7- 12; fragments of any of SEQ ID NOs:l, 3, 5, and 7-12; and a polynucleotide consisting of a partial sequence of a gene comprising one or more of SEQ ID NOs:l, 3, 5, and 7- 12; and/or complements thereof
• Some embodiments involve a recombinant host cell having in its genome a recombinant DNA encoding at least one iR A (e.g., dsR A) molecule(s) comprising all or part of SEQ ID NO:84, SEQ ID NO:85 or SEQ ID NO:91 (e.g., at least one polyribonucleotide selected from a group comprising SEQ ID NOs:86-90 and 92).
• iR A e.g., dsR A
• SEQ ID NO:91 e.g., at least one polyribonucleo
• the iRNA molecule(s) may silence or inhibit the expression of a target prp8 DNA (e.g., a DNA comprising all or part of a polynucleotide selected from the group consisting of SEQ ID NOs:l, 3, 5, and 7- 12) in the pest, and thereby result in cessation of growth, development, and/or feeding in the pest.
• a target prp8 DNA e.g., a DNA comprising all or part of a polynucleotide selected from the group consisting of SEQ ID NOs:l, 3, 5, and 7- 12
• a recombinant host cell having in its genome at least one recombinant DNA encoding at least one RNA molecule capable of fonning a dsRNA molecule may be a transformed plant cell.
• Some embodiments involve transgenic plants comprising such a transformed plant cell.
• progeny plants of any transgenic plant generation, transgenic seeds, and transgenic plant products, are all provided, each of which comprises recombinant DNA(s).
• an RNA molecule capable of fonning a dsRNA molecule may be expressed in a transgenic plant cell. Therefore, in these and other embodiments, a dsRNA molecule may be isolated from a transgenic plant cell.
• the transgenic plant is a plant selected from the group comprising corn (Zea mays), soybean (Glycine max), cotton, plants of the family Poaceae, and rapeseed (Brassica sp.).
• a nucleic acid molecule may be provided, wherein the nucleic acid molecule comprises a polynucleotide encoding an RNA molecule capable of foirning a dsRNA molecule.
• a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule may be operatively linked to a promoter, and may also be operatively linked to a transcription termination sequence.
• a method for modulating the expression of a target gene in an insect pest cell may comprise: (a) transforming a plant cell with a vector comprising a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule; (b) culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture comprising a plurality of transformed plant cells; (c) selecting for a transformed plant cell that has integrated the vector into its genome; and (d) determining that the selected transformed plant cell comprises the RNA molecule capable of forming a dsRNA molecule encoded by the polynucleotide of the vector.
• a plant may be regenerated from a plant cell that has the vector integrated in its genome and comprises the dsRNA molecule encoded by the polynucleotide of the vector.
• a transgenic plant comprising a vector having a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule integrated in its genome, wherein the transgenic plant comprises the dsRNA molecule encoded by the polynucleotide of the vector.
• expression of an RNA molecule capable of forming a dsRNA molecule in the plant is sufficient to modulate the expression of a target gene in a cell of an insect (e.g., coleopteran) pest that contacts the transformed plant or plant cell (for example, by feeding on the transformed plant, a part of the plant (e.g. , root) or plant cell), such that growth and/or survival of the pest is inhibited.
• insect e.g., coleopteran
• Transgenic plants disclosed herein may display resistance and/or enhanced tolerance to insect pest infestations. Particular transgenic plants may display resistance and/or enhanced protection from one or more coleopteran pest(s) selected from the group consisting of WCR; NCR; SCR; MCR; D. balteata LeConte; D. u. tenella; Meligethes aeneus Fabricius; and D. u. undecimpunctata Mannerheim.
• coleopteran pest(s) selected from the group consisting of WCR; NCR; SCR; MCR; D. balteata LeConte; D. u. tenella; Meligethes aeneus Fabricius; and D. u. undecimpunctata Mannerheim.
• control agents such as an iRNA molecule
• an insect pest e.g., coleopteran
• control agents may cause, directly or indirectly, an impairment in the ability of an insect pest population to feed, grow or otherwise cause damage in a host.
• a method is provided comprising delivery of a stabilized dsRN A molecule to an insect pest to suppress at least one target gene in the pest, thereby causing RNAi and reducing or eliminating plant damage in a pest host.
• a method of inhibiting expression of a target gene in the insect pest may result in cessation of growth, survival, and/or development in the pest.
• compositions e.g., a topical composition
• an iRNA e.g., dsR A
• the composition may be a nutritional composition or food source to be fed to the insect pest.
• Some embodiments comprise making the nutritional composition or food source available to the pest.
• Ingestion of a composition comprising iRNA molecules may result in the uptake of the molecules by one or more cells of the pest, which may in turn result in the inhibition of expression of at least one target gene in cell(s) of the pest.
• Ingestion of or damage to a plant or plant cell by an insect pest infestation may be limited or eliminated in or on any host tissue or environment in which the pest is present by providing one or more compositions comprising an iRNA molecule in the host of the pest.
• compositions and methods disclosed herein may be used together in combinations with other methods and compositions for controlling damage by insect (e.g., coleopteran) pests.
• insect e.g., coleopteran
• an iRNA molecule as described herein for protecting plants from insect pests may be used in a method comprising the additional use of one or more chemical agents effective against an insect pest, biopesticides effective against such a pest, crop rotation, recombinant genetic techniques that exhibit features different from the features of RNAi- mediated methods and RNAi compositions (e.g. , recombinant production of proteins in plants that are harmful to an insect pest (e.g., Bt toxins and PIP- 1 polypeptides (See U.S. Patent Publication No. US 2014/0007292 Al)), and/or recombinant expression of other iRNA molecules.
• Bt toxins and PIP- 1 polypeptides See U.S. Patent Publication No. US 2014/0007292 Al
• siPv A small inhibitory ribonucleic acid
• Coleopteran pest refers to pest insects of the order Coleoptera, including pest insects in the genus Diabrotica, which feed upon agricultural crops and crop products, including corn and other true grasses.
• a coleopteran pest is selected from a list comprising D. v. virgifera LeConte (WCR); D. barberi Smith and Lawrence (NCR); D. u. howardi (SCR); D. v. zeae (MCR); D. balteata LeConte; D. u. tenella, D. u. undecimpunctata Mannerheim; D. speciosa Germar; and Meligethes aeneus Fabricius (PB).
• WCR D. v. virgifera LeConte
• NCR D. barberi Smith and Lawrence
• SCR D. u. howardi
• MCR D. v. zeae
• PB Meligethes aeneus Fabricius
• contact with an organism: As used herein, the term "contact with” or “uptake by” an organism (e.g., a coleopteran pest), with regard to a nucleic acid molecule, includes internalization of the nucleic acid molecule into the organism, for example and without limitation: ingestion of the molecule by the organism (e.g., by feeding); contacting the organism with a composition comprising the nucleic acid molecule; and soaking of organisms with a solution comprising the nucleic acid molecule.
• an organism e.g., a coleopteran pest
• Contig refers to a DNA sequence that is reconstructed from a set of overlapping DNA segments derived from a single genetic source.
• Corn plant As used herein, the term “corn plant” refers to a plant of the species, Zea mays (maize).
• expression of a coding polynucleotide refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., gDNA or cDNA) is converted into an operational, non- operational, or structural part of a cell, often including the synthesis of a protein.
• Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein.
• Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof
• Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, northern blot, RT-PCR, western blot, or in vitro, in situ, or in vivo protein activity assay(s).
• Genetic material includes all genes, and nucleic acid molecules, such as DNA and RNA.
• Inhibition when used to describe an effect on a coding polynucleotide (for example, a gene), refers to a measurable decrease in the cellular level of mRNA transcribed from the coding polynucleotide and/or peptide, polypeptide, or protein product of the coding polynucleotide. In some examples, expression of a coding polynucleotide may be inhibited such that expression is approximately eliminated. "Specific inhibition” refers to the inhibition of a target coding polynucleotide without consequently affecting expression of other coding polynucleotides ⁇ e.g., genes) in the cell wherein the specific inhibition is being accomplished.
• Insect pest As used herein with regard to pests, the term “insect pest” specifically includes coleopteran insect pests.
• Isolated An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs ⁇ i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins), while effecting a chemical or functional change in the component ⁇ e.g., a nucleic acid may be isolated from a chromosome by breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome).
• Nucleic acid molecules and proteins that have been "isolated” include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically-synthesized nucleic acid molecules, proteins, and peptides.
• nucleic acid molecule may refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, gDNA, and synthetic forms and mixed polymers of the above.
• a nucleotide or nucleobase may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide.
• a “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide. " A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified.
• nucleotide sequence of a nucleic acid molecule is read from the 5' to the 3' end of the molecule.
• the "complement" of a nucleic acid molecule refers to a polynucleotide having nucleobases that may form base pairs with the nucleobases of the nucleic acid molecule (z.e., A-T/U, and G-C).
• nucleic acids comprising a template DNA that is transcribed into an RNA molecule that is the complement of an mRNA molecule.
• the complement of the nucleic acid transcribed into the mRNA molecule is present in the 5' to 3' orientation, such that RNA polymerase (which transcribes DNA in the 5' to 3' direction) will transcribe a nucleic acid from the complement that can hybridize to the mRNA molecule.
• the term “complement” therefore refers to a polynucleotide having nucleobases, from 5' to 3', that may form base pairs with the nucleobases of a reference nucleic acid.
• the "reverse complement" of a nucleic acid refers to the complement in reverse orientation. The foregoing is demonstrated in the following illustration:
• Some embodiments of the invention may include hairpin RNA-fonning RNAi molecules.
• RNAi molecules both the complement of a nucleic acid to be targeted by RNA interference and the reverse complement may be found in the same molecule, such that the single- stranded RNA molecule may "fold over" and hybridize to itself over the region comprising the complementary and reverse complementary polynucleotides.
• Nucleic acid molecules include all polynucleotides, for example: single- and double- stranded forms of DNA; single- stranded forms of R A; and double- stranded forms of RNA (dsRNA).
• nucleotide sequence or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex.
• ribonucleic acid (RNA) is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), shRNA (small hairpin RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), IRNA (transfer RNAs, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA).
• DNA deoxyribonucleic acid
• polynucleotide and “nucleic acid,” and “fragments” thereof will be understood by those in the art as a term that includes both gDNAs, ribosomal RNAs, transfer RNAs, messenger RNAs, operons, and smaller engineered polynucleotides that encode or may be adapted to encode, peptides, polypeptides, or proteins.
• Oligonucleotide An oligonucleotide is a short nucleic acid polymer.
• Oligonucleotides may be formed by cleavage of longer nucleic acid segments, or by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred bases in length. Because oligonucleotides may bind to a complementary nucleic acid, they may be used as probes for detecting DNA or RNA. Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be used in PCR, a technique for the amplification of DNAs. In PCR, the oligonucleotide is typically referred to as a "primer," which allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.
• a nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
• Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art.
• nucleic acid molecule also includes any topological conformation, including single- stranded, double- stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
• coding polynucleotide As used herein with respect to DNA, the term “coding polynucleotide, " "structural polynucleotide, “ or “structural nucleic acid molecule” refers to a polynucleotide that is ultimately translated into a polypeptide, via transcription and mRNA, when placed under the control of appropriate regulatory elements. With respect to RNA, the term “coding polynucleotide” refers to a polynucleotide that is translated into a peptide, polypeptide, or protein. The boundaries of a coding polynucleotide are detennined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-teirninus. Coding polynucleotides include, but are not limited to: gDNA; cDNA; EST; and recombinant polynucleotides.
• transcripts of mRNA molecules such as 5'UTR, 3'UTR, and intron segments that are not translated into a peptide, polypeptide, or protein.
• transcribed non-coding polynucleotide refers to a nucleic acid that is transcribed into an RNA that functions in the cell, for example, structural RNAs ⁇ e.g., ribosomal RNA (rRNA) as exemplified by 5S rRNA, 5.8S rRNA, 16S rRNA, 18S rRNA, 23S rRNA, and 28S rRNA, and the like); transfer RNA (tRNA); and snRNAs such as U4, U5, U6, and the like.
• structural RNAs ⁇ e.g., ribosomal RNA (rRNA) as exemplified by 5S rRNA, 5.8S rRNA, 16S rRNA, 18S rRNA, 23S rRNA, and 28S rRNA, and the like
• Transcribed non-coding polynucleotides also include, for example and without limitation, small RNAs (sRNA), which term is often used to describe small bacterial non-coding RNAs; small nucleolar RNAs (snoRNA); microRNAs (miRNA); small interfering RNAs (siRNA); Piwi- interacting RNAs (piRNA); and long non-coding RNAs.
• sRNA small RNAs
• sRNA small nucleolar RNAs
• miRNA microRNAs
• siRNA small interfering RNAs
• piRNA Piwi- interacting RNAs
• long non-coding RNAs long non-coding RNAs.
• “transcribed non- coding polynucleotide” refers to a polynucleotide that may natively exist as an intragenic "spacer" in a nucleic acid and which is transcribed into an RNA molecule.
• Lethal RNA interference refers to RNA interference that results in death or a reduction in viability of the subject individual to which, for example, a dsRNA, miRNA, siRNA, shRNA, and/or hpRNA is delivered.
• Genome refers to chromosomal DNA found within the nucleus of a cell, and also refers to organelle DNA found within subcellular components of the cell.
• a DNA molecule may be introduced into a plant cell, such that the DNA molecule is integrated into the genome of the plant cell.
• the DNA molecule may be either integrated into the nuclear DNA of the plant cell, or integrated into the DNA of the chloroplast or mitochondrion of the plant cell.
• a DNA molecule may be introduced into a bacterium such that the DNA molecule is integrated into the genome of the bacterium.
• the DNA molecule may be either chromosomally-integrated or located as or in a stable plasmid.
• sequence identity refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window.
• the term "percentage of sequence identity” may refer to the value detennined by comparing two optimally aligned sequences (e.g. , nucleic acid sequences or polypeptide sequences) of a molecule over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
• a sequence that is identical at every position in comparison to a reference sequence is said to be 100% identical to the reference sequence, and vice- versa.
• Methods for aligning sequences for comparison are well-known in the art.
• Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73 :237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.
• NCBI National Center for Biotechnology Information
• BLASTTM Basic Local Alignment Search Tool
• Bethesda, MD National Center for Biotechnology Information
• Blastn Blastn
• Specifically hybridizable/Specifically complementary are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the nucleic acid molecule and a target nucleic acid molecule.
• Hybridization between two nucleic acid molecules involves the formation of an anti-parallel alignment between the nucleobases of the two nucleic acid molecules. The two molecules are then able to form hydrogen bonds with corresponding bases on the opposite strand to form a duplex molecule that, if it is sufficiently stable, is detectable using methods well known in the art.
• a polynucleotide need not be 100% complementary to its target nucleic acid to be specifically hybridizable.
• Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acids. Generally, the temperature of hybridization and the ionic strength (especially the Na + and/orMg ++ concentration) of the hybridization buffer will detennine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are known to those of ordinary skill in the art, and are discussed, for example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory Manual 2 nd ed., vol.
• stringent conditions encompass conditions under which hybridization will only occur if there is less than 20% mismatch between the sequence of the hybridization molecule and a homologous polynucleotide within the target nucleic acid molecule.
• Stringent conditions include further particular levels of stringency.
• “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize; and conditions of "very high stringency” are those under which sequences with more than 5% mismatch will not hybridize.
• High Stringency condition detects polynucleotides that share at least 90% sequence identity: Hybridization in 5x SSC buffer at 65 °C for 16 hours; wash twice in 2x SSC buffer at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer at 65 °C for 20 minutes each.
• Moderate Stringency condition detects polynucleotides that share at least 80% sequence identity: Hybridization in 5x-6x SSC buffer at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer at room temperature for 5-20 minutes each; and wash twice in lx SSC buffer at 55-70 °C for 30 minutes each.
• Non- stringent control condition polynucleotides that share at least 50% sequence identity will hybridize: Hybridization in 6x SSC buffer at room temperature to 55 °C for 16-20 hours; wash at least twice in 2x-3x SSC buffer at room temperature to 55 °C for 20-30 minutes each.
