US 20170218391A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0218391 A1 Narva et al. (43) Pub. Date: Aug. 3, 2017
(54) GAWKY (GW) NUCLEIC ACID MOLECULES Publication Classification TO CONTROL INSECT PESTS (51) Int. Cl. (71) Applicants: Dow AgroSciences LLC, Indianapolis, CI2N 5/82 (2006.01) IN (US); Fraunhofer-Gesellschaft zur CI2O I/68 (2006.01) Forderung der angewandten C07K I4/325 (2006.01) C07K I4/435 (2006.01) Forschung e.V., Munchen (DE) AOIN 57/6 (2006.01) (72) Inventors: Kenneth E. Narva, Zionsville, IN CI2N IS/II3 (2006.01) (US); Sarah Worden, Indianapolis, IN AOIH 4/00 (2006.01) (US); Meghan Frey, Greenwood, IN (52) U.S. Cl. (US); Murugesan Rangasamy, CPC ...... CI2N 15/8286 (2013.01); C12N 15/I13 Zionsville, IN (US); Premchand (2013.01); C12N 15/8218 (2013.01); CI2N Gandra, Indianapolis, IN (US); Wendy 15/8261 (2013.01); C12O I/6895 (2013.01); Lo, Indianapolis, IN (US); Elane A0IH 4/008 (2013.01); C07K 14/43563 Fishilevich, Indianapolis, IN (US); (2013.01); A0IN 57/16 (2013.01); C07K Rainer Fischer, Munchen (DE); 14/325 (2013.01); C12O 2600/158 (2013.01); Andreas Vilcinskas, Giessen (DE); C12O 2600/13 (2013.01); C12N 23 10/14 Eileen Knorr, Giessen (DE) (2013.01) (57) ABSTRACT (21) Appl. No.: 15/421,233 This disclosure concerns nucleic acid molecules and meth ods of use thereof for control of insect pests through RNA (22) Filed: Jan. 31, 2017 interference-mediated inhibition of target coding and tran scribed non-coding sequences in insect pests, including coleopteran and/or hemipteran pests. The disclosure also Related U.S. Application Data concerns methods for making transgenic plants that express (60) Provisional application No. 62/290,852, filed on Feb. nucleic acid molecules useful for the control of insect pests, 3, 2016. and the plant cells and plants obtained thereby. Patent Application Publication Aug. 3, 2017. Sheet 1 of 2 US 2017/0218391 A1
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GAWKY (GW) NUCLEIC ACID MOLECULES results in the pruning of roots all the way to the base of the TO CONTROL INSECT PESTS corn stalk. Severe root injury interferes with the roots ability to transport water and nutrients into the plant, reduces TECHNICAL FIELD OF THE DISCLOSURE plant growth, and results in reduced grain production, thereby often drastically reducing overall yield. Severe root 0001. The present invention relates generally to genetic injury also often results in lodging of corn plants, which control of plant damage caused by insect pests (e.g., cole makes harvest more difficult and further decreases yield. opteran pests and hemipteran pests). In particular embodi Furthermore, feeding by adults on the corn reproductive ments, the present invention relates to identification of target tissues can result in pruning of silks at the ear tip. If this “silk coding and non-coding polynucleotides, and the use of clipping is severe enough during pollen shed, pollination recombinant DNA technologies for post-transcriptionally may be disrupted. repressing or inhibiting expression of target coding and 0006 Control of corn rootworms may be attempted by non-coding polynucleotides in the cells of an insect pest to crop rotation, chemical insecticides, biopesticides (e.g., the provide a plant protective effect. spore-forming gram-positive bacterium, Bacillus thuringi ensis), transgenic plants that express Bt toxins, or a combi BACKGROUND nation thereof. Crop rotation suffers from the disadvantage 0002. The western corn rootworm (WCR), Diabrotica of placing unwanted restrictions upon the use of farmland. virgifera virgifera LeConte, is one of the most devastating Moreover, oviposition of some rootworm species may occur corn rootworm species in North America and is a particular in soybean fields, thereby mitigating the effectiveness of concern in corn-growing areas of the Midwestern United crop rotation practiced with corn and Soybean. States. The northern corn rootworm (NCR), Diabrotica 0007 Chemical insecticides are the most heavily relied barberi Smith and Lawrence, is a closely-related species upon strategy for achieving corn rootworm control. Chemi that co-inhabits much of the same range as WCR. There are cal insecticide use, though, is an imperfect corn rootworm several other related subspecies of Diabrotica that are sig control strategy; over S1 billion may be lost in the United nificant pests in the Americas: the Mexican corn rootworm States each year due to corn rootworm when the costs of the (MCR). D. virgifera zeae Krysan and Smith; the southern chemical insecticides are added to the costs of the rootworm corn rootworm (SCR), D. undecimpunctata howardi Barber; damage that may occur despite the use of the insecticides. D. balteata LeConte; D. undecimpunctata tenella, D. spe High populations of larvae, heavy rains, and improper ciosa Germar, and D. u. undecimpunctata Mannerheim. The application of the insecticide(s) may all result in inadequate United States Department of Agriculture has estimated that corn rootworm control. Furthermore, the continual use of corn rootworms cause S1 billion in lost revenue each year, insecticides may select for insecticide-resistant rootworm including S800 million in yield loss and S200 million in strains, as well as raise significant environmental concerns treatment COStS. due to the toxicity to non-target species. 0003. Both WCR and NCR eggs are deposited in the soil 0008 Stink bugs and other hemipteran insects (heterop during the Summer. The insects remain in the egg stage tera) are another important agricultural pest complex. throughout the winter. The eggs are oblong, white, and less Worldwide over 50 closely related species of stink bugs are than 0.004 inches in length. The larvae hatch in late May or known to cause crop damage (McPherson & McPherson, R. early June, with the precise timing of egg hatching varying M. (2000) Stink bugs of economic importance in America from year to year due to temperature differences and loca north of Mexico CRC Press). These insects are present in a tion. The newly hatched larvae are white worms that are less large number of important crops including maize, soybean, than 0.125 inches in length. Once hatched, the larvae begin fruit, vegetables, and cereals. to feed on corn roots. Corn rootworms go through three 0009 Stink bugs go through multiple nymph stages larval instars. After feeding for several weeks, the larvae before reaching the adult stage. These insects develop from molt into the pupal stage. They pupate in the soil, and then eggs to adults is about 30-40 days. Both nymphs and adults emerge from the soil as adults in July and August. Adult feed on Sap from Soft tissues into which they also inject rootworms are about 0.25 inches in length. digestive enzymes causing extra-oral tissue digestion and 0004 Corn rootworm larvae complete development on necrosis. Digested plant material and nutrients are then corn and several other species of grasses. Larvae reared on ingested. Depletion of water and nutrients from the plant yellow foxtail emerge later and have a Smaller head capsule vascular system results in plant tissue damage. Damage to size as adults than larvae reared on corn. Ellsbury et al. developing grain and seeds is the most significant as yield (2005) Environ. Entomol. 34:627–34. WCR adults feed on and germination are significantly reduced. Multiple genera corn silk, pollen, and kernels on exposed ear tips. If WCR tions occur in warm climates resulting in significant insect adults emerge before corn reproductive tissues are present, pressure. Current management of Stink bugs relies on insec they may feed on leaf tissue, thereby slowing plant growth ticide treatment on an individual field basis. Therefore, and occasionally killing the host plant. However, the adults alternative management strategies are urgently needed to will quickly shift to preferred silks and pollen when they minimize ongoing crop losses. become available. NCR adults also feed on reproductive 0010 RNA interference (RNAi) is a process utilizing tissues of the corn plant, but in contrast rarely feed on corn endogenous cellular pathways, whereby an interfering RNA leaves. (iRNA) molecule (e.g., a dsRNA molecule) that is specific 0005 Most of the rootworm damage in corn is caused by for all, or any portion of adequate size, of a target gene larval feeding. Newly hatched rootworms initially feed on results in the degradation of the mRNA encoded thereby. fine corn root hairs and burrow into root tips. As the larvae RNAi has been used to perform gene “knockdown” in a grow larger, they feed on and burrow into primary roots. number of species and experimental systems; for example, When corn rootworms are abundant, larval feeding often Caenorhabditis elegans, plants, insect embryos, and cells in US 2017/0218391 A1 Aug. 3, 2017
tissue culture. See, e.g., Fire et al. (1998) Nature 391:806 use any particular sequence of the more than nine hundred 11; Martinez et al. (2002) Cell 110:563-74; McManus and sequences listed therein for RNA interference, other than the Sharp (2002) Nature Rev. Genetics 3:737-47. particular partial sequence of a charged multivesicular body 0011 RNAi accomplishes degradation of mRNA through protein 4b gene. Furthermore, U.S. Pat. No. 7,943,819 an endogenous pathway including the DICER protein com provides no guidance as to which other of the over nine plex. DICER cleaves long dsRNA molecules into short hundred sequences provided would be lethal, or even oth fragments of approximately 20 nucleotides, termed Small erwise useful, in species of corn rootworm when used as interfering RNA (siRNA). The siRNA is unwound into two dsRNA or siRNA. U.S. Patent Application Publication No. single-stranded RNAS: the passenger strand and the guide U.S. 2013/04.0173 and PCT Application Publication No. Strand. The passenger Strand is degraded, and the guide WO 2013/169923 describe the use of a sequence derived Strand is incorporated into the RNA-induced silencing com from a Diabrotica virgifera Snf7 gene for RNA interference plex (RISC). Micro ribonucleic acids (miRNAs) are struc in maize. turally very similar molecules that are cleaved from precur 0014. The overwhelming majority of sequences comple Sor molecules containing a polynucleotide "loop' mentary to corn rootworm DNAS (Such as the foregoing) do connecting the hybridized passenger and guide Strands, and not provide a plant protective effect from species of corn they may be similarly incorporated into RISC. Post-tran rootworm when used as dsRNA or siRNA. For example, Scriptional gene silencing occurs when the guide Strand Baum et al. (2007) Nature Biotechnology 25:1322-1326, binds specifically to a complementary mRNA molecule and describe the effects of inhibiting several WCR gene targets induces cleavage by Argonaute, the catalytic component of by RNAi. These authors reported that 8 of the 26 target the RISC complex. This process is known to spread sys genes they tested were not able to provide experimentally temically throughout the organism despite initially limited significant coleopteran pest mortality at a very high iPNA concentrations of siRNA and/or miRNA in some eukaryotes (e.g., dsRNA) concentration of more than 520 ng/cm. Such as plants, nematodes, and some insects. 0015 The authors of U.S. Pat. No. 7,612,194 and U.S. 0012 U.S. Pat. No. 7,612, 194 and U.S. Patent Publica Patent Publication No. 2007/0050860 made the first report tion Nos. 2007/0050860, 2010/0192265, and 2011/0154545 of in planta RNAi in corn plants targeting the western corn disclose a library of 91 12 expressed sequence tag (EST) rootworm. Baum et al. (2007) Nat. Biotechnol. 25(11): 1322 sequences isolated from D. v. virgifera LeConte pupae. It is 6. These authors describe a high-throughput in vivo dietary suggested in U.S. Pat. No. 7,612,194 and U.S. Patent RNAi system to screen potential target genes for developing Publication No. 2007/0050860 to operably link to a pro transgenic RNAi maize. Of an initial gene pool of 290 moter a nucleic acid molecule that is complementary to one targets, only 14 exhibited larval control potential. One of the of several particular partial sequences of D. v. virgifera most effective double-stranded RNAs (dsRNA) targeted a vacuolar-type Ht ATPase (V-ATPase) disclosed therein for gene encoding vacuolar ATPase subunit A (V-ATPase), the expression of anti-sense RNA in plant cells. U.S. Patent resulting in a rapid suppression of corresponding endog Publication No. 2010/0192265 suggests operably linking a enous mRNA and triggering a specific RNAi response with promoter to a nucleic acid molecule that is complementary low concentrations of dsRNA. Thus, these authors docu to a particular partial sequence of a D. v. virgifera gene of mented for the first time the potential for in planta RNAi as unknown and undisclosed function (the partial sequence is a possible pest management tool, while simultaneously stated to be 58% identical to C56C 10.3 gene product in C. demonstrating that effective targets could not be accurately elegans) for the expression of anti-sense RNA in plant cells. identified a priori, even from a relatively small set of U.S. Patent Publication No. 2011/0154545 suggests oper candidate genes. ably linking a promoter to a nucleic acid molecule that is complementary to two particular partial sequences of D. v. SUMMARY OF THE DISCLOSURE virgifera coatomer beta subunit genes for the expression of 0016 Disclosed herein are nucleic acid molecules (e.g., anti-sense RNA in plant cells. Further, U.S. Pat. No. 7,943, target genes, DNAs, dsRNAs, siRNAs, miRNAs, shRNAs, 819 discloses a library of 906 expressed sequence tag (EST) and hpRNAs), and methods of use thereof, for the control of sequences isolated from D. v. virgifera LeConte larvae, insect pests, including, for example, coleopteran pests, such pupae, and dissected midguts, and Suggests operably linking as D. v. virgifera LeConte (western corn rootworm, a promoter to a nucleic acid molecule that is complementary “WCR); D. barberi Smith and Lawrence (northern corn to a particular partial sequence of a D. v. virgifera charged rootworm, “NCR); D. u, howardi Barber (southern corn multivesicular body protein 4b gene for the expression of rootworm, “SCR); D. v. zeae Krysan and Smith (Mexican double-stranded RNA in plant cells. corn rootworm, “MCR); D. balteata LeConte; D. u. 0013 No further suggestion is provided in U.S. Pat. No. tenella, D. u. undecimpunctata Mannerheim; and D. Spe 7,612,194, and U.S. Patent Publication Nos. 2007/0050860, ciosa Germar, and hemipteran pests, such as Euschistus 2010/0192265, and 2011/0154545 to use any particular heros (Fabr) (Neotropical Brown Stink Bug, “BSB); E. sequence of the more than nine thousand sequences listed servus (Say) (Brown Stink Bug); Nezara viridula (L.) therein for RNA interference, other than the several particu (Southern Green Stink Bug); Piezodorus guildinii (West lar partial sequences of V-ATPase and the particular partial wood) (Red-banded Stink Bug); Halyomorpha halys (Stal) sequences of genes of unknown function. Furthermore, none (Brown Marmorated Stink Bug); Chinavia hilare (Say) of U.S. Pat. No. 7,612,194, and U.S. Patent Publication Nos. (Green Stink Bug); C. marginatum (Palisot de Beauvois); 2007/0050860, 2010/0192265, and 2011/0154545 provides Dichelops melacanthus (Dallas); D. furcatus (F.); Edessa any guidance as to which other of the over nine thousand meditabunda (F.); Thyanta perditor (F.) (Neotropical Red sequences provided would be lethal, or even otherwise Shouldered Stink Bug); Horcias nobilellus (Berg) (Cotton useful, in species of corn rootworm when used as dsRNA or Bug); Taedia Stigmosa (Berg); Dysdercus peruvianus siRNA. U.S. Pat. No. 7,943,819 provides no suggestion to (Guérin-Méneville); Neomegalotomus parvus (Westwood); US 2017/0218391 A1 Aug. 3, 2017
Leptoglossus zonatus (Dallas); Niesthreasidae (F.); Lygus an RNA transcribed from a coleopterangw gene comprising hesperus (Knight) (Western Tarnished Plant Bug); and L. any of SEQ ID NOS:3-5. A gw means for providing cole lineolaris (Palisot de Beauvois). In particular examples, opteran pest protection to a plant includes a DNA molecule exemplary nucleic acid molecules are disclosed that may be comprising a polynucleotide encoding a gw means for homologous to at least a portion of one or more native inhibiting expression of an essential gene in a coleopteran nucleic acids in an insect pest. pest operably linked to a promoter, wherein the DNA 0017. In these and further examples, the native nucleic molecule is capable of being integrated into the genome of acid sequence may be a target gene, the product of which a plant. may be, for example and without limitation: involved in a 0021. Further disclosed are gw means for inhibiting metabolic process or involved in larval/nymph development. expression of an essential gene in a hemipteran pest, and gw In Some examples, post-transcriptional inhibition of the means for providing hemipteran pest protection to a plant. A expression of a target gene by a nucleic acid molecule gw means for inhibiting expression of an essential gene in a comprising a polynucleotide homologous thereto may be hemipteran pest includes a single-stranded RNA molecule lethal to an insect pest or result in reduced growth and/or consisting of a polynucleotide selected from the group viability of an insect pest. In specific examples, the protein consisting of SEQ ID NO:84 and the complements and involved in silencing of mRNA through miRNA transla reverse complements thereof. Functional equivalents of gw tional repression, gawky gene (referred to herein as gw), or means for inhibiting expression of an essential gene in a a gw homolog or ortholog may be selected as a target gene hemipteran pest include single- or double-stranded RNA for post-transcriptional silencing. In particular examples, a molecules that are substantially homologous to all or part of target gene useful for post-transcriptional inhibition is a gw an RNA transcribed from a hemipterangw gene comprising gene selected from the group consisting of SEQ ID NO:1 SEQID NO:73. Agw means for providing hemipteran pest and SEQ ID NO:71. An isolated nucleic acid molecule protection to a plant includes a DNA molecule comprising a comprising the polynucleotide of SEQ ID NO:1; the polynucleotide encoding a gw means for inhibiting expres complement and/or reverse complement of SEQ ID NO:1; sion of an essential gene in a hemipteran pest operably SEQID NO:71; the complement and/or reverse complement linked to a promoter, wherein the DNA molecule is capable of SEQ ID NO:71; and/or fragments comprising at least 15 of being integrated into the genome of a plant. contiguous nucleotides of either of the foregoing (e.g., SEQ 0022. Additionally disclosed are methods for controlling ID NOS:3-5 and SEQ ID NO:73) is therefore disclosed a population of an insect pest (e.g., a coleopteran or herein. hemipteran pest), comprising providing to an insect pest 0018. Also disclosed are nucleic acid molecules compris (e.g., a coleopteran or hemipteran pest) an iRNA (e.g., ing a polynucleotide that encodes a polypeptide that is at dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecule least about 85% identical to an amino acid sequence within that functions upon being taken up by the pest to inhibit a a target gene product (for example, the product of a gw biological function within the pest. gene). For example, a nucleic acid molecule may comprise 0023. In some embodiments, a method for controlling a a polynucleotide encoding a polypeptide that is at least 85% population of a coleopteran pest comprises providing to the identical to SEQ ID NO:2 (D. virgifera GW); SEQ ID coleopteran pest an iRNA molecule that comprises all or a NO:72 (E. heros GW); and/or an amino acid sequence fragment comprising at least 15 contiguous nucleotides of a within a product of a gw gene. Further disclosed are nucleic polynucleotide selected from the group consisting of: SEQ acid molecules comprising a polynucleotide that is the ID NO:79; the complement or reverse complement of SEQ complement or reverse complement of a polynucleotide that ID NO:79; SEQ ID NO:80; the complement or reverse encodes a polypeptide at least 85% identical to an amino complement of SEQID NO:80: SEQID NO:81; the comple acid sequence within a target gene product. ment or reverse complement of SEQ ID NO:81; SEQ ID 0019. Also disclosed are cDNA polynucleotides that may NO:82; the complement or reverse complement of SEQ ID be used for the production of iRNA (e.g., dsRNA, siRNA, NO:82; a polynucleotide that hybridizes to a native gw shRNA, miRNA, and hpRNA) molecules that are comple polynucleotide of a coleopteran pest (e.g., WCR) compris mentary to all or part of an insect pest target gene, for ing any of SEQ ID NOs: 1 and 3-5; the complement or example, a gw gene. In particular embodiments, dsRNAS, reverse complement of a polynucleotide that hybridizes to a siRNAs, shRNAs, miRNAs, and/or hpRNAs may be pro native gw polynucleotide of a coleopteran pest comprising duced in vitro or in Vivo by a genetically-modified organism, any of SEQ ID NOS:1 and 3-5; a polynucleotide that Such as a plant or bacterium. In particular examples, cDNA hybridizes to a fragment comprising at least 15 contiguous molecules are disclosed that may be used to produce iRNA nucleotides of a native coding polynucleotide of a Dia molecules that are complementary to all or part of agw gene brotica organism (e.g., WCR) comprising any of SEQ ID (e.g., SEQ ID NO:1 and SEQ ID NO:71). NOs: 1 and 3-5; and the complement or reverse complement 0020. Further disclosed are gw means for inhibiting of a polynucleotide that hybridizes to a fragment comprising expression of an essential gene in a coleopteran pest, and gw at least 15 contiguous nucleotides of a native coding poly means for providing coleopteran pest protection to a plant. nucleotide of a Diabrotica organism comprising any of SEQ Agw means for inhibiting expression of an essential gene in ID NOS:1 and 3-5. a coleopteran pest includes a single-stranded RNA molecule 0024. In some embodiments, a method for controlling a consisting of a polynucleotide selected from the group population of a hemipteran pest comprises providing to the consisting of SEQID NOS:80-82; and the complements and hemipteran pest an iRNA molecule that comprises all or a reverse complements thereof. Functional equivalents of gw fragment comprising at least 15 contiguous nucleotides of a means for inhibiting expression of an essential gene in a polynucleotide selected from the group consisting of: SEQ coleopteran pest include single- or double-stranded RNA ID NO:83; the complement or reverse complement of SEQ molecules that are substantially homologous to all or part of ID NO:83: SEQ ID NO:84; the complement or reverse US 2017/0218391 A1 Aug. 3, 2017
complement of SEQ ID NO:84; a polynucleotide that acid molecules of the invention may be WCR, NCR, SCR, hybridizes to a native gw polynucleotide of a hemipteran MCR, BSB, D. balteata, D. u. tenella, D. speciosa, D. u. pest (e.g., BSB) comprising SEQID NO:71 and/or SEQ ID undecimpunctata, E. servus, Piezodorus guildinii, Halvo NO:73; the complement or reverse complement of a poly morpha haly's, Nezara viridula, Chinavia hilare, C. margi– nucleotide that hybridizes to a native gw polynucleotide of natum, Dichelops melacanthus, D. furcatus, Edessa medit a hemipteran pest comprising SEQ ID NO:71 and/or SEQ abunda, Thyanta perditor, Horcias nobilellus, Taedia ID NO:73; a polynucleotide that hybridizes to a fragment Stigmosa, Dysdercus peruvianus, NeOmegalotomus parvus, comprising at least 15 contiguous nucleotides of a native Leptoglossus zonatus, Niesthreasidae, Lygus hesperus, and/ coding polynucleotide of a hemipteran organism comprising or Lygus lineolaris. SEQ ID NO:71 and/or SEQID NO:73; and the complement 0027. The foregoing and other features will become more or reverse complement of a polynucleotide that hybridizes to apparent from the following Detailed Description of several a fragment comprising at least 15 contiguous nucleotides of embodiments, which proceeds with reference to the accom a native coding polynucleotide of a hemipteran organism panying FIGS. 1-2. comprising SEQ ID NO:71 and/or SEQ ID NO:73. 0025. In particular embodiments, an iRNA that functions BRIEF DESCRIPTION OF THE FIGURES upon being taken up by an insect pest to inhibit a biological 0028 FIG. 1 includes a depiction of a strategy used to function within the pest is transcribed from a DNA com generate dsRNA from a single transcription template with a prising all or part of a polynucleotide selected from the single pair of primers. group consisting of: SEQ ID NO:1; the complement or 0029 FIG. 2 includes a depiction of a strategy used to reverse complement of SEQ ID NO:1; SEQ ID NO:3: the generate dsRNA from two transcription templates. complement or reverse complement of SEQID NO:3: SEQ ID NO:4; the complement or reverse complement of SEQID SEQUENCE LISTING NO:4: SEQ ID NO:5; the complement or reverse comple ment of SEQID NO:5; SEQID NO:71; the complement or 0030 The nucleic acid sequences listed in the accompa reverse complement of SEQID NO:71; SEQID NO:73; the nying sequence listing are shown using standard letter complement or reverse complement of SEQ ID NO:73; a abbreviations for nucleotide bases, as defined in 37 C.F.R. native coding polynucleotide of a Diabrotica organism (e.g., S1.822. The nucleic acid and amino acid sequences listed WCR) comprising any of SEQ ID NOS:1 and 3-5; the define molecules (i.e., polynucleotides and polypeptides, complement or reverse complement of a native coding respectively) having the nucleotide and amino acid mono polynucleotide of a Diabrotica organism comprising any of mers arranged in the manner described. The nucleic acid and SEQ ID NOs: 1 and 3-5; a fragment comprising at least 15 amino acid sequences listed also each define a genus of contiguous nucleotides of a native coding polynucleotide of polynucleotides or polypeptides that comprise the nucleo a Diabrotica organism comprising any of SEQ ID NOs: 1 tide and amino acid monomers arranged in the manner and 3-5; the complement or reverse complement of a frag described. In view of the redundancy of the genetic code, it ment comprising at least 15 contiguous nucleotides of a will be understood that a nucleotide sequence including a native coding polynucleotide of a Diabrotica organism coding sequence also describes the genus of polynucleotides comprising any of SEQID NOS:1 and 3-5; a native coding encoding the same polypeptide as a polynucleotide consist polynucleotide of a hemipteran organism (e.g., BSB) com ing of the reference sequence. It will further be understood prising SEQ ID NO:71 and/or SEQID NO:73; the comple that an amino acid sequence describes the genus of poly ment or reverse complement of a native coding polynucle nucleotide ORFs encoding that polypeptide. otide of a hemipteran organism comprising SEQ ID NO:71 0031. Only one strand of each nucleic acid sequence is and/or SEQ ID NO:73; a fragment comprising at least 15 shown, but the complementary Strand is understood as contiguous nucleotides of a native coding polynucleotide of included by any reference to the displayed strand. As the a hemipteran organism comprising SEQ ID NO:71 and/or complement and reverse complement of a primary nucleic SEQ ID NO:73; and a fragment comprising at least 15 acid sequence are necessarily disclosed by the primary contiguous nucleotides of the complement or reverse sequence, the complementary sequence and reverse comple complement of a native coding polynucleotide of a mentary sequence of a nucleic acid sequence are included by hemipteran organism comprising SEQ ID NO:71 and/or any reference to the nucleic acid sequence, unless it is SEQ ID NO:73. explicitly stated to be otherwise (or it is clear to be otherwise 0026. Also disclosed herein are methods wherein dsR from the context in which the sequence appears). Further NAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be more, as it is understood in the art that the nucleotide provided to an insect pest in a diet-based assay, or in sequence of an RNA strand is determined by the sequence of genetically-modified plant cells expressing the dsRNAS, the DNA from which it was transcribed (but for the substi siRNAs, shRNAs, miRNAs, and/or hpRNAs. In these and tution of uracil (U) nucleobases for thymine (T)), an RNA further examples, the dsRNAs, siRNAs, shRNAs, miRNAs, sequence is included by any reference to the DNA sequence and/or hpRNAs may be ingested by the pest. Ingestion of encoding it. In the accompanying sequence listing: dsRNAs, siRNA, shRNAs, miRNAs, and/or hpRNAs of the 0032 SEQID NO:1 shows a contig containing an exem invention may then result in RNAi in the pest, which in turn plary WCR gw DNA, referred to herein in some places as may result in silencing of a gene essential for viability of the WCR gw or WCR gw-1: pest and leading ultimately to mortality. Thus, methods are disclosed wherein nucleic acid molecules comprising exem plary polynucleotide(s) useful for control of insect pests are GCACCATTATCAAAGAACTATGGGTGAATCCACAATTTTACAAACATAAC provided to an insect pest. In particular examples, a cole ATTTGACCAAAATGTTATCCAAAAGTTAAATTTGTATTATTCTGGAATTT opteran and/or hemipteran pest controlled by use of nucleic