• nucleic acid refers to a polynucleotide having contiguous nucleobases that hybridize under stringent conditions to the reference nucleic acid.
• nucleic acids that are substantially homologous to a reference nucleic acid of any of SEQ ID NOs:l, 3, 5, and 7- 12 are those nucleic acids that hybridize under stringent conditions (e.g., the Moderate Stringency conditions set forth, supra) to the reference nucleic acid.
• Substantially homologous polynucleotides may have at least 80%) sequence identity.
• substantially homologous polynucleotides may have from about 80%> to 100%> sequence identity, such as 79%; 80%>; about 81%>; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%.
• the property of substantial homology is closely related to specific hybridization.
• a nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to non- target polynucleotides under conditions where specific binding is desired, for example, under stringent hybridization conditions.
• ortholog refers to a gene in two or more species that has evolved from a common ancestral nucleic acid, and may retain the same function in the two or more species.
• nucleic acid molecules are said to exhibit "complete complementarity" when every nucleotide of a polynucleotide read in the 5' to 3' direction is complementary to every nucleotide of the other polynucleotide when read in the 3' to 5' direction.
• a polynucleotide that is complementary to a reference polynucleotide will exhibit a sequence identical to the reverse complement of the reference polynucleotide.
• a first polynucleotide is operably linked with a second polynucleotide when the first polynucleotide is in a functional relationship with the second polynucleotide.
• operably linked polynucleotides are generally contiguous, and, where necessary to join two protein- coding regions, in the same reading frame (e.g. , in a translationally fused ORF).
• nucleic acids need not be contiguous to be operably linked.
• operably linked when used in reference to a regulatory genetic element and a coding polynucleotide, means that the regulatory element affects the expression of the linked coding polynucleotide.
• regulatory elements or “control elements,” refer to polynucleotides that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding polynucleotide. Regulatory elements may include promoters; translation leaders; introns; enhancers; stem-loop structures; repressor binding polynucleotides; polynucleotides with a termination sequence; polynucleotides with a polyadenylation recognition sequence; etc.
• Particular regulatory elements may be located upstream and/or downstream of a coding polynucleotide operably linked thereto. Also, particular regulatory elements operably linked to a coding polynucleotide may be located on the associated complementary strand of a double- stranded nucleic acid molecule.
• promoter refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
• a promoter may be operably linked to a coding polynucleotide for expression in a cell, or a promoter may be operably linked to a polynucleotide encoding a signal peptide which may be operably linked to a coding polynucleotide for expression in a cell.
• a "plant promoter” may be a promoter capable of initiating transcription in plant cells.
• promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue-preferred”. Promoters which initiate transcription only in certain tissues are referred to as "tissue-specific”. A "cell type- specific" promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
• An "inducible" promoter may be a promoter which may be under environmental control. Examples of environmental conditions that may initiate transcription by inducible promoters include anaerobic conditions and the presence of light.
• Tissue-specific, tissue- preferred, cell type specific, and inducible promoters constitute the class of "non- constitutive" promoters.
• a “constitutive” promoter is a promoter which may be active under most environmental conditions or in most tissue or cell types.
• any inducible promoter can be used in some embodiments of the invention. See Ward et al. (1993) Plant Mol. Biol. 22:361-366. With an inducible promoter, the rate of transcription increases in response to an inducing agent.
• exemplary inducible promoters include, but are not limited to: Promoters from the ACEI system that respond to copper, In2 gene from maize that responds to benzenesulfonamide herbicide safeners; Tet repressor from TnlO; and the inducible promoter from a steroid hormone gene, the transcriptional activity of which may be induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:0421).
• Exemplary constitutive promoters include, but are not limited to: Promoters from plant viruses, such as the 35S promoter from Cauliflower Mosaic Virus (CaMV); promoters from rice actin genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter; and the ALS promoter, Xbal/Ncol fragment 5' to the Brassica napus ALS3 structural gene (or a polynucleotide similar to said Xbal/Ncol fragment) (International PCT Publication No. WO96/30530).
• Promoters from plant viruses such as the 35S promoter from Cauliflower Mosaic Virus (CaMV); promoters from rice actin genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter; and the ALS promoter, Xbal/Ncol fragment 5' to the Brassica napus ALS3 structural gene (or a polynucleotide similar to said Xbal/Ncol fragment)
• tissue-specific or tissue- preferred promoter may be utilized in some embodiments of the invention. Plants transformed with a nucleic acid molecule comprising a coding polynucleotide operably linked to a tissue- specific promoter may produce the product of the coding polynucleotide exclusively, or preferentially, in a specific tissue.
• tissue-specific or tissue-preferred promoters include, but are not limited to: A seed-preferred promoter, such as that from the phaseolin gene; a leaf- specific and light-induced promoter such as that from cab or rubisco; an anther- specific promoter such as that from LAT52; a pollen- specific promoter such as that from ZmI3; and a microspore-preferred promoter such as that from apg.
• transformation refers to the transfer of one or more nucleic acid molecule(s) into a cell.
• a cell is "transformed” by a nucleic acid molecule transduced into the cell when the nucleic acid molecule becomes stably replicated by the cell, either by incorporation of the nucleic acid molecule into the cellular genome, or by episomal replication.
• transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al.
• Transgene An exogenous nucleic acid.
• atransgene may be a DNA that encodes one or both strand(s) of an RNA capable of fonning a dsRNA molecule that comprises a polyribonucleotide that is complementary to a nucleic acid molecule found in a coleopteran pest.
• a transgene may be a gene ⁇ e.g. , a herbicide-tolerance gene, a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable agricultural trait).
• a transgene may contain regulatory elements operably linked to a coding polynucleotide of the transgene ⁇ e.g., a promoter).
• a nucleic acid molecule as introduced into a cell for example, to produce a transformed cell.
• a vector may include genetic elements that permit it to replicate in the host cell, such as an origin of replication. Examples of vectors include, but are not limited to: a plasmid; cosmid; bacteriophage; or virus that carries exogenous DNA into a cell.
• a vector may also include one or more genes, including ones that produce antisense molecules, and/or selectable marker genes and other genetic elements known in the art.
• a vector may transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecules and/or proteins encoded by the vector.
• a vector optionally includes materials to aid in achieving entry of the nucleic acid molecule into the cell (e.g., a liposome, protein coating, etc.).
• Yield A stabilized yield of about 100% or greater relative to the yield of check varieties in the same growing location growing at the same time and under the same conditions.
• improved yield or “improving yield” means a cultivar having a stabilized yield of 105% or greater relative to the yield of check varieties in the same growing location containing significant densities of the coleopteran pests that are injurious to that crop growing at the same time and under the same conditions, which are targeted by the compositions and methods herein.
• nucleic acid molecules useful for the control of insect pests.
• the insect pest is a coleopteran insect pest.
• Described nucleic acid molecules include target polynucleotides (e.g., native genes, and non-coding polynucleotides), dsRNAs, siRNAs, shRNAs, hpRNAs, and miRNAs.
• target polynucleotides e.g., native genes, and non-coding polynucleotides
• dsRNAs e.g., native genes, and non-coding polynucleotides
• siRNAs e.g., native genes, and non-coding polynucleotides
• shRNAs e.g., shRNAs, hpRNAs
• miRNAs e.g., miRNA, shRNA, and/or hpRNA molecules
• miRNAs e.g., miRNA, shRNA, and/or hpRNA molecules
• the native nucleic acid(s) may be one or more target gene(s), the product of
• Nucleic acid molecules described herein when introduced into a cell comprising at least one native nucleic acid(s) to which the nucleic acid molecules are specifically complementary, may initiate RNAi in the cell, and consequently reduce or eliminate expression of the native nucleic acid(s). In some examples, reduction or elimination of the expression of a target gene by a nucleic acid molecule specifically complementary thereto may result in reduction or cessation of growth, development, and/or feeding in the coleopteran pest.
• At least one target gene in an insect pest may be selected, wherein the target gene comprises a coleopteran prp8 polynucleotide.
• a target gene comprising a coleopteran prp8 polynucleotide is selected, wherein the target gene comprises a Diabrotica polynucleotide selected from among SEQ ID NOs:l, 3, and 7- 11.
• a target gene comprising a coleopteran prp8 polynucleotide is selected, wherein the target gene comprises a Meligethes polynucleotide selected from among SEQ ID NOs:5 and 12.
• a target gene may be a nucleic acid molecule comprising a polynucleotide that can be reverse translated in silico to a polypeptide comprising a contiguous amino acid sequence that is at least about 85% identical (e.g., at least 84%>, 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%), or 100%) identical) to the amino acid sequence of a protein product of a prp8 polynucleotide.
• a target gene may be any prp8 polynucleotide in an insect pest, the post-transcriptional inhibition of which has a deleterious effect on the growth and/or survival of the pest, for example, to provide a protective benefit against the pest to a plant.
• a target gene is a nucleic acid molecule comprising a polynucleotide that can be reverse translated in silico to a polypeptide comprising a contiguous amino acid sequence that is at least about 85%> identical, about 90% identical, about 95%) identical, about 96% identical, about 97% identical, about 98% identical, about 99%) identical, about 100% identical, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, and 6.
• RNAs the expression of which results in an RNA molecule comprising a polynucleotide that is specifically complementary to all or part of a native RNA molecule that is encoded by a coding polynucleotide in an insect (e.g., coleopteran) pest.
• insect e.g., coleopteran
• down-regulation of the coding polynucleotide in cells of the pest may be obtained.
• down- regulation of the coding polynucleotide in cells of the insect pest results in a deleterious effect on the growth and/or development of the pest.
• target polynucleotides include transcribed non-coding RNAs, such as 5'UTRs; 3'UTRs; spliced leaders; introns; outrons (e.g., 5'UTR RNA subsequently modified in trans splicing); donatrons (e.g., non- coding RNA required to provide donor sequences for trans splicing); and other non-coding transcribed RNA of target insect pest genes.
• RNAs such as 5'UTRs; 3'UTRs; spliced leaders; introns; outrons (e.g., 5'UTR RNA subsequently modified in trans splicing); donatrons (e.g., non- coding RNA required to provide donor sequences for trans splicing); and other non-coding transcribed RNA of target insect pest genes.
• Such polynucleotides may be derived from both mono-cistronic and poly-cistronic genes.
• iRNA molecules e.g., dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs
• iRNA molecules that comprise at least one polynucleotide that is specifically complementary to all or part of a target nucleic acid in an insect (e.g., coleopteran) pest.
• an iRNA molecule may comprise polynucleotide(s) that are complementary to all or part of a plurality of target nucleic acids; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids.
• an iRNA molecule may be produced in vitro, or in vivo by a genetically- modified organism, such as a plant or bacterium.
• a genetically- modified organism such as a plant or bacterium.
• cDNAs that may be used for the production of dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary to all or part of a target nucleic acid in an insect pest.
• recombinant DNA constructs for use in achieving stable transformation of particular host targets. Transformed host targets may express effective levels of dsRNA, siRNA, miRNA, shRNA, and/or hpRNA molecules from the recombinant DNA constructs.
• a plant transformation vector comprising at least one polynucleotide operably linked to a heterologous promoter functional in a plant cell, wherein expression of the polynucleotide(s) results in an RNA molecule comprising a string of contiguous nucleobases that is specifically complementary to all or part of a target nucleic acid in an insect pest.
• nucleic acid molecules useful for the control of insect (e.g., coleopteran) pests may include: all or part of a native nucleic acid isolated from Diabrotica comprising aprp8 polynucleotide (e.g., any of SEQ ID NOs , 3, and 7-11); DNAs that when expressed result in an RNA molecule comprising a polynucleotide that is specifically complementary to all or part of a native R A molecule that is encoded by Diabrotica prp8; iR A molecules (e.g., dsRNAs, siR As, miR As, shR As, and hpRNAs) that comprise at least one polynucleotide that is specifically complementary to all or part of Diabrotica prp8; cDNAs that may be used for the production of dsR A molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary to all or part of Diabrotica prp8; all or part
• dsRN As siRNAs, miRNAs, shRNAs, and hpRNAs
• siRNAs miRNAs, shRNAs, and hpRNAs
• cDNAs that may be used for the production of dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary to all or part of Meligethes prp8
• recombinant DNA constructs for use in achieving stable transformation of particular host targets, wherein a transformed host target comprises one or more of the foregoing nucleic acid molecules.
• the present invention provides, inter alia, iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules that inhibit target gene expression in a cell, tissue, or organ of an insect (e.g., coleopteran) pest; and DNA molecules capable of being expressed as an iRNA molecule in a cell or microorganism to inhibit target gene expression in a cell, tissue, or organ of an insect pest.
• iRNA e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA
• Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of any of SEQ ID NOs:l, 3, and 5; the complement of
• a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:l, 3, and 5 e.g., any of SEQ ID NOs:7-12
• the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:l, 3, and 5 a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising any of SEQ ID NO s:7- 11
• the complement of a native coding polynucleotide of a Diabrotica organism comprising any of SEQ ID NO s:7- l 1
• a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising any of SEQ ID NOs:7- l l
• contact with or uptake by an insect ⁇ e.g. , coleopteran pest of an iRNA transcribed from the isolated polynucleotide inhibits the growth, development, and/or feeding of the pest.
• contact with or uptake by the insect occurs via feeding on plant material comprising the iRNA.
• contact with or uptake by the insect occurs via spraying of a plant comprising the insect with a composition comprising the iRNA.
• an isolated nucleic acid molecule of the invention may comprise at least one ⁇ e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO :84; the complement of SEQ IDNO:84; SEQ ID NO:85; the complement of SEQ ID NO:85; SEQ ID NO:86; the complement of SEQ ID NO:86; SEQ ID NO :87; the complement of SEQ ID NO :87; SEQ ID NO :88; the complement of SEQ ID NO:88; SEQ ID NO:89; the complement of SEQ ID NO:89; SEQ ID NO:90; the complement of SEQ ID NO:90; SEQ ID NO:91; the complement of SEQ ID NO:91; SEQ ID NO :92; the complement of SEQ ID NO :92; a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:84, 85, and 91; the complement of a fragment of at least
• contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, and/or feeding of the pest.
• contact with or uptake by the insect occurs via feeding on plant material or bait comprising the iR A.
• contact with or uptake by the coleopteran pest occurs via spraying of a plant comprising the insect with a composition comprising the iRNA.
• dsRNA molecules provided by the invention comprise polynucleotides complementary to a transcript from a target gene comprising any of SEQ ID NOs:l, 3, 5, and 7-12, and fragments thereof, the inhibition of which target gene in an insect pest results in the reduction or removal of a polypeptide or polynucleotide agent that is essential for the pest's growth, development, or other biological function.
• a selected polynucleotide may exhibit from about 80% to about 100% sequence identity to any of SEQ ID NOs:l, 3, 5, and 7- 12; a contiguous fragment of any of SEQ ID NOs:l, 3, 5, and 7- 12; and the complement of any of the foregoing.
• a selected polynucleotide may exhibit 79%; 80%; about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; or about 100% sequence identity to any of SEQ ID NOs:l, 3, 5, and 7- 12; a contiguous fragment of any of SEQ ID NOs:l, 3, 5, and 7- 12; and the complement of any of the foregoing.
• a dsRNA molecule is transcribed from a polynucleotide containing a sense nucleotide sequence that is substantially identical or identical to a contiguous fragment of any of SEQ ID NOs:l, 3, 5, and 7- 12; an antisense nucleotide sequence that is at least substantially the reverse complement of the sense nucleotide sequence; and an intervening nucleotide sequence positioned between the sense and the antisense sequences, such that the sense and antisense polyribonucleotides transcribed from the respective nucleotide sequences hybridize to form a "stem" structure in the dsRNA, and polyribonucleotide transcribed from the intervening sequence forms a "loop."
• a dsR A molecule may be referred to as a hairpin R A (hpR A) molecule.
• a DN A molecule capable of being expressed as an iR A molecule in a cell or microorganism to inhibit target gene expression may comprise a single polynucleotide that is specifically complementary to all or part of a native polynucleotide found in one or more target insect pest species (e.g., a coleopteran pest species), or the DNA molecule can be constructed as a chimera from a plurality of such specifically complementary polynucleotides.
• target insect pest species e.g., a coleopteran pest species
• a nucleic acid molecule may comprise a first and a second polynucleotide separated by a "spacer."
• a spacer may be a region comprising any sequence of nucleotides that facilitates secondary structure formation between the first and second polynucleotides, where this is desired.
• the spacer is part of a sense or antisense coding polynucleotide for mR A.
• the spacer may alternatively comprise any combination of nucleotides or homologues thereof that are capable of being linked covalently to a nucleic acid molecule.
• the spacer may be an intron (e.g., as ST-LS 1 intron or a RTM1 intron).
• the DNA molecule may comprise a polynucleotide coding for one or more different iRNA molecules, wherein each of the different iRNA molecules comprises a first polyribonucleotide and a second polyribonucleotide, wherein the first and second polyribonucleotides are complementary to each other.
• the first and second polyribonucleotides may be connected within an RNA molecule by a spacer.
• the spacer may constitute part of the first polyribonucleotide or the second polyribonucleotide.
• RNA molecule comprising the first and second polyribonucleotides
• expression of a RNA molecule may lead to the formation of a dsRNA molecule by specific intramolecular base-pairing of the first and second polyribonucleotides.
• the first polyribonucleotide or the second polyribonucleotide may be substantially identical to a polyribonucleotide (e.g., a transcript of a target gene or transcribed non-coding polynucleotide) native to an insect pest, a derivative thereof, or a complementary polynucleotide thereto.
• dsR A nucleic acid molecules comprise double strands of polymerized ribonucleotides, and may include modifications to either the phosphate- sugar backbone or the nucleoside. Modifications in RNA structure may be tailored to allow specific inhibition.
• dsRN A molecules may be modified through a ubiquitous enzymatic process so that siR A molecules may be generated. This enzymatic process may utilize an RNase ⁇ enzyme, such as DICER in eukaryotes, either in vitro or in vivo. See Elbashir et al. (2001) Nature 411 :494-8; and Hamilton and Baulcombe (1999) Science 286(5441):950-2.
• DICER or functionally-equivalent RNase ⁇ enzymes cleave larger dsRNA strands and/or hpRNA molecules into smaller oligonucleotides ⁇ e.g., siRNAs), each of which is about 19-25 nucleotides in length.
• the siRNA molecules produced by these enzymes have 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini.
• the siRNA molecules generated by RNase ⁇ enzymes are unwound and separated into single- stranded RNA in the cell. The siRNA molecules then specifically hybridize with RNAs transcribed from a target gene, and both RNA molecules are subsequently degraded by an inherent cellular RNA- degrading mechanism.
• siRNA molecules produced by endogenous RNase ⁇ enzymes from heterologous nucleic acid molecules may efficiently mediate the down- regulation of target genes in insect pests.
• a nucleic acid molecule may include at least one non- naturally occurring polynucleotide that can be transcribed into a single- stranded RNA molecule capable of forming a dsRNA molecule in vivo through intermolecular hybridization.
• dsRNAs typically self- assemble, and can be provided in the nutrition source of an insect ⁇ e.g., coleopteran) pest to achieve the post- transcriptional inhibition of a target gene.
• a nucleic acid molecule may comprise two different non-naturally occurring polynucleotides, each of which is specifically complementary to a different target gene in an insect pest.
• the dsRN A molecule inhibits the expression of at least two different target genes in the pest.
• a variety of polynucleotides in insect (e.g., coleopteran) pests may be used as targets for the design of nucleic acid molecules, such as iRNAs and DNA molecules encoding iRNAs. Selection of native polynucleotides is not, however, a straight- forward process. For example, only a small number of native polynucleotides in a coleopteran pest will be effective targets. It cannot be predicted with certainty whether a particular native polynucleotide can be effectively down- regulated by nucleic acid molecules of the invention, or whether down- regulation of a particular native polynucleotide will have a detrimental effect on the growth, viability, feeding, and/or survival of an insect pest.
• nucleic acid molecules e.g., dsRNA molecules to be provided in the host plant of an insect pest
• target cDNAs that encode proteins or parts of proteins essential for pest development and/or survival, such as polypeptides involved in metabolic or catabolic biochemical pathways, cell division, energy metabolism, digestion, host plant recognition, and the like.
• ingestion of compositions by a target pest organism containing one or more dsRNAs, at least one segment of which is specifically complementary to at least a substantially identical segment of RNA produced in the cells of the target pest organism can result in the death or other inhibition of the target.
• a polynucleotide, either DNA or RNA, derived from an insect pest can be used to construct plant cells protected against infestation by the pests.
• the host plant of the coleopteran pest (e.g., Z mays or Brassica sp.), for example, can be transformed to contain one or more polynucleotides derived from the coleopteran pest as provided herein.
• the polynucleotide transformed into the host may encode one or more R As that form into a dsRNA structure in the cells or biological fluids within the transformed host, thus making the dsRNA available if/when the pest forms a nutritional relationship with the transgenic host. This may result in the suppression of expression of one or more genes in the cells of the pest, and ultimately death or inhibition of its growth or development.
• a gene is targeted that is essentially involved in the growth and development of an insect ⁇ e.g., coleopteran) pest.
• Other target genes for use in the present invention may include, for example, those that play important roles in pest viability, movement, migration, growth, development, infectivity, and establishment of feeding sites.
• a target gene may therefore be a housekeeping gene or a transcription factor.
• a native insect pest polynucleotide for use in the present invention may also be derived from a homolog ⁇ e.g., an ortholog), of a plant, viral, bacterial or insect gene, the function of which is known to those of skill in the art, and the polynucleotide of which is specifically hybridizable with a target gene in the genome of the target pest.
• a homolog ⁇ e.g., an ortholog
• the invention provides methods for obtaining a nucleic acid molecule comprising a polynucleotide for producing an iRNA ⁇ e.g., dsRNA, siRNA, rniRNA, shRNA, and hpRNA) molecule.
• iRNA e.g., dsRNA, siRNA, rniRNA, shRNA, and hpRNA
• One such embodiment comprises: (a) analyzing one or more target gene(s) for their expression, function, and phenotype upon dsR A- mediated gene suppression in an insect ⁇ e.g., coleopteran) pest; (b) probing a cDNA or gDNA library with a probe comprising all or a portion of a polynucleotide or a homolog thereof from a targeted pest that displays an altered ⁇ e.g., reduced) growth or development phenotype in a dsRNA-mediated suppression analysis; (c) identifying a DNA clone that specifically hybridizes with the probe; (d) isolating the DNA clone identified in step (b); (e) sequencing the cDNA or gDNA fragment that comprises the clone isolated in step (d), wherein the sequenced nucleic acid molecule comprises all or a substantial portion of the RNA or a homolog thereof; and (f) chemically synthesizing all or a substantial portion of a gene, or an
• a method for obtaining a nucleic acid fragment comprising a polynucleotide for producing a substantial portion of an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule includes: (a) synthesizing first and second oligonucleotide primers specifically complementary to a portion of a native polynucleotide from a targeted insect (e.g., coleopteran) pest; and (b) amplifying a cDNA or gDNA insert present in a cloning vector using the first and second oligonucleotide primers of step (a), wherein the amplified nucleic acid molecule comprises a substantial portion of a siRNA, miRNA, hpRNA, mRNA, shRNA, or dsRNA molecule.
• a target insect e.g., coleopteran
• Nucleic acids can be isolated, amplified, or produced by a number of approaches.
• an iRNA e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA
• a target polynucleotide e.g., a target gene or a target transcribed non-coding polynucleotide
• DNA or RNA may be extracted from a target organism, and nucleic acid libraries may be prepared therefrom using methods known to those ordinarily skilled in the art.
• gDNA or cDNA libraries generated from a target organism may be used for PCR amplification and sequencing of target genes.
• a confirmed PCR product may be used as a template for in vitro transcription to generate sense and antisense RNA with minimal promoters.
• nucleic acid molecules may be synthesized by any of a number of techniques (See, e.g., Oza i et al. (1992) Nucleic Acids Research, 20: 5205- 5214; and Agrawal et al. (1990) Nucleic Acids Research, 18: 5419-5423), including use of an automated DNA synthesizer (for example, a P.E. Biosystems, Inc. (Foster City, Calif) model 392 or 394 DNA/RNA Synthesizer), using standard chemistries, such as phosphoramidite chemistry.
• RNA, dsRNA, siRNA, miRNA, shRNA, orhpRNA molecule of the present invention may be produced chemically or enzymatically by one skilled in the art through manual or automated reactions, or in vivo in a cell comprising a nucleic acid molecule comprising a polynucleotide encoding the RNA, dsRNA, siRNA, miRNA, shRNA, or hpRN A molecule.
• RNA may also be produced by partial or total organic synthesis- any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
• An R A molecule may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase ⁇ e.g., T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase).
• Expression constructs useful for the cloning and expression of polynucleotides are known in the art. See, e.g., International PCT Publication No. WO97/32016; and U.S. Patents 5,593,874, 5,698,425, 5,712, 135, 5,789,214, and 5,804,693.
• RNA molecules that are synthesized chemically or by in vitro enzymatic synthesis may be purified prior to introduction into a cell.
• RNA molecules can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof
• RNA molecules that are synthesized chemically or by in vitro enzymatic synthesis may be used with no or a minimum of purification, for example, to avoid losses due to sample processing.
• the RNA molecules may be dried for storage or dissolved in an aqueous solution.
• the solution may contain buffers or salts to promote annealing, and/or stabilization of dsRNA molecule duplex strands.
• a dsRNA molecule may be formed by a single self- complementary RNA strand or from two complementary RNA strands. dsRNA molecules may be synthesized either in vivo or in vitro.
• An endogenous RNA polymerase of the cell may mediate transcription of the one or two RNA strands in vivo, or cloned RNA polymerase may be used to mediate transcription in vivo or in vitro.
• Post- transcriptional inhibition of a target gene in an insect pest may be host-targeted by specific transcription in an organ, tissue, or cell type of the host ⁇ e.g. , by using a tissue- specific promoter); stimulation of an environmental condition in the host ⁇ e.g. , by using an inducible promoter that is responsive to infection, stress, temperature, and/or chemical inducers); and/or engineering transcription at a developmental stage or age of the host
• RNA strands that form a dsRNA molecule may or may not be polyadenylated, and may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
• the invention also provides a DNA molecule for introduction into a cell ⁇ e.g., a bacterial cell, a yeast cell, or a plant cell), wherein the DNA molecule comprises a polynucleotide that, upon expression to RNA and ingestion by an insect ⁇ e.g., coleopteran) pest, achieves suppression of a target gene in a cell, tissue, or organ of the pest.
• a recombinant nucleic acid molecule comprising a polynucleotide capable of being expressed as an iRNA ⁇ e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule in a plant cell to inhibit target gene expression in an insect pest.
• such recombinant nucleic acid molecules may comprise one or more regulatory elements, which regulatory elements may be operably linked to the polynucleotide capable of being expressed as an iRNA.
• Methods to express a gene suppression molecule in plants are known, and may be used to express a polynucleotide of the present invention. See, e.g., International PC T Publication No. WO06/073727; and U.S. Patent Publication No. 2006/0200878 Al)
• a recombinant DNA molecule of the invention may comprise a polynucleotide encoding an RNA that may form a dsRNA molecule.
• Such recombinant DNA molecules may encode RNAs that may form dsRNA molecules capable of inhibiting the expression of endogenous target gene(s) in an insect ⁇ e.g., coleopteran) pest cell upon ingestion.
• a transcribed RNA may form a dsRNA molecule that may be provided in a stabilized form; e.g., as a hairpin and stem and loop structure.
• one strand of a dsRNA molecule may be formed by transcription from a polynucleotide which is substantially homologous to a polynucleotide selected from the group consisting of SEQ ID NOs:l, 3, and 5; the complements of SEQ ID NOs:l, 3, and 5; a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:l, 3, and 5 ⁇ e.g., SEQ ID NOs:7-12);the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:l, 3, and 5; a native coding polynucleotide of a Diabrotica organism ⁇ e.g., WCR) comprising any of SEQ ID NOs:7- l l; the complement of a native coding polynucleotide of a Diabrotica organism comprising any of SEQ ID NOs:7-l l; a fragment of at least 15 contiguous
• one strand of a dsRNA molecule may be formed by transcription from a polynucleotide that is substantially homologous to a polynucleotide selected from the group consisting of SEQ IDNOs:7-12; the complement of any of SEQ ID NOs:7-12; fragments of at least 15 contiguous nucleotides of any of SEQ ID NOs:7- 12; and the complements of fragments of at least 15 contiguous nucleotides of any of SEQ ID NOs:7- 12.
• a recombinant DN A molecule encoding an RN A that may form a dsRNA molecule may comprise a coding region wherein at least two polynucleotides are arranged such that one polynucleotide is in a sense orientation, and the other polynucleotide is in an antisense orientation, relative to at least one promoter, wherein the sense polynucleotide and the antisense polynucleotide are linked or connected by a spacer of, for example, from about five ( ⁇ 5) to about one thousand (-1000) nucleotides. The spacer may form a loop between the sense and antisense polynucleotides.
• the sense polynucleotide or the antisense polynucleotide may be substantially homologous to a target gene (e.g., a prp8 gene comprising any of SEQ ID
• a recombinant DNA molecule may encode an RNA that may form a dsRNA molecule without a spacer.
• a sense coding polynucleotide and an antisense coding polynucleotide may be different lengths. Polynucleotides identified as having a deleterious effect on an insect pest or a plant- protective effect with regard to the pest may be readily incorporated into expressed dsRNA molecules through the creation of appropriate expression cassettes in a recombinant nucleic acid molecule of the invention.
• such polynucleotides may be expressed as a hairpin with stem and loop structure by taking a first segment corresponding to a target gene polynucleotide (e.g. , a prp8 gene comprising any of SBQ ID NOs:l, 3, 5, and 7- 12, and fragments of any of the foregoing); linking this polynucleotide to a second segment spacer region that is not homologous or complementary to the first segment; and linking this to a third segment, wherein at least a portion of the third segment is substantially complementary to the first segment.
• a target gene polynucleotide e.g. , a prp8 gene comprising any of SBQ ID NOs:l, 3, 5, and 7- 12, and fragments of any of the foregoing
• the transcript of such a construct forms a stem and loop structure by intramolecular base- pairing of the polyribonucleotide encoded by the first segment with the polyribonucleotide encoded by the third segment, wherein the loop structure forms comprising the polyribonucleotide encoded by the second segment.
• the loop structure forms comprising the polyribonucleotide encoded by the second segment.
• a dsRNA molecule may be generated, for example, in the form of a double- stranded structure such as a stem-loop structure ⁇ e.g., hairpin), whereby production of siR A targeted for a native insect ⁇ e.g., coleopteran) pest polynucleotide is enhanced by co-expression of a fragment of the targeted gene, for instance on an additional plant expressible cassette, that leads to enhanced siRNA production, or reduces methylation to prevent transcriptional gene silencing of the dsRNA hairpin promoter.
• a stem-loop structure ⁇ e.g., hairpin
• Certain embodiments of the invention include introduction of a recombinant nucleic acid molecule of the present invention into a plant ⁇ i.e., transformation) to achieve insect ⁇ e.g., coleopteran) pest- inhibitory levels of expression of one or more iRNA molecules.
• a recombinant DNA molecule may, for example, be a vector, such as a linear or a closed circular plasmid.
• the vector system may be a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of a host.
• a vector may be an expression vector.
• Polynucleotides of the invention can, for example, be suitably inserted into a vector under the control of a suitable promoter that functions in one or more hosts to drive expression of a linked coding polynucleotide or other DNA element.
• a suitable promoter that functions in one or more hosts to drive expression of a linked coding polynucleotide or other DNA element.