US 2017/0218391 A1 Aug. 3, 2017
- Continued - Continued
CATTATGTTTACAACATGGTCCGCTCTTAAGTTTCCATCTATACTTACAC ALAKYSSREEAIKAQTTLNNCVLGNTTILAENPTDWDANTLLQQVASOOS
CAAGGCTTTGCACTTGCCAAATATTCATCCCGTGAGGAAGCTATCAAAGC GSSGAWRGSSKOPTGADTWSTGWPNNSSSTSLWAAPOLDNSDPARGTPSS
TCAGACCACCCTCAACAACTGTGTACTCGGTAACACAACAATACTAGCCG LNSFLPNDLLGGESM
AAAATCCAACCGATTGGGATGCAAACACTTTGCTCCAACAAGTAGCAAGT 0034 SEQID NO:3 shows an exemplary WCRgw DNA, referred to herein in some places as WCR gw-1 reg1 (region CAACAGAGCGGCTCTTCCGGCGCATGGCGAGGTTCAAGCAAACAACCCAC 1), which is used in Some examples for the production of a TGGGGCAGACACCTGGAGTACCGGCTGGCCCAACAATTCAAGCAGCACCA dsRNA:
GTTTGTGGGCAGCTCCTCAACTCGACAACTCAGATCCCGCTCGTGGAACC ACGCAACAACTACGGATGTTGGTGCAACAAATACAGATGGCAGTTCAGGC CCATCTAGTCTAAATTCTTTTCTTCCTAACGACCTCTTAGGTGGTGAGTC AGGGTATCTCAATCACCAGATTCTTAATCAACCTTTGGCGCCACAAACGT CATGTAAGTTAAGGATGAAACCAAAATAATTCCATCTTAGTTACAAGTGT TGGTTCTTCTAAATCAACTGTTGCAACAGATCAAGAATTTACAGCAGCTC TGATATCTCTCTCTGCGCTATTTCACTATAAAAGTTTTATTGAATGTTTT ATATCACAACAATCAATAACTGGTACGCCTATCAACGGAAAACAGAATAA TAATGTTTTATAATATTAAATTTAACAATTG CGCTTATATGCAGTTTTCAGTACTCATCACAAAAACAAAACAATCAATTG 0033 SEQID NO:2 shows the amino acid sequence of a GW polypeptide encoded by an exemplary WCR gw DNA, CCAATTTACAGAATCAAATCGCTGCTCAACAAGCGACTTACGTTAAGCAA referred to herein in some places as WCR GW or WCR CAACAACACCAAAGCAGCATGGGTGCCTATGACTCATTTAAAACGAATCC GW-1 : CATGCATGATTCGATAAACGCTTTACAAACCAATTTTGGTGACTTAGGCA
TTAACAAAGAGCCT CAAATGAACCCACAACAATCACGACT CACCCAGTGG MRAPTPSEPKSTFPTYOWPOKSAMRGSAPPVOVAGPSWGGRADPPSSTRC ATAAGTAAAGATAAGGATG ADEGALSVISGSSCRSIDNSNIRMOSVTENCLLNSWTVPNMORLDHGMVT 0035) SEQID NO:4 shows a further exemplary WCRgw HNNSFKLVSKFGALLPGRDIPNOKSDDLELLRDDLNWLNSTKYDTKTLCD DNA, referred to herein in some places as WCR gw-1 V1 NNDEKDDHDAYOMSNIETHTCTNNDNSYOELYKPLRLRGGGESSLSTGTS (version 1), which is used in Some examples for the pro duction of a dsRNA: GWGTPPSOSGNNNANKSNGOOPPTSOSNNTGWGOPGTKTANNNAMPPNSO
PPTSTANSONNNGPSNNTKOOLEOLNSMREAIFSODGWGGOHVNODTNWD AAAACGAATCCCATGCATGATTCGATAAACGCTTTACAAACCAATTTTGG
IPSSPEPPIKMDGSGGPPPWKPAVNNGTELWEANLRNGGOPPPOPOOKTP TGACTTAGGCATTAACAAAGAGCCT CAAATGAACCCACAACAATCACGAC
WGHTPSTNIGGTWGEDDDADTSNVWTGVPSNOPOWGGAGGNTNNGAMWGG TCACCCAGTGGATAAGTAAAGATAAGGATG PKKENDWGTGASNTGGWGDPRAADPROTGMDPREIRPELRDMRAGNTETM 0036 SEQID NO:5 shows a further exemplary WCRgw DNA, referred to herein in some places as WCR gw-1 V2 RIMDPRETMROMSNSDMRGDPRGITGRLNGAGAEAFWGOGTPHAASOPIH (version 2), which is used in Some examples for the pro HHNKMPVPPGNGTGGWEEPSPPSORRNMPNYDDGTSLWGNPOOGSHWKDL duction of a dsRNA: PTGGSMGRGGNPAGPPGMNOARGMKOPEGSMWGGHGRNGSWDETGPGAAW
DEPNSWAKOKMPDPLWDESEWGHKOOSKPOLTKEMVWNSKOFRMLVDMGH TCAATCACCAGATTCTTAATCAACCTTTGGCGCCACAAACGTTGGTTCTT
KKEDVENALRLRAMNVEEALDLLSPMRNNRANDGWNTRHDDHYEHPPFCO CTAAATCAACTGTTGCAACAGATCAAGAATTTACAGCAGCTCATATCACA
RGFSTGPGGOLTGFOPGNNAPNLLNNMSNPGTNNSLINNIAPAVWOKLLT ACAATCAATAAC 0037 SEQ ID NO:6 shows a the nucleotide sequence of QQOGGGSQGFGGSSANAGRNIQPQSQPSTOOLRMLWOOIQMAVOAGYLNH T7 phage promoter. QILNOPLAPOTLVLLNOLLOOIKNLOOLISQQSITGTPINGKONNAYMO 0038 SEQ ID NO:7 shows a fragment of an exemplary YFP coding region. SWLITKTKOSIANLONOIAAQQATYWKOOOHOSSMGAYDSFKTNPMHDSI 0039 SEQ ID NOS:8-13 show primers used to amplify NALOTNFGDLGINKEPOMNPOOSRLTOWISKDKDDGGEFSRAPGSSSKPP portions of exemplary WCR gw sequences comprising gw-1 reg1, gW-1 V1, and gw-1 V2, used in some examples for NTSPNMNPLVLNPSDGPWSTGRTGDTGWPDSSANDNSNDWKDAOWSTTTO dsRNA production. PSLTDLVPEFEPGKPWKGNOIKIEDDPSITPGSVVRSPLSIATIKDNELF 0040 SEQ ID NO:14 shows an exemplary YFP gene. 0041 SEQID NO:15 shows a DNA sequence of annexin NMNPSKSPPATDGIOSLSLSSSTWSFNPSGTSTSSAFTSPPGKLPTSKAL region 1. 0042 SEQID NO:16 shows a DNA sequence of annexin GDLNPSTAWTSELWGAPKSSRGPPPGLSAKGSGAISNGWSAVNTMPWGPG region 2. GQRTSGNWGGSSQWLLLRNLTAQIDGSTLRTLCLOHGPLLSFHLYLHOGF 0043 SEQ ID NO:17 shows a DNA sequence of beta spectrin 2 region 1. US 2017/0218391 A1 Aug. 3, 2017
0044 SEQ ID NO:18 shows a DNA sequence of beta - Continued spectrin 2 region 2. ACCAACGATGATACCGATCAAGATGTTAAAAGCTTTAAAATCTGTGATTA 0045 SEQ ID NO:19 shows a DNA sequence of mtRP L4 region 1. TTATACTCGGTGGGGAATACCACGAAACTTGAAACTGTTAGGAGGAGGAG 0046 SEQ ID NO:20 shows a DNA sequence of mtRP AGAGTTCACTTACTACAGGGACTACCGGTTGGGGATCCCCACCTTCAAAT L4 region 2. 0047 SEQ ID NOS:21-48 show primers used to amplify CAAGGTGGTAGTACTGGTTGGAACAGTGCTAATACTACTAGTGGAAGTAA gene regions of annexin, beta spectrin 2. mtRP-L4, and YFP TAGTTCTTCAGGACAAGGACAAGCAGGTACTGGGCAAAGCCCAGCTCCTG for dsRNA synthesis. 0048 SEQ ID NO:49 shows a maize DNA sequence CCTCTGCTGGACAAACTTGGGGTAGTTCCCAAAATAATACCAACAACAGC encoding a TIP41-like protein. 0049 SEQ ID NO:50 shows the nucleotide sequence of AATAGTAATAGTAACAACAATAATGGATCTCGCAGTTCTGTTAGCCAGCA a T20VN primer oligonucleotide. AGGTGGAGGTAGCACACAACAGCAACCAGGAGGGGGGCCACCTAGTCAGT 0050 SEQID NOS:51-55 show primers and probes used for dsRNA transcript expression analyses in maize. CCACGGCTCCACCTGTAGCAACTGTGTCTACATCAACTGTTACAACTGCT 0051 SEQ ID NO:56 shows a nucleotide sequence of a portion of a SpecR coding region used for binary vector CCAGCCTCTTCAGCAACAAATACGTCCAATATTAACACTGCTACTACATC backbone detection. AGCTTCTCAACAAAATGGTTCAGCTAGTGGCAACCAAGTGGTAGGAAGTG 0052 SEQID NO:57 shows a nucleotide sequence of an AAD1 coding region used for genomic copy number analy GTTCTACCTGGGCAACTGCTGTTGGTAAAGGGCTTCCTCCGACAAGCACA S1S GTTTCAACTCCAACTTCAAGTGGAAGCACATCTACTAAGCAACAAATGGA 0053 SEQID NO:58 shows a DNA sequence of a maize invertase gene. ACAGCTAAACACAATGAGAGAAGCCCTTTACAGTCAAGATGGATGGGGTG 0054 SEQID NOS:59-67 show the nucleotide sequences GTCAAAATGTTAACCAAGATAGCAATTGGGATATACCAGGTTCCCCAGAA of DNA oligonucleotides used for gene copy number deter minations and binary vector backbone detection. CCAGGCACAAAAGATAGCAACAATGCAGCTCCTGTTCCTCTTTGGAAACT 0055 SEQID NOs:68-70 show primers and probes used for maize dsRNA transcript expression analyses. GCCTATCAATAATGGTACTGATCTTTGGGAGGCTAATCTGAGAAATGGCG 0056 SEQ ID NO:71 shows an exemplary Neotropical GTGTTCCTCCTCCTGTAAGCCAACAGAGTCAGAAAACACCTTGGGTTCAC Brown Stink Bug (Euschistus heros) gw DNA, referred to herein in some places as BSB gw-1: ACTCCAAGCACCAACATTGGTGGAACATGGGGTGAAGATGATGAAGGTGA TGCTTCTAATGTGTGGACTGGTGTTCCTCAAGCACAGACTGGATGCGGTC
AGTAATGGCGTGCAAGAAAGTTTTGGAAGTGTGCTATGCTTAAATTACAG CT CAATGGCCAGCTCAACCACCTCCTATTTGGCCTGCTACTAAGAAAGAA
ATTAAAAAAATATAGTTACATTGATGTTTTGATATTAATTAAGAGTTCTT GGAGATTGGGGAGGGCCTAACTGGAATGATCAACGTGACACAAGAGATCT
GTGTGATCAAAAACATTAGTTTTTCATTTTTTGTTTCCCCCTTTCCTAAA TCGCCACAGTGATATGAGACAAATGATGGATGCTAGAGATCATATGAGAC
ATACAAGTATTTGCTTCATCTTGACTGATAGTATTATCGAACTTTTTGGA CAACTTCTATTGATCACAGATCAATGGGAGGCAATGATGTTATAATGCGA
AAGCCTTGTCCAAGCTTGATCATCACACTTGTAAAAAACTTTTCTTACCA GGTGACCCACGCGGAATCAGCGGTAGGCTTAATGGCGTAACGAGTGAGGC
ACATTGAGCAGACCTTTCTTCTTTAACT CACCAAGTGACATTGGCTGTGG CATGTGGCCTGGTCCAGGTCCTCATCACCATATACCCCATCATCAAGGAA
GACATCCTCATTCTAACAATGACTAAAAAATAGCTCAATCTGCATATTTA AATTGCCTTCTCAACCTAATCAACCAGTTAATCAATGGAGCAGCTCTGGA
TCCATCATGTATTATAAACAAAAGTTAAACTGAGCAGAAGAGGATTAAGT CCCCCAATGAAGGACATGACTGGTCTTGGTGGTAAATCAACTGGTTGGGA
GCTGTAAAGTATTTCTTTAAAGATTTCTGCCACAATGAGAAAAGCCCAAG GGAGCCTTCACCTCCAGCTCAACGGAGGAATATGCCTAATTATGATGATG
ATAATTAATTAGTCTATAAGACTTTGGTTTTTACATATTGCCTGCCAAAG GAACATCACTTTGGGGCCCACAGCATCCCAGACCTACCATCCAAGGTCAA
ACGTACTGAGAGCCAATGTTTCGAAACAATTCTAGTTCAAATGAGATTTC AATAAAGTTTCTCATTGGAAAGAAATGCCGGCTCCTGGAATAGGGCGAGG
TTCTAAAACTAATGCCTTTGTACAAAATAAAGACGAGGAGGACAAATCTG TGGTTTACAGTGTCCCCCAGGCCGTGCTAACCCTACAATGAAACCAGATC
AGAGCTTGTTAAGAGGTATGGCGCAGCCTCCCAAGCCTACGAGTCCTACT AACCTTTATGGCCTCATCATCCCAGAAATGAACGGGGATGGGAAGGAGGA
CATCAAGTGCCTGAGAAAAGGGACGTAATGGTGGTAGATATTGGGGTGAG ATGGATAGTGGACCCTGGGGAGATGAAAAACCAACTCCTGCTGCTGCACC
AGAAGATGATGGCCCCGTCCTGACTGTGATAACCAACCATCCGTCCCAAG TTGGATGGACCAAGGTCTAGCTCCTTCATCATGGCAAGGTGGACCAAAAC
CGCCCGCCAAGATTTTCTCATCAGAAATTGGTGAAAGTGAATCTGACGGT ATAAACCAGCATGGGATGGATCTGATTTAGATCCCACTTCTTGGGTTCAC
TCTTCCAAAATGCCACTAGCAGAGACACAAGGAACGGGTGCTCTTTGCTT TCAAAACAGCCCTCTAAGTCCGTTTCAAAGGAATTTATTTGGACAAGCAA
AGATAGCATTAAGTCTATTAGTGTTAATGAATCATTTAGTGTTAAGGATA GCAGTTTCGTATTTTGTCTGAAATGGGTTTCAAGAAAGAAGATATAGAAA
AATTTATTTGCCCGAGCAAGAGTTTAATTCTGCCGCCAAACATTCCAAAT GTGCATTAAGAAGTTCCGGAATGAGCCTTGAAGATGCATTAGATCAGCTT
US 2017/0218391 A1 Aug. 3, 2017
0058 SEQ ID NO:73 shows an exemplary BSB gw transcription elongation factor sptiS RNAi targets, as DNA, referred to herein in some places as BSB gw-1 reg1 described in U.S. Patent Application No. 62/168,613), and (region 1), which is used in some examples for the produc transcription elongation factor sptó RNAi targets, as tion of a dsRNA: described in U.S. Patent Application No. 62/168,606), the potential to affect multiple target sequences, for example, with multiple modes of action, may increase opportunities to GTATGTTAAGCATCAACAGCATACACCACCTACTTCTGAGTTTTTCAAGA develop Sustainable approaches to insect pest management GTTCATTACATGAACCAATTTCTGCACTTCATCCTAATTTTTCTGATCTT involving RNAi technologies. 0064 Disclosed herein are methods and compositions for TCTCTTAAAGATCCCCCGACCAGTGGAACTAGCCAGCAATCACGATTAAA genetic control of insect (e.g., coleopteran and/or hemipteran) pest infestations. Methods for identifying one TCAGTGGAAGTTACCTGCCCTGGAAAAAGACTCAGATATTGGGACAGGTG or more gene(s) essential to the lifecycle of an insect pest for AATTTTCTAGAGCTCCAGGTACAACAGCTAAGTCAGCTCAAGGCTCTTCT use as a target gene for RNAi-mediated control of an insect pest population are also provided. DNA plasmid vectors TCACCTAATACAAATTTATTACTTGGGCAGGCTGATGGTACTTGGTCTTC encoding an RNA molecule may be designed to suppress TGTAAATCGTGAATCTAGTTGGCCTGATTCATCCGGTGATGATGCTTCTG one or more target gene(s) essential for growth, Survival, and/or development. In some embodiments, the RNA mol GCAAGGATTGGCCAAATTCCAGTCAAC CTCCATCTCAAGCATTCTCTGAT ecule may be capable of forming dsRNA molecules. In some embodiments, methods are provided for post-transcriptional CTTGTTCCTGAGTTTGAACCAGGAAAGCCTTGGAAGGGAAACCCACTAAA repression of expression or inhibition of a target gene via AAGCATCGAGGATGATCCAAGCCTTACACCTGGTTCGGTTGTG nucleic acid molecules that are complementary to a coding or non-coding sequence of the target gene in an insect pest. 0059 SEQ ID NOs:74-75 show primers used to amplify In these and further embodiments, a pest may ingest one or portions of exemplary BSB gw sequences comprising gw-1 more dsRNA, siRNA, shRNA, miRNA, and/or hpRNA reg 1 used in Some examples for dsRNA production. molecules transcribed from all or a portion of a nucleic acid 0060 SEQID NO:76 shows an exemplary YFPv2 DNA, molecule that is complementary to a coding or non-coding which is used in some examples for the production of the sequence of a target gene, thereby providing a plant-protec sense strand of a dsRNA. tive effect. 0061 SEQ ID NOS:77-78 show primers used for PCR 0065 Thus, some embodiments involve sequence-spe amplification of YFP sequence YFP V2, used in some cific inhibition of expression of target gene products, using examples for dsRNA production. dsRNA, siRNA, shRNA, miRNA and/or hpRNA that is 0062 SEQ ID NOS:79-84 show exemplary RNAs tran complementary to coding and/or non-coding sequences of scribed from nucleic acids comprising exemplary gw poly the target gene(s) to achieve at least partial control of an nucleotides and fragments thereof. insect (e.g., coleopteran and/or hemipteran) pest. Disclosed is a set of isolated and purified nucleic acid molecules DETAILED DESCRIPTION comprising a polynucleotide, for example, as set forth in one of SEQ ID NOS:1 and 71; and fragments of at least 15 I. Overview of Several Embodiments contiguous nucleotides thereof. In some embodiments, a 0063. We developed RNA interference (RNAi) as a tool stabilized dsRNA molecule may be expressed from these for insect pest management, using one of the most likely polynucleotides, fragments thereof, or a gene comprising target pest species for transgenic plants that express dsRNA; one of these polynucleotides, for the post-transcriptional the western corn rootworm. Thus far, most genes proposed silencing or inhibition of a target gene. In certain embodi as targets for RNAi in rootworm larvae do not actually ments, isolated and purified nucleic acid molecules comprise achieve their purpose. Herein, we describe RNAi-mediated all or at least 15 contiguous nucleotides of either SEQ ID knockdown of gw in the exemplary insect pests, western NO:1 and 71 (e.g., SEQ ID NOS:3-5 and 73), and/or a corn rootworm and neotropical brown Stink bug, which is complement or reverse complement thereof. shown to have a lethal phenotype when, for example, iRNA 0066. Some embodiments involve a recombinant host molecules are delivered via ingested or injected gw dsRNA. cell (e.g., a plant cell) having in its genome at least one In embodiments herein, the ability to deliver gw dsRNA by recombinant DNA encoding at least one iRNA (e.g., feeding to insects confers a RNAi effect that is very useful dsRNA) molecule(s). In particular embodiments, a dsRNA for insect (e.g., coleopteran and hemipteran) pest manage molecule may be provided when ingested by an insect (e.g., ment. By combining gw-mediated RNAi with other useful coleopteran and/or hemipteran) pest to post-transcriptionally RNAi targets (for example and without limitation, ROP silence or inhibit the expression of a target gene in the pest. RNAi targets, as described in U.S. patent application Ser. The recombinant DNA may comprise, for example, any of No. 14/577,811, RNA polymerase I1 RNAi targets, as SEQ ID NOS:1, 3-5, 71, and 73; fragments of at least 15 described in U.S. Patent Application No. 62/133,214, RNA contiguous nucleotides of any of SEQ ID NOs: 1, 3-5, 71, polymerase II 140 RNAi targets, as described in U.S. patent and 73; and a polynucleotide consisting of a partial sequence application Ser. No. 14/577,854, RNA polymerase II215 of a gene comprising one of SEQID NOs: 1, 3-5, 71, and 73: RNAi targets, as described in U.S. Patent Application No. and/or complements or reverse complements thereof. 62/133,202, RNA polymerase II33 RNAi targets, as 0067. Some embodiments involve a recombinant host described in U.S. Patent Application No. 62/133,210), incm cell having in its genome a recombinant DNA encoding at RNAi targets (U.S. Patent Application No. 62/095,487), least one iRNA (e.g., dsRNA) molecule(s) comprising all or dre4 (U.S. Patent Application No. 61/989,843), snap25 at least 15 contiguous nucleotides of either of SEQ ID RNAi targets (U.S. Patent Application No. 62/193.502), NO:79 and 83 (e.g., at least one polynucleotide selected US 2017/0218391 A1 Aug. 3, 2017
from a group comprising SEQID NOS:80-82 and 84), or the display protection and/or enhanced protection to insect pest complement or reverse complement thereof. When ingested infestations. Particular transgenic plants may display pro by an insect (e.g., coleopteran and/or hemipteran) pest, the tection and/or enhanced protection to one or more coleop iRNA molecule(s) may silence or inhibit the expression of a teran and/or hemipteran pest(s) selected from the group target gw DNA (e.g., a DNA comprising all or at least 15 consisting of WCR; BSB; NCR; SCR; MCR: D. balteata contiguous nucleotides of a polynucleotide selected from the LeConte; D. u. tenella, D. u. undecimpunctata Mannerheim; group consisting of SEQ ID NOs: 1, 3-5, 71, and 73) in the D. speciosa Germar, E. servus (Say); Nezara viridula (L.); pest, and thereby result in cessation of growth, development, Piezodorus guildinii (Westwood); Halyomorpha haly's viability, and/or feeding in the pest. (Stal); Chinavia hilare (Say); C. marginatum (Palisot de 0068. In some embodiments, a recombinant host cell Beauvois); Dichelops melacanthus (Dallas); D. furcatus having in its genome at least one recombinant DNA encod (F.); Edessa meditabunda (F.); Thyanta perditor (F.); Hor ing at least one RNA molecule capable of forming a dsRNA cias nobilellus (Berg): Taedia Stigmosa (Berg); Dysdercus molecule may be a transformed plant cell. Some embodi peruvianus (Guérin-Méneville); NeOmegalotomus parvus ments involve transgenic plants comprising Such a trans (Westwood); Leptoglossus Zonatus (Dallas); Niesthreasidae formed plant cell. In addition to such transgenic plants, (F.); Lygus hesperus (Knight); and L. lineolaris (Palisot de progeny plants of any transgenic plant generation, trans Beauvois). genic seeds, and transgenic plant products, are all provided, 0071 Also disclosed herein are methods for delivery of each of which comprises recombinant DNA(s). In particular control agents, such as an iRNA molecule, to an insect (e.g., embodiments, an RNA molecule capable of forming a coleopteran or hemipteran) pest. Such control agents may dsRNA molecule may be expressed in a transgenic plant cause, directly or indirectly, impairment in the ability of an cell. Therefore, in these and other embodiments, a dsRNA insect pest population to feed, grow, or otherwise cause molecule may be isolated from a transgenic plant cell. In damage in a host. In some embodiments, a method is particular embodiments, the transgenic plant is a plant provided comprising delivery of a stabilized dsRNA mol selected from the group comprising corn (Zea mays), Soy ecule to an insect pest to Suppress at least one target gene in bean (Glycine max), cotton, and plants of the family the pest, thereby causing RNAi and reducing or eliminating Poaceae. plant damage in a pest host. In some embodiments, a method 0069. Some embodiments involve a method for modu of inhibiting expression of a target gene in the insect pest lating the expression of a target gene in an insect (e.g., may result in cessation of growth, Survival, and/or devel coleopteran or hemipteran) pest cell. In these and other opment in the pest. embodiments, a nucleic acid molecule may be provided, 0072. In some embodiments, compositions (e.g., a topical wherein the nucleic acid molecule comprises a polynucle composition) are provided that comprise an iRNA (e.g., otide encoding an RNA molecule capable of forming a dsRNA) molecule for use in plants, animals, and/or the dsRNA molecule. In particular embodiments, a polynucle environment of a plant or animal to achieve the elimination otide encoding an RNA molecule capable of forming a or reduction of an insect (e.g., coleopteran or hemipteran) dsRNA molecule may be operatively linked to a promoter, pest infestation. In particular embodiments, the composition and may also be operatively linked to a transcription termi may be a nutritional composition or food source to be fed to nation sequence. In particular embodiments, a method for the insect pest. A nutritional composition or food source to modulating the expression of a target gene in an insect pest be fed to the insect pest may be, for example and without cell may comprise: (a) transforming a plant cell with a vector limitation, a RNAi bait or a plant cell or tissue comprising comprising a polynucleotide encoding an RNA molecule an iRNA molecule. Some embodiments comprise making capable of forming a dsRNA molecule; (b) culturing the the nutritional composition or food source available to the transformed plant cell under conditions sufficient to allow pest. Ingestion of a composition comprising iRNA mol for development of a plant cell culture comprising a plurality ecules may result in the uptake of the molecules by one or of transformed plant cells; (c) selecting for a transformed more cells of the pest, which may in turn result in the plant cell that has integrated the vector into its genome; and inhibition of expression of at least one target gene in cell(s) (d) determining that the selected transformed plant cell of the pest. Ingestion of or damage to a plant or plant cell by comprises the RNA molecule capable of forming a dsRNA an insect pest infestation may be limited or eliminated in or molecule encoded by the polynucleotide of the vector. A on any host tissue or environment in which the pest is plant may be regenerated from a plant cell that has the vector present by providing one or more compositions comprising integrated in its genome and comprises the dsRNA molecule an iRNA molecule in the host of the pest. encoded by the polynucleotide of the vector. 0073. RNAi baits are formed when the dsRNA is mixed 0070 Thus, also disclosed is a transgenic plant compris with food or an attractant, or both. When the pests eat the ing a vector having a polynucleotide encoding an RNA bait, they also consume the dsRNA. Baits may take the form molecule capable of forming a dsRNA molecule integrated of granules, gels, flowable powders, liquids, or Solids. In in its genome, wherein the transgenic plant comprises the another embodiment, gW may be incorporated into a bait dsRNA molecule encoded by the polynucleotide of the formulation such as that described in U.S. Pat. No. 8,530, vector. In particular embodiments, expression of an RNA 440 which is hereby incorporated by reference. Generally, molecule capable of forming a dsRNA molecule in the plant with baits, the baits are placed in or around the environment is sufficient to modulate the expression of a target gene in a of the insect pest, for example, WCR can come into contact cell of an insect (e.g., coleopteran or hemipteran) pest that with, and/or be attracted to, the bait. contacts the transformed plant or plant cell (for example, by 0074 The compositions and methods disclosed herein feeding on the transformed plant, a part of the plant (e.g., may be used together in combinations with other methods root) or plant cell), such that growth and/or survival of the and compositions for controlling damage by insect (e.g., pest is inhibited. Transgenic plants disclosed herein may coleopteran or hemipteran) pests. For example, an iRNA US 2017/0218391 A1 Aug. 3, 2017
molecule as described herein for protecting plants from acid molecule, includes internalization of the nucleic acid insect pests may be used in a method comprising the molecule into the organism, for example and without limi additional use of one or more chemical agents effective tation: ingestion of the molecule by the organism (e.g., by against an insect pest, biopesticides effective against Such a feeding); contacting the organism with a composition com pest, crop rotation, recombinant expression of other iRNA prising the nucleic acid molecule; and soaking of organisms molecules, and/or recombinant genetic techniques that with a solution comprising the nucleic acid molecule. exhibit features different from the features of RNAi-medi 0100 Contig: As used herein the term “contig refers to ated methods and RNAi compositions (e.g., recombinant a DNA sequence that is reconstructed from a set of over production of proteins in plants that are harmful to an insect lapping DNA segments derived from a single genetic source. pest (e.g., Bt toxins, PIP-1 polypeptides (See, e.g., U.S. 0101 Corn plant: As used herein, the term “corn plant” Patent Publication No. US 2014/0007292 A1), and/or AflP refers to a plant of the species, Zea mays (maize). polypeptides (See, e.g., U.S. Patent Publication No. US 0102 Expression: As used herein, “expression of a 2104/0033361 A1)). coding polynucleotide (for example, a gene or a transgene) refers to the process by which the coded information of a II. Abbreviations nucleic acid transcriptional unit (including, e.g., gDNA or 0075 BSB neotropical brown stink bug (Euschistus cDNA) is converted into an operational, non-operational, or heros) structural part of a cell, often including the synthesis of a 0076 dsRNA double-stranded ribonucleic acid protein. Gene expression can be influenced by external (0077 GI growth inhibition signals; for example, exposure of a cell, tissue, or organism (0078 NCBI National Center for Biotechnology Infor to an agent that increases or decreases gene expression. mation Expression of a gene can also be regulated anywhere in the 0079 g|DNA genomic deoxyribonucleic acid pathway from DNA to RNA to protein. Regulation of gene 0080 iRNA inhibitory ribonucleic acid expression occurs, for example, through controls acting on I0081 ORF open reading frame transcription, translation, RNA transport and processing, 0082 RNAi ribonucleic acid interference degradation of intermediary molecules such as mRNA, or 0083 miRNA micro ribonucleic acid through activation, inactivation, compartmentalization, or I0084 shRNA small hairpin ribonucleic acid degradation of specific protein molecules after they have I0085 siRNA small inhibitory ribonucleic acid been made, or by combinations thereof. Gene expression can I0086 hpRNA hairpin ribonucleic acid be measured at the RNA level or the protein level by any I0087 UTR untranslated region method known in the art, including, without limitation, I0088. WCR western corn rootworm (Diabrotica vir northern blot, RT-PCR, western blot, or in vitro, in situ, or gifera virgifera LeConte) in vivo protein activity assay(s). I0089 NCR northern corn rootworm (Diabrotica bar 0103 Genetic material: As used herein, the term “genetic beri Smith and Lawrence) material includes all genes, and nucleic acid molecules, (0090 MCR Mexican corn rootworm (Diabrotica vir such as DNA and RNA. gifera zeae Krysan and Smith) 0104 Hemipteran pest: As used herein, the term (0091 PCR polymerase chain reaction “hemipteran pest” refers to pest insects of the order Hemip 0092 qPCR quantitative polymerase chain reaction tera, including, for example and without limitation, insects (0093 RISC RNA-induced Silencing Complex in the families Pentatomidae, Miridae, Pyrrhocoridae, Cor eidae, Alydidae, and Rhopalidae, which feed on a wide 0094 SCR southern corn rootworm (Diabrotica range of host plants and have piercing and Sucking mouth undecimpunctata howardi Barber) parts. In particular examples, a hemipteran pest is selected 0095 SEM standard error of the mean from the list comprising Euschistus heros (Fabr.) (Neotro (0096 YFP yellow florescent protein pical Brown Stink Bug), Nezara viridula (L.) (Southern Green Stink Bug), Piezodorus guildinii (Westwood) (Red III. Terms banded Stink Bug), Halyomorpha halys (Stal) (Brown Mar 0097. In the description and tables which follow, a num morated Stink Bug), Chinavia hilare (Say) (Green Stink ber of terms are used. In order to provide a clear and Bug), Euschistus servus (Say) (Brown Stink Bug). Dich consistent understanding of the specification and claims, elops melacanthus (Dallas), Dichelops fircatus (F), Edessa including the scope to be given Such terms, the following meditabunda (F.), Thyanta perditor (F) (Neotropical Red definitions are provided: Shouldered Stink Bug), Chinavia marginatum (Palisot de 0098 Coleopteran pest: As used herein, the term “cole Beauvois), Horcias nobilellus (Berg) (Cotton Bug), Taedia opteran pest” refers to pest insects of the order Coleoptera, Stigmosa (Berg), Dysdercus peruvianus (Guérin-Méneville). including pest insects in the genus Diabrotica, which feed NeOmegalotomus parvus (Westwood), Leptoglossus Zonatus upon agricultural crops and crop products, including corn (Dallas), Niesthrea Sidae (F), Lygus hesperus (Knight) and other true grasses. In particular examples, a coleopteran (Western Tarnished Plant Bug), and Lygus lineolaris (Palisot pest is selected from a list comprising D. v. virgifera de Beauvois). LeConte (WCR); D. barberi Smith and Lawrence (NCR); D. 0105. Inhibition: As used herein, the term “inhibition, u. howardi (SCR); D. v. zeae (MCR); D. balteata LeConte; when used to describe an effect on a coding polynucleotide D. u. tenella, D. u. undecimpunctata Mannerheim; and D. (for example, a gene), refers to a measurable decrease in the speciosa Germar. cellular level of mRNA transcribed from the coding poly 0099 Contact (with an organism): As used herein, the nucleotide and/or peptide, polypeptide, or protein product of term "contact with or "uptake by an organism (e.g., a the coding polynucleotide. In some examples, expression of coleopteran or hemipteran pest), with regard to a nucleic a coding polynucleotide may be inhibited Such that expres US 2017/0218391 A1 Aug. 3, 2017 sion is approximately eliminated. “Specific inhibition” refers to the inhibition of a target coding polynucleotide ATGATGATG polynucleotide without consequently affecting expression of other coding polynucleotides (e.g., genes) in the cell wherein the specific TACTACTAC complement" of the polynucleotide inhibition is being accomplished. CATCATCAT reverse complement" of the 0106 Insect: As used herein with regard to pests, the term polynucleotide “insect pest” specifically includes coleopteran insect pests. 