• Many vectors are available for this purpose, and selection of the appropriate vector will depend mainly on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector.
• Each vector contains various components depending on its function (e.g., amplification of DNA or expression of DNA) and the particular host cell with which it is compatible.
• a recombinant DNA may, for example, be transcribed into an iRNA molecule (e.g., an RNA molecule that forms a dsRNA molecule) within the tissues or fluids of the recombinant plant.
• An iRNA molecule may comprise a polyribonucleotide that is substantially homologous and specifically hybridizable to a corresponding transcribed polyribonucleotide within an insect pest that may cause damage to the host plant species.
• the pest may contact the iRNA molecule that is transcribed in cells of the transgenic host plant, for example, by ingesting cells or fluids of the transgenic host plant that comprise the iRNA molecule.
• expression of a target gene is suppressed by the iRNA molecule within insect pests that infest the transgenic host plant.
• suppression of expression of the target gene in a target coleopteran pest may result in the plant being protected against attack by the pest.
• a recombinant nucleic acid molecule may comprise a polynucleotide of the invention operably linked to one or more regulatory elements, such as a heterologous promoter element that functions in a host cell, such as a bacterial cell wherein the nucleic acid molecule is to be amplified, and a plant cell wherein the nucleic acid molecule is to be expressed.
• Promoters suitable for use in nucleic acid molecules of the invention include those that are inducible, viral, synthetic, or constitutive, all of which are well known in the art.
• Non- limiting examples describing such promoters include U.S. Patents 6,437,217 (maize RS81 promoter); 5,641,876 (rice actin promoter); 6,426,446 (maize
• RS324 promoter 6,429,362 (maize PR- 1 promoter); 6,232,526 (maize A3 promoter); 6, 177,611 (constitutive maize promoters); 5,322,938, 5,352,605, 5,359, 142, and 5,530,196 (CaMV 35S promoter); 6,433,252 (maize L3 oleosin promoter); 6,429,357 (rice actin 2 promoter, and rice actin 2 intron); 6,294,714 (light- inducible promoters); 6, 140,078 (salt- inducible promoters); 6,252, 138 (pathogen- inducible promoters); 6, 175,060 (phosphorous deficiency- inducible promoters); 6,388, 170 (bidirectional promoters); 6,635,806 (gamma-coixin promoter); and U.S.
• Patent Publication No. 2009/757,089 (maize chloroplast aldolase promoter).
• Additional promoters include the nopaline synthase (NOS) promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-9) and the octopine synthase (OCS) promoters (which are carried on tumor- inducing plasmids of Agrobacterium tumefaciens); the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-24); the CaMV 35S promoter (Odell et al.
• NOS nopaline synthase
• OCS octopine synthase
• nucleic acid molecules of the invention comprise a tissue- specific promoter, such as a root- specific promoter.
• Root- specific promoters drive expression of operably-linked coding polynucleotides exclusively or preferentially in root tissue. Examples of root-specific promoters are known in the art. See, e.g., U.S. Patents 5,110,732; 5,459,252 and 5,837,848; and Opperman et al. (1994) Science 263 :221-3; and Hirel et al. (1992) Plant Mol. Biol. 20:207- 18.
• a polynucleotide or fragment for coleopteran pest control according to the invention may be cloned between two root-specific promoters oriented in opposite transcriptional directions relative to the polynucleotide or fragment, and which are operable in a transgenic plant cell and expressed therein to produce RNA molecules in the transgenic plant cell that subsequently may form dsRN A molecules, as described, supra.
• the iRNA molecules expressed in plant tissues may be ingested by an insect pest so that suppression of target gene expression is achieved.
• Additional regulatory elements that may optionally be operably linked to a nucleic acid include 5'UTRs located between a promoter element and a coding polynucleotide that function as a translation leader element.
• the translation leader element is present in fully-processed mRNA, and it may affect processing of the primary transcript, and/or RNA stability.
• Examples of translation leader elements include maize and petunia heat shock protein leaders (U.S. Patent 5,362,865), plant virus coat protein leaders, plant rubisco leaders, and others. See, e.g., Turner and Foster (1995) Molecular Biotech. 3(3)225-36.
• Non-limiting examples of 5'UTRs include GmHsp (U.S. Patent 5,659,122); PhDnaK (U.S.
• Patent 5,362,865 AtAntl; TEV (Carrington and Freed (1990) J. Virol. 64:1590-7); and AGRtunos (GenBankTM Accession No. V00087; and Bevan et al. (1983) Nature 304:184-7).
• Additional regulatory elements that may optionally be operably linked to a nucleic acid also include 3' non- translated elements, 3' transcription teirnination regions, or polyadenylation regions. These are genetic elements located downstream of a polynucleotide, and include polynucleotides that provide polyadenylation signal, and/or other regulatory signals capable of affecting transcription or mRNA processing.
• the polyadenylation signal functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the mRNA precursor.
• the polyadenylation element can be derived from a variety of plant genes, or from T-DNA genes.
• a non-limiting example of a 3' transcription tenriination region is the nopaline synthase 3' region (nos 3'; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7).
• An example of the use of different 3' non-translated regions is provided in Ingelbrecht et al, (1989) Plant Cell 1 :671-80.
• Non-limiting examples of polyadenylation signals include one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. (1984) EMBO J. 3 :1671-9) and AGRtu.nos (GenBankTM Accession No. E01312).
• Some embodiments may include a plant transformation vector that comprises an isolated and purified DNA molecule comprising at least one of the above-described regulatory elements operatively linked to one or more polynucleotides of the present invention.
• the one or more polynucleotides result in one or more iRNA molecule(s) comprising a polyribonucleotide that is specifically complementary to all or part of a native RNA molecule in an insect pest.
• the polynucleotide(s) may comprise a segment encoding all or part of a polyribonucleotide present within a targeted RNA transcript in the insect pest, and may comprise inverted repeats of all or a part of a targeted pest transcript.
• a plant transformation vector may contain polynucleotides specifically complementary to more than one target polynucleotide, thus allowing production of more than one dsRNA for inhibiting expression of two or more genes in cells of one or more populations or species of target insect pests. Segments of polynucleotides specifically complementary to polynucleotides present in different genes can be combined into a single composite nucleic acid molecule for expression in a transgenic plant. Such segments may be contiguous or separated by a spacer.
• a plasmid of the present invention already containing at least one polynucleotide(s) of the invention can be modified by the sequential insertion of additional polynucleotide(s) in the same plasmid, wherein the additional polynucleotide(s) are operably linked to the same regulatory elements as the original at least one polynucleotide(s).
• a nucleic acid molecule may be designed for the inhibition of multiple target genes.
• the multiple genes to be inhibited can be obtained from the same insect (e.g., coleopteran) pest species, which may enhance the effectiveness of the nucleic acid molecule.
• the genes can be derived from different insect pests, which may broaden the range of pests against which the agent(s) is/are effective.
• a polycistronic DNA element can be engineered.
• a recombinant nucleic acid molecule or vector of the present invention may comprise a selectable marker that confers a selectable phenotype on a transformed cell, such as a plant cell.
• Selectable markers may also be used to select for plants or plant cells that comprise a recombinant nucleic acid molecule of the invention.
• the marker may encode biocide resistance, antibiotic resistance (e.g., kanamycin, Geneticin (G418), bleomycin, hygromycin, etc.), or herbicide tolerance (e.g., glyphosate, etc.).
• selectable markers include, but are not limited to: a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene which encodes glyphosate tolerance; a nitrilase gene which confers resistance to bromoxynil; a mutant acetolactate synthase (ALS) gene which confers imidazolinone or sulfonylurea resistance; and a methotrexate resistant DHFR gene.
• a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc
• a bar gene which codes for bialaphos resistance
• a mutant EPSP synthase gene which encodes glyphosate tolerance
• a nitrilase gene which confers resistance to bromoxynil
• ALS acetolactate synthase
• selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, streptomycin and tetracycline, and the like. Examples of such selectable markers are illustrated in, e.g., U.S. Patents 5,550,318; 5,633,435; 5,780,708 and 6,118,047.
• a recombinant nucleic acid molecule or vector of the present invention may also include a screenable marker.
• Screenable markers may be used to monitor expression.
• Exemplary screenable markers include a ⁇ -glucuronidase or uidA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known (Jefferson et al. (1987) Plant Mol. Biol. Rep. 5:387-405); an R- locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al.
• GUS ⁇ -glucuronidase or uidA gene
• recombinant nucleic acid molecules may be used in methods for the creation of transgenic plants and expression of heterologous nucleic acids in plants to prepare transgenic plants that exhibit reduced susceptibility to insect ⁇ e.g., coleopteran) pests.
• Plant transformation vectors can be prepared, for example, by inserting nucleic acid molecules encoding iR A molecules into plant transformation vectors and introducing these into plants.
• Suitable methods for transformation of host cells include any method by which DNA can be introduced into a cell, such as by transformation of protoplasts ⁇ See, e.g., U.S. Patent 5,508,184), by desiccation/inhibition- mediated DNA uptake ⁇ See, e.g., Potrykus et al. (1985) Mol. Gen. Genet. 199:183-8), by electroporation ⁇ See, e.g., U.S. Patent 5,384,253), by agitation with silicon carbide fibers ⁇ See, e.g., U.S. Patents 5,302,523 and 5,464,765), by Agrobacterium -mediated transformation ⁇ See, e.g., U.S.
• transgenic cells may be regenerated into a transgenic organism. Any of these techniques may be used to produce a transgenic plant, for example, comprising one or more nucleic acids encoding one or more iRNA molecules in the genome of the transgenic plant.
• A. turn efaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells.
• the Ti and Ri plasmids of tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant.
• the Ti (tumor-inducing)-plasmids contain a large segment, known as T-DNA, which is transferred to transformed plants.
• Another segment of the Ti plasmid, the Vir region is responsible for T-DNA transfer.
• the T-DNA region is bordered by tenninal repeats.
• the tumor- inducing genes have been deleted, and the functions of the Vir region are utilized to transfer foreign DNA bordered by the T-DNA border elements.
• the T- region may also contain a selectable marker for efficient recovery of transgenic cells and plants, and a multiple cloning site for inserting polynucleotides for transfer such as a dsR A encoding nucleic acid.
• a plant transformation vector is derived from a Ti plasmid of A. tumefaciens ⁇ See, e.g., U.S. Patents 4,536,475, 4,693,977, 4,886,937, and 5,501,967; and European Patent No. EP 0 122 791) or a Ri plasmid of A. rhizogenes.
• Additional plant transformation vectors include, for example and without limitation, those described by Herrera-Estrella et al. (1983)Nature 303 :209-13; Bevan et al. (1983) Nature 304:184-7; Klee et al. (1985)Bio/Technol.
• transformed cells After providing exogenous DNA to recipient cells, transformed cells are generally identified for further culturing and plant regeneration. In order to improve the ability to identify transformed cells, one may desire to employ a selectable or screenable marker gene, as previously set forth, with the transformation vector used to generate the transformant. In the case where a selectable marker is used, transformed cells are identified within the potentially transformed cell population by exposing the cells to a selective agent or agents. In the case where a screenable marker is used, cells may be screened for the desired marker gene trait.
• Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay may be cultured in media that supports regeneration of plants.
• any suitable plant tissue culture media ⁇ e.g., MS and N6 media
• Tissue may be maintained on a basic medium with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration ⁇ e.g., at least 2 weeks), then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoots are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturation.
• a variety of assays may be performed.
• assays include, for example: molecular biological assays, such as Southern and northern blotting, PCR, and nucleic acid sequencing; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISA and/or western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and analysis of the phenotype of the whole regenerated plant.
• molecular biological assays such as Southern and northern blotting, PCR, and nucleic acid sequencing
• biochemical assays such as detecting the presence of a protein product, e.g., by immunological means (ELISA and/or western blots) or by enzymatic function
• plant part assays such as leaf or root assays
• analysis of the phenotype of the whole regenerated plant for example: molecular biological assays, such as Southern and northern blotting,
• Integration events may be analyzed, for example, by PCR amplification using, e.g., oligonucleotide primers specific for a nucleic acid molecule of interest.
• PCR genotyping is understood to include, but not be limited to, polymerase- chain reaction (PCR) amplification of gDNA derived from isolated host plant callus tissue predicted to contain a nucleic acid molecule of interest integrated into the genome, followed by standard cloning and sequence analysis of PCRamplification products. Methods of PCR genotyping have been well described (for example, Rios, G. et al. (2002)Plant J. 32:243- 53) and may be applied to gDNA derived from any plant species ⁇ e.g., Z. mays, cotton, soybean, and B. napus) or tissue type, including cell cultures.
• a transgenic plant formed using Agrobacterium-dependert transformation methods typically contains a single recombinant DNA inserted into one chromosome.
• the polynucleotide of the single recombinant DNA is referred to as a "transgenic event" or "integration event".
• Such transgenic plants are heterozygous for the inserted exogenous polynucleotide.
• a transgenic plant homozygous with respect to a transgene may be obtained by sexually mating (selling) an independent segregant transgenic plant that contains a single exogenous gene to itself for example a To plant, to produce ⁇ seed.
• One fourth of the ⁇ seed produced will be homozygous with respect to the transgene.
• Genriinating ⁇ seed results in plants that can be tested for heterozygosity, typically using an SNP assay or a thermal amplification assay that allows for the distinction between heterozygotes and homozygotes (i.e., a zygosity assay).
• at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different iRNA molecules are produced in a plant cell that have an insect (e.g., coleopteran) pest- inhibitory effect.
• the iRNA molecules e.g., dsRN A molecules
• a plurality of iRNA molecules are expressed under the control of a single promoter. In other embodiments, a plurality of iRNA molecules are expressed under the control of multiple promoters.
• Single iRNA molecules may be expressed that comprise multip le polynucleotides that are each homologous to different loci within one or more insect pests (for example, the loci defined by SEQ ID NOs:l, 3, and 5), both in different populations of the same species of insect pest, or in different species of insect pests.
• transgenic plants can be prepared by crossing a first plant having at least one transgenic event with a second plant lacking such an event.
• a recombinant nucleic acid molecule comprising a polynucleotide that encodes an iRNA molecule may be introduced into a first plant line that is amenable to transformation to produce a transgenic plant, which transgenic plant may be crossed with a second plant line to introgress the polynucleotide that encodes the iRNA molecule into the second plant line.
• seeds and commodity products produced by transgenic plants derived from transformed plant cells are included, wherein the seeds or commodity products comprise a detectable amount of a nucleic acid of the invention.
• such commodity products may be produced, for example, by obtaining transgenic plants and preparing food or feed from them.
• Commodity products comprising one or more of the polynucleotides of the invention includes, for example and without limitation: meals, oils, crushed or whole grains or seeds of a plant, and any food product comprising any meal, oil, or crushed or whole grain of a recombinant plant or seed comprising one or more of the nucleic acid molecules of the invention.
• a commodity product is a bait composition or formulation comprising one or more of the nucleic acid molecules of the invention.
• detection of one or more of the polynucleotides of the invention in one or more commodity or commodity products is de facto evidence that the commodity or commodity product is produced from a transgenic plant designed to express one or more of the iRNA molecules of the invention for the purpose of controlling insect pests.
• a transgenic plant or seed comprising a nucleic acid molecule of the invention also may comprise at least one other transgenic event in its genome, including without limitation: a transgenic event from which is transcribed an iRNA molecule targeting a locus in a coleopteran pest other than the one defined by SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, such as, for example, one or more loci selected from the group consisting of Caf 1-180 (U.S. Patent Application Publication No. 2012/0174258), VatpaseC (U.S. Patent Application Publication No. 2012/0174259), Rhol (U.S. Patent Application Publication No. 2012/0174260), VatpaseH (U.S.
• Patent Application Publication No. 62/133,210 transcription elongation factor spt5 (U.S. Patent Application No. 62/168,613), transcription elongation factor spt6 (U.S. Patent Application No. 62/168,606), ncm (U.S. Patent Application No. 62/095487), dre4 (U.S. Patent Application No. 14/705,807), COPI alpha (U.S. Patent Application No. 62/063,199), COPIbeta (V.S. Patent Application No. 62/063,203), COPI gamma (U.S. Patent Application No. 62/063, 192), and COPI delta (U.S. Patent Application No.