0110. Some embodiments of the invention may include In some examples, the term “insect pest” specifically refers hairpin RNA-forming RNAi molecules. In these RNAi to a coleopteran pest in the genus Diabrotica selected from molecules, both the complement of a nucleic acid to be a list comprising D. v. virgifera LeConte (WCR); D. barberi targeted by RNA interference and the reverse complement Smith and Lawrence (NCR); D. u. howardi (SCR); D. v. Zeae may be found in the same molecule. Such that the single (MCR); D. balteata LeConte; D. u. tenella, D. u. undecim stranded RNA molecule may “fold over and hybridize to punctata Mannerheim; and D. speciosa Germar. In some itself over the region comprising the complementary and embodiments, the term also includes some other insect reverse complementary polynucleotides. pests; e.g., hemipteran insect pests. 0111 "Nucleic acid molecules' include all polynucle 0107 Isolated: An "isolated' biological component (such otides, for example: single- and double-stranded forms of as a nucleic acid or protein) has been Substantially separated, DNA; single-stranded forms of RNA; and double-stranded produced apart from, or purified away from other biological forms of RNA (dsRNA). The term “nucleotide sequence' or components in the cell of the organism in which the com “nucleic acid sequence” refers to both the sense and anti ponent naturally occurs (i.e., other chromosomal and extra sense strands of a nucleic acid as either individual single chromosomal DNA and RNA, and proteins), while effecting strands or in the duplex. The term “ribonucleic acid (RNA) a chemical or functional change in the component (e.g., a is inclusive of iRNA (inhibitory RNA), dsRNA (double nucleic acid may be isolated from a chromosome by break stranded RNA), siRNA (small interfering RNA), shRNA ing chemical bonds connecting the nucleic acid to the (small hairpin RNA), mRNA (messenger RNA), miRNA remaining DNA in the chromosome). Nucleic acid mol (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer ecules and proteins that have been "isolated include nucleic RNAS, whether charged or discharged with a corresponding acid molecules and proteins purified by standard purification acylated amino acid), and cFNA (complementary RNA). methods. The term also embraces nucleic acids and proteins The term “deoxyribonucleic acid” (DNA) is inclusive of prepared by recombinant expression in a host cell, as well as cDNA, gDNA, and DNA-RNA hybrids. The terms “poly chemically-synthesized nucleic acid molecules, proteins, nucleotide' and “nucleic acid,” and “fragments’ thereof will and peptides. be understood by those in the art as a term that includes both 0108 Nucleic acid molecule: As used herein, the term gDNAs, ribosomal RNAs, transfer RNAs, messenger “nucleic acid molecule' may refer to a polymeric form of RNAS, operons, and Smaller engineered polynucleotides that nucleotides, which may include both sense and anti-sense encode or may be adapted to encode, peptides, polypeptides, strands of RNA, cDNA, gDNA, and synthetic forms and or proteins. mixed polymers of the above. A nucleotide or nucleobase 0112 Oligonucleotide: An oligonucleotide is a short may refer to a ribonucleotide, deoxyribonucleotide, or a nucleic acid polymer. Oligonucleotides may be formed by modified form of either type of nucleotide. A “nucleic acid cleavage of longer nucleic acid segments, or by polymeriZ molecule' as used herein is synonymous with “nucleic acid ing individual nucleotide precursors. Automated synthesiz and “polynucleotide.” A nucleic acid molecule is usually at ers allow the synthesis of oligonucleotides up to several least 10 bases in length, unless otherwise specified. By hundred bases in length. Because oligonucleotides may bind convention, the nucleotide sequence of a nucleic acid mol to a complementary nucleic acid, they may be used as probes ecule is read from the 5' to the 3' end of the molecule. The for detecting DNA or RNA. Oligonucleotides composed of “complement of a nucleic acid molecule refers to a poly DNA (oligodeoxyribonucleotides) may be used in PCR, a nucleotide having nucleobases that may form base pairs with technique for the amplification of DNAs. In PCR, the the nucleobases of the nucleic acid molecule (i.e., A-T/U. oligonucleotide is typically referred to as a “primer,” which and G-C). allows a DNA polymerase to extend the oligonucleotide and 0109 Some embodiments include nucleic acids compris replicate the complementary strand. ing a template DNA that is transcribed into an RNA mol 0113. A nucleic acid molecule may include either or both ecule that is the complement of an mRNA molecule. In these naturally occurring and modified nucleotides linked together embodiments, the complement of the nucleic acid tran by naturally occurring and/or non-naturally occurring scribed into the mRNA molecule is present in the 5' to 3' nucleotide linkages. Nucleic acid molecules may be modi orientation, such that RNA polymerase (which transcribes fied chemically or biochemically, or may contain non DNA in the 5' to 3’ direction) will transcribe a nucleic acid natural or derivatized nucleotide bases, as will be readily from the complement that can hybridize to the mRNA appreciated by those of skill in the art. Such modifications molecule. Unless explicitly stated otherwise, or it is clear to include, for example, labels, methylation, Substitution of one be otherwise from the context, the term “complement or more of the naturally occurring nucleotides with an therefore refers to a polynucleotide having nucleobases, analog, internucleotide modifications (e.g., uncharged link from 5' to 3', that may form base pairs with the nucleobases ages: for example, methyl phosphonates, phosphotriesters, of a reference nucleic acid. Similarly, unless it is explicitly phosphoramidates, carbamates, etc., charged linkages: for stated to be otherwise (or it is clear to be otherwise from the example, phosphorothioates, phosphorodithioates, etc.; pen context), the “reverse complement of a nucleic acid refers dent moieties: for example, peptides; intercalators: for to the complement in reverse orientation. The foregoing is example, acridine, psoralen, etc.; chelators; alkylators; and demonstrated in the following illustration: modified linkages: for example, alpha anomeric nucleic US 2017/0218391 A1 Aug. 3, 2017
acids, etc.). The term “nucleic acid molecule' also includes 0119) As used herein, the term “percentage of sequence any topological conformation, including single-stranded, identity” may refer to the value determined by comparing double-stranded, partially duplexed, triplexed, hairpinned, two optimally aligned sequences (e.g., nucleic acid circular, and padlocked conformations. sequences or polypeptide sequences) of a molecule over a 0114. As used herein with respect to DNA, the term comparison window, wherein the portion of the sequence in “coding polynucleotide,” “structural polynucleotide,” or the comparison window may comprise additions or deletions “structural nucleic acid molecule” refers to a polynucleotide (i.e., gaps) as compared to the reference sequence (which that is ultimately translated into a polypeptide, via transcrip does not comprise additions or deletions) for optimal align tion and mRNA, when placed under the control of appro ment of the two sequences. The percentage is calculated by priate regulatory elements. With respect to RNA, the term determining the number of positions at which the identical “coding polynucleotide' refers to a polynucleotide that is nucleotide or amino acid residue occurs in both sequences to translated into a peptide, polypeptide, or protein. The bound yield the number of matched positions, dividing the number aries of a coding polynucleotide are determined by a trans of matched positions by the total number of positions in the lation start codon at the 5'-terminus and a translation stop comparison window, and multiplying the result by 100 to codon at the 3'-terminus. Coding polynucleotides include, yield the percentage of sequence identity. A sequence that is but are not limited to:gDNA, cDNA; EST; and recombinant identical at every position in comparison to a reference polynucleotides. sequence is said to be 100% identical to the reference 0115. As used herein, “transcribed non-coding poly sequence, and Vice-versa. nucleotide' refers to segments of mRNA molecules such as 0120 Methods for aligning sequences for comparison are 5' UTR, 3'UTR, and intron segments that are not translated well-known in the art. Various programs and alignment into a peptide, polypeptide, or protein. Further, “transcribed algorithms are described in, for example: Smith and Water non-coding polynucleotide' refers to a nucleic acid that is man (1981) Adv. Appl. Math. 2:482; Needleman and Wun transcribed into an RNA that functions in the cell, for sch (1970).J. Mol. Biol. 48:443; Pearson and Lipman (1988) example, structural RNAs (e.g., ribosomal RNA (rRNA) as Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp exemplified by 5S rRNA, 5.8S rRNA, 16S rRNA, 18 S (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS rRNA, 23 S rRNA, and 28S rRNA, and the like); transfer 5:151-3: Corpet et al. (1988) Nucleic Acids Res. 16:10881 RNA (tRNA); and snRNAs such as U4, U5, U6, and the like. 90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Transcribed non-coding polynucleotides also include, for Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana example and without limitation, small RNAs (sRNA), which et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed term is often used to describe Small bacterial non-coding consideration of sequence alignment methods and homology RNAs; small nucleolar RNAs (snoRNA); microRNAs: calculations can be found in, e.g., Altschul et al. (1990) J. small interfering RNAs (siRNA); Piwi-interacting RNAs Mo1. Biol. 215:403-10. (piRNA); and long non-coding RNAs. Further still, “tran I0121 The National Center for Biotechnology Informa scribed non-coding polynucleotide' refers to a polynucle tion (NCBI) Basic Local Alignment Search Tool otide that may natively exist as an intragenic 'spacer” in a (BLASTTM; Altschulet al. (1990)) is available from several nucleic acid and which is transcribed into an RNA molecule. sources, including the National Center for Biotechnology 0116 Lethal RNA interference: As used herein, the term Information (Bethesda, Md.), and on the internet, for use in “lethal RNA interference refers to RNA interference that connection with several sequence analysis programs. A results in death or a reduction in viability of the subject description of how to determine sequence identity using this individual to which, for example, a dsRNA, miRNA, program is available on the internet under the “help' section siRNA, shRNA, and/or hpRNA is delivered. for BLASTTM. For comparisons of nucleic acid sequences, 0117 Genome: As used herein, the term “genome' refers the “Blast 2 sequences” function of the BLASTTM (Blastin) to chromosomal DNA found within the nucleus of a cell, and program may be employed using the default BLOSUM62 also refers to organelle DNA found within subcellular com matrix set to default parameters. Nucleic acids with even ponents of the cell. In some embodiments of the invention, greater sequence similarity to the sequences of the reference a DNA molecule may be introduced into a plant cell, such polynucleotides will show increasing percentage identity that the DNA molecule is integrated into the genome of the when assessed by this method. plant cell. In these and further embodiments, the DNA 0.122 Specifically hybridizable/Specifically complemen molecule may be either integrated into the nuclear DNA of tary: As used herein, the terms “Specifically hybridizable' the plant cell, or integrated into the DNA of the chloroplast and “Specifically complementary are terms that indicate a or mitochondrion of the plant cell. The term “genome,” as it Sufficient degree of complementarity Such that stable and applies to bacteria, refers to both the chromosome and specific binding occurs between the nucleic acid molecule plasmids within the bacterial cell. In some embodiments of and a target nucleic acid molecule. Hybridization between the invention, a DNA molecule may be introduced into a two nucleic acid molecules involves the formation of an bacterium such that the DNA molecule is integrated into the anti-parallel alignment between the nucleobases of the two genome of the bacterium. In these and further embodiments, nucleic acid molecules. The two molecules are then able to the DNA molecule may be either chromosomally-integrated form hydrogen bonds with corresponding bases on the or located as or in a stable plasmid. opposite strand to form a duplex molecule that, if it is 0118 Sequence identity: The term “sequence identity” or sufficiently stable, is detectable using methods well known “identity,” as used herein in the context of two polynucle in the art. A polynucleotide need not be 100% complemen otides or polypeptides, refers to the residues in the sequences tary to its target nucleic acid to be specifically hybridizable. of the two molecules that are the same when aligned for However, the amount of complementarity that must exist for maximum correspondence over a specified comparison win hybridization to be specific is a function of the hybridization dow. conditions used. US 2017/0218391 A1 Aug. 3, 2017
0123 Hybridization conditions resulting in particular Stringency conditions set forth, Supra) to the reference degrees of stringency will vary depending upon the nature of nucleic acid. Substantially homologous polynucleotides the hybridization method of choice and the composition and may have at least 80% sequence identity. For example, length of the hybridizing nucleic acids. Generally, the tem Substantially homologous polynucleotides may have from perature of hybridization and the ionic strength (especially about 80% to 100% sequence identity, such as 79%; 80%: the Na" and/or Mg" concentration) of the hybridization about 81%; about 82%; about 83%; about 84%; about 85%: buffer will determine the stringency of hybridization, though about 86%; about 87%; about 88%; about 89%; about 90%; wash times also influence stringency. Calculations regarding about 91%; about 92%; about 93%; about 94% about 95%: hybridization conditions required for attaining particular about 96%; about 97%; about 98%; about 98.5%; about degrees of stringency are known to those of ordinary skill in 99%; about 99.5%; and about 100%. The property of sub the art, and are discussed, for example, in Sambrook et al. stantial homology is closely related to specific hybridization. (ed.) Molecular Cloning: A Laboratory Manual, 2" ed., vol. For example, a nucleic acid molecule is specifically hybrid 1-3, Cold Spring Harbor Laboratory Press, Cold Spring izable when there is a sufficient degree of complementarity Harbor, N.Y., 1989, chapters 9 and 11; and Hames and to avoid non-specific binding of the nucleic acid to non Higgins (eds.) Nucleic Acid Hybridization, IRL Press, target polynucleotides under conditions where specific bind Oxford, 1985. Further detailed instruction and guidance with ing is desired, for example, under stringent hybridization regard to the hybridization of nucleic acids may be found, conditions. for example, in Tijssen, “Overview of principles of hybrid 0.130. As used herein, the term “ortholog” refers to a gene ization and the strategy of nucleic acid probe assays.” in in two or more species that has evolved from a common Laboratory Techniques in Biochemistry and Molecular Biol ancestral nucleic acid, and may retain the same function in ogy Hybridization with Nucleic Acid Probes, Part I, Chap the two or more species. ter 2, Elsevier, NY, 1993; and Ausubel et al., Eds. Current 0.131. As used herein, two nucleic acid molecules are said Protocols in Molecular Biology, Chapter 2, Greene Publish to exhibit “complete complementarity” when every nucleo ing and Wiley-Interscience, NY, 1995. tide of a polynucleotide read in the 5' to 3’ direction is 0124 AS used herein, 'stringent conditions' encompass complementary to every nucleotide of the other polynucle conditions under which hybridization will only occur if there otide when read in the 3' to 5' direction. A polynucleotide is less than 20% mismatch between the sequence of the that is complementary to a reference polynucleotide will hybridization molecule and a homologous polynucleotide exhibit a sequence identical to the reverse complement of within the target nucleic acid molecule. “Stringent condi the reference polynucleotide. These terms and descriptions tions’ include further particular levels of stringency. Thus, are well defined in the art and are easily understood by those as used herein, "moderate stringency' conditions are those of ordinary skill in the art. under which molecules with more than 20% sequence I0132) Operably linked: A first polynucleotide is operably mismatch will not hybridize; conditions of “high stringency’ linked with a second polynucleotide when the first poly are those under which sequences with more than 10% nucleotide is in a functional relationship with the second mismatch will not hybridize; and conditions of “very high polynucleotide. When recombinantly produced, operably stringency are those under which sequences with more than linked polynucleotides are generally contiguous, and, where 5% mismatch will not hybridize. necessary to join two protein-coding regions, in the same 0.125. The following are representative, non-limiting reading frame (e.g., in a translationally fused ORF). How hybridization conditions. ever, nucleic acids need not be contiguous to be operably 0126 High Stringency condition (detects polynucle linked. otides that share at least 90% sequence identity): Hybrid I0133. The term, “operably linked,” when used in refer ization in 5xSSC buffer at 65° C. for 16 hours; wash twice ence to a regulatory genetic element and a coding poly in 2xSSC buffer at room temperature for 15 minutes each; nucleotide, means that the regulatory element affects the and wash twice in 0.5xSSC buffer at 65° C. for 20 minutes expression of the linked coding polynucleotide. "Regulatory each. elements.’’ or “control elements.” refer to polynucleotides 0127. Moderate Stringency condition (detects polynucle that influence the timing and level/amount of transcription, otides that share at least 80% sequence identity): Hybrid RNA processing or stability, or translation of the associated ization in 5x-6xSSC buffer at 65-70° C. for 16-20 hours: coding polynucleotide. Regulatory elements may include wash twice in 2xSSC buffer at room temperature for 5-20 promoters; translation leaders; introns; enhancers; stem-loop minutes each; and wash twice in 1xSSC buffer at 55-70° C. structures; repressor binding polynucleotides; polynucle for 30 minutes each. otides with a termination sequence; polynucleotides with a 0128. Non-stringent control condition (polynucleotides polyadenylation recognition sequence; etc. Particular regu that share at least 50% sequence identity will hybridize): latory elements may be located upstream and/or downstream Hybridization in 6xSSC buffer at room temperature to 55° of a coding polynucleotide operably linked thereto. Also, C. for 16-20 hours; wash at least twice in 2x-3XSSC buffer particular regulatory elements operably linked to a coding at room temperature to 55° C. for 20-30 minutes each. polynucleotide may be located on the associated comple 0129. As used herein, the term “substantially homolo mentary strand of a double-stranded nucleic acid molecule. gous' or 'substantial homology,’ with regard to a nucleic 0.134 Promoter: As used herein, the term “promoter' acid, refers to a polynucleotide having contiguous nucle refers to a region of DNA that may be upstream from the obases that hybridize under stringent conditions to the start of transcription, and that may be involved in recogni reference nucleic acid. For example, nucleic acids that are tion and binding of RNA polymerase and other proteins to Substantially homologous to a reference nucleic acid of any initiate transcription. A promoter may be operably linked to of SEQ ID NOS:1 and 71 are those nucleic acids that a coding polynucleotide for expression in a cell, or a hybridize under stringent conditions (e.g., the Moderate promoter may be operably linked to a polynucleotide encod US 2017/0218391 A1 Aug. 3, 2017 ing a signal peptide which may be operably linked to a molecule into the cellular genome, or by episomal replica coding polynucleotide for expression in a cell. A "plant tion. As used herein, the term “transformation' encompasses promoter may be a promoter capable of initiating transcrip all techniques by which a nucleic acid molecule can be tion in plant cells. Examples of promoters under develop introduced into Such a cell. Examples include, but are not mental control include promoters that preferentially initiate limited to: transfection with viral vectors; transformation transcription in certain tissues, such as leaves, roots, seeds, with plasmid vectors; electroporation (Fromm et al. (1986) fibers, xylem vessels, tracheids, or Sclerenchyma. Such Nature 319:791-3); lipofection (Feigner et al. (1987) Proc. promoters are referred to as “tissue-preferred. Promoters Natl. Acad. Sci. USA84:7413-7); microinjection (Mueller et which initiate transcription only in certain tissues are al. (1978) Cell 15:579-85); Agrobacterium-mediated trans referred to as “tissue-specific''. A "cell type-specific' pro fer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA80:4803 moter primarily drives expression in certain cell types in one 7); direct DNA uptake; and microprojectile bombardment or more organs, for example, vascular cells in roots or (Klein et al. (1987) Nature 327:70). leaves. An “inducible' promoter may be a promoter which 0140 Transgene: An exogenous nucleic acid. In some may be under environmental control. Examples of environ examples, a transgene may be a DNA that encodes one or mental conditions that may initiate transcription by induc both strand(s) of an RNA capable of forming a dsRNA ible promoters include anaerobic conditions and the pres molecule that comprises a polynucleotide that is comple ence of light. Tissue-specific, tissue-preferred, cell type mentary to a nucleic acid molecule found in a coleopteran specific, and inducible promoters constitute the class of and/or hemipteran pest. In some examples, a transgene may “non-constitutive' promoters. A “constitutive' promoter is a be an antisense polynucleotide, wherein expression of the promoter which may be active under most environmental antisense polynucleotide inhibits expression of a target conditions or in most tissue or cell types. nucleic acid, thereby producing an RNAi phenotype. In 0135 Any inducible promoter can be used in some Some examples, a transgene may be a structural gene (e.g., embodiments of the invention. See Ward et al. (1993) Plant a herbicide-tolerance gene, a gene encoding an industrially Mol. Biol. 22:361-366. With an inducible promoter, the rate or pharmaceutically useful compound, or a gene encoding a of transcription increases in response to an inducing agent. desirable agricultural trait). In these and other examples, a Exemplary inducible promoters include, but are not limited transgene may contain regulatory elements operably linked to: Promoters from the ACEI system that respond to copper; to a coding polynucleotide of the transgene (e.g., a pro Int gene from maize that responds to benzenesulfonamide moter). herbicide safeners; Tet repressor from Tn10; and the induc 0141 Vector: A nucleic acid molecule as introduced into ible promoter from a steroid hormone gene, the transcrip a cell, for example, to produce a transformed cell. A vector tional activity of which may be induced by a glucocorticos may include genetic elements that permit it to replicate in the teroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. host cell. Such as an origin of replication. Examples of USA 88:0421). vectors include, but are not limited to: a plasmid; cosmid; 0.136 Exemplary constitutive promoters include, but are bacteriophage; or virus that carries exogenous DNA into a not limited to: Promoters from plant viruses, such as the 35S cell. A vector may also include one or more genes, including promoter from Cauliflower Mosaic Virus (CaMV); promot ones that produce antisense molecules, and/or selectable ers from rice actin genes; ubiquitin promoters; pEMU; marker genes and other genetic elements known in the art. MAS; maize H3 histone promoter; and the ALS promoter, A vector may transduce, transform, or infect a cell, thereby Xbal/NcoI fragment 5' to the Brassica napus ALS3 struc causing the cell to express the nucleic acid molecules and/or tural gene (or a polynucleotide similar to said Xbal/NcoI proteins encoded by the vector. A vector optionally includes fragment) (International PCT Publication No. WO96/ materials to aid in achieving entry of the nucleic acid 30530). molecule into the cell (e.g., a liposome, protein coating, 0.137 Additionally, any tissue-specific or tissue-preferred etc.). promoter may be utilized in some embodiments of the 0142. Yield: A stabilized yield of about 100% or greater invention. Plants transformed with a nucleic acid molecule relative to the yield of check varieties in the same growing comprising a coding polynucleotide operably linked to a location growing at the same time and under the same tissue-specific promoter may produce the product of the conditions. In particular embodiments, “improved yield' or coding polynucleotide exclusively, or preferentially, in a “improving yield’ means a cultivar having a stabilized yield specific tissue. Exemplary tissue-specific or tissue-preferred of 105% or greater relative to the yield of check varieties in promoters include, but are not limited to: A seed-preferred the same growing location containing significant densities of promoter. Such as that from the phaseolin gene; a leaf the coleopteran and/or hemipteran pests that are injurious to specific and light-induced promoter Such as that from cab or that crop growing at the same time and under the same rubisco; an anther-specific promoter such as that from conditions, which are targeted by the compositions and LAT52; a pollen-specific promoter such as that from Zm 13: methods herein. and a microspore-preferred promoter Such as that from apg. 0.143 Unless specifically indicated or implied, the terms 0138 Soybean plant: As used herein, the term “soybean “a,” “an, and “the signify “at least one,” as used herein. plant” refers to a plant of the species Glycine; for example, 0144. Unless otherwise specifically explained, all tech Glycine max. nical and Scientific terms used herein have the same meaning 0139 Transformation: As used herein, the term “trans as commonly understood by those of ordinary skill in the art formation' or “transduction” refers to the transfer of one or to which this disclosure belongs. Definitions of common more nucleic acid molecule(s) into a cell. A cell is “trans terms in molecular biology can be found in, for example, formed by a nucleic acid molecule transduced into the cell Lewin's Genes X. Jones & Bartlett Publishers, 2009 (ISBN when the nucleic acid molecule becomes stably replicated 10 0763766321); Krebs et al. (eds.), The Encyclopedia of by the cell, either by incorporation of the nucleic acid Molecular Biology, Blackwell Science Ltd., 1994 (ISBN US 2017/0218391 A1 Aug. 3, 2017