• a gene encoding an insecticidal protem e.g., a Bacillus thuringiensis sectici
• polynucleotides encoding iRNA molecules of the invention may be combined with other insect control and disease traits in a plant to achieve desired traits for enhanced control of plant disease and insect damage.
• genes encoding pesticidal proteins may be combined, including, for example and without limitation: isolated or recombinant nucleic acid molecules encoding Alcaligenes Insecticidal Protein- 1 A and Alcaligenes Insecticidal Protein- IB (AflP- lA and AflP- lB) polypeptides (U.S. Patent Application Publication No. 2014/0033361); or isolated or recombinant nucleic acid molecules encoding PIP polypeptides (WO 2015038734).
• Combining insect control traits that employ distinct modes- of- action may provide protected transgenic plants with superior durability over plants harboring a single control trait, for example, because of the reduced probability that resistance to the trait(s) will develop in the field.
• At least one nucleic acid molecule useful for the control of insect (e.g., coleopteran) pests may be provided to an insect pest, wherein the nucleic acid molecule leads to RNAi- mediated gene silencing in the pest.
• an iRNA molecule e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA
• a nucleic acid molecule useful for the control of insect pests may be provided to a pest by contacting the nucleic acid molecule with the pest.
• a nucleic acid molecule useful for the control of insect pests may be provided in a feeding substrate of the pest, for example, a nutritional composition.
• a nucleic acid molecule useful for the control of an insect pest may be provided through ingestion of plant material comprising the nucleic acid molecule that is ingested by the pest.
• the nucleic acid molecule is present in plant material through expression of a recombinant nucleic acid introduced into the plant material, for example, by transformation of a plant cell with a vector comprising the recombinant nucleic acid and regeneration of a plant material or whole plant from the transformed plant cell.
• a pest is contacted with the nucleic acid molecule that leads to RNAi- mediated gene silencing in the pest through contact with a topical composition (e.g., a composition applied by spraying) or an RNAi bait.
• RNAi baits are formed when the dsRNA is mixed with food or an attractant or both. When the pests eat the bait, they also consume the dsRNA.
• Baits may take the form of granules, gels, flowable powders, liquids, or solids.
• iR A molecules targeting prp8 may be incorporated into a bait formulation such as that described in U.S. Patent No. 8,530,440 which is hereby incorporated by reference.
• the baits are placed in or around the environment of the insect pest, for example, such that the insect pest can come into contact with, and/or be attracted to, the bait.
• the invention provides iRNA molecules ⁇ e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) that may be designed to target essential native polynucleotides ⁇ e.g., essential genes) in the transcriptome of an insect pest (for example, a coleopteran ⁇ e.g. , WCR, SCR, NCR, and PB) pest), for example, by designing an iRNA molecule that comprises at least one strand comprising a polynucleotide that is specifically complementary to the target polynucleotide.
• the sequence of an iRNA molecule so designed may be identical to that of the target polynucleotide, or may incorporate mismatches that do not prevent specific hybridization between the iRNA molecule and its target polynucleotide.
• iRNA molecules of the invention may be used in methods for gene suppression in an insect ⁇ e.g., coleopteran) pest, thereby reducing the level or incidence of damage caused by the pest on a plant (for example, a protected transformed plant comprising an iRNA molecule).
• gene suppression refers to any of the well- known methods for reducing the levels of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA, including the reduction of protein expression from a gene or a coding polynucleotide including post- transcriptional inhibition of expression and transcriptional suppression.
• Post- transcriptional inhibition is mediated by specific homology between all or a part of an mRNA transcribed from a gene targeted for suppression and the corresponding iRNA molecule used for suppression. Additionally, post-transcriptional inhibition refers to the substantial and measurable reduction of the amount of mRNA available in the cell for binding by ribosomes.
• an iRNA molecule is a dsRNA molecule
• the dsRN A molecule may be cleaved by the enzyme, DICER, into short siRNA molecules (approximately 20 nucleotides in length).
• the double- stranded siRNA molecule generated by DICER activity upon the dsRNA molecule may be separated into two single- stranded siRNAs; the "passenger strand" and the "guide strand.”
• the passenger strand may be degraded, and the guide strand may be incorporated into RISC.
• Post- transcriptional inhibition occurs by specific hybridization of the guide strand with a specifically complementary polynucleotide of an mRNA molecule, and subsequent cleavage by the enzyme, Argonaute (catalytic component of the RISC complex).
• any form of iRNA molecule may be used.
• dsRNA molecules typically are more stable during preparation and during the step of providing the iRNA molecule to a cell than are single- stranded RN A molecules, and are typically also more stable in a cell.
• siRNA and miRNA molecules may be equally effective in some embodiments, a dsRNA molecule may be chosen due to its stability.
• a nucleic acid molecule that comprises a polynucleotide, which polynucleotide may be expressed in vitro to produce an iRNA molecule that comprises a polyribonucleotide that is substantially homologous to a polyribonucleotide of an RNA molecule encoded by a polynucleotide within the genome of an insect pest.
• the in vitro transcribed iRNA molecule may be a stabilized dsRNA molecule that comprises a stem-loop structure. After an insect pest contacts the in vitro transcribed iRNA molecule, post- transcriptional inhibition of a target gene in the pest (for example, an essential gene) may occur.
• expression of a nucleic acid molecule comprising at least 15 contiguous nucleotides ⁇ e.g., at least 19 contiguous nucleotides) of a polynucleotide are used in a method for post-transcriptional inhibition of a target gene in an insect ⁇ e.g., coleopteran) pest, wherein the polynucleotide is selected from the group consisting of: SEQ ID NO:l; the complement of SEQ ID NO:l; SEQ ID NO:3; the complement of SEQ ID NO:3; SEQ ID NO:5; the complement of SEQ ID NO:5; SEQ ID NO:7; the complement of SEQ ID NO:7; SEQ ID NO:8; the complement of
• a nucleic acid molecule that is at least about 80% identical (e.g. , 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, and 100%)) with any of the foregoing may be used.
• a nucleic acid molecule may be expressed that specifically hybridizes to a RNA molecule present in at least one cell of a coleopteran insect (e.g., Diabrotica and Meligethes) pest.
• a coleopteran insect e.g., Diabrotica and Meligethes
• the RNAi post- transcriptional inhibition system is able to tolerate sequence variations among target genes that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
• the introduced nucleic acid molecule may not need to be absolutely homologous to either a primary transcription product or a fully-processed mRNA of a target gene, so long as the introduced nucleic acid molecule is specifically hybridizable to either a primary transcription product or a fully-processed mRNA of the target gene.
• the introduced nucleic acid molecule may not need to be full- length, relative to either a primary transcription product or a fully processed mRNA of the target gene.
• Inhibition of a target gene using the iRNA technology of the present invention is sequence- specific; i.e., polynucleotides substantially homologous to the iRNA molecule(s) are targeted for genetic inhibition.
• an RNA molecule comprising a polynucleotide with a nucleotide sequence that is identical to that of a portion of a target gene may be used for inhibition.
• an RNA molecule comprising a polynucleotide with one or more insertion, deletion, and/or point mutations relative to a target polynucleotide may be used.
• an iRNA molecule and a portion of a target gene may share, for example, at least from about 80%, at least from about 81%>, at least from about 82%, at least from about 83%), at least from about 84%>, at least from about 85%>, at least from about 86%>, at least from about 87%, at least from about 88%>, at least from about 89%>, at least from about 90%), at least from about 91%, at least from about 92%, at least from about 93%, at least from about 94%, at least from about 95%, at least from about 96%, at least from about 97%), at least from about 98%, at least from about 99%, at least from about 100%, and 100%) sequence identity.
• the duplex region of a dsRN A molecule may be specifically hybridizable with a portion of a target gene transcript.
• a less than full length polynucleotide exhibiting a greater homology compensates for a longer, less homologous polynucleotide.
• the length of the polynucleotide of a duplex region of a dsRNA molecule that is identical to a portion of a target gene transcript may be at least about 25, 50, 100, 200, 300, 400, 500, or at least about 1000 bases.
• a polynucleotide of greater than 20- 100 nucleotides may be used.
• a polynucleotide of greater than about 200-300 nucleotides may be used.
• a polynucleotide of greater than about 500- 1000 nucleotides may be used, depending on the size of the target gene.
• expression of a target gene in a pest may be inhibited by at least 10%; at least 33%; at least 50%; or at least 80% within a cell of the pest, such that a significant inhibition takes place.
• Significant inhibition refers to inhibition over a threshold that results in a detectable phenotype (e.g., cessation of growth, cessation of feeding, cessation of development, induced mortality, etc.), or a detectable decrease in RNA and/or gene product corresponding to the target gene being inhibited.
• a detectable phenotype e.g., cessation of growth, cessation of feeding, cessation of development, induced mortality, etc.
• inhibition occurs in substantially all cells of the pest, in other embodiments inhibition occurs only in a subset of cells expressing the target gene.
• transcriptional suppression is mediated by the presence in a cell of a dsR A molecule exhibiting substantial sequence identity to a promoter DNA or the complement thereof to effect what is referred to as "promoter trans suppression.”
• Gene suppression may be effective against target genes in an insect pest that may ingest or contact such dsRNA molecules, for example, by ingesting or contacting plant material containing the dsRNA molecules.
• dsRNA molecules for use in promoter trans suppression may be specifically designed to inhibit or suppress the expression of one or more homologous or complementary polynucleotides in the cells of the insect pest.
• Post-transcriptional gene suppression by antisense or sense oriented RNA to regulate gene expression in plant cells is disclosed in U.S. Patents 5,107,065; 5,759,829; 5,283,184; and 5,231,020.
• RNAi-mediated gene inhibition in an insect (e.g., coleopteran) pest may be carried out in any one of many in vitro or in vivo formats.
• the iRNA molecules may then be provided to an insect pest, for example, by contacting the iRNA molecules with the pest, or by causing the pest to ingest or otherwise internalize the iRNA molecules.
• Some embodiments include transformed host plants of a coleopteran pest, transformed plant cells, and progeny of transformed plants.
• the transformed plant cells and transformed plants may be engineered to express one or more of the iRNA molecules, for example, under the control of a heterologous promoter, to provide a pest-protective effect.
• the pest when a transgenic plant or plant cell is consumed by an insect pest during feeding, the pest may ingest iRNA molecules expressed in the transgenic plants or cells.
• the polynucleotides of the present invention may also be introduced into a wide variety of prokaryotic and eukaryotic microorganism hosts to produce iRNA molecules.
• the term "microorganism" includes prokaryotic and eukaryotic species, such as bacteria and fungi.
• Modulation of gene expression may include partial or complete suppression of such expression.
• a method for suppression of gene expression in an insect (e.g., coleopteran) pest comprises providing in the tissue of the host of the pest a gene- suppressive amount of at least one dsRNA molecule formed following transcription of a polynucleotide as described herein, at least one segment of which is complementary to an mR A within the cells of the insect pest.
• a dsR A molecule including its modified form such as an siRNA, miRNA, shRNA, or hpRNA molecule, ingested by an insect pest may be at least from about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%) identical to an RN A molecule transcribed from a prp8 DN A molecule, for example, comprising a polynucleotide selected from the group consisting of SEQ ID NOs:l, 3, 5, and 7- 12.
• Isolated and substantially purified nucleic acid molecules including, but not limited to, non-naturally occurring polynucleotides and recombinant DNA constructs for providing dsRNA molecules are therefore provided, which suppress or inhibit the expression of an endogenous coding polynucleotide or a target coding polynucleotide in an insect pest when introduced thereto.
• a delivery system for the delivery of iRNA molecules for the post- transcriptional inhibition of one or more target gene(s) in an insect ⁇ e.g., coleopteran) plant pest and control of a population of the plant pest.
• the delivery system comprises ingestion of a host transgenic plant cell or contents of the host cell comprising RNA molecules transcribed in the host cell.
• a transgenic plant cell or a transgenic plant is created that contains a recombinant DNA construct providing a stabilized dsRNA molecule of the invention.
• Transgenic plant cells and transgenic plants comprising nucleic acids encoding a particular iRNA molecule may be produced by employing recombinant DNA technologies (which basic technologies are well-known in the art) to construct a plant transformation vector comprising a polynucleotide encoding an iRNA molecule of the invention (e.g. , a stabilized dsRNA molecule); to transform a plant cell or plant; and to generate the transgenic plant cell or the transgenic plant that contains the transcribed iR A molecule.
• a plant transformation vector comprising a polynucleotide encoding an iRNA molecule of the invention (e.g. , a stabilized dsRNA molecule)
• a recombinant DN A molecule may, for example, be transcribed into an iRNA molecule, such as a dsRNA molecule, a siRNA molecule, a miRNA molecule, a shRNA molecule, or a hpRNA molecule.
• a RNA molecule transcribed from a recombinant DNA molecule may form a dsRNA molecule within the tissues or fluids of the recombinant plant.
• Such a dsRNA molecule may comprise in part a polyribonucleotide that is identical to a corresponding polyribonucleotide transcribed from a DNA within an insect pest of a type that may infest the host plant. Expression of a target gene within the pest is suppressed by the dsRNA molecule, and the suppression of expression of the target gene in the pest results in the transgenic plant being protected against the pest.
• the modulatory effects of dsRNA molecules have been shown to be applicable to a variety of genes expressed in pests, including, for example, endogenous genes responsible for cellular metabolism or cellular transformation, including house-keeping genes; transcription factors; molting- related genes; and other genes which encode polypeptides involved in cellular metabolism or normal growth and development.
• a regulatory region ⁇ e.g., promoter, enhancer, silencer, and polyadenylation signal
• a polynucleotide for use in producing iRNA molecules may be operably linked to one or more promoter elements functional in a plant host cell.
• the promoter may be an endogenous promoter, normally resident in the host genome.
• the polynucleotide of the present invention, under the control of an operably linked promoter element, may further be flanked by additional elements that advantageously affect its transcription and/or the stability of a resulting transcript.
• Such elements may be located upstream of the operably linked promoter, downstream of the 3' end of the expression construct, and may occur both upstream of the promoter and downstream of the 3' end of the expression construct.
• Some embodiments provide methods for reducing the damage to a host plant (e.g., a corn plant) caused by an insect (e.g., coleopteran) pest that feeds on the plant, wherein the method comprises providing in the host plant a transformed plant cell expressing at least one nucleic acid molecule of the invention, wherein the nucleic acid molecule(s) functions upon being taken up by the pest(s) to inhibit the expression of a target polynucleotide within the pest(s), which inhibition of expression results in mortality and/or reduced growth of the pest(s), thereby reducing the damage to the host plant caused by the pest(s).
• the nucleic acid molecule(s) comprise dsRNA molecules. In these and further embodiments, the nucleic acid molecule(s) comprise dsRNA molecules that each comprise more than one polyribonucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell. In some embodiments, the nucleic acid molecule(s) consist of one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in an insect pest cell.
• a method for increasing the yield of a crop comprises introducing into a plant at least one nucleic acid molecule comprising a polynucleotide of the invention; cultivating the plant to allow the expression of an iRNA molecule from the polynucleotide, wherein expression of the iRNA molecule inhibits insect pest damage and/or growth, thereby reducing or eliminating a loss of yield due to pest infestation.
• the iRNA molecule is a dsRNA molecule.
• the dsRNA molecules may each comprise more than one polyribonucleotide that is specifically hybridizable to a nucleic acid molecule expressed in an insect pest cell.
• specifically polyribonucleotides of a dsRNA molecule may be expressed from one or more nucleotide sequences within a polynucleotide of the invention.
• a method for modulating the expression of a target gene in an insect pest comprising: transforming a plant cell with a vector comprising a polynucleotide encoding at least one iRNA molecule of the invention, wherein the polynucleotide is operatively-linked to a promoter and a transcription termination element; culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture including a plurality of transformed plant cells; selecting for transformed plant cells that have integrated the polynucleotide into their genomes; screening the transformed plant cells for expression of an iRNA molecule encoded by the integrated polynucleotide; selecting a transgenic plant cell that expresses the iRNA molecule; and feeding the selected transgenic plant cell to the insect pest.
• Plants may also be regenerated from transgenic plant cells that express an iRNA molecule encoded by the integrated nucleic acid molecule.
• the iRNA molecule is a dsRNA molecule comprising a polyribonucleotide that is specifically hybridizable to the transcript of a target gene in the insect pest.
• the dsRN A molecules comprise more than one polyribonucleotide that is transcribed from a nucleotide sequence within the polynucleotide encoding the dsRNA molecule.
• iRNA molecules of the invention can be incorporated within the seeds of a plant species (e.g. , corn and canola), either as a product of expression from a recombinant gene incorporated into a genome of the plant cells, or as incorporated into a coating or seed treatment that is applied to the seed before planting.
• a plant cell comprising a recombinant gene is considered to be a transgenic event.
• delivery systems for the delivery of iRNA molecules to insect (e.g., coleopteran) pests for example, the iRNA molecules of the invention may be directly introduced into the cells ofa pest(s).
• Methods for introduction may include direct mixing of iRNA with plant tissue from a host for the insect pest(s), as well as application of compositions comprising iRNA molecules of the invention to host plant tissue.
• iRNA molecules may be sprayed onto a plant surface.
• an iRNA molecule may be expressed by a microorganism, and the microorganism may be applied onto the plant surface, or introduced into a root or stem by a physical means such as an injection.
• a transgenic plant may also be genetically engineered to express at least one iRNA molecule in an amount sufficient to kill the insect pests known to infest the plant.
• iRNA molecules produced by chemical or enzymatic synthesis may also be formulated in a manner consistent with common agricultural practices, and used as spray-on or bait products for controlling plant damage by an insect pest.
• the formulations may include the appropriate adjuvants (e.g., stickers and wetters) required for efficient foliar coverage, as well as UV protectants to protect iRNA molecules (e.g., dsRNA molecules) from UV damage.
• adjuvants e.g., stickers and wetters
• UV protectants to protect iRNA molecules (e.g., dsRNA molecules) from UV damage.
• Such additives are commonly used in the bioinsecticide industry, and are well known to those skilled in the art.
• Such applications may be combined with other spray-on insecticide applications (biologically based or otherwise) to enhance plant protection from the pests.
• dsRNA molecules including those corresponding to prp8-l
• the purified dsRNA molecules were prepared in TE buffer, and all bioassays contained a control treatment consisting of this buffer, which served as a background check for mortality or growth inhibition of WCR (Diabrotica virgifera virgiferaLeCorte).
• the concentrations of dsRNA molecules in the bioassay buffer were measured using a NANODROPTM 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, DE). Samples were tested for insect activity in bioassays conducted with neonate insect larvae on artificial insect diet.
• WCR eggs were obtained from CROP CHARACTERISTIC S, INC. (Farmington, MN).
• the bioassays were conducted in 128- well plastic trays specifically designed for insect bioassays (C-D INTERNATIONAL, Pitman, NJ). Each well contained approximately 1.0 mL of an artificial diet designed for growth of coleopteran insects. A 60 uL aliquot of dsRNA sample was delivered by pipette onto the surface of the diet of each well (40 ⁇ /cm 2 ). dsRNA sample concentrations were calculated as the amount of dsRNAper square centimeter (ng/cm 2 ) of surface area (1.5 cm 2 ) in the well. The treated trays were held in a fume hood until the liquid on the diet surface evaporated or were absorbed into the diet.
• GI [1 - (TWIT/TNIT)/(TWIBC/TNIBC)], where TWIT is the Total Weight of live Insects in the Treatment;
• TNIT is the Total Number of Insects in the Treatment
• TWIBC is the Total Weight of live Insects in the Background Check (Buffer control).
• TNIBC is the Total Number of Insects in the Background Check (Buffer control).
• the statistical analysis was done using JMPTM software (S AS, Cary, NC).
• the LC50 Lethal Concentration
• the GI50 Crowth Inhibition
• the mean growth (e.g. live weight) of the test insects is 50% of the mean value seen in Background Check samples. Replicated bioassays demonstrated that ingestion of particular samples resulted in a surprising and unexpected mortality and growth inhibition of corn rootworm larvae.
• Insects from multiple stages ofWCR (Diabrotica virgifera virgif era LeConte) development were selected for pooled transcriptome analysis to provide candidate target gene sequences for control by RNAi transgenic plant insect protection technology.
• total RNA was isolated from about 0.9 gm whole first- instar WCR larvae; (4 to 5 days post- hatch; held at 16 °C), and purified using the following phenol/TRI REAGENT ® -based method (MOLECULAR RESEARCH CENTER, Cincinnati, OH):
• RNA concentration was detennined by measuring the absorbance (A) at 260 nm and 280 nm. A typical extraction from about 0.9 gm of larvae yielded over 1 mg of total RNA, with an A260/A280 ratio of 1.9. The RNA thus extracted was stored at -80 °C until further processed.
• RNA quality was determined by running an aliquot through a 1% agarose gel.
• the agarose gel solution was made using autoclaved lOx TAE buffer (Tris- acetate EDTA; lx concentration is 0.04 M Tris-acetate, 1 mM EDTA (ethylenediamine tetra- acetic acid sodium salt), pH 8.0) diluted with DEPC (diethyl pyrocarbonate)-treated water in an autoclaved container, lx TAE was used as the running buffer. Before use, the electrophoresis tank and the well-forming comb were cleaned with RNaseAwayTM (IN VITRO GEN INC., Carlsbad, CA).
• RNA sample buffer 10 mM Tris HCl pH 7.0; 1 mM EDTA
• RNA sample buffer 10 uL
• the sample was heated at 70 °C for 3 rnin, cooled to room temperature, and 5 uL (containing 1 ug to 2 ug RNA) were loaded per well.
• RNA molecular weight markers were simultaneously run in separate wells for molecular size comparison. The gel was run at 60 volts for 2 hrs.
• a normalized cDNA library was prepared from the larval total RNA by a commercial service provider (EUROFINS MWG Operon, Huntsville, AL), using random priming.
• the normalized larval cDNA library was sequenced at 1/2 plate scale by GS FLX 454 TitaniumTM series chemistry at EUROFINS MWG Operon, which resulted in over 600,000 reads with an average read length of 348 bp. 350,000 reads were assembled into over 50,000 contigs. Both the unassembled reads and the contigs were converted into BLASTable databases using the publicly available program, FORMATDB (available from NCBI).
• RNA and normalized cDNA libraries were similarly prepared from materials harvested at other WCR developmental stages.
• a pooled transcriptome library for target gene screening was constructed by combining cDNA library members representing the various developmental stages.
• RNAi targeting was hypothesized to be essential for survival and growth in pest insects. Selected target gene homologs were identified in the transcriptome sequence database, as described below. Full-length or partial sequences of the target genes were amplified by PCR to prepare templates for double- stranded RNA (dsRNA) production.
• dsRNA double- stranded RNA
• TBLASTN searches using candidate protein coding sequences were run against BLASTable databases containing the unassembled Diabrotica sequence reads or the assembled contigs. Significant hits to a Diabrotica sequence (defined as better than e "20 for contigs homologies and better than e "10 for unassembled sequence reads homologies) were confirmed using BLASTX against the NCBI non-redundant database. The results of this BLASTX search confirmed that the Diabrotica homolog candidate gene sequences identified in the TBLASTN search indeed comprised Diabrotica genes, or were the best hit to the non-Diabrotica candidate gene sequence present in the Diabrotica sequences.
• Tribolium candidate genes which were annotated as encoding a protein gave an unambiguous sequence homology to a sequence or sequences in the Diabrotica transcriptome sequences.
• sequences or unassembled sequence reads selected by homology to a non-Diabrotica candidate gene overlapped, and that the assembly of the contigs had failed to join these overlaps.
• SequencherTM v4.9 GENE CODES CORPORATION, Ann Arbor, MI was used to assemble the sequences into longer contigs.
• Diabrotica prp8 SEQ ID NO:l and SEQ ID NO:3 were identified as genes that may lead to coleopteran pest mortality, inhibition of growth, inhibition of development, and/or inhibition of feeding in WCR.
• the Drosophila prp8 gene consists of a NusG amino-terminal (NGN) domain and a C-tenninal Kyprides-Onzonis-Woese (KOW) domain, acting as a dual transcriptional regulator that functions as both a negative and positive elongation factor.
• NGN NusG amino-terminal
• KOW Kyprides-Onzonis-Woese
• the KOW domain in eukaryotic is thought to allow the recruitment of a larger number of transcription factors.
• prp8 may also participate in the regulation of pre-mRNA processing, as it interacts with the capping enzyme.
• prp8 builds the heterodimeric complex DSIF (DRB (5,6-dichloro- l-P-D- ribofuranosylbenzimidazole) sensitivity- inducing factor).
• DRB 5,6-dichloro- l-P-D- ribofuranosylbenzimidazole
• sequences SEQ ID NO:l and SEQ ID NO:3 are novel.
• the sequences are not provided in public databases, and are not disclosed in PCT International Patent Publication No. WO/2011/025860; U.S. Patent Application No. 20070124836; U.S. Patent Application No. 20090306189; U.S. Patent Application No. US20070050860;
• WCRprp8-l (SEQ ID NO:l) is somewhat related to a fragment of a sequence from Tribolium castaneum (GEN BANK Accession No. XM 961838.2).
• WCRprp8-2 (SEQ ID NO:3) is somewhat related to a fragment of a sequence from Oryctolagus cuniculus (GENBANK Accession No. NM_144353.4).
• the closest homolog of the WCR PRP8- 1 amino acid sequence (SEQ ID NO:2) is a Tribolium casetanum protein having GENBANK Accession No.
• XP 966931.1 (99% similar; 98% identical over the homology region).
• the closest homolog of the WCR PRP8-2 amino acid sequence (SEQ ID NO:4) is a Gregarina niphandrodes protein having GENBANK Accession No. XP_011131272.1 (85% similar; 76% identical over the homology region).
• Prp8 dsRNA transgenes can be combined with other dsRNA molecules to provide redundant RNAi targeting and synergistic RNAi effects. Transgenic corn events expressing dsRNA that targets prp8 are useful for preventing root feeding damage by corn rootworm. Prp8 dsRNA transgenes represent new modes of action for combining with Bacillus thuringiensis insecticidal protein technology in Insect Resistance Management gene pyramids to mitigate against the development of rootworm populations resistant to either of these rootworm control technologies.
• YFP yellow fluorescent protein
• Template preparation by PCR and dsRNA synthesis A strategy used to provide specific templates for prp8 and YFP dsRN A production is shown in FIG. 1.
• Template DN As intended for use in prp8 dsRN A synthesis were prepared by PCRusing the primer pairs in Table 1 and (as PCR template) first- strand cDN A prepared from total RNA isolated from WCR eggs, first- instar larvae, or adults.
• PCR amplifications introduced a T7 promoter sequence at the 5' ends of the amplified sense and antisense strands (the YFP segment was amplified from a DNA clone of the YFP coding region).
• the two PCR amplified fragments for each region of the target genes were then mixed in approximately equal amounts, and the mixture was used as transcription template for dsRNA production. See FIG. 1.
• the sequences of the dsRN A templates amplified with the particular primer pairs were: SEQ ID NO :7 (prp8-J gr ), SEQ ID NO :8 (prp8-2 gr ), SEQ ID NO :9 (prp8-3 regl ), SEQ JD NOA0 (prp8-3 v ⁇ SEQ IDNO:l l (prp8-3 w2 ⁇ and SEQ ID NO :14 (YFP).
• Double- stranded RNA for insect bioassay was synthesized and purified using an AMBION ® MEGASCRIPT ® RNAi kit following the manufacturer's instructions (IN VITRO GEN) or HiScribe ® T7 In Vitro Transcription Kit following the manufacturer's instructions (New England Biolabs, Ipswich, MA).
• concentrations of dsRN As were measured using a NANODROPTM 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, DE).
• Entry vectors harboring a target gene construct for hairpin formation comprising segments of prp8 (SEQ ID NO:l and SEQ ID NO :3) are assembled using a combination of chemically synthesized fragments (DNA2.0, Menlo Park, CA) and standard molecular cloning methods.
• Intramolecular hairpin formation by RNA primary transcripts is facilitated by arranging (within a single transcription unit) two copies of the prp8 target gene segment in opposite orientation to one another, the two segments being separated by a linker polynucleotide ⁇ e.g., an ST- LSI intron; Vancanneyt et al. (1990) Mol. Gen. Genet. 220(2)245-50).
• the primary mRNA transcript contains the two prp8 gene segment sequences as large inverted repeats of one another, separated by the intron sequence.
• a copy of a maize ubiquitin 1 promoter (U.S. Patent 5,510,474) is used to drive production of the primary mRNA hairpin transcript, and a fragment comprising a 3' untranslated region from a maize peroxidase 5 gene (ZmPer5 3'UTR v2; U.S. Patent 6,699,984)is used to tenninate transcription of the hairpin- RNA- expressing gene.
• a negative control binary vector which comprises a gene that expresses a YFP hairpin dsRNA, is constructed by means of standard GATEWAY ® recombination reactions with a typical binary destination vector and entry vector.
• the binary destination vector comprises a herbicide tolerance gene
• a synthetic 5'UTR sequence comprised of sequences from a Maize Streak Virus (MSV) coat protein gene 5'UTR and intron 6 from a maize Alcohol Dehydrogenase 1 (ADHl) gene, is positioned between the 3' end of the SCBV promoter segment and the start codon of the AAD- 1 coding region.
• a fragment comprising a 3' untranslated region from a maize lipase gene (ZmLip 3'UTR; U.S. Patent 7, 179,902) is used to teirninate transcription of the AAD- 1 mRN A.
• a further negative control binary vector which comprises a gene that expresses a YFP protein, is constructed by means of standard GATEWAY ® recombination reactions with a typical binary destination vector and entry vector.
• the binary destination vector comprises a herbicide tolerance gene (aryloxyalknoate dioxygenase; AAD- 1 v3) (as above) under the expression regulation of a maize ubiquitin 1 promoter (as above) and a fragment comprising a 3' untranslated region from a maize lipase gene (ZmLip 3'UTR; as above).
• the entry vector comprises a YFP coding region (SEQ ID NO:27) under the expression control of a maize ubiquitin 1 promoter (as above) and a fragment comprising a 3' untranslated region from a maize peroxidase 5 gene (as above).
• Synthetic dsRNA designed to inhibit target gene sequences identified in
• EXAMPLE 2 caused mortality and growth inhibition when administered to WCR in diet-based assays.
• annexin, beta spectrin 2, and mtRP-L4 were each suggested in U. S. Patent 7,612, 194 to be efficacious in RNAi-mediated insect control.
• SEQ ID NO:28 is the DN A sequence of annexin region 1 (Reg 1 ) and SEQ ID NO :29 is the DN A sequence of annexin region 2 (Reg 2).
• SEQ ID NO:30 is the DNA sequence of beta spectrin 2 region 1 (Reg 1) and SEQ ID NO:31 is the DNA sequence of beta spectrin ! region 2 (Reg2).
• SEQ ID NO 32 is the DNA sequence oimtRP-L4 region 1 (Reg 1) and SEQ ID NO:33 is the DNA sequence of mtRP-L4 region 2 (Reg 2).
• a YFP sequence (SEQ ID NO: 14) was also used to produce dsRNA as a negative control.
• Double- stranded RNA was synthesized and purified using an AMBION ® MEGAscript ® RNAi kit following the manufacturer's instructions (IN VITRO GEN) .
• concentrations of dsRNAs were measured using a NANODROPTM 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, DE) and the dsRNAs were each tested by the same diet-based bioassay methods described above.
• Table 4 lists the sequences of the primers used to produce the annexin Regl , annexin Reg2, beta spectrin 2 Regl, beta spectrin 2 Reg2, mtRP-L4 Regl, mtRP-L4 Reg2, and YFP dsRNA molecules.