0-632-02182-9); and Meyers R. A. (ed.), Molecular Biology 0149 Provided according to the invention are DNAs, the and Biotechnology: A Comprehensive Desk Reference, VCH expression of which results in an RNA molecule comprising Publishers, Inc., 1995 (ISBN 1-56081-569-8). All percent a polynucleotide that is specifically complementary to all or ages are by weight and all solvent mixture proportions are by part of a native RNA molecule that is encoded by a coding Volume unless otherwise noted. All temperatures are in polynucleotide in an insect (e.g., coleopteran and/or degrees Celsius. hemipteran) pest. In some embodiments, after ingestion of the expressed RNA molecule by an insect pest, down IV Nucleic Acid Molecules Comprising an Insect regulation of the coding polynucleotide in cells of the pest Pest Sequence may be obtained. In particular embodiments, down-regula (0145 A. Overview tion of the coding polynucleotide in cells of the insect pest 0146 Described herein are nucleic acid molecules useful may result in a deleterious effect on the growth and/or for the control of insect pests. In some examples, the insect development of the pest. pest is a coleopteran (e.g., species of the genus Diabrotica) 0150. In some embodiments, target polynucleotides or hemipteran (e.g., species of the genus Euschistus) insect include transcribed non-coding RNAs, such as 5'UTRs: pest. Described nucleic acid molecules include target poly 3'UTRs; spliced leaders; introns; outrons (e.g., 5'UTR RNA nucleotides (e.g., native genes, and non-coding polynucle Subsequently modified in trans splicing); donatrons (e.g., otides), dsRNAs, siRNAs, shRNAs, hpRNAs, and miRNAs. non-coding RNA required to provide donor sequences for For example, dsRNA, siRNA, miRNA, shRNA, and/or trans splicing); and other non-coding transcribed RNA of hpRNA molecules are described in some embodiments that target insect pest genes. Such polynucleotides may be may be specifically complementary to all or part of one or derived from both mono-cistronic and poly-cistronic genes. more native nucleic acids in a coleopteran and/or hemipteran 0151. Thus, also described herein in connection with pest. In these and further embodiments, the native nucleic some embodiments are iRNA molecules (e.g., dsRNAs, acid(s) may be one or more target gene(s), the product of siRNAs, miRNAs, shRNAs, and hpRNAs) that comprise at which may be, for example and without limitation: involved least one polynucleotide that is specifically complementary in a metabolic process or involved in larval/nymph devel to all or part of a target nucleic acid in an insect (e.g., opment. Nucleic acid molecules described herein, when coleopteran and/or hemipteran) pest. In some embodiments introduced into a cell comprising at least one native nucleic an iRNA molecule may comprise polynucleotide(s) that are acid(s) to which the nucleic acid molecules are specifically complementary to all or part of a plurality of target nucleic complementary, may initiate RNAi in the cell, and conse acids; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target quently reduce or eliminate expression of the native nucleic nucleic acids. In particular embodiments, an iRNA molecule acid(s). In some examples, reduction or elimination of the may be produced in vitro or in vivo by a genetically expression of a target gene by a nucleic acid molecule modified organism, Such as a plant or bacterium. Also specifically complementary thereto may result in reduction disclosed are cDNAs that may be used for the production of or cessation of growth, development, and/or feeding in the dsRNA molecules, siRNA molecules, miRNA molecules, pest. shRNA molecules, and/or hpRNA molecules that are spe 0147 In some embodiments, at least one target gene in an cifically complementary to all or part of a target nucleic acid insect pest may be selected, wherein the target gene com in an insect pest. Further described are recombinant DNA prises a gw polynucleotide. In particular examples, a target constructs for use in achieving stable transformation of gene in a coleopteran pest is selected, wherein the target particular host targets. Transformed host targets may express gene comprises a polynucleotide selected from among SEQ effective levels of dsRNA, siRNA, miRNA, shRNA, and/or ID NOs: 1 and 3-5. In particular examples, a target gene in hpRNA molecules from the recombinant DNA constructs. a hemipteran pest is selected, wherein the target gene Therefore, also described is a plant transformation vector comprises the polynucleotide of SEQID NOS:71 and/or the comprising at least one polynucleotide operably linked to a polynucleotide of SEQ ID NO:73. heterologous promoter functional in a plant cell, wherein 0148. In some embodiments, a target gene may be a expression of the polynucleotide(s) results in an RNA mol nucleic acid molecule comprising a polynucleotide that can ecule comprising a string of contiguous nucleobases that is be reverse translated in silico to a polypeptide comprising a specifically complementary to all or part of a target nucleic contiguous amino acid sequence that is at least about 85% acid in an insect pest. identical (e.g., at least 84%, 85%, about 90%, about 95%, 0152. In particular examples, nucleic acid molecules use about 96%, about 97%, about 98%, about 99%, about 100%, ful for the control of insect (e.g., coleopteran and/or or 100% identical) to the amino acid sequence of a protein hemipteran) pests may include: all or at least 15 contiguous product of a gw polynucleotide. A target gene may be any nucleotides of a native nucleic acid isolated from Diabrotica gw polynucleotide in an insect pest, the post-transcriptional comprising a gw polynucleotide (e.g., SEQ ID NO: 1); inhibition of which has a deleterious effect on the growth, DNAs that when expressed result in an RNA molecule survival, and/or viability of the pest, for example, to provide comprising a polynucleotide that is specifically complemen a protective benefit against the pest to a plant. In particular tary to all or at least 15 contiguous nucleotides of a native examples, a target gene is a nucleic acid molecule compris RNA molecule that is encoded by Diabrotica gw: iRNA ing a polynucleotide that can be reverse translated in silico molecules (e.g., dsRNAs, siRNAs, miRNAs, shRNAs, and to a polypeptide comprising a contiguous amino acid hpRNAs) that comprise at least one polynucleotide that is sequence that is at least about 85% identical, about 90% specifically complementary to all or at least 15 contiguous identical, about 95% identical, about 96% identical, about nucleotides of Diabroticagw; c)NAs that may be used for 97% identical, about 98% identical, about 99% identical, the production of dsRNA molecules, siRNA molecules, about 100% identical, or 100% identical to the amino acid miRNA molecules, shRNA molecules, and/or hpRNA mol sequence of SEQ ID NO:2 or SEQ ID NO:72. ecules that are specifically complementary to all or at least US 2017/0218391 A1 Aug. 3, 2017
15 contiguous nucleotides of Diabrotica gw; all or at least iRNA transcribed from the isolated polynucleotide inhibits 15 contiguous nucleotides of a native nucleic acid isolated the growth, development, and/or feeding of the pest. In some from Euschistus heros comprising a gw polynucleotide (e.g., embodiments, contact with or uptake by the insect occurs via SEQ ID NO:71); DNAs that when expressed result in an feeding on plant material or bait comprising the iRNA RNA molecule comprising a polynucleotide that is specifi (“RNAi bait). In some embodiments, contact with or cally complementary to all or at least 15 contiguous nucleo uptake by the insect occurs via spraying of a plant compris tides of a native RNA molecule that is encoded by E. heros ing the insect with a composition comprising the iRNA. gw; iRNA molecules that comprise at least one polynucle 0158. In some embodiments, an isolated nucleic acid otide that is specifically complementary to all or at least 15 molecule of the invention may comprise at least one (e.g., contiguous nucleotides of E. heros gw; cDNAS that may be one, two, three, or more) polynucleotide(s) selected from the used for the production of dsRNA molecules, siRNA mol group consisting of: SEQ ID NO:79; the complement or ecules, miRNA molecules, shRNA molecules, and/or reverse complement of SEQID NO:79: SEQID NO:80; the hpRNA molecules that are specifically complementary to all complement or reverse complement of SEQID NO:80: SEQ or part of E. heros gw; and recombinant DNA constructs for ID NO:81; the complement or reverse complement of SEQ use in achieving stable transformation of particular host ID NO:81; SEQ ID NO:82; the complement or reverse targets, wherein a transformed host target comprises one or complement of SEQID NO:82; SEQID NO:83; the comple more of the foregoing nucleic acid molecules. ment or reverse complement of SEQ ID NO:83: SEQ ID 0153. B. Nucleic Acid Molecules NO:84; the complement or reverse complement of SEQ ID 0154 Embodiments include, inter alia, iRNA molecules NO:84; a fragment of at least 15 contiguous nucleotides of (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) that any of SEQ ID NOS:79-84; the complement or reverse inhibit target gene expression in a cell, tissue, or organ of an complement of a fragment of at least 15 contiguous nucleo insect (e.g., coleopteran and/or hemipteran) pest, and DNA tides of any of SEQ ID NOS:79-84; a native coding poly molecules capable of being expressed as an iRNA molecule nucleotide of a Diabrotica organism comprising any of SEQ in a cell or microorganism to inhibit target gene expression ID NOS:80-82; the complement or reverse complement of a in a cell, tissue, or organ of an insect pest. native coding polynucleotide of a Diabrotica organism 0155 Some embodiments of the invention provide an comprising any of SEQID NOs:80-82; a fragment of at least isolated nucleic acid molecule comprising at least one (e.g., 15 contiguous nucleotides of a native coding polynucleotide one, two, three, or more) polynucleotide(s) selected from the of a Diabrotica organism comprising any of SEQ ID NOs: group consisting of SEQ ID NO:1; the complement or 80-82; the complement or reverse complement of a fragment reverse complement of SEQID NO:1; a fragment of at least of at least 15 contiguous nucleotides of a native coding 15 contiguous nucleotides of SEQ ID NO:1 (e.g., any of polynucleotide of a Diabrotica organism comprising any of SEQ ID NOS:3-5); the complement or reverse complement SEQ ID NOS:80-82; a native coding polynucleotide of a of a fragment of at least 15 contiguous nucleotides of SEQ Euschistus organism comprising SEQ ID NO:84; the ID NO:1; a native coding polynucleotide of a Diabrotica complement or reverse complement of a native coding organism (e.g., WCR) comprising any of SEQID NOS:3-5; polynucleotide of a Euschistus organism comprising SEQ the complement or reverse complement of a native coding ID NO:84; a fragment of at least 15 contiguous nucleotides polynucleotide of a Diabrotica organism comprising any of of a native coding polynucleotide of a Euschistus organism SEQ ID NOS:3-5; a fragment of at least 15 contiguous comprising SEQ ID NO:84; and the complement or reverse nucleotides of a native coding polynucleotide of a Dia complement of a fragment of at least 15 contiguous nucleo brotica organism comprising any of SEQ ID NOS:3-5; and tides of a native coding polynucleotide of a Euschistus the complement or reverse complement of a fragment of at organism comprising SEQ ID NO:84. least 15 contiguous nucleotides of a native coding poly 0159. In certain embodiments, dsRNA molecules pro nucleotide of a Diabrotica organism comprising any of SEQ vided by the invention comprise polynucleotides comple ID NOS:3-5. mentary to a transcript from a target gene comprising either 0156 Some embodiments of the invention provide an of SEQ ID NO:1 and SEQ ID NO:71, and fragments of at isolated nucleic acid molecule comprising at least one (e.g., least 15 contiguous nucleotides thereof, the inhibition of one, two, three, or more) polynucleotide(s) selected from the which target gene in an insect pest results in the reduction or group consisting of: SEQ ID NO:71; the complement or removal of a polypeptide or polynucleotide agent that is reverse complement of SEQ ID NO:71; a fragment of at essential for the pest’s growth, development, or other bio least 15 contiguous nucleotides of SEQID NO:71 (e.g., SEQ logical function. A selected polynucleotide may exhibit from ID NO:73); the complement or reverse complement of a about 80% to about 100% sequence identity to either of SEQ fragment of at least 15 contiguous nucleotides of SEQ ID ID NOS:1 and 71; a fragment of at least 15 contiguous NO:71; a native coding polynucleotide of a hemipteran nucleotides of either of SEQ ID NOS:1 and 71; and the organism (e.g., BSB) comprising SEQ ID NO:73; the complement or reverse complement of any of the foregoing. complement or reverse complement of a native coding For example, a selected polynucleotide may exhibit 79%; polynucleotide of a hemipteran organism comprising SEQ 80%; about 81%; about 82%; about 83%; about 84%; about ID NO:73; a fragment of at least 15 contiguous nucleotides 85%; about 86%; about 87%; about 88%; about 89%; about of a native coding polynucleotide of a hemipteran organism 90%; about 91%; about 92%; about 93%; about 94% about comprising SEQID NO:73; and the complement or reverse 95%; about 96%; about 97%; about 98%; about 98.5%: complement of a fragment of at least 15 contiguous nucleo about 99%; about 99.5%; or about 100% sequence identity tides of a native coding polynucleotide of a hemipteran to any of either of SEQ ID NOS:1 and 71; a fragment of at organism comprising SEQ ID NO:73. least 15 contiguous nucleotides of either of SEQ ID NOs:1 0157. In particular embodiments, contact with or uptake and 71 (e.g., SEQID NOS:3-5 and 73); and the complement by an insect (e.g., coleopteran and/or hemipteran) pest of an or reverse complement of any of the foregoing. US 2017/0218391 A1 Aug. 3, 2017
0160. In some embodiments, a DNA molecule capable of ecules produced by endogenous RNase III enzymes from being expressed as an iRNA molecule in a cell or microor heterologous nucleic acid molecules may efficiently mediate ganism to inhibit target gene expression may comprise a the down-regulation of target genes in insect pests. single polynucleotide that is specifically complementary to (0164. In some embodiments, a nucleic acid molecule all or part of a native polynucleotide found in one or more may include at least one non-naturally occurring polynucle target insect pest species (e.g., a coleopteran or hemipteran otide that can be transcribed into a single-stranded RNA pest species), or the DNA molecule can be constructed as a molecule capable of forming a dsRNA molecule in vivo chimera from a plurality of Such specifically complementary through intermolecular hybridization. Such dsRNAs typi polynucleotides. cally self-assemble, and can be provided in the nutrition 0161 In other embodiments, a nucleic acid molecule may Source of an insect (e.g., coleopteran or hemipteran) pest to comprise a first and a second polynucleotide separated by a achieve the post-transcriptional inhibition of a target gene. 'spacer.” A spacer may be a region comprising any sequence In these and further embodiments, a nucleic acid molecule of nucleotides that facilitates secondary structure formation may comprise two different non-naturally occurring poly between the first and second polynucleotides, where this is nucleotides, each of which is specifically complementary to desired. In one embodiment, the spacer is part of a sense or a different target gene in an insect pest. When such a nucleic antisense coding polynucleotide for mRNA. The spacer may acid molecule is provided as a dsRNA molecule to, for alternatively comprise any combination of nucleotides or example, a coleopteran and/or hemipteran pest, the dsRNA homologues thereof that are capable of being linked cova molecule inhibits the expression of at least two different lently to a nucleic acid molecule. In some examples, the target genes in the pest. spacer may be an intron (e.g., as ST-LS1 intron). (0165 C. Obtaining Nucleic Acid Molecules 0162 For example, in some embodiments, the DNA 0166 A variety of polynucleotides in insect (e.g., cole molecule may comprise a polynucleotide coding for one or opteran and hemipteran) pests may be used as targets for the more different iRNA molecules, wherein each of the differ design of nucleic acid molecules, such as iRNAs and DNA ent iRNA molecules comprises a first polynucleotide and a molecules encoding iRNAs. Selection of native polynucle second polynucleotide, wherein the first and second poly otides is not, however, a straight-forward process. For nucleotides are complementary to each other. The first and example, only a small number of native polynucleotides in second polynucleotides may be connected within an RNA a coleopteran or hemipteran pest will be effective targets. It molecule by a spacer. The spacer may constitute part of the cannot be predicted with certainty whether a particular first polynucleotide or the second polynucleotide. Expres native polynucleotide can be effectively down-regulated by sion of a RNA molecule comprising the first and second nucleic acid molecules of the invention, or whether down nucleotide polynucleotides may lead to the formation of a regulation of a particular native polynucleotide will have a dsRNA molecule, by specific intramolecular base-pairing of detrimental effect on the growth, viability, feeding, and/or the first and second nucleotide polynucleotides. The first Survival of an insect pest. The vast majority of native polynucleotide or the second polynucleotide may be Sub coleopteran and hemipteran pest polynucleotides, such as stantially identical to a polynucleotide (e.g., a target gene, or ESTs isolated therefrom (for example, the coleopteran pest transcribed non-coding polynucleotide) native to an insect polynucleotides listed in U.S. Pat. No. 7,612,194), do not pest (e.g., a coleopteran or hemipteran pest), a derivative have a detrimental effect on the growth and/or viability of thereof, or a complementary polynucleotide thereto. the pest. Neither is it predictable which of the native 0163 dsRNA nucleic acid molecules comprise double polynucleotides that may have a detrimental effect on an Strands of polymerized ribonucleotides, and may include insect pest are able to be used in recombinant techniques for modifications to either the phosphate-sugar backbone or the expressing nucleic acid molecules complementary to Such nucleoside. Modifications in RNA structure may be tailored native polynucleotides in a host plant and providing the to allow specific inhibition. In one embodiment, dsRNA detrimental effect on the pest upon feeding without causing molecules may be modified through a ubiquitous enzymatic harm to the host plant. process so that siRNA molecules may be generated. This (0167. In some embodiments, nucleic acid molecules enzymatic process may utilize an RNase III enzyme. Such as (e.g., dsRNA molecules to be provided in the host plant of DICER in eukaryotes, either in vitro or in vivo. See Elbashir an insect (e.g., coleopteran or hemipteran) pest) are selected et al. (2001) Nature 411:494-8; and Hamilton and Baul to target cDNAS that encode proteins or parts of proteins combe (1999) Science 286(5441):950-2. DICER or func essential for pest development, such as polypeptides tionally-equivalent RNase III enzymes cleave larger dsRNA involved in metabolic or catabolic biochemical pathways, strands and/or hpRNA molecules into smaller oligonucle cell division, energy metabolism, digestion, host plant rec otides (e.g., siRNAs), each of which is about 19-25 nucleo ognition, and the like. As described herein, ingestion of tides in length. The siRNA molecules produced by these compositions by a target pest organism containing one or enzymes have 2 to 3 nucleotide 3' overhangs, and 5' phos more dsRNAs, at least one segment of which is specifically phate and 3' hydroxyl termini. The siRNA molecules gen complementary to at least a substantially identical segment erated by RNase III enzymes are unwound and separated of RNA produced in the cells of the target pest organism, can into single-stranded RNA in the cell. The siRNA molecules result in the death or other inhibition of the target. A then specifically hybridize with RNAs transcribed from a polynucleotide, either DNA or RNA, derived from an insect target gene, and both RNA molecules are Subsequently pest can be used to construct plant cells protected against degraded by an inherent cellular RNA-degrading mecha infestation by the pests. The host plant of the coleopteran nism. This process may result in the effective degradation or and/or hemipteran pest (e.g., Z. mays or G. max), for removal of the RNA encoded by the target gene in the target example, can be transformed to contain one or more poly organism. The outcome is the post-transcriptional silencing nucleotides derived from the coleopteran and/or hemipteran of the targeted gene. In some embodiments, siRNA mol pest as provided herein. The polynucleotide transformed US 2017/0218391 A1 Aug. 3, 2017