• Table 5 presents the results of diet-based feeding bioassays of WCR larvae following 9-day exposure to these dsRNA molecules. Replicated bioassays demonstrated that ingestion of these dsRNAs resulted in no mortality or growth inhibition of western corn rootworm larvae above that seen with control samples of TE buffer, Water, or YFP protein.
• Beta-spect2 G C AGAT GAACAC CAGC GAGAAA (SEQ ID NO: 1
• Beta-spect2 T TAATACGACT CACT ATAGG GAGAC TGGGCAG
• Agrobacterium -mediated Transformatio n Transgenic maize cells, tissues, and plants that produce one or more insecticidal dsRN A molecules for example, at least one dsRNA molecule including a dsRNA molecule targeting a gene comprising prp8 ⁇ e.g., SEQ ID NO:l and SEQ ID NO:3)
• dsRN A molecules for example, at least one dsRNA molecule including a dsRNA molecule targeting a gene comprising prp8 ⁇ e.g., SEQ ID NO:l and SEQ ID NO:3
• Maize transformation methods employing superbinary or binary transformation vectors are known in the art, as described, for example, in U.S. Patent 8,304,604, which is herein incorporated by reference in its entirety.
• Transformed tissues are selected by their ability to grow on Haloxyfop- containing medium and are screened for dsRNA production, as appropriate. Portions of such transformed tissue cultures may be presented to neonate corn rootworm larvae for bioassay, essentially as described in EXAMPLE 1.
• Glycerol stocks of Agrobacterium strain DAtl3192 cells (PCT International Publication No. WO 2012/016222 A2) harboring a binary transformation vector described above (EXAMPLE 4) are streaked on AB minimal medium plates (Watson, et al. (1975) J. Bacterid. 123 :255-264) containing appropriate antibiotics, and are grown at 20 °C for 3 days.
• the cultures are then streaked onto YEP plates (gm/L: yeast extract, 10; Peptone, 10; NaCl, 5) containing the same antibiotics and are incubated at 20 °C for 1 day.
• Inoculation Medium (Frame et al. (2011) Genetic Transformation Using Maize Immature Zygotic Embryos. IN Plant Embryo Culture Methods and Protocols: Methods in Molecular Biology. T. A. Thorpe and E. C. Yeung, (Eds), Springer Science and Business Media, LLC.
• pp 327-341) contains: 2.2 gm/L MS salts; IX ISU Modified MS Vitamins (Frame et al, ibid.) 68.4 gm/L sucrose; 36 gm/L glucose; 115 mg/L L-proline; and 100 mg/L myo-inositol; atpH 5.4.) Acetosyringone is added to the flask containing Inoculation Medium to a final concentration of 200 ⁇ from a 1 M stock solution in 100% dimethyl sulfoxide, and the solution is thoroughly mixed.
• 1 or 2 inoculating loops- full of Agrobacterium from the YEP plate are suspended in 15 mL Inoculation Medium/acetosyringone stock solution in a sterile, disposable, 50 mL centrifuge tube, and the optical density of the solution at 550 nm (OD550) is measured in a spectrophotometer.
• the suspension is then diluted to OD550 of 0.3 to 0.4 using additional Inoculation Medium/acetosyringone mixtures.
• the tube of Agrobacterium suspension is then placed horizontally on a platform shaker set at about 75 rpm at room temperature and shaken for 1 to 4 hours while embryo dissection is performed.
• Maize immature embryos are obtained from plants of Zea mays inbred line B104 (Hallauer et al. (1997) Crop Science 37:1405- 1406), grown in the greenhouse and self- or sib-pollinated to produce ears. The ears are harvested approximately 10 to 12 days post-pollination. On the experimental day, de- husked ears are surface- sterilized by immersion in a 20% solution of commercial bleach (ULTRA CLOROX® Germicidal Bleach, 6.15% sodium hypochlorite; with two drops of TWEEN 20) and shaken for 20 to 30 rnin, followed by three rinses in sterile deionized water in a laminar flow hood.
• ULTRA CLOROX® Germicidal Bleach 6.15% sodium hypochlorite; with two drops of TWEEN 20
• Immature zygotic embryos (1.8 to 2.2 mm long) are aseptically dissected from each ear and randomly distributed into microcentrifuge tubes containing 2.0 mL of a suspension of appropriate Agrobacterium cells in liquid Inoculation Medium with 200 ⁇ acetosyringone, into which 2 uL of 10% BREAK- THRU ® S233 surfactant (EVONIK INDUSTRIES; Essen, Germany) is added.
• BREAK- THRU ® S233 surfactant EVONIK INDUSTRIES; Essen, Germany
• Co-cultivation Medium which contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH (3,6- dichloro-o- anisic acid or 3,6-dichloro-2-methoxybenzoic acid); 100 mg/L myo-inositol; 100 mg L Casein Enzymatic Hydrolysate; 15 mg/L AgNO3; 200
• the liquid Agrobacterium suspension is removed with a sterile, disposable, transfer pipette.
• the embryos are then oriented with the scutellum facing up using sterile forceps with the aid of a microscope.
• the plate is closed, sealed with 3MTM MICROPORETM medical tape, and placed in an incubator at 25 °C with continuous light at approximately 60 ⁇ rrrV 1 of Photosynthetically Active Radiation (PAR).
• PAR Photosynthetically Active Radiation
• Resting Medium which is composed of 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L L- proline; 3.3 mg L Dicamba in KOH; 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgN0 3 ; 0.5 gm/L MES (2-(N- morpholino)ethanesulfonic acid monohydrate; PHYTO TECHNOLOGIES LABR; Lenexa, KS); 250 mg/L Carbenicillin; and 2.3 gm/L GELZANTM; at pH 5.8.
• Resting Medium which is composed of 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L L- proline; 3.3 mg L Dicamba in KOH; 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolys
• No more than 36 embryos are moved to each plate.
• the plates are placed in a clear plastic box and incubated at 27 °C with continuous light at approximately 50 ⁇ rrrV 1 PAR for 7 to 10 days.
• Callused embryos are then transferred ( ⁇ 18/plate) onto Selection Medium I, which is comprised of Resting Medium (above) with 100 nM R- Haloxyfop acid (0.0362 mg/L; for selection of calli harboring the AAD-1 gene).
• the plates are returned to clear boxes and incubated at 27 °C with continuous light at approximately 50 ⁇ m " 1 PAR for 7 days.
• Callused embryos are then transferred ( ⁇ 12/plate) to Selection Medium II, which is comprised of Resting Medium (above) with 500 nM R-Haloxyfop acid (0.181 mg/L).
• Selection Medium II which is comprised of Resting Medium (above) with 500 nM R-Haloxyfop acid (0.181 mg/L).
• the plates are returned to clear boxes and incubated at 27 °C with continuous light at approximately 50 ⁇ rrrV 1 PAR for 14 days. This selection step allows transgenic callus to further proliferate and differentiate.
• Pre-Regeneration Medium contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 45 gm/L sucrose; 350 mg/L L-proline; 100 mg/L myo-inositol; 50 mg/L Casein Enzymatic Hydrolysate; 1.0 mg/L AgN0 3 ; 0.25 gm/L MES; 0.5 mg/L naphthaleneacetic acid in NaOH; 2.5 mg/L abscisic acid in ethanol; 1 mg/L 6- benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L GELZANTM; and 0.181 mg/L Haloxyfop acid; at pH 5.8.
• the plates are stored in clear boxes and incubated at 27 °C with continuous light at approximately 50 ⁇ rrrV 1 PAR for 7 days. Regenerating calli are then transferred ( ⁇ 6/plate) to Regeneration Medium in PHYTATRAYSTM (SIGMA- ALDRICH) and incubated at 28 °C with 16 hours light/8 hours dark per day (at approximately 160 ⁇ rrrV 1 PAR) for 14 days or until shoots and roots develop.
• PHYTATRAYSTM SIGMA- ALDRICH
• Regeneration Medium contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 60 gm/L sucrose; 100 mg L myo-inositol; 125 mg/L Carbenicillin; 3 gm/L GELLANTM gum; and 0.181 mg/L R-Haloxyfop acid; atpH 5.8. Small shoots with primary roots are then isolated and transferred to Elongation Medium without selection.
• Elongation Medium contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; and 3.5 gm/L GELRITETM: at pH 5.8.
• Transformed plant shoots selected by their ability to grow on medium containing Haloxyfop are transplanted from PHYTATRAYSTM to small pots filled with growing medium (PROMIX BX; PREMIER TECH HORTICULTURE), covered with cups or HUMI-DOMES (ARCO PLASTICS), and then hardened- off in a CONVIRON growth chamber (27 °C day/24 °C night, 16-hour photoperiod, 50-70% RH, 200 ⁇ nrV PAR).
• putative transgenic plantlets are analyzed for transgene relative copy number by quantitative real-time PCR assays using primers designed to detect the AAD1 herbicide tolerance gene integrated into the maize genome. Further, RNA qPCR assays are used to detect the presence of the linker sequence in expressed dsRNAs of putative transformants. Selected transformed plantlets are then moved into a greenhouse for further growth and testing.
• Plants to be used for insect bioassays are transplanted from small pots to
• ROOTRATNERS ® S PEN CER-LEM AIRE INDUSTRIES, Acheson, Alberta, Canada;
• ROOTRAINER ® one plant per event perROOTRAINER ® .
• Plants of the T ⁇ generation are obtained by pollinating the silks of To transgenic plants with pollen collected from plants of non- transgenic elite inbred line B 104 or other appropriate pollen donors, and planting the resultant seeds. Reciprocal crosses are performed when possible.
• EXAMPLE 7 Molecular Analyses of Trans enic Maize Tissues Molecular analyses (e.g. RNA qPCR) of maize tissues are performed on samples from leaves and roots that were collected from greenhouse grown plants on the same days that root feeding damage is assessed.
• RNA qPCR RNA qPCR
• RNA qPCR assays for the Per5 3'UTR are used to validate expression of hairpin transgenes.
• a low level of Per5 3'UTR detection is expected in non- transformed maize plants, since there is usually expression of the endogenous Per5 gene in maize tissues.
• Results ofRNAqPCR assays for intervening sequence between repeat sequences (which is integral to the formation of dsRNA hairpin molecules) in expressed RNAs are used to validate the presence of hairpin transcripts.
• Transgene RNA expression levels are measured relative to the RNA levels of an endogenous maize gene.
• DNA qPCR analyses to detect a portion of the AAD1 coding region in gDN A are used to estimate transgene insertion copy number. Samples for these analyses are collected from plants grown in environmental chambers. Results are compared to DNA qPCR results of assays designed to detect a portion of a single- copy native gene, and simple events (having one or two copies of prp8 transgenes) are advanced for further studies in the greenhouse.
• qPCR assays designed to detect a portion of the spectinomycin- resistance gene (SpecR; harbored on the binary vector plasmids outside of the T-DNA) are used to determine if the transgenic plants contain extraneous integrated plasmid backbone sequences.
• RNA transcript expression level Per 5 3'UTR qPCR. Callus cell events or transgenic plants are analyzed by real time quantitative PCR (qPCR) of the Per 5 3'UTR sequence to detennine the relative expression level of the full length hairpin transcript, as compared to the transcript level of an internal maize gene (for example, GENBANK Accession No. BT069734), which encodes a TIP41-like protein (i.e. a maize homolog of GENBANK Accession No. AT4G34270; having a tBLASTX score of 74% identity;
• qPCR real time quantitative PCR
• RNA is isolated using an RNeasyTM 96 kit (QIAGEN, Valencia, CA). Following elution, the total RNA is subjected to a DNasel treatment according to the kit's suggested protocol. The RNA is then quantified on a NANODROP 8000 spectrophotometer (THERMO SCIENTIFIC) and the concentration is normalized to 25 ng/nL.
• First strand cDNA is prepared using a HIGH CAPACITY cDNA SYNTHESIS KIT (IN VITRO GEN) in a 10 L reaction volume with 5 denatured RNA, substantially according to the manufacturer's recommended protocol.
• the protocol is modified slightly to include the addition of 10
• IDTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN T20VN oligonucleotide
• samples are diluted 1 :3 with nuclease-free water, and stored at -20 °C until assayed.
• Transcript size and integrity Northern Blot Assay.
• additional molecular characterization of the transgenic plants is obtained by the use of Northern Blot (RNA blot) analysis to determine the molecular size of the prp8 hairpin dsRNA in transgenic plants expressing a prp8 hairpin dsRNA.
• Tissue samples (100 mg to 500 mg) are collected in 2 mL S AFELOCK EPPENDORF tubes, disrupted with a KLECKOTM tissue pulverizer (GARCIA MANUFACTURING, Visalia, C A) with three tungsten beads in 1 mL TRIZOL (FN VITRO GEN) for 5 min, then incubated at room temperature (RT) for 10 min.
• a KLECKOTM tissue pulverizer GARCIA MANUFACTURING, Visalia, C A
• TRIZOL FN VITRO GEN
• the samples are centrifuged for 10 min at 4 °C at 11,000 rpm and the supernatant is transferred into a fresh 2 mL SAFELOCK EPPENDORF tube. After 200 uL chloroform are added to the homogenate, the tube is mixed by inversion for 2 to
• the top phase is transferred into a sterile 1.5 mL EPPENDORF tube, 600 uL of 100% isopropanol are added, followed by incubation at RT for 10 min to 2 hr, and then centrifuged at 12,000 x g for 10 min at 4 °C to 25 °C. The supernatant is discarded and the RNA pellet is washed twice with 1 mL 70% ethanol, with centrifugation at 7,500 x g for 10 min at 4 °C to 25 °C between washes. The ethanol is discarded and the pellet is briefly air dried for 3 to 5 min before resuspending in 50 uL of nuclease-free water.
• RNA is quantified using the NANODROP 8000 ® (THERMO-FISHER) and samples are normalized to 5 ug/10 uL. 10 pL of glyoxal (AMBION/INVITROGEN) are then added to each sample. Five to 14 ng of DIG RNA standard marker mix (ROCHE APPLIED SCIENCE, Indianapolis, IN) are dispensed and added to an equal volume of glyoxal.
• RNAs are denatured at 50 °C for 45 min and stored on ice until loading on a 1.25% SEAKEM GOLD agarose (LONZA, Allendale, NJ) gel in NORTHERNMAX 10 X glyoxal running buffer (AMBION/INVITROGEN). RNAs are separated by electrophoresis at 65 volts/30 mA for 2 hours and 15 minutes.
• the gel is rinsed in 2X SSC for 5 min and imaged on a GEL DOC station (BIORAD, Hercules, CA), then the RNA is passively transferred to a nylon membrane (MILLIPORE) overnight atRT, using 10X SSC as the transfer buffer (20X SSC consists of 3 sodium chloride and 300 mM trisodium citrate, pH 7.0).
• 10X SSC consists of 3 sodium chloride and 300 mM trisodium citrate, pH 7.0.
• the membrane is rinsed in 2X SSC for 5 minutes, the RNA is UV-crosslinked to the membrane (AGILENT/STRATAGENE), and the membrane is allowed to dry at room temperature for up to 2 days.
• the membrane is pre-hybridized in ULTRAHYBTM buffer (AMBION/INVITROGEN) for 1 to 2 hr.
• the probe consists of a PCRamplified product containing the sequence of interest, (for example, the antisense sequence portion ofSEQ ID NOs:7- l l, as appropriate) labeled with digoxygenin by means of a ROCHE APPLIED SCIENCE DIG procedure.
• Hybridization in recommended buffer is overnight at a temperature of 60 °C in hybridization tubes.
• the blot is subjected to DIG washes, wrapped, exposed to film for 1 to 30 minutes, then the film is developed, all by methods recommended by the supplier of the DIG kit.
• Transgene copy number determination Maize leaf pieces approximately equivalent to 2 leaf punches are collected in 96-well collection plates (QIAGEN). Tissue disruption is performed with a KLECKOTM tissue pulverizer (GARCIA MANUFACTURING, Visalia, CA) in BIOSPRINT96 API lysis buffer (supplied with a BIOSPRFNT96 PLANT KIT; QIAGEN) with one stainless steel bead. Following tissue maceration, gDNA is isolated in high throughput format using a BIOSPRINT96 PLANT KIT and a BIOSPRINT96 extraction robot. gDNA is diluted 2:3 DNA:water prior to setting up the qPCR reaction.
• KLECKOTM tissue pulverizer GARCIA MANUFACTURING, Visalia, CA
• BIOSPRINT96 API lysis buffer supplied with a BIOSPRFNT96 PLANT KIT; QIAGEN
• gDNA is isolated in high throughput format using a BIOSPRINT96 PLANT KIT and a BIOSPRINT96 extraction
• Transgene detection by hydrolysis probe assay is performed by real-time PCR using a LIGHTC YCLER ® 480 system.
• Oligonucleotides to be used in hydrolysis probe assays to detect the linker sequence, or to detect a portion of the SpecR gene are designed using LIGHTC YCLER® PROBE DESIGN SOFTWARE 2.0.
• oligonucleotides to be used in hydrolysis probe assays to detect a segment of the AAD-1 herbicide tolerance gene (SEQ ID NO :70; GAADl oligonucleotides in Table 9) are designed using PPJMER EXPRESS software (APPLIED BIO SYSTEMS). Table 9 shows the sequences of the primers and probes. Assays are multiplexed with reagents for an endogenous maize chromosomal gene (Invertase (SEQ ID NO:71; GENBANK Accession No: Ul 6123; referred to herein as IVRl), which serves as an internal reference sequence to ensure gDNA is present in each assay.
• IVRl endogenous maize chromosomal gene
• LIGHTCYCLER®480 PROBES MASTER mix (ROCHE APPLIED SCIENCE) is prepared at lx final concentration in a 10 uL volume multip lex reaction containing 0.4 ⁇ of each primer and 0.2 ⁇ of each probe (Table 10).
• a two- step amplification reaction is performed as outlined in Table 11. Fluorophore activation and emission for the FAM- and HEX-labeled probes are as described above; CY5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm.
• Cp scores (the point at which the fluorescence signal crosses the background threshold) are deterinined from the real time PCR data using the fit points algorithm (LIGHTC YCLER ® SOFTWARE release 1.5) and the Relative Quant module (based on the ⁇ method). Data are handled as described previously (above; RNA qPCR).
• CY5 Cyanine-5 Table 10. Reaction components for gene copy number analyses and plasmid backbone detection.
• Bioactivity of dsRNA of the subject invention produced in plant cells is demonstrated by bioassay methods. See, e.g., Baum et al. (2007) Nat. Biotechnol. 25(11):1322-1326.
• One is able to demonstrate efficacy, for example, by feeding various plant tissues or tissue pieces derived from a plant producing an insecticidal dsRNA to target insects in a controlled feeding environment.
• extracts are prepared from various plant tissues derived from a plant producing the insecticidal dsRN A, and the extracted nucleic acids are dispensed on top of artificial diets for bioassays as previously described herein.
• the soil around the maize plants growing in ROOTRANERS ® is infested with 150 to 200 WCR eggs.
• the insects are allowed to feed for 2 weeks, after which time a "Root Rating" is given to each plant.
• a Node-Injury Scale is utilized for grading, essentially according to Oleson et al. (2005) J. Econ. Entomol. 98:1-8. Plants passing this bioassay, showing reduced injury, are transplanted to 5-gallon pots for seed production. Transplants are treated with insecticide to prevent iurther rootworm damage and insect release in the greenhouses. Plants are hand pollinated for seed production. Seeds produced by these plants are saved for evaluation at the T ⁇ and subsequent generations of plants.
• Greenhouse bioassays include two kinds of negative control plants.
• Transgenic negative control plants are generated by transformation with vectors harboring genes designed to produce a yellow fluorescent protein (YFP) or a YFP hairpin dsRNA ⁇ See EXAMPLE 4).
• Non-transformed negative control plants are grown from seeds of parental corn varieties from which the transgenic plants were produced.
• Bioassays are conducted on two separate dates, with negative controls included in each set of plant materials.
• hairpin dsRNA comprise a portion of SEQ ID NO:l and/or SEQ ID NO:3. Additional hairpin dsR As are derived, for example, from coleopteran pest sequences such as, for example, Caf 1-180 (U. S. Patent Application Publication No. 2012/0174258), VatpaseC (U.S. Patent Application Publication No. 2012/0174259), Rhol (U.S. Patent Application Publication No. 2012/0174260), VatpaseH (U.S. Patent Application Publication No.
• transcription elongation factor spt5 U.S. Patent Application No. 62/168,613
• transcription elongation factor spt 6 U.S. Patent Application No. 62/168,606
• ncm U. S . Patent Application No. 62/095487
• dre4 U.S. Patent Application No. 14/705,807
• COP I alpha U.S. Patent Application No. 62/063, 199
• COPIbeta V.S. Patent Application No. 62/063,203
• COPI gamma U.S. Patent Application No. 62/063,192
• COPI delta U.S. Patent Application No. 62/063,216
• Total RNA preparations from selected independent ⁇ lines are optionally used for RT-PCR with primers designed to bind in the linker of the hairpin expression cassette in each of the RNAi constructs.
• specific primers for each target gene in an RNAi construct are optionally used to amplify and confirm the production of the pre- processed mRNA required for siRNA production in planta.
• the amplification of the desired bands for each target gene confirms the expression of the hairpin RNA in each transgenic Zea mays plant. Processing of the dsRNA hairpin of the target genes into siRNA is subsequently optionally confirmed in independent transgenic lines using RNA blot hybridizations.
• RNAi molecules having mismatch sequences with more than 80% sequence identity to target genes affect corn rootworms in a way similar to that seen with RNAi molecules having 100% sequence identity to the target genes.
• the pairing of mismatch sequence with native sequences to form a hairpin dsRNA in the same RNAi construct delivers plant-processed siRNAs capable of affecting the growth, development, and viability of feeding coleopteran pests.
• RNA-mediated gene silencing In planta delivery of dsRNA, siRNA, or miRNA corresponding to target genes and the subsequent uptake by coleopteran pests through feeding results in down- regulation of the target genes in the coleopteran pest through RNA-mediated gene silencing.
• the function of a target gene is important at one or more stages of development, the growth and/or development of the coleopteran pest is affected, and in the case of at least one of WCR, NCR, SCR, MCR, D. balteataLeConte, D. speciosa Germar, D. u. tenella, and D. u. undecimpunctata Mannerheim, leads to failure to successfully infest, feed, and/or develop, or leads to death of the coleopteran pest.
• the choice of target genes and the successful application of RNAi are then used to control coleopteran pests.
• RNAi lines and nontransformed Zea mays Phenotypic comparison of transgenic RNAi lines and nontransformed Zea mays.
• Target coleopteran pest genes or sequences selected for creating hairpin dsRNA have no similarity to any known plant gene sequence.
• it is not expected that the production or the activation of (systemic) RNAi by constructs targeting these coleopteran pest genes or sequences will have any deleterious effect on transgenic plants.
• development and morphological characteristics of transgenic lines are compared with non-transformed plants, as well as those of transgenic lines transformed with an "empty" vector having no hairpin-expressing gene. Plant root, shoot, foliage and reproduction characteristics are compared. There is no observable difference in root length and growth patterns of transgenic and non-transformed plants.
• Plant shoot characteristics such as height, leaf numbers and sizes, time of flowering, floral size and appearance are similar. In general, there are no observable morphological differences between transgenic lines and those without expression of target iRNA molecules when cultured in vitro and in soil in the glasshouse.
• a transgenic Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets an organism other than a coleopteran pest is secondarily transformed via Agrobacterium or WHISKERSTM methodologies (see Petolino and Arnold (2009) Methods Mol. Biol. 526:59-67) to produce one or more insecticidal dsRNA molecules (for example, at least one dsRNA molecule including a dsRN A molecule targeting a gene comprising SEQ IDNO:l and/or SEQ ID NO:3).
• Plant transformation plasmid vectors prepared essentially as described in EXAMPLE 4 are delivered via Agrobacterium or WHISKERSTM-mediated transformation methods into maize suspension cells or immature maize embryos obtained from a transgenic Hi ⁇ or B 104 Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets an organism other than a coleopteran pest.
• EXAMPLE 11 Transgenic Zea mays Comprising an RNAi Construct and
• a transgenic Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets a coleopteran pest organism (for example, at least one dsRNA molecule including a dsRNA molecule targeting a gene comprising SEQ ID NO:l and/or SEQ ID NO:3) is secondarily transformed via Agrobacterium or WHISKERSTM methodologies (see Petolino and Arnold (2009) Methods Mol. Biol. 526:59-67) to produce one or more insecticidal protein molecules, for example, Cry3, Cry34 and Cry35 insecticidal proteins.
• Plant transformation plasmid vectors prepared essentially as described in EXAMPLE 4 are delivered via Agrobacterium or WHISKERSTM-mediated transformation methods into maize suspension cells or immature maize embryos obtained from a transgenic B104
• Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets a coleopteran pest organism. Doubly- transformed plants are obtained that produce iRNA molecules and insecticidal proteins for control of coleopteran pests.
• EXAMPLE 12 Pollen Beetle Transcriptome
• the mixture was introduced ventrolaterally by pricking the abdomen of pollen beetle imagoes using a dissecting needle dipped in an aqueous solution of lOmg ml LPS (purified E. coli endotoxin; SIGMA, Taufkirchen, Germany) and the bacterial and yeast cultures.
• lOmg ml LPS purified E. coli endotoxin; SIGMA, Taufkirchen, Germany
• RNA isolation Total RNA was extracted 8 h after immunization from frozen beetles and larvae using TriReagent (Molecular Research Centre, Cincinnati, OH, USA) and purified using the RNeasy Micro Kit (Qiagen, Hilden, Germany) in each case following the manufacturers' guidelines. The integrity of the RNA was verified using an Agilent 2100 Bioanalyzer and a RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, CA, USA). The quantity of RNA was determined using a Nanodrop ND-1000 spectrophotometer. RNA was extracted from each of the adult immune-induced treatment groups, adult control groups, and larval groups individually and equal amounts of total RNA were subsequently combined in one pool per sample (immune-challenged adults, control adults and larvae) for sequencing.
• RNA- Seq data generation and assembly Single-read 100-bp RNA- Seq was carried out separately on 5 pg total RNA isolated from immune- challenged adult beetles, naive (control) adult beetles, and untreated larvae. Sequencing was carried out by EUROFINS MWG Operon using the Illumina HiSeq-2000 platform. This yielded 20.8 million reads for the adult control beetle sample, 21.5 million reads for the LPS -challenged adult beetle sample and 25.1 million reads for the larval sample. The pooled reads (67.5 million) were assembled using Velvet/Oases assembler software (Schulz et al. (2012) Bioinformatics. 28:1086-92; Zerbino and Birney (2008) Genome Res. 18:821-9). The transcriptome contained 55,648 sequences.
• PCR fragments were cloned in the pGEM T easy vector according to the manufacturer's protocol and sent to a sequencing company to verify the sequence.
• the dsRNA was then produced by the T7 RNA polymerase (MEGAscript® RNAi Kit, Applied Biosystems) from a PCR construct generated from the sequenced plasmid according to the manufacturer's protocol.
• Feeding Bioassay Beetles were kept without access to water in empty falcon tubes 24 h before treatment. A droplet of dsRNA ( ⁇ 5
• Agrobacterium Preparation The Agrobacterium strain containing the binary plasmid is streaked out on YEP media (Bacto PeptoneTM 20.0 gm/L and Yeast Extract 10.0 gm/L) plates containing streptomycin (lOO mg/ml) and spectinomycin (50 mg/mL) and incubated for 2 days at 28°C. The propagated Agrobacterium strain containing the binary plasmid is scraped from the 2-day streak plate using a sterile inoculation loop.
• the scraped Agrobacterium strain containing the binary plasmid is then inoculated into 150 mL modified YEP liquid with streptomycin (100 mg/ml) and spectinomycin (50 mg ml) into sterile 500 mL baffled flask(s) and shaken at 200 rpm at 28°C.
• the cultures are centrifuged and resuspended in M-medium (LS salts, 3% glucose, modified B5 vitamins, 1 ⁇ , 1 ⁇ 2,4-D, pH 5.8) and diluted to the appropriate density (50 Klett Units as measured using a spectrophotometer) prior to transformation of canola hypocotyls.
• Canola Transformation Seed germination: Canola seeds (var. NEXERA 710TM) are surface- sterilized in 10% CloroxTMfor 10 minutes and rinsed three times with sterile distilled water (seeds are contained in steel strainers during this process). Seeds are planted for germination on 1 ⁇ 2 MS Canola medium (1/2 MS, 2% sucrose, 0.8% agar) contained in PhytatraysTM (25 seeds per PhytatrayTM) and placed in a PercivalTM growth chamber with growth regime set at 25°C, photoperiod of 16 hours light and 8 hours dark for 5 days of germination
• hypocotyl segments of about 3 mm in length are aseptically excised, the remaining root and shoot sections are discarded (drying of hypocotyl segments is prevented by immersing the hypocotyls segments into lO mL of sterile milliQTM water during the excision process).
• Hypocotyl segments are placed horizontally on sterile filter paper on callus induction medium, MSK1D1 (MS, 1 mg/L kinetin, 1 mg/L 2,4-D, 3.0% sucrose, 0.7% phytagar) for 3 days pre-treatment in a PercivalTM growth chamber with growth regime set at 22-23 °C, and a photoperiod of 16 hours light, 8 hours dark.
• hypocotyl segments are treated for 30 minutes with periodic swirling of the PetriTM dish so that the hypocotyl segments remained immersed in the Agrobacterium solution.
• the Agrobacterium solution is pipetted into a waste beaker; autoclaved and discarded (the Agrobacterium solution is completely removed to prevent Agrobacterium overgrowth).
• the treated hypocotyls are transferred with forceps back to the original plates containing MSK1D1 media overlaid with filter paper (care is taken to ensure that the segments did not dry).
• the transformed hypocotyl segments and non-transformed control hypocotyl segments are returned to the PercivalTM growth chamber under reduced light intensity (by covering the plates with aluminum foil), and the treated hypocotyl segments are co- cultivated with Agrobacterium for 3 days.
• MSB3Z1H3 MS, 3 mg/L BAP, 1 mg/L Zeatin, 0.5 gm/L MES, 5 mg/L AgN0 3 , 300 mgfl TimentinTM, 200 mg/L carbenicillin, 3 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar
• MSB3Z1H3 MS, 3 mg/L BAP, 1 mg/L Zeatin, 0.5 gm/L MES, 5 mg/L AgN0 3
• 300 mgfl TimentinTM 200 mg/L carbenicillin, 3 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar
• Root induction After 14 days of culturing on the shoot elongation medium, the isolated shoots are transferred to MSMEST medium (MS, 0.5 g/L MES, 300 mg/L TimentinTM, 2% sucrose, 0.7% TC Agar) for root induction at 22-26 °C. Any shoots which do not produce roots after incubation in the first transfer to MSMEST medium are transferred for a second or third round of incubation on MSMEST medium until the shoots develop roots.
• MSMEST medium MS, 0.5 g/L MES, 300 mg/L TimentinTM, 2% sucrose, 0.7% TC Agar
• Prp8 dsRNA transgenes are combined with other dsRNA molecules in transgenic plants to provide redundant RNAi targeting and synergistic RNAi effects.
• Transgenic plants including, for example and without limitation, corn, soybean, and cotton expressing dsRNA that targets prp8 are useful for preventing feeding damage by coleopteran insects.
• Prp8 dsRNA transgenes are also combined in plants with Bacillus thuringiensis insecticidal protein technology to represent new modes of action in Insect Resistance Management gene pyramids. When combined with other dsRN A molecules that target insect pests and/or with insecticidal proteins in transgenic plants, a synergistic insecticidal effect is observed that also mitigates the development of resistant insect populations.

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