into the host may encode one or more RNAs that form into from a target organism, and nucleic acid libraries may be a dsRNA structure in the cells or biological fluids within the prepared therefrom using methods known to those ordinarily transformed host, thus making the dsRNA available if/when skilled in the art. gldNA or cDNA libraries generated from the pest forms a nutritional relationship with the transgenic a target organism may be used for PCR amplification and host. This may result in the Suppression of expression of one sequencing of target genes. A confirmed PCR product may or more genes in the cells of the pest, and ultimately death be used as a template for in vitro transcription to generate or inhibition of its growth or development. sense and antisense RNA with minimal promoters. Alterna 0.168. In particular embodiments, a gene is targeted that is tively, nucleic acid molecules may be synthesized by any of essentially involved in the growth and development of an a number of techniques (See, e.g., Ozaki et al. (1992) insect (e.g., coleopteran or hemipteran) pest. Other target Nucleic Acids Research, 20: 5205-5214; and Agrawal et al. genes for use in the present invention may include, for (1990) Nucleic Acids Research, 18: 5419-5423), including example, those that play important roles in pest viability, use of an automated DNA synthesizer (for example, a P.E. movement, migration, growth, development, infectivity, and Biosystems, Inc. (Foster City, Calif.) model 392 or 394 establishment of feeding sites. A target gene may therefore DNA/RNA Synthesizer), using standard chemistries, such as be a housekeeping gene or a transcription factor. Addition phosphoramidite chemistry. See, e.g., Beaucage et al. (1992) ally, a native insect pest polynucleotide for use in the present Tetrahedron, 48: 2223-2311; U.S. Pat. Nos. 4,980,460, invention may also be derived from a homolog (e.g., an 4,725,677, 4,415,732, 4,458,066, and 4,973,679. Alternative ortholog), of a plant, viral, bacterial or insect gene, the chemistries resulting in non-natural backbone groups, such function of which is known to those of skill in the art, and as phosphorothioate, phosphoramidate, and the like, can also the polynucleotide of which is specifically hybridizable with be employed. a target gene in the genome of the target pest. Methods of 0172 An RNA, dsRNA, siRNA, miRNA, shRNA, or identifying a homolog of a gene with a known nucleotide hpRNA molecule of the present invention may be produced sequence by hybridization are known to those of skill in the chemically or enzymatically by one skilled in the art through art manual or automated reactions, or in vivo in a cell com 0169. In some embodiments, the invention provides prising a nucleic acid molecule comprising a polynucleotide methods for obtaining a nucleic acid molecule comprising a encoding the RNA, dsRNA, siRNA, miRNA, shRNA, or polynucleotide for producing an iRNA (e.g., dsRNA, hpRNA molecule. RNA may also be produced by partial or siRNA, miRNA, shRNA, and hpRNA) molecule. One such total organic synthesis—any modified ribonucleotide can be embodiment comprises: (a) analyzing one or more target introduced by in vitro enzymatic or organic synthesis. An gene(s) for their expression, function, and phenotype upon RNA molecule may be synthesized by a cellular RNA dsRNA-mediated gene Suppression in an insect (e.g., cole polymerase or a bacteriophage RNA polymerase (e.g., T3 opteran or hemipteran) pest; (b) probing a cDNA or gldNA RNA polymerase, T7 RNA polymerase, and SP6 RNA library with a probe comprising all or a portion of a polymerase). Expression constructs useful for the cloning polynucleotide or a homolog thereof from a targeted pest and expression of polynucleotides are known in the art. See, that displays an altered (e.g., reduced) growth or develop e.g., International PCT Publication No. WO97/32016; and ment phenotype in a dsRNA-mediated Suppression analysis; U.S. Pat. Nos. 5,593.874, 5,698,425, 5,712,135, 5,789,214, (c) identifying a DNA clone that specifically hybridizes with and 5,804,693. RNA molecules that are synthesized chemi the probe; (d) isolating the DNA clone identified in step (b): cally or by in vitro enzymatic synthesis may be purified prior (e) sequencing the cDNA org)NA fragment that comprises to introduction into a cell. For example, RNA molecules can the clone isolated in step (d), wherein the sequenced nucleic be purified from a mixture by extraction with a solvent or acid molecule comprises all or a substantial portion of the resin, precipitation, electrophoresis, chromatography, or a RNA or a homolog thereof; and (f) chemically synthesizing combination thereof. Alternatively, RNA molecules that are all or a substantial portion of a gene, oran siRNA, miRNA, synthesized chemically or by in vitro enzymatic synthesis hpRNA, mRNA, shRNA, or dsRNA. may be used with no or a minimum of purification, for 0170 In further embodiments, a method for obtaining a example, to avoid losses due to sample processing. The nucleic acid fragment comprising a polynucleotide for pro RNA molecules may be dried for storage or dissolved in an ducing a substantial portion of an iRNA (e.g., dsRNA, aqueous solution. The solution may contain buffers or salts siRNA, miRNA, shRNA, and hpRNA) molecule includes: to promote annealing, and/or stabilization of dsRNA mol (a) synthesizing first and second oligonucleotide primers ecule duplex strands. specifically complementary to a portion of a native poly 0173. In embodiments, a dsRNA molecule may be nucleotide from a targeted insect (e.g., coleopteran or formed by a single self-complementary RNA strand or from hemipteran) pest; and (b) amplifying a cDNA or gldNA two complementary RNA strands. dsRNA molecules may be insert present in a cloning vector using the first and second synthesized either in vivo or in vitro. An endogenous RNA oligonucleotide primers of step (a), wherein the amplified polymerase of the cell may mediate transcription of the one nucleic acid molecule comprises a Substantial portion of a or two RNA strands in vivo, or cloned RNA polymerase may siRNA, miRNA, hpRNA, mRNA, shRNA, or dsRNA mol be used to mediate transcription in vivo or in vitro. Post ecule. transcriptional inhibition of a target gene in an insect pest 0171 Nucleic acids can be isolated, amplified, or pro may be host-targeted by specific transcription in an organ, duced by a number of approaches. For example, an iRNA tissue, or cell type of the host (e.g., by using a tissue-specific (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) mol promoter); Stimulation of an environmental condition in the ecule may be obtained by PCR amplification of a target host (e.g., by using an inducible promoter that is responsive polynucleotide (e.g., a target gene or a target transcribed to infection, stress, temperature, and/or chemical inducers); non-coding polynucleotide) derived from a gldNA or cDNA and/or engineering transcription at a developmental stage or library, or portions thereof. DNA or RNA may be extracted age of the host (e.g., by using a developmental stage-specific US 2017/0218391 A1 Aug. 3, 2017 20 promoter). RNA strands that form a dsRNA molecule, fragment of at least 15 contiguous nucleotides of a native whether transcribed in vitro or in vivo, may or may not be coding polynucleotide of a hemipteran organism comprising polyadenylated, and may or may not be capable of being SEQ ID NO:73. translated into a polypeptide by a cells translational appa 0.178 In some embodiments, one strand of a dsRNA ratuS. molecule may be formed by transcription from a polynucle 0174 D. Recombinant Vectors and Host Cell Transfor otide that is Substantially homologous to a polynucleotide mation selected from the group consisting of SEQID NOS:3-5 and 0.175. In some embodiments, the invention also provides 73; the complement or reverse complement of any of SEQ a DNA molecule for introduction into a cell (e.g., a bacterial ID NOS:3-5 and 73; a fragment of at least 15 contiguous cell, a yeast cell, or a plant cell), wherein the DNA molecule nucleotides of either of SEQ ID NOS:1 and 71; and the comprises a polynucleotide that, upon expression to RNA complement or reverse complement of a fragment of at least and ingestion by an insect (e.g., coleopteran and/or 15 contiguous nucleotides of either of SEQ ID NOS:1 and hemipteran) pest, achieves Suppression of a target gene in a 71. cell, tissue, or organ of the pest. Thus, some embodiments 0179. In particular embodiments, a recombinant DNA provide a recombinant nucleic acid molecule comprising a molecule encoding an RNA that may form a dsRNA mol polynucleotide capable of being expressed as an iRNA (e.g., ecule may comprise a coding region wherein at least two dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule in polynucleotides are arranged Such that one polynucleotide is a plant cell to inhibit target gene expression in an insect pest. in a sense orientation, and the other polynucleotide is in an In order to initiate or enhance expression, such recombinant antisense orientation, relative to at least one promoter, nucleic acid molecules may comprise one or more regula wherein the sense polynucleotide and the antisense poly tory elements, which regulatory elements may be operably nucleotide are linked or connected by a spacer of for linked to the polynucleotide capable of being expressed as example, from about five (-5) to about one thousand an iRNA. Methods to express a gene Suppression molecule (~1000) nucleotides. The spacer may form a loop between in plants are known, and may be used to express a poly the sense and antisense polynucleotides. The sense poly nucleotide of the present invention. See, e.g., International nucleotide or the antisense polynucleotide may be substan PCT Publication No. WO06/073727; and U.S. Patent Pub tially homologous to a target gene (e.g., a gW gene com lication No. 2006/0200878 A1) prising either of SEQ ID NOS:1 and 71) or a fragment (0176). In specific embodiments, a recombinant DNA mol comprising at least 15 contiguous nucleotides thereof. In ecule of the invention may comprise a polynucleotide some embodiments, however, a recombinant DNA molecule encoding an RNA that may form a dsRNA molecule. Such may encode an RNA that may form a dsRNA molecule recombinant DNA molecules may encode RNAs that may without a spacer. In embodiments, a sense coding polynucle form dsRNA molecules capable of inhibiting the expression otide and an antisense coding polynucleotide may be dif of endogenous target gene(s) in an insect (e.g., coleopteran ferent lengths. and/or hemipteran) pest cell upon ingestion. In many 0180 Polynucleotides identified as having a deleterious embodiments, a transcribed RNA may form a dsRNA mol effect on an insect pest or a plant-protective effect with ecule that may be provided in a stabilized form; e.g., as a regard to the pest may be readily incorporated into expressed hairpin and stem and loop structure. dsRNA molecules through the creation of appropriate 0177. In some embodiments, one strand of a dsRNA expression cassettes in a recombinant nucleic acid molecule molecule may be formed by transcription from a polynucle of the invention. For example, Such polynucleotides may be otide which is Substantially homologous to a polynucleotide expressed as a hairpin with stem and loop structure by taking selected from the group consisting of SEQID NOs: 1 and 71; a first segment corresponding to a target gene polynucleotide the complement or reverse complement of either of SEQID (e.g., a gw gene comprising either of SEQID NOS:1 and 71, NOs: 1 and 71; a fragment of at least 15 contiguous nucleo and a fragment comprising at least 15 contiguous nucleo tides of either of SEQ ID NOS:1 and 71 (e.g., SEQ ID tides of any of the foregoing); linking this polynucleotide to NOs:3-5 and 73); the complement or reverse complement of a second segment spacer region that is not homologous or a fragment of at least 15 contiguous nucleotides of either of complementary to the first segment; and linking this to a SEQ ID NOs: 1 and 71; a native coding polynucleotide of a third segment, wherein at least a portion of the third segment Diabrotica organism (e.g., WCR) comprising any of SEQID is Substantially complementary to the first segment. Such a NOs:3-5; the complement or reverse complement of a native construct forms a stem and loop structure by intramolecular coding polynucleotide of a Diabrotica organism comprising base-pairing of the first segment with the third segment, any of SEQID NOS:3-5; a fragment of at least 15 contiguous wherein the loop structure forms comprising the second nucleotides of a native coding polynucleotide of a Dia segment. See, e.g., U.S. Patent Publication Nos. 2002/ brotica organism comprising any of SEQ ID NOS:3-5; the 0048814 and 2003/0018993; and International PCT Publi complement or reverse complement of a fragment of at least cation NOS. WO94/O1550 and WO98/05770. A dsRNA 15 contiguous nucleotides of a native coding polynucleotide molecule may be generated, for example, in the form of a of a Diabrotica organism comprising any of SEQ ID NOs: double-stranded structure such as a stem-loop structure (e.g., 3-5; a native coding polynucleotide of a hemipteran organ hairpin), whereby production of siRNA targeted for a native ism (e.g., BSB) comprising SEQID NO:73; the complement insect (e.g., coleopteran and/or hemipteran) pest polynucle or reverse complement of a native coding polynucleotide of otide is enhanced by co-expression of a fragment of the a hemipteran organism comprising SEQ ID NO:73; a frag targeted gene, for instance on an additional plant expressible ment of at least 15 contiguous nucleotides of a native coding cassette, that leads to enhanced siRNA production, or polynucleotide of a hemipteran organism comprising SEQ reduces methylation to prevent transcriptional gene silenc ID NO:73; and the complement or reverse complement of a ing of the dsRNA hairpin promoter. US 2017/0218391 A1 Aug. 3, 2017
0181 Some embodiments of the invention include intro 252 (maize L3 oleosin promoter); U.S. Pat. No. 6,429,357 duction of a recombinant nucleic acid molecule of the (rice actin 2 promoter, and rice actin 2 intron); U.S. Pat. No. present invention into a plant (i.e., transformation) to 6.294.714 (light-inducible promoters); U.S. Pat. No. 6,140. achieve insect (e.g., coleopteran and/or hemipteran) pest 078 (salt-inducible promoters); U.S. Pat. No. 6,252,138 inhibitory levels of expression of one or more iRNA mol (pathogen-inducible promoters); U.S. Pat. No. 6,175,060 ecules. A recombinant DNA molecule may, for example, be (phosphorous deficiency-inducible promoters); U.S. Pat. a vector, Such as a linear or a closed circular plasmid. The No. 6,388,170 (bidirectional promoters); U.S. Pat. No. vector system may be a single vector or plasmid, or two or 6,635,806 (gamma-coixin promoter); and U.S. Patent Pub more vectors or plasmids that together contain the total lication No. 2009/757,089 (maize chloroplast aldolase pro DNA to be introduced into the genome of a host. In addition, a vector may be an expression vector. Nucleic acids of the moter). Additional promoters include the nopaline synthase invention can, for example, be suitably inserted into a vector (NOS) promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. under the control of a suitable promoter that functions in one USA 84(16):5745-9) and the octopine synthase (OCS) pro or more hosts to drive expression of a linked coding poly moters (which are carried on tumor-inducing plasmids of nucleotide or other DNA element. Many vectors are avail Agrobacterium tumefaciens); the caulimovirus promoters able for this purpose, and selection of the appropriate vector such as the cauliflower mosaic virus (CaMV) 19S promoter will depend mainly on the size of the nucleic acid to be (Lawton et al. (1987) Plant Mol. Biol. 9:315-24); the CaMV inserted into the vector and the particular host cell to be 35S promoter (Odell et al. (1985) Nature 313:810-2: the transformed with the vector. Each vector contains various figwort mosaic virus 35S-promoter (Walker et al. (1987) components depending on its function (e.g., amplification of Proc. Natl. Acad. Sci. USA 84(19):6624-8); the sucrose DNA or expression of DNA) and the particular host cell with synthase promoter (Yang and Russell (1990) Proc. Natl. which it is compatible. Acad. Sci. USA 87:4144-8); the R gene complex promoter 0182 To impart protection from an insect (e.g., coleop (Chandler et al. (1989) Plant Cell 1:1175-83); the chloro teran and/or hemipteran) pest to a transgenic plant, a recom phyll a?b binding protein gene promoter; CaMV 35S (U.S. binant DNA may, for example, be transcribed into an iRNA Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196): molecule (e.g., a RNA molecule that forms a dsRNA mol FMV 35S (U.S. Pat. Nos. 6,051,753, and 5,378,619); a ecule) within the tissues or fluids of the recombinant plant. PC1SV promoter (U.S. Pat. No. 5,850,019); the SCP1 An iRNA molecule may comprise a polynucleotide that is promoter (U.S. Pat. No. 6,677.503); and AGRtu.nos pro Substantially homologous and specifically hybridizable to a moters (GenBankTM Accession No. V00087; Depicker et al. corresponding transcribed polynucleotide within an insect (1982) J. Mol. Appl. Genet. 1:561-73; Bevan et al. (1983) pest that may cause damage to the host plant species. The Nature 304:184-7). pest may contact the iRNA molecule that is transcribed in cells of the transgenic host plant, for example, by ingesting 0185. In particular embodiments, nucleic acid molecules cells or fluids of the transgenic host plant that comprise the of the invention comprise a tissue-specific promoter. Such as iRNA molecule. Thus, in particular examples, expression of a root-specific promoter. Root-specific promoters drive a target gene is suppressed by the iRNA molecule within expression of operably-linked coding polynucleotides exclu coleopteran and/or hemipteran pests that infest the trans sively or preferentially in root tissue. Examples of root genic host plant. In some embodiments, suppression of specific promoters are known in the art. See, e.g., U.S. Pat. expression of the target gene in a target coleopteran and/or Nos. 5,110,732; 5.459.252 and 5,837,848; and Opperman et hemipteran pest may result in the plant being protected al. (1994) Science 263:221-3; and Hirel et al. (1992) Plant against attack by the pest. Mol. Biol. 20:207-18. In some embodiments, a polynucle 0183. In order to enable delivery of iRNA molecules to otide or fragment for coleopteran pest control according to an insect pest in a nutritional relationship with a plant cell the invention may be cloned between two root-specific that has been transformed with a recombinant nucleic acid promoters oriented in opposite transcriptional directions molecule of the invention, expression (i.e., transcription) of relative to the polynucleotide or fragment, and which are iRNA molecules in the plant cell is required. Thus, a operable in a transgenic plant cell and expressed therein to recombinant nucleic acid molecule may comprise a poly produce RNA molecules in the transgenic plant cell that nucleotide of the invention operably linked to one or more subsequently may form dsRNA molecules, as described, regulatory elements, such as a heterologous promoter ele Supra. The iRNA molecules expressed in plant tissues may ment that functions in a host cell. Such as a bacterial cell be ingested by an insect pest so that Suppression of target wherein the nucleic acid molecule is to be amplified, and a gene expression is achieved. plant cell wherein the nucleic acid molecule is to be 0186. Additional regulatory elements that may optionally expressed. be operably linked to a nucleic acid include 5' UTRs located 0184 Promoters suitable for use in nucleic acid mol between a promoter element and a coding polynucleotide ecules of the invention include those that are inducible, viral, that function as a translation leader element. The translation synthetic, or constitutive, all of which are well known in the leader element is present in fully-processed mRNA, and it art. Non-limiting examples describing Such promoters may affect processing of the primary transcript, and/or RNA include U.S. Pat. No. 6,437.217 (maize RS81 promoter): stability. Examples of translation leader elements include U.S. Pat. No. 5,641,876 (rice actin promoter); U.S. Pat. No. maize and petunia heat shock protein leaders (U.S. Pat. No. 6,426,446 (maize RS324 promoter); U.S. Pat. No. 6,429,362 5.362,865), plant virus coat protein leaders, plant rubisco (maize PR-1 promoter); U.S. Pat. No. 6,232,526 (maize A3 leaders, and others. See, e.g., Turner and Foster (1995) promoter); U.S. Pat. No. 6,177,611 (constitutive maize pro Molecular Biotech. 3(3):225-36. Non-limiting examples of moters); U.S. Pat. Nos. 5,322.938, 5,352,605, 5,359,142, 5'UTRs include GmHsp (U.S. Pat. No. 5,659,122); PhDnaK and 5,530,196 (CaMV 35S promoter); U.S. Pat. No. 6,433, (U.S. Pat. No. 5,362,865); AtAnt1; TEV (Carrington and US 2017/0218391 A1 Aug. 3, 2017 22
Freed (1990) J. Virol. 64:1590-7); and AGRtunos (Gen 0190. A recombinant nucleic acid molecule or vector of BankTM Accession No. V00087; and Bevan et al. (1983) the present invention may comprise a selectable marker that Nature 304:184-7). confers a selectable phenotype on a transformed cell. Such as a plant cell. Selectable markers may also be used to select for 0187. Additional regulatory elements that may optionally plants or plant cells that comprise a recombinant nucleic be operably linked to a nucleic acid also include 3' non acid molecule of the invention. The marker may encode translated elements, 3' transcription termination regions, or biocide resistance, antibiotic resistance (e.g., kanamycin, polyadenylation regions. These are genetic elements located Geneticin (G418), bleomycin, hygromycin, etc.), or herbi downstream of a polynucleotide, and include polynucle cide tolerance (e.g., glyphosate, etc.). Examples of select otides that provide polyadenylation signal, and/or other able markers include, but are not limited to: a neo gene regulatory signals capable of affecting transcription or which codes for kanamycin resistance and can be selected mRNA processing. The polyadenylation signal functions in for using kanamycin, G418, etc.; a bar gene which codes for plants to cause the addition of polyadenylate nucleotides to bialaphos resistance; a mutant EPSP synthase gene which the 3' end of the mRNA precursor. The polyadenylation encodes glyphosate tolerance; a nitrilase gene which confers element can be derived from a variety of plant genes, or from resistance to bromoxynil; a mutant acetolactate synthase T-DNA genes. A non-limiting example of a 3' transcription (ALS) gene which confers imidazolinone or Sulfonylurea termination region is the nopaline synthase 3' region (nos. 3'; tolerance; and a methotrexate resistant DHFR gene. Mul Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7). tiple selectable markers are available that confer resistance An example of the use of different 3' non-translated regions to amplicillin, bleomycin, chloramphenicol, gentamycin, is provided in Ingelbrecht et al., (1989) Plant Cell 1:671-80. hygromycin, kanamycin, lincomycin, methotrexate, phos Non-limiting examples of polyadenylation signals include phinothricin, puromycin, spectinomycin, rifampicin, Strep one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; tomycin and tetracycline, and the like. Examples of Such Coruzzi et al. (1984) EMBO J. 3:1671-9) and AGRtu.nos selectable markers are illustrated in, e.g., U.S. Pat. Nos. (GenBankTM Accession No. E01312). 5,550,318; 5,633,435; 5,780,708 and 6,118,047. 0188 Some embodiments may include a plant transfor (0191) A recombinant nucleic acid molecule or vector of mation vector that comprises an isolated and purified DNA the present invention may also include a screenable marker. molecule comprising at least one of the above-described Screenable markers may be used to monitor expression. regulatory elements operatively linked to one or more poly Exemplary screenable markers include a B-glucuronidase or nucleotides of the present invention. When expressed, the uidA gene (GUS) which encodes an enzyme for which one or more polynucleotides result in one or more iRNA various chromogenic Substrates are known (Jefferson et al. molecule(s) comprising a polynucleotide that is specifically (1987) Plant Mol. Biol. Rep. 5:387-405); an R-locus gene, complementary to all or part of a native RNA molecule in an which encodes a product that regulates the production of insect (e.g., coleopteran and/or hemipteran) pest. Thus, the anthocyanin pigments (red color) in plant tissues (Dellaporta polynucleotide(s) may comprise a segment encoding all or et al. (1988) “Molecular cloning of the maize R-njallele by part of a polyribonucleotide present within a targeted cole transposon tagging with Ac.” In 18' Stadler Genetics Sym opteran and/or hemipteran pest RNA transcript, and may posium, P. Gustafson and R. Appels, eds. (New York: comprise inverted repeats of all or a part of a targeted pest Plenum), pp. 263-82); a 3-lactamase gene (Sutcliffe et al. transcript. A plant transformation vector may contain poly (1978) Proc. Natl. Acad. Sci. USA 75:3737-41); a gene nucleotides specifically complementary to more than one which encodes an enzyme for which various chromogenic target polynucleotide, thus allowing production of more than Substrates are known (e.g., PADAC, a chromogenic cepha one dsRNA for inhibiting expression of two or more genes losporin); a luciferase gene (Ow et al. (1986) Science in cells of one or more populations or species of target insect 234:856-9); an xylE gene that encodes a catechol dioxy pests. Segments of polynucleotides specifically complemen genase that can convert chromogenic catechols (Zukowski et tary to polynucleotides present in different genes can be al. (1983) Gene 46(2-3):247-55); an amylase gene (Ikatu et combined into a single composite nucleic acid molecule for al. (1990) Bio/Technol. 8:241-2); a tyrosinase gene which expression in a transgenic plant. Such segments may be encodes an enzyme capable of oxidizing tyrosine to DOPA contiguous or separated by a spacer. and dopaquinone which in turn condenses to melanin (Katz 0189 In other embodiments, a plasmid of the present et al. (1983) J. Gen. Microbiol. 129:2703-14); and an invention already containing at least one polynucleotide(s) O-galactosidase. of the invention can be modified by the sequential insertion 0.192 In some embodiments, recombinant nucleic acid of additional polynucleotide(s) in the same plasmid, wherein molecules, as described, Supra, may be used in methods for the additional polynucleotide(s) are operably linked to the the creation of transgenic plants and expression of heterolo same regulatory elements as the original at least one poly gous nucleic acids in plants to prepare transgenic plants that nucleotide(s). In some embodiments, a nucleic acid mol exhibit reduced Susceptibility to insect (e.g., coleopteran ecule may be designed for the inhibition of multiple target and/or hemipteran) pests. Plant transformation vectors can genes. In some embodiments, the multiple genes to be be prepared, for example, by inserting nucleic acid mol inhibited can be obtained from the same insect (e.g., cole ecules encoding iRNA molecules into plant transformation opteran or hemipteran) pest species, which may enhance the vectors and introducing these into plants. effectiveness of the nucleic acid molecule. In other embodi 0193 Suitable methods for transformation of host cells ments, the genes can be derived from different insect pests, include any method by which DNA can be introduced into which may broaden the range of pests against which the a cell. Such as by transformation of protoplasts (See, e.g., agent(s) is/are effective. When multiple genes are targeted U.S. Pat. No. 5,508,184), by desiccation/inhibition-medi for Suppression or a combination of expression and Suppres ated DNA uptake (See, e.g., Potrykus et al. (1985) Mol. Gen. Sion, a polycistronic DNA element can be engineered. Genet. 199:183-8), by electroporation (See, e.g., U.S. Pat. US 2017/0218391 A1 Aug. 3, 2017
No. 5,384.253), by agitation with silicon carbide fibers (See, agent or agents. In the case where a screenable marker is e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), by Agrobac used, cells may be screened for the desired marker gene trait. terium-mediated transformation (See, e.g., U.S. Pat. Nos. (0197) Cells that survive the exposure to the selective 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; and agent, or cells that have been scored positive in a screening 6,384.301) and by acceleration of DNA-coated particles assay, may be cultured in media that Supports regeneration (See, e.g., U.S. Pat. Nos. 5,015,580: 5,550,318; 5.538,880; of plants. In some embodiments, any suitable plant tissue 6,160,208; 6.399,861; and 6,403,865), etc. Techniques that culture media (e.g., MS and N6 media) may be modified by are particularly useful for transforming corn are described, including further Substances, such as growth regulators. for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616; and Tissue may be maintained on a basic medium with growth International PCT Publication WO95/06722. Through the regulators until Sufficient tissue is available to begin plant application of techniques such as these, the cells of virtually regeneration efforts, or following repeated rounds of manual any species may be stably transformed. In some embodi selection, until the morphology of the tissue is suitable for ments, transforming DNA is integrated into the genome of regeneration (e.g., at least 2 weeks), then transferred to the host cell. In the case of multicellular species, transgenic media conducive to shoot formation. Cultures are trans cells may be regenerated into a transgenic organism. Any of ferred periodically until sufficient shoot formation has these techniques may be used to produce a transgenic plant, occurred. Once shoots are formed, they are transferred to for example, comprising one or more nucleic acids encoding media conducive to root formation. Once sufficient roots are one or more iRNA molecules in the genome of the trans formed, plants can be transferred to soil for further growth genic plant. and maturation. 0194 The most widely utilized method for introducing an 0198 To confirm the presence of a nucleic acid molecule expression vector into plants is based on the natural trans of interest (for example, a DNA encoding one or more iRNA formation system of Agrobacterium. A. tumefaciens and A. molecules that inhibit target gene expression in a coleop rhizogenes are plant pathogenic soil bacteria which geneti teran and/or hemipteran pest) in the regenerating plants, a cally transform plant cells. The Ti and Ri plasmids of A. variety of assays may be performed. Such assays include, for tumefaciens and A. rhizogenes, respectively, carry genes example: molecular biological assays, such as Southern and responsible for genetic transformation of the plant. The Ti northern blotting, PCR, and nucleic acid sequencing, bio (tumor-inducing)-plasmids contain a large segment, known chemical assays, such as detecting the presence of a protein as T-DNA, which is transferred to transformed plants. product, e.g., by immunological means (ELISA and/or west Another segment of the Ti plasmid, the Vir region, is ern blots) or by enzymatic function; plant part assays, such responsible for T-DNA transfer. The T-DNA region is bor as leaf or root assays; and analysis of the phenotype of the dered by terminal repeats. In modified binary vectors, the whole regenerated plant. tumor-inducing genes have been deleted, and the functions 0199 Integration events may be analyzed, for example, of the Vir region are utilized to transfer foreign DNA by PCR amplification using, e.g., oligonucleotide primers bordered by the T-DNA border elements. The T-region may specific for a nucleic acid molecule of interest. PCR geno also contain a selectable marker for efficient recovery of typing is understood to include, but not be limited to, transgenic cells and plants, and a multiple cloning site for polymerase-chain reaction (PCR) amplification of gldNA inserting polynucleotides for transfer such as a dsRNA derived from isolated host plant callus tissue predicted to encoding nucleic acid. contain a nucleic acid molecule of interest integrated into the genome, followed by Standard cloning and sequence analy 0.195. In particular embodiments, a plant transformation sis of PCR amplification products. Methods of PCR geno vector is derived from a Ti plasmid of A. tumefaciens (See, typing have been well described (for example, Rios, G. et al. e.g., U.S. Pat. Nos. 4,536,475, 4,693.977, 4,886,937, and (2002) Plant J. 32:243-53) and may be applied to g|DNA 5,501,967; and European Patent No. EP 0122 791) or a Ri derived from any plant species (e.g., Z. mays) or tissue type, plasmid of A. rhizogenes. Additional plant transformation including cell cultures. vectors include, for example and without limitation, those 0200. A transgenic plant formed using Agrobacterium described by Herrera-Estrella et al. (1983) Nature 303:209 dependent transformation methods typically contains a 13; Bevan et al. (1983) Nature 304:184-7; Klee et al. (1985) single recombinant DNA inserted into one chromosome. Bio/Technol. 3:637-42; and in European Patent No. EP 0 The polynucleotide of the single recombinant DNA is 120516, and those derived from any of the foregoing. Other referred to as a “transgenic event' or “integration event.” bacteria such as Sinorhizobium, Rhizobium, and Mesorhizo Such transgenic plants are heterozygous for the inserted bium that interact with plants naturally can be modified to exogenous polynucleotide. In some embodiments, a trans mediate gene transfer to a number of diverse plants. These genic plant homozygous with respect to a transgene may be plant-associated Symbiotic bacteria can be made competent obtained by sexually mating (selfing) an independent Ser for gene transfer by acquisition of both a disarmed Ti geant transgenic plant that contains a single exogenous gene plasmid and a suitable binary vector. to itself, for example a To plant, to produce T seed. One 0196. After providing exogenous DNA to recipient cells, fourth of the T seed produced will be homozygous with transformed cells are generally identified for further cultur respect to the transgene. Germinating T seed results in ing and plant regeneration. In order to improve the ability to plants that can be tested for heterozygosity, typically using identify transformed cells, one may desire to employ a an SNP assay or a thermal amplification assay that allows for selectable or screenable marker gene, as previously set forth, the distinction between heterozygotes and homozygotes with the transformation vector used to generate the trans (i.e., a Zygosity assay). formant. In the case where a selectable marker is used, 0201 In particular embodiments, at least 2, 3, 4, 5, 6, 7, transformed cells are identified within the potentially trans 8, 9 or 10 or more different iRNA molecules are produced formed cell population by exposing the cells to a selective in a plant cell that have an insect (e.g., coleopteran and/or US 2017/0218391 A1 Aug. 3, 2017 24 hemipteran) pest-inhibitory effect. The iRNA molecules 214, RNA polymerase II 140 RNAi targets, as described in (e.g., dsRNA molecules) may be expressed from multiple U.S. patent application Ser. No. 14/577,854, RNA poly nucleic acids introduced in different transformation events, merase II215 RNAi targets, as described in U.S. Patent or from a single nucleic acid introduced in a single trans Application No. 62/133,202, RNA polymerase II33 RNAi formation event. In some embodiments, a plurality of iRNA targets, as described in U.S. Patent Application No. 62/133, molecules are expressed under the control of a single 210), incm (U.S. Patent Application No. 62/095,487), Drea. promoter. In other embodiments, a plurality of iRNA mol (U.S. patent application Ser. No. 14/705,807), transcription ecules are expressed under the control of multiple promot elongation factor sptiS RNAi targets, as described in U.S. ers. Single iRNA molecules may be expressed that comprise Patent Application No. 62/168,613), and histone chaperone multiple polynucleotides that are each homologous to dif sptó (U.S. Patent Application No. 62/168,606); a transgenic ferent loci within one or more insect pests (for example, the event from which is transcribed an iRNA molecule targeting loci defined by SEQ ID NOS:1 and 71), both in different a gene in an organism other than a coleopteran and/or populations of the same species of insect pest, or in different hemipteran pest (e.g., a plant-parasitic nematode); a gene species of insect pests. encoding an insecticidal protein (e.g., a Bacillus thuringi 0202 In addition to direct transformation of a plant with ensis insecticidal protein, a PIP-1 polypeptide, and an AflP a recombinant nucleic acid molecule, transgenic plants can polypeptide); a herbicide tolerance gene (e.g., a gene pro be prepared by crossing a first plant having at least one viding tolerance to glyphosate); and a gene contributing to transgenic event with a second plant lacking such an event. a desirable phenotype in the transgenic plant, such as For example, a recombinant nucleic acid molecule compris increased yield, altered fatty acid metabolism, or restoration ing a polynucleotide that encodes an iRNA molecule may be of cytoplasmic male sterility. In particular embodiments, introduced into a first plant line that is amenable to trans polynucleotides encoding iRNA molecules of the invention formation to produce a transgenic plant, which transgenic may be combined with other insect control and disease traits plant may be crossed with a second plant line to introgress in a plant to achieve desired traits for enhanced control of the polynucleotide that encodes the iRNA molecule into the plant disease and insect damage. Combining insect control second plant line. traits that employ distinct modes-of-action may provide 0203. In some aspects, seeds and commodity products protected transgenic plants with Superior durability over produced by transgenic plants derived from transformed plants harboring a single control trait, for example, because plant cells are included, wherein the seeds or commodity of the reduced probability that resistance to the trait(s) will products comprise a detectable amount of a nucleic acid of develop in the field. the invention. In some embodiments, such commodity prod ucts may be produced, for example, by obtaining transgenic V. Target Gene Suppression in an Insect Pest plants and preparing food or feed from them. Commodity 0205 A. Overview products comprising one or more of the polynucleotides of 0206. In some embodiments of the invention, at least one the invention includes, for example and without limitation: nucleic acid molecule useful for the control of insect (e.g., meals, oils, crushed or whole grains or seeds of a plant, and coleopteran and/or hemipteran) pests may be provided to an any food product comprising any meal, oil, or crushed or insect pest, wherein the nucleic acid molecule leads to whole grain of a recombinant plant or seed comprising one RNAi-mediated gene silencing in the pest. In particular or more of the nucleic acids of the invention. The detection embodiments, an iRNA molecule (e.g., dsRNA, siRNA, of one or more of the polynucleotides of the invention in one miRNA, shRNA, and hpRNA) may be provided to a cole or more commodity or commodity products is de facto opteran and/or hemipteran pest. In some embodiments, a evidence that the commodity or commodity product is nucleic acid molecule useful for the control of insect pests produced from a transgenic plant designed to express one or may be provided to a pest by contacting the nucleic acid more of the iRNA molecules of the invention for the purpose molecule with the pest. In these and further embodiments, a of controlling insect (e.g., coleopteran and/or hemipteran) nucleic acid molecule useful for the control of insect pests pests. may be provided in a feeding substrate of the pest, for 0204. In some embodiments, a transgenic plant or seed example, a nutritional composition. In these and further comprising a nucleic acid molecule of the invention also embodiments, a nucleic acid molecule useful for the control may comprise at least one other transgenic event in its of an insect pest may be provided through ingestion of plant genome, including without limitation: a transgenic event material comprising the nucleic acid molecule that is from which is transcribed an iRNA molecule targeting a ingested by the pest. In certain embodiments, the nucleic locus in a coleopteran or hemipteran pest other than the one acid molecule is present in plant material through expression defined by SEQ ID NO:1 and SEQ ID NO:71, such as, for of a recombinant nucleic acid introduced into the plant example, one or more loci selected from the group consist material, for example, by transformation of a plant cell with ing of Caf1-180 (U.S. Patent Application Publication No. a vector comprising the recombinant nucleic acid and regen 2012/0174258), VatpaseC (U.S. Patent Application Publica eration of a plant material or whole plant from the trans tion No. 2012/0174259), Rhol (U.S. Patent Application formed plant cell. Publication No. 2012/0174260), VatpaseH (U.S. Patent 0207 B. RNAi-Mediated Target Gene Suppression Application Publication No. 2012/0198586), PPI-87B (U.S. 0208. In some embodiments, the invention provides Patent Application Publication No. 2013/009 1600), RPA70 iRNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, (U.S. Patent Application Publication No. 2013/009 1601), and hpRNA) that may be designed to target essential native RPS6 (U.S. Patent Application Publication No. 2013/ polynucleotides (e.g., essential genes) in the transcriptome 0097730), ROP RNAi targets, as described in U.S. patent of an insect pest (for example, a coleopteran (e.g., WCR, application Ser. No. 14/577,811, RNA polymerase I1 RNAi NCR, and SCR) or hemipteran (e.g., BSB) pest), for targets, as described in U.S. Patent Application No. 62/133, example by designing an iRNA molecule that comprises at US 2017/0218391 A1 Aug. 3, 2017 least one strand comprising a polynucleotide that is specifi and/or hemipteran) pest, wherein the polynucleotide is cally complementary to the target polynucleotide. The selected from the group consisting of: SEQ ID NO:79; the sequence of an iRNA molecule so designed may be identical complement or reverse complement of SEQID NO:79; SEQ to that of the target polynucleotide, or may incorporate ID NO:83; the complement or reverse complement of SEQ mismatches that do not prevent specific hybridization ID NO:83; an RNA expressed from a native coding poly between the iRNA molecule and its target polynucleotide. nucleotide of a Diabrotica organism comprising SEQ ID 0209 iRNA molecules of the invention may be used in NO:1; the complement or reverse complement of an RNA methods for gene Suppression in an insect (e.g., coleopteran expressed from a native coding polynucleotide of a Dia and/or hemipteran) pest, thereby reducing the level or inci brotica organism comprising SEQ ID NO:1; an RNA dence of damage caused by the pest on a plant (for example, expressed from a native coding polynucleotide of a Euschis a protected transformed plant comprising an iRNA mol tus heros organism comprising SEQ ID NO:71; and the ecule). As used herein the term 'gene Suppression” refers to complement or reverse complement of an RNA expressed any of the well-known methods for reducing the levels of from a native coding polynucleotide of a E. heros organism protein produced as a result of gene transcription to mRNA comprising SEQ ID NO:71. Nucleic acid molecules com and Subsequent translation of the mRNA, including the prising at least 15 contiguous nucleotides of the foregoing reduction of protein expression from a gene or a coding polynucleotides include, for example and without limitation, polynucleotide including post-transcriptional inhibition of fragments comprising at least 15 contiguous nucleotides of expression and transcriptional Suppression. Post-transcrip a polynucleotide selected from the group consisting of SEQ tional inhibition is mediated by specific homology between ID NOS:80-82 and 84. In certain embodiments, expression all or a part of an mRNA transcribed from a gene targeted of a nucleic acid molecule that is at least about 80% identical for Suppression and the corresponding iRNA molecule used (e.g., 79%, about 80%, about 81%, about 82%, about 83%, for Suppression. Additionally, post-transcriptional inhibition about 84%, about 85%, about 86%, about 87%, about 88%, refers to the substantial and measurable reduction of the about 89%, about 90%, about 91%, about 92%, about 93%, amount of mRNA available in the cell for binding by about 94%, about 95%, about 96%, about 97%, about 98%, ribosomes. about 99%, about 100%, and 100%) with any of the fore 0210. In embodiments wherein an iRNA molecule is a going may be used. In these and further embodiments, a dsRNA molecule, the dsRNA molecule may be cleaved by nucleic acid molecule may be expressed that specifically the enzyme, DICER, into short siRNA molecules (approxi hybridizes to an RNA molecule present in at least one cell mately 20 nucleotides in length). The double-stranded of an insect (e.g., coleopteran and/or hemipteran) pest. siRNA molecule generated by DICER activity upon the 0214. In some embodiments, an iRNA molecule is pro dsRNA molecule may be separated into two single-stranded vided in a nutritional composition referred to herein as a siRNAs; the “passenger strand' and the “guide strand.” The “RNAi bait.” A RNAi bait may be formed in particular passenger Strand may be degraded, and the guide Strand may embodiments when an iRNA molecule (e.g., a dsRNA) is be incorporated into RISC. Post-transcriptional inhibition mixed with a food of the target insect, an attractant of the occurs by specific hybridization of the guide strand with a insect, or both. When the insect eats a RNAi bait, the insect specifically complementary polynucleotide of an mRNA may consume the iRNA molecule. A RNAi bait may be, for molecule, and Subsequent cleavage by the enzyme, Argo example and without limitation, a granule, gel, flowable naute (catalytic component of the RISC complex). powder, liquid, or Solid. In particular embodiments, an 0211. In embodiments of the invention, any form of iRNA molecule may be incorporated into a bait formulation iRNA molecule may be used. Those of skill in the art will such as that described in U.S. Pat. No. 8,530,440, the understand that dsRNA molecules typically are more stable contents of which are incorporated in their entirety herein by during preparation and during the step of providing the this reference. In some examples, a RNAi bait is placed in iRNA molecule to a cell than are single-stranded RNA or around the environment of an insect pest, such that, for molecules, and are typically also more stable in a cell. Thus, example, the pest can come into contact with and/or be while siRNA and miRNA molecules, for example, may be attracted to the RNAi bait. equally effective in some embodiments, a dsRNA molecule 0215. It is an important feature of some embodiments may be chosen due to its stability. herein that the RNAi post-transcriptional inhibition system 0212. In particular embodiments, a nucleic acid molecule is able to tolerate sequence variations among target genes is provided that comprises a polynucleotide, which poly that might be expected due to genetic mutation, Strain nucleotide may be expressed in vitro to produce an iRNA polymorphism, or evolutionary divergence. The introduced molecule that is substantially homologous to a nucleic acid nucleic acid molecule may not need to be absolutely molecule encoded by a polynucleotide within the genome of homologous to either a primary transcription product or a an insect (e.g., coleopteran and/or hemipteran) pest. In fully-processed mRNA of a target gene, so long as the certain embodiments, the in vitro transcribed iRNA mol introduced nucleic acid molecule is specifically hybridizable ecule may be a stabilized dsRNA molecule that comprises a to either a primary transcription product or a fully-processed stem-loop structure. After an insect pest contacts the in vitro mRNA of the target gene. Moreover, the introduced nucleic transcribed iRNA molecule, post-transcriptional inhibition acid molecule may not need to be full-length, relative to of a target gene in the pest (for example, an essential gene) either a primary transcription product or a fully processed may occur. mRNA of the target gene. 0213. In some embodiments of the invention, expression 0216. Inhibition of a target gene using the iRNA tech of a nucleic acid molecule comprising at least 15 contiguous nology of the present invention is sequence-specific; i.e., nucleotides (e.g., at least 19 contiguous nucleotides) of a polynucleotides substantially homologous to the iRNA mol polynucleotide are used in a method for post-transcriptional ecule(s) are targeted for genetic inhibition. In some embodi inhibition of a target gene in an insect (e.g., coleopteran ments, an RNA molecule comprising a polynucleotide with US 2017/0218391 A1 Aug. 3, 2017 26 a nucleotide sequence that is identical to that of a portion of hemipteran) pest may be carried out in any one of many in a target gene may be used for inhibition. In these and further vitro or in vivo formats. The iRNA molecules may then be embodiments, an RNA molecule comprising a polynucle provided to an insect pest, for example, by contacting the otide with one or more insertion, deletion, and/or point iRNA molecules with the pest, or by causing the pest to mutations relative to a target polynucleotide may be used. In ingest or otherwise internalize the iRNA molecules. Some particular embodiments, an iRNA molecule and a portion of embodiments include transformed host plants of a coleop a target gene may share, for example, at least from about teran and/or hemipteran pest, transformed plant cells, and 80%, at least from about 81%, at least from about 82%, at progeny of transformed plants. The transformed plant cells least from about 83%, at least from about 84%, at least from and transformed plants may be engineered to express one or about 85%, at least from about 86%, at least from about more of the iRNA molecules, for example, under the control 87%, at least from about 88%, at least from about 89%, at of a heterologous promoter, to provide a pest-protective least from about 90%, at least from about 91%, at least from about 92%, at least from about 93%, at least from about effect. Thus, when a transgenic plant or plant cell is con 94%, at least from about 95%, at least from about 96%, at Sumed by an insect pest during feeding, the pest may ingest least from about 97%, at least from about 98%, at least from iRNA molecules expressed in the transgenic plants or cells. about 99%, at least from about 100%, and 100% sequence The polynucleotides of the present invention may also be identity. Alternatively, the duplex region of a dsRNA mol introduced into a wide variety of prokaryotic and eukaryotic ecule may be specifically hybridizable with a portion of a microorganism hosts to produce iRNA molecules. The term target gene transcript. In specifically hybridizable mol “microorganism' includes prokaryotic and eukaryotic spe ecules, a less than full length polynucleotide exhibiting a cies, such as bacteria and fungi. greater homology compensates for a longer, less homolo 0221 Modulation of gene expression may include partial gous polynucleotide. The length of the polynucleotide of a or complete Suppression of Such expression. In another duplex region of a dsRNA molecule that is identical to a embodiment, a method for Suppression of gene expression in portion of a target gene transcript may be at least about 25, an insect (e.g., coleopteran and/or hemipteran) pest com 50, 100, 200, 300, 400, 500, or at least about 1000 bases. In prises providing in the tissue of the host of the pest a some embodiments, a polynucleotide of greater than 20-100 gene-suppressive amount of at least one dsRNA molecule nucleotides may be used. In particular embodiments, a formed following transcription of a polynucleotide as polynucleotide of greater than about 200-300 nucleotides described herein, at least one segment of which is comple may be used. In particular embodiments, a polynucleotide of mentary to a mRNA within the cells of the insect pest. A greater than about 500-1000 nucleotides may be used, dsRNA molecule, including its modified form such as a depending on the size of the target gene. siRNA, miRNA, shRNA, or hpRNA molecule, ingested by 0217. In certain embodiments, expression of a target gene an insect pest may be at least from about 80%, 81%, 82%, in a pest (e.g., coleopteran or hemipteran) may be inhibited 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, by at least 10%; at least 33%;at least 50%; or at least 80% 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% within a cell of the pest, such that a significant inhibition identical to an RNA molecule transcribed from a gw DNA takes place. Significant inhibition refers to inhibition over a molecule, for example, comprising a polynucleotide threshold that results in a detectable phenotype (e.g., cessa selected from the group consisting of SEQ ID NOS:1, 3-5, tion of growth, cessation of feeding, cessation of develop 71, and 73. Isolated and substantially purified nucleic acid ment, induced mortality, etc.), or a detectable decrease in molecules including, but not limited to, non-naturally occur RNA and/or gene product corresponding to the target gene ring polynucleotides and recombinant DNA constructs for being inhibited. Although, in certain embodiments of the providing dsRNA molecules are therefore provided, which invention, inhibition occurs in substantially all cells of the Suppress or inhibit the expression of an endogenous coding pest, in other embodiments inhibition occurs only in a Subset polynucleotide or a target coding polynucleotide in an insect of cells expressing the target gene. pest when introduced thereto. 0218. In some embodiments, transcriptional Suppression 0222 Particular embodiments provide a delivery system is mediated by the presence in a cell of a dsRNA molecule for the delivery of iRNA molecules for the post-transcrip exhibiting substantial sequence identity to a promoter DNA tional inhibition of one or more target gene(s) in an insect or the complement thereof to effect what is referred to as (e.g., coleopteran and/or hemipteran) plant pest and control “promoter trans Suppression. Gene Suppression may be of a population of the plant pest. In some embodiments, the effective against target genes in an insect pest that may delivery system comprises ingestion of a host transgenic ingest or contact Such dsRNA molecules, for example, by plant cell or contents of the host cell comprising RNA ingesting or contacting plant material containing the dsRNA molecules transcribed in the host cell. In these and further molecules. dsRNA molecules for use in promoter trans embodiments, a transgenic plant cell or a transgenic plant is Suppression may be specifically designed to inhibit or Sup created that contains a recombinant DNA construct provid press the expression of one or more homologous or comple ing a stabilized dsRNA molecule of the invention. Trans mentary polynucleotides in the cells of the insect pest. genic plant cells and transgenic plants comprising nucleic Post-transcriptional gene Suppression by antisense or sense acids encoding a particular iRNA molecule may be produced oriented RNA to regulate gene expression in plant cells is by employing recombinant DNA technologies (which basic disclosed in U.S. Pat. Nos. 5,107,065; 5,759,829; 5,283, 184: technologies are well-known in the art) to construct a plant and 5,231,020. transformation vector comprising a polynucleotide encoding 0219. C. Expression of iRNA Molecules Provided to an an iRNA molecule of the invention (e.g., a stabilized dsRNA Insect Pest molecule); to transform a plant cell or plant; and to generate 0220 Expression of iRNA molecules for RNAi-mediated the transgenic plant cell or the transgenic plant that contains gene inhibition in an insect (e.g., coleopteran and/or the transcribed iRNA molecule. US 2017/0218391 A1 Aug. 3, 2017 27
0223 To impart insect (e.g., coleopteran and/or acid molecule of the invention; cultivating the corn plant to hemipteran) pest protection to a transgenic plant, a recom allow the expression of an iRNA molecule comprising the binant DNA molecule may, for example, be transcribed into nucleic acid, wherein expression of an iRNA molecule an iRNA molecule, such as a dsRNA molecule, a siRNA comprising the nucleic acid inhibits insect (e.g., coleopteran molecule, a miRNA molecule, a shRNA molecule, or a and/or hemipteran) pest damage and/or growth, thereby hpRNA molecule. In some embodiments, a RNA molecule reducing or eliminating a loss of yield due to pest infesta transcribed from a recombinant DNA molecule may form a tion. In some embodiments, the iRNA molecule is a dsRNA dsRNA molecule within the tissues or fluids of the recom molecule. In these and further embodiments, the nucleic acid binant plant. Such a dsRNA molecule may be comprised in molecule(s) comprise dsRNA molecules that each comprise part of a polynucleotide that is identical to a corresponding more than one polynucleotide that is specifically hybridiz polynucleotide transcribed from a DNA within an insect pest able to a nucleic acid molecule expressed in an insect pest of a type that may infest the host plant. Expression of a target cell. In some examples, the nucleic acid molecule(s) com gene within the pest is suppressed by the dsRNA molecule, prises a polynucleotide that is specifically hybridizable to a and the Suppression of expression of the target gene in the nucleic acid molecule expressed in a coleopteran and/or pest results in the transgenic plant being protected against hemipteran pest cell. the pest. The modulatory effects of dsRNA molecules have 0227. In some embodiments, a method for modulating been shown to be applicable to a variety of genes expressed the expression of a target gene in an insect (e.g., coleopteran in pests, including, for example, endogenous genes respon and/or hemipteran) pest is provided, the method comprising: sible for cellular metabolism or cellular transformation, transforming a plant cell with a vector comprising a poly including house-keeping genes; transcription factors; molt nucleotide encoding at least one iRNA molecule of the ing-related genes; and other genes which encode polypep invention, wherein the polynucleotide is operatively-linked tides involved in cellular metabolism or normal growth and to a promoter and a transcription termination element; development. culturing the transformed plant cell under conditions Sufi 0224 For transcription from a transgene in vivo or an cient to allow for development of a plant cell culture expression construct, a regulatory region (e.g., promoter, including a plurality of transformed plant cells; selecting for enhancer, silencer, and polyadenylation signal) may be used transformed plant cells that have integrated the polynucle in some embodiments to transcribe the RNA strand (or otide into their genomes; Screening the transformed plant Strands). Therefore, in Some embodiments, as set forth, cells for expression of an iRNA molecule encoded by the Supra, a polynucleotide for use in producing iRNA mol integrated polynucleotide; selecting a transgenic plant cell ecules may be operably linked to one or more promoter that expresses the iRNA molecule; and feeding the selected elements functional in a plant host cell. The promoter may transgenic plant cell to the insect pest. Plants may also be be an endogenous promoter, normally resident in the host regenerated from transformed plant cells that express an genome. The polynucleotide of the present invention, under iRNA molecule encoded by the integrated nucleic acid the control of an operably linked promoter element, may molecule. In some embodiments, the iRNA molecule is a further be flanked by additional elements that advanta dsRNA molecule. In these and further embodiments, the geously affect its transcription and/or the stability of a nucleic acid molecule(s) comprise dsRNA molecules that resulting transcript. Such elements may be located upstream each comprise more than one polynucleotide that is specifi of the operably linked promoter, downstream of the 3' end of cally hybridizable to a nucleic acid molecule expressed in an the expression construct, and may occur both upstream of insect pest cell. In some examples, the nucleic acid molecule the promoter and downstream of the 3' end of the expression (s) comprises a polynucleotide that is specifically hybridiz COnStruct. able to a nucleic acid molecule expressed in a coleopteran 0225. Some embodiments provide methods for reducing and/or hemipteran pest cell. the damage to a host plant (e.g., a corn plant) caused by an 0228 iRNA molecules of the invention can be incorpo insect (e.g., coleopteran and/or hemipteran) pest that feeds rated within the seeds of a plant species (e.g., corn), either on the plant, wherein the method comprises providing in the as a product of expression from a recombinant gene incor host plant a transformed plant cell expressing at least one porated into a genome of the plant cells, or as incorporated nucleic acid molecule of the invention, wherein the nucleic into a coating or seed treatment that is applied to the seed acid molecule(s) functions upon being taken up by the before planting. A plant cell comprising a recombinant gene pest(s) to inhibit the expression of a target polynucleotide is considered to be a transgenic event. Also included in within the pest(s), which inhibition of expression results in embodiments of the invention are delivery systems for the mortality and/or reduced growth of the pest(s), thereby delivery of iRNA molecules to insect (e.g., coleopteran reducing the damage to the host plant caused by the pest(s). and/or hemipteran) pests. For example, the iRNA molecules In some embodiments, the nucleic acid molecule(s) com of the invention may be directly introduced into the cells of prise dsRNA molecules. In these and further embodiments, a pest(s). Methods for introduction may include direct the nucleic acid molecule(s) comprise dsRNA molecules mixing of iRNA with plant tissue from a host for the insect that each comprise more than one polynucleotide that is pest(s), as well as application of compositions comprising specifically hybridizable to a nucleic acid molecule iRNA molecules of the invention to host plant tissue. For expressed in a coleopteran and/or hemipteran pest cell. In example, iRNA molecules may be sprayed onto a plant Some embodiments, the nucleic acid molecule(s) consist of surface. Alternatively, an iRNA molecule may be expressed one polynucleotide that is specifically hybridizable to a by a microorganism, and the microorganism may be applied nucleic acid molecule expressed in an insect pest cell. onto the plant Surface, or introduced into a root or stem by 0226. In some embodiments, a method for increasing the a physical means such as an injection. As discussed, Supra, yield of a corn crop is provided, wherein the method a transgenic plant may also be genetically engineered to comprises introducing into a corn plant at least one nucleic express at least one iRNA molecule in an amount sufficient US 2017/0218391 A1 Aug. 3, 2017 28 to kill the insect pests known to infest the plant. iRNA deposited on the treated diet (one or two larvae per well). molecules produced by chemical or enzymatic synthesis The infested wells of the 128-well plastic trays were then may also be formulated in a manner consistent with common sealed with adhesive sheets of clear plastic, and vented to agricultural practices, and used as spray-on or bait products allow gas exchange. Bioassay trays were held under con for controlling plant damage by an insect pest. The formu trolled environmental conditions (28° C., ~40% Relative lations may include the appropriate Stickers and wetters Humidity, 16:8 (Light: Dark)) for 9 days, after which time required for efficient foliar coverage, as well as UV pro the total number of insects exposed to each sample, the tectants to protect iRNA molecules (e.g., dsRNA molecules) number of dead insects, and the weight of Surviving insects from UV damage. Such additives are commonly used in the were recorded. Average percent mortality and average bioinsecticide industry, and are well known to those skilled growth inhibition were calculated for each treatment. in the art. Such applications may be combined with other Growth inhibition (GI) was calculated as follows: spray-on insecticide applications (biologically based or oth erwise) to enhance plant protection from the pests. 0229. All references, including publications, patents, and 0236 where TWIT is the Total Weight of live Insects in patent applications, cited herein are hereby incorporated by the Treatment; reference to the extent they are not inconsistent with the 0237 TNIT is the Total Number of Insects in the Treat explicit details of this disclosure, and are so incorporated to ment; the same extent as if each reference were individually and 0238 TWIBC is the Total Weight of live Insects in the specifically indicated to be incorporated by reference and Background Check (Buffer control); and were set forth in its entirety herein. The references discussed 0239 TNIBC is the Total Number of Insects in the herein are provided solely for their disclosure prior to the Background Check (Buffer control). filing date of the present application. Nothing herein is to be 0240. The statistical analysis was done using JMPTM construed as an admission that the inventors are not entitled software (SAS, Cary, N.C.). to antedate such disclosure by virtue of prior invention. 0241 The LCs (Lethal Concentration) is defined as the 0230. The following EXAMPLES are provided to illus dosage at which 50% of the test insects are killed. The GIs trate certain particular features and/or aspects. These (Growth Inhibition) is defined as the dosage at which the EXAMPLES should not be construed to limit the disclosure mean growth (e.g. live weight) of the test insects is 50% of to the particular features or aspects described. the mean value seen in Background Check samples. 0242 Replicated bioassays demonstrated that ingestion Examples of particular samples resulted in a Surprising and unexpected mortality and growth inhibition of corn rootworm larvae. Example 1: Materials and Methods 0231 Sample Preparation and Bioassays Example 2: Identification of Candidate Target 0232 A number of dsRNA molecules (including those Genes corresponding to gw-1 reg1 (SEQID NO:3), gw-1 V1 (SEQ 0243 Insects from multiple stages of WCR (Diabrotica ID NO:4), and gw-1 V2 (SEQ ID NO:5) were synthesized virgifera virgifera LeConte) development were selected for and purified using a MEGASCRIPTR. T7 RNAi kit (LIFE pooled transcriptome analysis to provide candidate target TECHNOLOGIES, Carlsbad, Calif.) or T7 Quick High gene sequences for control by RNAi transgenic plant insect Yield RNA Synthesis Kit (NEW ENGLAND BIOLABS, protection technology. Whitby, Ontario). The purified dsRNA molecules were 0244. In one exemplification, total RNA was isolated prepared in TE buffer, and all bioassays contained a control from about 0.9 gm whole first-instar WCR larvae; (4 to 5 treatment consisting of this buffer, which served as a back days post-hatch; held at 16° C.), and purified using the ground check for mortality or growth inhibition of WCR following phenol/TRI REAGENTR)-based method (MO (Diabrotica virgifera virgifera LeConte). The concentrations LECULAR RESEARCH CENTER, Cincinnati, Ohio): of dsRNA molecules in the bioassay buffer were measured 0245 Larvae were homogenized at room temperature in using a NANODROPTM 8000 spectrophotometer a 15 mL homogenizer with 10 mL of TRI REAGENTR) until (THERMO SCIENTIFIC, Wilmington, Del.). a homogenous Suspension was obtained. Following 5 min. 0233 Samples were tested for insect activity in bioassays incubation at room temperature, the homogenate was dis conducted with neonate insect larvae on artificial insect diet. pensed into 1.5 mL microfuge tubes (1 mL per tube), 200 uL WCR eggs were obtained from CROP CHARACTERIS of chloroform was added, and the mixture was vigorously TICS, INC. (Farmington, Minn.). shaken for 15 seconds. After allowing the extraction to sit at 0234. The bioassays were conducted in 128-well plastic room temperature for 10 min, the phases were separated by trays specifically designed for insect bioassays (C-D centrifugation at 12,000xg at 4°C. The upper phase (com INTERNATIONAL Pitman, N.J.). Each well contained prising about 0.6 mL) was carefully transferred into another approximately 1.0 mL of an artificial diet designed for sterile 1.5 mL tube, and an equal Volume of room tempera growth of coleopteran insects. A 60 uL aliquot of dsRNA ture isopropanol was added. After incubation at room tem sample was delivered by pipette onto the surface of the diet perature for 5 to 10 min, the mixture was centrifuged 8 min of each well (40 uL/cm). dsRNA sample concentrations at 12,000xg (4° C. or 25° C.). were calculated as the amount of dsRNA per square centi 0246 The supernatant was carefully removed and dis meter (ng/cm) of surface area (1.5 cm) in the well. The carded, and the RNA pellet was washed twice by vortexing treated trays were held in a fume hood until the liquid on the with 75% ethanol, with recovery by centrifugation for 5 min diet surface evaporated or were absorbed into the diet. at 7,500xg (4° C. or 25°C.) after each wash. The ethanol 0235. Within a few hours of eclosion, individual larvae was carefully removed, the pellet was allowed to air-dry for were picked up with a moistened camel hair brush and 3 to 5 min, and then was dissolved in nuclease-free sterile US 2017/0218391 A1 Aug. 3, 2017 29 water. RNA concentration was determined by measuring the assembly of the contigs had failed to join these overlaps. In absorbance (A) at 260 nm and 280 nm. A typical extraction those cases, SequencherTM v4.9 (GENE CODES CORPO from about 0.9 gm of larvae yielded over 1 mg of total RNA, RATION, Ann Arbor, Mich.) was used to assemble the with an A260/A280 ratio of 1.9. The RNA thus extracted sequences into longer contigs. was stored at -80° C. until further processed. 0247 RNA quality was determined by running an aliquot 0252. A candidate target gene encoding Diabrotica gw through a 1% agarose gel. The agarose gel Solution was (SEQ ID NO: 1) was identified as a gene that may lead to made using autoclaved 10xTAE buffer (Tris-acetate EDTA; coleopteran pest mortality, inhibition of growth, inhibition 1x concentration is 0.04 M Tris-acetate, 1 mM EDTA of development, and/or inhibition of feeding in WCR. (ethylenediamine tetra-acetic acid sodium salt), pH 8.0) diluted with DEPC (diethyl pyrocarbonate)-treated water in 0253 Gawky (gw) is a protein that is involved in silenc an autoclaved container. 1XTAE was used as the running ing of mRNA through miRNA translational repression. buffer. Before use, the electrophoresis tank and the well (0254 The sequence of SEQ ID NO:1 is novel. The forming comb were cleaned with RNaseAwayTM (INVIT sequence is not provided in public databases, and is not ROGEN INC., Carlsbad, Calif.). Two uL of RNA sample were mixed with 8 uIl of TE buffer (10 mM Tris HCl pH 7.0; disclosed in PCT International Patent Publication No. 1 mM EDTA) and 10 uL of RNA sample buffer (NOVA WO/2011/025860; U.S. Patent Application No. GENR) Catalog No. 70606; EMD4 Bioscience, Gibbstown, 20070124836; U.S. Patent Application No. 20090306189: N.J.). The sample was heated at 70° C. for 3 min, cooled to U.S. Patent Application No. US20070050860; U.S. Patent room temperature, and 5 L (containing 1 g to 2 LugRNA) Application No. 20100192265; U.S. Pat. No. 7,612, 194; or were loaded per well. Commercially available RNA molecu U.S. Patent Application No. 2013 192256. WCR gw (SEQ lar weight markers were simultaneously run in separate ID NO: 1) is somewhat related to a fragment of a sequence wells for molecular size comparison. The gel was run at 60 from Tribolium castaneum (GENBANK Accession No. volts for 2 hrs. XM 008199035.1). The closest homolog of the WCRGW-1 0248. A normalized cDNA library was prepared from the amino acid sequence (SEQ ID NO:2) is a Tribolium cas larval total RNA by a commercial service provider (EURO taneum protein having GENBANK Accession No. FINS MWG Operon, Huntsville, Ala.), using random prim XP 008197256.1 (77% similar: 70% identical over the ing. The normalized larval cDNA library was sequenced at homology region). !/2 plate scale by GS FLX 454 TitaniumTM series chemistry at EUROFINS MWG Operon, which resulted in over 600, (0255 Gw dsRNA transgenes can be combined with other 000 reads with an average read length of 348 bp. 350,000 dsRNA molecules, for example, to provide redundant RNAi reads were assembled into over 50,000 contigs. Both the targeting and synergistic RNAi effects. Transgenic corn unassembled reads and the contigs were converted into events expressing dsRNA that targets gw are useful for BLASTable databases using the publicly available program, preventing root feeding damage by corn rootworm. Gw FORMATDB (available from NCBI). dsRNA transgenes represent new modes of action for com 0249 Total RNA and normalized cDNA libraries were bining with Bacillus thuringiensis, PIP, and/or AflP insecti similarly prepared from materials harvested at other WCR cidal protein technology in Insect Resistance Management developmental stages. A pooled transcriptome library for gene pyramids to mitigate the development of rootworm target gene Screening was constructed by combining cDNA populations resistant to either of these rootworm control library members representing the various developmental technologies. Stages. 0250 Candidate genes for RNAi targeting were hypoth Example 3: Amplification of Target Genes to esized to be essential for Survival and growth in pest insects. Selected target gene homologs were identified in the tran Produce dsRNA Scriptome sequence database, as described below. Full length or partial sequences of the target genes were ampli 0256 Full-length or partial clones of sequences of a fied by PCR to prepare templates for double-stranded RNA Diabrotica candidate gene, herein referred to as gw, were (dsRNA) production. used to generate PCR amplicons for dsRNA synthesis. 0251 TBLASTN searches using candidate protein cod Primers were designed to amplify portions of coding regions ing sequences were run against BLASTable databases con of each target gene by PCR. See Table 1. Where appropriate, a T7 phage promoter sequence (TTAATACGACTCAC taining the unassembled Diabrotica sequence reads or the TATAGGGAGA: SEQID NO:6) was incorporated into the assembled contigs. Significant hits to a Diabrotica sequence 5' ends of the amplified sense or antisense strands. See Table (defined as better thane' for contigs homologies and better 1. Total RNA was extracted from WCR using TRIZolR (Life than e' for unassembled sequence reads homologies) were Technologies, Grand Island, N.Y.), and was then used to confirmed using BLASTX against the NCBI non-redundant make first-strand cDNA with SuperScriptiII(R) First-Strand database. The results of this BLASTX search confirmed that Synthesis System and manufacturers Oligo dT primed the Diabrotica homolog candidate gene sequences identified instructions (Life Technologies, Grand Island, N.Y.). First in the TBLASTN search indeed comprised Diabrotica strand cDNA was used as template for PCR reactions using genes, or were the best hit to the non-Diabrotica candidate opposing primers positioned to amplify all or part of the gene sequence present in the Diabrotica sequences. In a few native target gene sequence. dsRNA was also amplified from cases, it was clear that some of the Diabrotica contigs or a DNA clone comprising the coding region for a yellow unassembled sequence reads selected by homology to a fluorescent protein (YFP) (SEQ ID NO:7: Shagin et al. non-Diabrotica candidate gene overlapped, and that the (2004) Mol. Biol. Evol. 21(5): 841-50). US 2017/0218391 A1 Aug. 3, 2017 30
TABLE 1. Primers and Primer Pairs used to amplify portions of coding regions of exemplary gW target gene and YFP negative contro Cele.
Gene ID Primer ID Sequence Pair 1 gw- 1 Dvv-gw-1 For TTAATACGACT CACTATAGGGAGAACGCAACAAC TACGGATGTTG (SEO ID NO : 8) Dvv-gw-1 Rev TTAATACGACT CACTATAGGGAGACATCCTTATC TTTACTTATCCACTGG (SEQ ID NO: 9) Pair 2 gw-1 v1 Dvv-gw- 1 v1. For TTAATACGACTCACTATAGGGAGAAAAACGAATC CCATGCATGATTC (SEO ID NO : 10) Dvv-gw-1 v1 Rev TAATACGACT CACTATAGGGAGACATCCTTATC TTTACTTATCCACTGG (SEQ ID NO: 11) Pair 3 gw-1 v2 Dvv-gw- 1 v2 For TTAATACGACTCACTATAGGGAGATCATCACCA GATTCTTAATCAACC (SEO ID NO: 12) Dvv-gw-1 v2. Rev AATACGACT CACTATAGGGAGAGTTATTGATT GTTGTGATATGAGCT G (SEQ ID NO: 13)
Pair 4 YFP YFP-F T7 TTAATACGACT CACTATAGGGAGACACCATGGGC TCCAGCGGCGCCC (SEO ID NO: 21) YFP-R T7 TTAATACGACT CACTATAGGGAGAAGATCTTGAA GGCGCTCTTCAGG (SEO ID NO. 24)
Example 4: RNAi Constructs et al. (1990) Mol. Gen. Genet. 220(2):245-50). Thus, the primary mRNA transcript contains the two gw gene segment 0257 Template Preparation by PCR and dsRNA Synthe sequences as large inverted repeats of one another, separated by the linker sequence. A copy of a promoter (e.g. maize 0258. The strategies used to provide specific templates ubiquitin 1, U.S. Pat. No. 5,510,474; 35S from Cauliflower for gw dsRNA and YFP dsRNA production are shown in Mosaic Virus (CaMV); Sugarcane bacilliform badnavirus FIG. 1 and FIG. 2. Template DNAs intended for use in gw (SchV) promoter, promoters from rice actin genes; ubiquitin dsRNA synthesis were prepared by PCR using the primer promoters; pl.MU; MAS; maize H3 histone promoter: ALS pairs in Table 1 and (as PCR template) first-strand cDNA promoter, phaseolin gene promoter; cab: rubisco; LAT52; prepared from total RNA isolated from WCR eggs, first Zm13; and/or apg) is used to drive production of the primary instar larvae, or adults. For each selected gw and YFP target mRNA hairpin transcript, and a fragment comprising a 3' gene region, PCR amplifications introduced a T7 promoter untranslated region (e.g., a maize peroxidase 5 gene sequence at the 5' ends of the amplified sense and antisense (ZmPerS 3'UTR v2; U.S. Pat. No. 6,699,984), Atubi10, strands (the YFP segment was amplified from a DNA clone AtEf1, or StPinII) is used to terminate transcription of the of the YFP coding region). The two PCR amplified frag hairpin-RNA-expressing gene. ments for each region of the target genes were then mixed in approximately equal amounts, and the mixture was used 0261 The binary destination vector comprises a herbi as transcription template for dsRNA production. See FIG. 1. cide tolerance gene (aryloxyalknoate dioxygenase; AAD-1 The sequences of the dsRNA templates amplified with the v3) (U.S. Pat. No. 7,838,733(B2), and Wright et al. (2010) particular primer pairs were: SEQ ID NO:3 (gw-1 reg1), Proc. Natl. Acad. Sci. U.S.A. 107:20240-5) under the regu SEQID NO:4 (gw-1 V1), SEQID NO:5 (gw-1 V2), and SEQ lation of a plant operable promoter (e.g., Sugarcane bacilli ID NO:7 (YFP). Double-stranded RNA for insect bioassay form badnavirus (SchBV) promoter (Schenk et al. (1999) was synthesized and purified using an AMBIONOR MEGA Plant Mol. Biol. 39:1221-30) or Zmubil (U.S. Pat. No. SCRIPTR) RNAi kit following the manufacturer's instruc 5,510.474)). A 5'UTR and linker are positioned between the tions (INVITROGEN) or HiScribe(RT7 In Vitro Transcrip 3' end of the promoter segment and the start codon of the tion Kit following the manufacturer's instructions (New AAD-1 coding region. A fragment comprising a 3' untrans England Biolabs, Ipswich, Mass.). The concentrations of lated region from a maize lipase gene (ZmLip 3'UTR; U.S. dsRNAs were measured using a NANODROPTM 8000 spec Pat. No. 7,179,902) is used to terminate transcription of the trophotometer (THERMO SCIENTIFIC, Wilmington, Del.). AAD-1 mRNA. 0259 Construction of Plant Transformation Vectors 0262. A negative control binary vector, which comprises 0260 Entry vectors harboring a target gene construct for a gene that expresses a YFP protein, is constructed by means hairpin formation comprising segments of gw (SEQ ID of standard GATEWAYOR) recombination reactions with a NO: 1) are assembled using a combination of chemically typical binary destination vector and entry vector. The synthesized fragments (DNA2.0, Menlo Park, Calif.) and binary destination vector comprises a herbicide tolerance standard molecular cloning methods. Intramolecular hairpin gene (aryloxyalknoate dioxygenase; AAD-1 V3) (as above) formation by RNA primary transcripts is facilitated by under the expression regulation of a maize ubiquitin 1 arranging (within a single transcription unit) two copies of promoter (as above) and a fragment comprising a 3' untrans the gw target gene segment in opposite orientation to one lated region from a maize lipase gene (ZmLip 3'UTR, as another, the two segments being separated by a linker above). The entry vector comprises a YFP coding region polynucleotide (e.g., a loop oran ST-LS1 intron; Vancanneyt (SEQ ID NO:14) under the expression control of a maize US 2017/0218391 A1 Aug. 3, 2017
ubiquitin 1 promoter (as above) and a fragment comprising for RNAi-mediated insect control are not efficacious in a 3' untranslated region from a maize peroxidase 5 gene (as controlling Diabrotica. It was also determined that above). sequences gw-1 regl, gW-1 V1, and gw-1 V2 dsRNA provide Surprising and unexpected Superior control of Diabrotica, Example 5: Screening of Candidate Target Genes compared to other genes suggested to have utility for 0263 Synthetic dsRNA designed to inhibit target gene RNAi-mediated insect control. sequences identified in EXAMPLE 2 caused mortality and 0266 For example, annexin, beta spectrin 2, and mtRP growth inhibition when administered to WCR in diet-based L4 were each suggested in U.S. Pat. No. 7,612,194 to be assayS. efficacious in RNAi-mediated insect control. SEQID NO:15 0264 Replicated bioassays demonstrated that ingestion is the DNA sequence of annexin region 1 (Reg. 1) and SEQ of dsRNA preparations derived from gw-1 reg1, gw-1 V1, ID NO:16 is the DNA sequence of annexin region 2 (Reg. 2). and gw-1 V2 resulted in mortality and/or growth inhibition SEQ ID NO:17 is the DNA sequence of beta spectrin 2 of western corn rootworm larvae. Table 2 shows the results region 1 (Reg. 1) and SEQID NO:18 is the DNA sequence of diet-based feeding bioassays of WCR larvae following of beta spectrin 2 region 2 (Reg2). SEQ ID NO:19 is the 9-day exposure to gw-1 reg1, gW-1 V1, and gw-1 V2 dsRNA, DNA sequence of mtRP-L4 region 1 (Reg. 1) and SEQ ID as well as the results obtained with a negative control sample NO:20 is the DNA sequence of mtRP-L4 region 2 (Reg. 2). of dsRNA prepared from a yellow fluorescent protein (YFP) AYFP sequence (SEQ ID NO:7) was also used to produce coding region (SEQID NO:14). Table 3 shows the LCs and dsRNA as a negative control. GIs results of exposure to gw-1 V1 dsRNA. 0267 Each of the aforementioned sequences was used to produce dsRNA by the methods of EXAMPLE 3. The TABLE 2 strategy used to provide specific templates for dsRNA Results of gw dsRNA diet feeding assays obtained with western corn production is shown in FIG. 2. Template DNAs intended for rootworm larvae after 9 days of feeding. ANOVA analysis found use in dsRNA synthesis were prepared by PCR using the significant differences in Mean % Mortality and Mean % Growth primer pairs in Table 4 and (as PCR template) first-strand Inhibition (GI). Means were separated using the Tukey-Kramer test. cDNA prepared from total RNA isolated from WCR first Dose Mean (% Mean instar larvae. (YFP was amplified from a DNA clone.) For Gene Name (ng/cm2) N Mortality) + SEM* (GI) + SEM each selected target gene region, two separate PCR ampli fications were performed. The first PCR amplification intro gW-1 Regl 500 18 56.49 it 4.46 (A) 0.83 + 0.03 (A) gw-1 V1 500 2 23.53 + 5.88 (AB) 0.70 + 0.10 (A) duced a T7 promoter sequence at the 5' end of the amplified gw-1 V2 500 2 31.37 + 1.96 (B) 0.55 + 0.10 (A) sense strands. The second reaction incorporated the T7 TE** O 20 12.21 + 2.32 (B) 0.06 + 0.03 (B) promoter sequence at the 5' ends of the antisense strands. WATER O 19 12.94 + 1.97 (B) -0.01 + 0.04 (B) The two PCR amplified fragments for each region of the YFP: 500 20 10.26 + 1.87 (B) 0.02 + 0.04 (B) target genes were then mixed in approximately equal *SEM = Standard Error of the Mean. Letters in parentheses designate statistical levels. amounts, and the mixture was used as transcription template Levels not connected by same letter are significantly different (P< 0.05). **TE = Tris HCl (1 mM) plus EDTA (0.1 mM) buffer, pH 7.2. for dsRNA production. See FIG. 2. Double-stranded RNA ***YFP = Yellow Fluorescent Protein was synthesized and purified using an AMBIONR) MiE GAScript(R) RNAi kit following the manufacturer's instruc tions (INVITROGEN). The concentrations of dsRNAs were TABLE 3 measured using a NANODROPTM 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, Del.) and the dsR Summary of oral potency of gw dsRNA on WCR larvae (ng/cm). NAs were each tested by the same diet-based bioassay Gene Name LCso Range GIso Range methods described above. Table 4 lists the sequences of the primers used to produce the annexin Regl, annexin Reg2, gw-1 V1 188 118.38-329.62 4.41 2.88-6.78 beta spectrin 2 Reg1, beta spectrin 2 Reg2, mtRP-L4 Reg1, mtRP-L4 Reg2, and YFP dsRNA molecules. Table 5 pres 0265. It has previously been suggested that certain genes ents the results of diet-based feeding bioassays of WCR of Diabrotica spp. may be exploited for RNAi-mediated larvae following 9-day exposure to these dsRNA molecules. insect control. See U.S. Patent Publication No. 2007/ Replicated bioassays demonstrated that ingestion of these 0.124836, which discloses 906 sequences, and U.S. Pat. No. dsRNAs resulted in no mortality or growth inhibition of 7,612,194, which discloses 9,112 sequences. However, it western corn rootworm larvae above that seen with control was determined that many genes suggested to have utility samples of TE buffer, water, or YFP protein. TABLE 4
Primers and Primer Pairs used to amplify portions of coding regions of genes.
Gene (Region) Primer ID Sequence
Pair 5 YFP TTAATACGACT CACTATAGGGAGACACCATGGGCTC CAGCGGCGCCC (SEO ID NO: 21) AGATCTTGAAGGCGCTCTTCAGG (SEO ID NO: 22) US 2017/0218391 A1 Aug. 3, 2017 32
TABLE 4 - continued Primers and Primer Pairs used to amplify portions of coding regions of Seles
Gene (Region) Primer ID Sequence
Pair 6 YFP YFP-F CACCATGGGCTCCAGCGGCGCCC (SEO ID NO. 23) YFP YFP-R T7 TTAATACGACT CACTATAGGGAGAAGATCTTGAAGG CGCTCTTCAGG (SEO ID NO: 24)
Pair 7 annexin Ann-F1 TT TTAATACGACT CACTATAGGGAGAGCTCCAACAGTG (Reg. 1) GTTCCTTATC (SEQ ID NO 25) annexin Ann-R1 CTAATAATTCTTTTTTAATGTTCCTGAGG (SEQ ID (Reg. 1) NO: 26)
Pair 8 annexin Ann-F1 GCTCCAACAGTGGTTCCTTATC (SEQ ID No. 27) (Reg. 1) annexin Ann-R1. T7 TTAATACGACT CACTATAGGGAGACTAATAATTCTT (Reg. 1) TTTTAATGTTCCTGAGG (SEQ ID NO: 28)
Pair 9 annexin Ann-F2 T7 TTAATACGACT CACTATAGGGAGATTGTTACAAGCT (Reg 2) GGAGAACTTCTC (SEO ID NO : 29) annexin Ann-R2 CTTAACCAACAACGGCTAATAAGG (SEQ ID NO: 3 O) (Reg 2)
Pair 10 annexin Ann-F2 TTGTTACAAGCTGGAGAACTTCTC (SEO ID NO : 31) (Reg 2) annexin Ann-R2Tf TTAATACGACT CACTATAGGGAGACTTAACCAACAA (Reg 2) CGGCTAATAAGG (SEQ ID NO: 32) Pair 11 beta-spect2 Betasp2-F1. T7 TTAATACGACTCACTATAGGGAGAAGATGTTGGCTG (Reg. 1) CATCTAGAGAA (SEQ ID NO: 33) beta- spect2 Beta sp2-R1 GTCCATTCGTCCATCCACTGCA (SEO ID NO. 34) (Reg. 1) Pair 12 beta- spect2 Beta sp2-F1. AGATGTTGGCTGCATCTAGAGAA (SEO ID NO : 35) (Reg. 1) beta-spect2 Betasp2-R1. T7 TTAATACGACTCACTATAGGGAGAGTCCATTCGTCC (Reg. 1) ATCCACTGCA (SEO ID NO : 36) Pair 13 beta-spect2 Betasp2-F2 T7 TTAATACGACTCACTATAGGGAGAGCAGATGAACAC (Reg 2) CAGCGAGAAA (SEQ ID NO : 37) beta- spect2 Beta sp2-R2 CTGGGCAGCTTCTTGTTTCCTC (SEQ ID NO: 38) (Reg 2)
Pair 14 beta- spect2 Beta sp2-F2 GCAGATGAACACCAGCGAGAAA (SEQ ID NO: 39) (Reg 2) beta-spect2 Betasp2-R2 T7 TTAATACGACTCACTATAGGGAGACTGGGCAGCTTC (Reg 2) TTGTTTCCTC (SEQ ID NO : 40)
Pair 15 mtRP-L14 L4-F1. T7 TTAATACGACT CACTATAGGGAGAAGTGAAATGTTA (Reg. 1) GCAAATATAACATCC (SEQ ID NO: 41) mtRP-L14 4-R1 ACCTCTCACTTCAAATCTTGACTTTG (SEQ ID (Reg. 1) NO: 42)
Pair 16 mtRP-L14 4 - F1 AGTGAAATGTTAGCAAATATAACATCC (SEO ID (Reg. 1) NO: 43) mtRP-L14 L4-R1. T7 TTAATACGACT CACTATAGGGAGAACCTCTCACTTC (Reg. 1) AAATCTTGACTTTG (SEO ID NO : 44)
Pair 17 mtRP-L14 L4-F2 T7 TTAATACGACTCACTATAGGGAGACAAAGTCAAGAT (Reg 2) TTGAAGTGAGAGGT (SEQ ID NO: 45) mtRP-L14 4- R2 CTACAAATAAAACAAGAAGGACCCC (SEQ ID NO: 46) (Reg 2)
Pair 18 mtRP-L14 4-F2 CAAAGTCAAGATTTGAAGTGAGAGGT (SEQ ID (Reg 2) NO: 47) mtRP-L14 L4-R2 T7 TTAATACGACTCACTATAGGGAGACTACAAATAAAA (Reg 2) CAAGAAGGACCCC (SEQ ID NO: 48) US 2017/0218391 A1 Aug. 3, 2017
TABLE 5 Medium to a final concentration of 200 uM from a 1 M stock solution in 100% dimethyl sulfoxide, and the solution is Results of diet feeding assays obtained with thoroughly mixed. Western corn rootworn larvae after 9 days. 0274 For each construct, 1 or 2 inoculating loops-full of Mean Live Agrobacterium from the YEP plate are suspended in 15 mL Dose Larval Mean %. Mean Growth Inoculation Medium/acetosyringone stock Solution in a ster Gene Name (ng/cm) Weight (mg) Mortality Inhibition ile, disposable, 50 mL centrifuge tube, and the optical annexin-Reg 1 OOO O.S45 O -0.262 density of the solution at 550 nm (ODsso) is measured in a annexin-Reg 2 OOO 0.565 O -O.301 spectrophotometer. The suspension is then diluted to ODsso beta spectrin2 OOO O.340 12 -0.014 of 0.3 to 0.4 using additional Inoculation Medium/acetosy Reg 1 beta spectrin2 OOO O465 18 -0.367 ringone mixtures. The tube of Agrobacterium suspension is Reg 2 then placed horizontally on a platform shaker set at about 75 mtRP-L4 Reg 1 OOO O.305 4 -0.168 rpm at room temperature and shaken for 1 to 4 hours while mtRP-L4 Reg 2 OOO O.305 7 -018O embryo dissection is performed. TE buffer O O430 13 O.OOO Water O 0.535 12 O.OOO (0275 Ear Sterilization and Embryo Isolation. YFP* * OOO O480 9 -0.386 0276 Maize immature embryos are obtained from plants of Zea mays inbred line B104 (Hallauer et al. (1997) Crop *TE = Tris HCl (10 mM) plus EDTA (1 mM) buffer, pH 8. Science 37: 1405-1406), grown in the greenhouse and self YFP = Yellow Fluorescent Protein or sib-pollinated to produce ears. The ears are harvested approximately 10 to 12 days post-pollination. On the experi Example 6: Production of Transgenic Maize mental day, de-husked ears are surface-sterilized by immer Tissues Comprising Insecticidal dsRNAs sion in a 20% solution of commercial bleach (ULTRA CLOROX(R) Germicidal Bleach, 6.15% sodium hypochlo 0268 Agrobacterium-Mediated Transformation. rite; with two drops of TWEEN 20) and shaken for 20 to 30 0269 Transgenic maize cells, tissues, and plants that min, followed by three rinses in sterile deionized water in a produce one or more insecticidal dsRNA molecules (for laminar flow hood. Immature Zygotic embryos (1.8 to 2.2 example, at least one dsRNA molecule including a dsRNA mm long) are aseptically dissected from each ear and molecule targeting a gene comprising gW (e.g., SEQ ID randomly distributed into microcentrifuge tubes containing NO: 1)) through expression of a chimeric gene stably-inte 2.0 mL of a suspension of appropriate Agrobacterium cells grated into the plant genome are produced following Agro in liquid Inoculation Medium with 200 uMacetosyringone, bacterium-mediated transformation. Maize transformation into which 2 uL of 10% BREAK-THRUR 5233 surfactant methods employing Superbinary or binary transformation (EVONIKINDUSTRIES: Essen, Germany) is added. For a vectors are known in the art, as described, for example, in given set of experiments, embryos from pooled ears are used U.S. Pat. No. 8.304,604, which is herein incorporated by for each transformation. reference in its entirety. Transformed tissues are selected by 0277 Agrobacterium Co-Cultivation. their ability to grow on Haloxyfop-containing medium and 0278. Following isolation, the embryos are placed on a are screened for dsRNA production, as appropriate. Portions rocker platform for 5 minutes. The contents of the tube are of Such transformed tissue cultures may be presented to then poured onto a plate of Co-cultivation Medium, which neonate corn rootworm larvae for bioassay, essentially as contains 4.33 gm/LMS salts; 1 xISU Modified MS Vitamins: described in EXAMPLE 1. 30gm/L Sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in 0270 Agrobacterium Culture Initiation. KOH (3,6-dichloro-o-anisic acid or 3,6-dichloro-2- 0271 Glycerol stocks of Agrobacterium strain DAt 13192 methoxybenzoic acid); 100 mg/L myo-inositol; 100 mg/L cells (PCT International Publication No. WO 2012/ Casein Enzymatic Hydrolysate; 15 mg/L AgNO, 200 uM 016222A2) harboring a binary transformation vector acetosyringone in DMSO; and 3 gm/L GELZANTM, at pH described above (EXAMPLE 4) are streaked on AB minimal 5.8. The liquid Agrobacterium suspension is removed with medium plates (Watson, et al. (1975) J. Bacteriol. 123:255 a sterile, disposable, transfer pipette. The embryos are then 264) containing appropriate antibiotics, and are grown at 20° oriented with the Scutellum facing up using sterile forceps C. for 3 days. The cultures are then streaked onto YEP plates with the aid of a microscope. The plate is closed, sealed with (gm/L: yeast extract, 10; Peptone, 10; NaCl, 5) containing 3MTM MICROPORETM medical tape, and placed in an the same antibiotics and are incubated at 20° C. for 1 day. incubator at 25°C. with continuous light at approximately 0272 Agrobacterium Culture. 60 umol mis' of Photosynthetically Active Radiation 0273. On the day of an experiment, a stock solution of (PAR). Inoculation Medium and acetosyringone is prepared in a 0279 Callus Selection and Regeneration of Transgenic Volume appropriate to the number of constructs in the Events. experiment and pipetted into a sterile, disposable, 250 mL 0280. Following the Co-Cultivation period, embryos are flask. Inoculation Medium (Frame et al. (2011) Genetic transferred to Resting Medium, which is composed of 4.33 Transformation Using Maize Immature Zygotic Embryos. IN gm/L MS salts: 1XISU Modified MS Vitamins; 30 gm/L Plant Embryo Culture Methods and Protocols: Methods in sucrose: 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH: Molecular Biology. T. A. Thorpe and E. C. Yeung, (Eds), 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydro Springer Science and Business Media, LLC. pp. 327-341) lysate; 15 mg/L AgNO: 0.5gm/L MES (2-(N-morpholino) contains: 2.2 gm/LMS salts; 1 xISU Modified MS Vitamins ethanesulfonic acid monohydrate; PHYTOTECHNOLO (Frame et al., ibid.) 68.4 gm/L Sucrose; 36 gm/L glucose: GIES LABR.: Lenexa, Kans.); 250 mg/L Carbenicillin; and 115 mg/L L-proline; and 100 mg/L myo-inositol; at pH 5.4.) 2.3 gm/L GELZANTM: at pH 5.8. No more than 36 embryos Acetosyringone is added to the flask containing Inoculation are moved to each plate. The plates are placed in a clear US 2017/0218391 A1 Aug. 3, 2017 34 plastic box and incubated at 27°C. with continuous light at 0285 Plants to be used for insect bioassays are trans approximately 50 umol ms' PAR for 7 to 10 days. planted from small pots to TINUSTM 350-4 ROOTRAIN Callused embryos are then transferred (<18/plate) onto ERS(R) (SPENCER-LEMAIRE INDUSTRIES, Acheson, Selection Medium I, which is comprised of Resting Medium Alberta, Canada) (one plant per event per ROOTRAINER(R). (above) with 100 nM R-Haloxyfop acid (0.0362 mg/L; for Approximately four days after transplanting to selection of calli harboring the AAD-1 gene). The plates are ROOTRAINERS(R), plants are infested for bioassay. returned to clear boxes and incubated at 27°C. with con 0286 Plants of the T generation are obtained by polli tinuous light at approximately 50 umol ms' PAR for 7 nating the silks of To transgenic plants with pollen collected days. Callused embryos are then transferred (<12/plate) to from plants of non-transgenic elite inbred line B104 or other Selection Medium II, which is comprised of Resting appropriate pollen donors, and planting the resultant seeds. Medium (above) with 500 nM R-Haloxyfop acid (0.181 Reciprocal crosses are performed when possible. mg/L). The plates are returned to clear boxes and incubated at 27°C. with continuous light at approximately 50 umol Example 7: Molecular Analyses of Transgenic m’s' PAR for 14 days. This selection step allows trans Maize Tissues genic callus to further proliferate and differentiate. 0287 Molecular analyses (e.g. RT-qPCR) of maize tis 0281 Proliferating, embryogenic calli are transferred Sues are performed on samples from leaves collected from (<9/plate) to Pre-Regeneration medium. Pre-Regeneration greenhouse grown plants on the same days that root feeding Medium contains 4.33 gm/LMS salts: 1XISU Modified MS damage is assessed. Vitamins; 45 gm/L Sucrose; 350 mg/L L-proline; 100 mg/L (0288 Results of RT-qPCR assays for the PerS 3'UTRor myo-inositol; 50 mg/L Casein Enzymatic Hydrolysate; 1.0 target gene are used to validate expression of the transgenes. mg/L. AgNO, 0.25 gm/L MES: 0.5 mg/L naphthaleneacetic (A low level of PerS 3'UTR detection is expected in non acid in NaOH: 2.5 mg/L abscisic acid in ethanol; 1 mg/L transformed maize plants, since there is usually expression 6-benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L of the endogenous PerS gene in maize tissues.) Results of GELZANTM; and 0.181 mg/L Haloxyfop acid; at pH 5.8. RT-qPCR assays for linker sequence (which is integral to the The plates are stored in clear boxes and incubated at 27°C. formation of dsRNA hairpin molecules) in expressed RNAs with continuous light at approximately 50 umolm's PAR are used to validate the presence of hairpin transcripts. for 7 days. Regenerating calli are then transferred (<6/plate) Transgene RNA expression levels are measured relative to to Regeneration Medium in PHYTATRAYSTM (SIGMA the RNA levels of an endogenous maize gene. ALDRICH) and incubated at 28°C. with 16 hours light/8 0289 DNA qPCR analyses to detect a portion of the hours dark per day (at approximately 160 umolm's PAR) AAD1 coding region in g|DNA are used to estimate trans for 14 days or until shoots and roots develop. Regeneration gene insertion copy number. Samples for these analyses are Medium contains 4.33 gm/LMS salts: 1XISU Modified MS collected from plants grown in environmental chambers. Vitamins; 60 gm/L Sucrose; 100 mg/L myo-inositol; 125 Results are compared to DNA qPCR results of assays mg/L Carbenicillin; 3 gm/L GELLANTM gum; and 0.181 designed to detect a portion of a single-copy native gene, mg/L R-Haloxyfop acid; at pH 5.8. Small shoots with and simple events (having one or two copies of gw trans primary roots are then isolated and transferred to Elongation genes) are advanced for further studies in the greenhouse. Medium without selection. Elongation Medium contains 0290 Additionally, qPCR assays designed to detect a 4.33 gm/LMS salts: 1XISU Modified MS Vitamins; 30 gm/L portion of the spectinomycin-resistance gene (SpecR; har sucrose; and 3.5gm/L GELRITETM: at pH 5.8. bored on the binary vector plasmids outside of the T-DNA) 0282. Transformed plant shoots selected by their ability are used to determine if the transgenic plants contain extra to grow on medium containing Haloxyfop are transplanted neous integrated plasmid backbone sequences. from PHYTATRAYSTM to small pots filled with growing 0291 RNA Transcript Expression Level: Per 5 3'UTRor medium (PROMIX BX; PREMIER TECH HORTICUL Target Gene qPCR. TURE), covered with cups or HUMI-DOMES (ARCO 0292 Callus cell events or transgenic plants are analyzed PLASTICS), and then hardened-off in a CONVIRON by real time quantitative PCR (qPCR) of the Per 53'UTRor growth chamber (27° C. day/24° C. night, 16-hour photo target sequence to determine the relative expression level of period, 50-70% RH, 200 umol ms' PAR). In some the full length hairpin transcript, as compared to the tran instances, putative transgenic plantlets are analyzed for script level of an internal maize gene (for example, GEN transgene relative copy number by quantitative real-time BANK Accession No. BTO69734), which encodes a TIP41 PCR assays using primers designed to detect the AAD1 like protein (i.e. a maize homolog of GENBANK Accession herbicide tolerance gene integrated into the maize genome. No. AT4G34270; having a tRLASTX score of 74% identity: Further, qPCR assays are used to detect the presence of the SEQ ID NO:49). RNA is isolated using an Norgen BioTek linker and/or target sequence in putative transformants. Total RNA. Isolation Kit (Norgen, Thorold, ON) or Selected transformed plantlets are then moved into a green RNeasyTM 96 kit (QIAGEN, Valencia, Calif.). The total house for further growth and testing. RNA is subjected to an On Column DNasel treatment according to the kits suggested protocol. The RNA is then 0283 Transfer and Establishment of To Plants in the quantified on a NANODROP 8000 spectrophotometer Greenhouse for Bioassay and Seed Production. (THERMO SCIENTIFIC) and the concentration is normal 0284. When plants reach the V3-V4 stage, they are ized to 25 or 50 ng/uL. First strand cDNA is prepared using transplanted into IE CUSTOM BLEND (PROFILE/ a HIGH CAPACITY cDNASYNTHESIS KIT (INVITRO METRO MIX 160) soil mixture and grown to flowering in GEN) in a 10 reaction volume with 5 uL denatured RNA, the greenhouse (Light Exposure Type: Photo or Assimila Substantially according to the manufacturer's recommended tion; High Light Limit: 1200 PAR; 16-hour day length; 27 protocol. The protocol is modified slightly to include the C. day/24°C. night). addition of 10 uL of 100 T20VN oligonucleotide (IDT) US 2017/0218391 A1 Aug. 3, 2017 35
(TTTTTTTTTTTTTTTTTTTTVN, where V is A, C, or G, TABLE 7-continued and N is A, C, G, or T: SEQID NO:50) into the 1 mL tube of random primer stock mix, in order to prepare a working PCR reaction recipes for transcript detection. stock of combined random primers and oligo dT. PerS 3'UTR TIP-like Gene 0293 Following cDNA synthesis, samples are diluted 1:3 Component Final Concentration with nuclease-free water, and stored at -20°C. until assayed. 0294 Separate real-time PCR assays for the PerS 3' cDNA (2.0 L) NA NA UTRor target gene and TIP41-like transcript are performed Water To 10 L To 10 L on a LIGHTCYCLERTM 480 (ROCHE DIAGNOSTICS, Indianapolis, Ind.) in 10 uL reaction volumes. For the Per5 TABLE 8 3'UTR assay, reactions are run with Primers P5U76S (F) (SEQ ID NO:51) and P5U76A (R) (SEQID NO:52), and an Thermocycler conditions for RNA qPCR. IDT Custom Oligo probe labeled with FAM and double PerS 3'UTR and TIP41-like Gene Detection quenched with Zen and Iowa Black quenchers or a ROCHE Process Temp. Time No. Cycles UNIVERSAL PROBETM (UPL76: Catalog No. Target Activation 95° C. 10 min 1 4889960001; labeled with FAM). For the TIP41-like refer Denature 95° C. 10 sec 40 ence gene assay, primers TIPmxF (SEQ ID NO:53) and Extend 60° C. 40 sec TIPmxR (SEQ ID NO:54), and Probe HXTIP (SEQ ID Acquire FAM or HEX 72° C. 1 Sec NO:55) labeled with HEX (hexachlorofluorescein) are used. Cool 40° C. 10 sec 1 0295 All assays include negative controls of no-template (mix only). For the standard curves, a blank (water in Source 0296 Data are analyzed using LIGHTCYCLERTM Soft well) is also included in the source plate to check for sample ware v1.5 by relative quantification using a second deriva cross-contamination. Primer and probe sequences are set tive max algorithm for calculation of Cq values according to forth in Table 6. Reaction components recipes for detection the Supplier's recommendations. For expression analyses, of the various transcripts are disclosed in Table 7, and PCR expression values are calculated using the AACt method reactions conditions are summarized in Table 8. The FAM (i.e., 2-(Cd TARGET-Cd REF)), which relies on the com (6-Carboxy Fluorescein Amidite) fluorescent moiety is parison of differences of Cq values between two targets, excited at 465 nm and fluorescence is measured at 510 nm, with the base value of 2 being selected under the assumption the corresponding values for the HEX (hexachlorofluores that, for optimized PCR reactions, the product doubles every cein) fluorescent moiety are 533 nm and 580 nm. cycle. TABLE 6 Oligonucleotide sequences used for molecular analyses of transcript levels in transcenic maize. Target Oligonucleotide Sequence Per5 3' UTR P5U76S (F) TTGTGATGTTGGTGGCGTAT (SEO ID NO. 51) Per5 3' UTR P5U76A (R) TGTTAAATAAAACCCCAAAGATCG (SEO ID NO : 52) Pers 3' UTR Roche UPL76 Roche Diagnostics Catalog Number 488996 OO1* * (FAM-Probe) TIP41 TIPmxF TGAGGGTAATGCCAACTGGTT (SEO ID NO : 53)
TIP41 TIPmixR GCAATGTAACCGAGTGTCTCTCAA (SEO ID NO. 54)
TIP41 HXTIP TTTTTGGCTTAGAGTTGATGGTGTACTGATGA (SEQ ID (HEX-Probe) NO: 55) *TIP41-like protein. **NAV Sequence Not Available from the Supplier.
TABLE 7 0297 Transcript Size and Integrity: Northern Blot Assay. 0298. In some instances, additional molecular character PCR reaction recipes for transcript detection. ization of the transgenic plants is obtained by the use of PerS 3'UTR TIP-like Gene Northern Blot (RNA blot) analysis to determine the molecu Component Final Concentration lar size of the gw hairpin dsRNA in transgenic plants expressing a gw dsRNA. Roche Buffer 1 X 1X PSU76S (F) 0.4 M O 0299 All materials and equipment are treated with PSU76A (R) 0.4 M O RNaseZAP (AMBION/INVITROGEN) before use. Tissue Roche UPL76 (FAM) 0.2 M O samples (100 mg to 500 mg) are collected in 2 mL SAFE HEXtipZM F O 0.4 M LOCK EPPENDORF tubes, disrupted with a KLECKOTM HEXtipZM R O 0.4 M tissue pulverizer (GARCIA MANUFACTURING, Visalia, HEXtipZMP (HEX) O 0.2 M Calif.) with three tungsten beads in 1 mL TRIZOL (INVIT ROGEN) for 5 min, then incubated at room temperature US 2017/0218391 A1 Aug. 3, 2017 36
(RT) for 10 min. Optionally, the samples are centrifuged for washes, wrapped, exposed to film for 1 to 30 minutes, then 10 min at 4° C. at 11,000 rpm and the supernatant is the film is developed, all by methods recommended by the transferred into a fresh 2 mL SAFELOCK EPPENDORF supplier of the DIG kit. tube. After 200 uL chloroform are added to the homogenate, 0303 Transgene Copy Number Determination. the tube is mixed by inversion for 2 to 5 min, incubated at 0304 Maize leaf pieces approximately equivalent to 2 RT for 10 minutes, and centrifuged at 12,000xg for 15 min leaf punches are collected in 96-well collection plates (QIA at 4°C. The top phase is transferred into a sterile 1.5 mL GEN). Tissue disruption is performed with a KLECKOTM EPPENDORF tube, 600 uL of 100% isopropanol are added, tissue pulverizer (GARCIA MANUFACTURING, Visalia, followed by incubation at RT for 10 minutes to 2 hours, and Calif.) in BIOSPRINT96 AP1 lysis buffer (supplied with a then centrifuged at 12,000xg for 10 min at 4°C. to 25°C. BIOSPRINT96 PLANT KIT: QIAGEN) with one stainless The supernatant is discarded and the RNA pellet is washed steel bead. Following tissue maceration, gDNA is isolated in twice with 1 mL 70% ethanol, with centrifugation at high throughput format using a BIOSPRINT96 PLANT KIT 7,500xg for 10 min at 4°C. to 25° C. between washes. The and a BIOSPRINT96 extraction robot, gDNA is diluted 2:3 ethanol is discarded and the pellet is briefly air dried for 3 DNA: water prior to setting up the qPCR reaction. to 5 min before resuspending in 50 u, of nuclease-free (0305 qPCR. Analysis. Water. 0306 Transgene detection by hydrolysis probe assay is 0300 Total RNA is quantified using the NANODROP performed by real-time PCR using a LIGHTCYCLERR480 8000(R) (THERMO-FISHER) and samples are normalized to system. Oligonucleotides to be used in hydrolysis probe 5 g/10 uL. 10 uL glyoxal (AMBION/INVITROGEN) are assays to detect the target gene, the linker sequence, or to then added to each sample. Five to 14 ng DIG RNA standard detect a portion of the SpecR gene (i.e. the spectinomycin marker mix (ROCHE APPLIED SCIENCE, Indianapolis, resistance gene borne on the binary vector plasmids; SEQID Ind.) is dispensed and added to an equal Volume of glyoxal. NO:56: SPC1 oligonucleotides in Table 9), are designed Samples and marker RNAs are denatured at 50° C. for 45 using LIGHTCYCLERR PROBE DESIGN SOFTWARE min and stored on ice until loading on a 1.25% SEAKEM 2.0. Further, oligonucleotides to be used in hydrolysis probe GOLD agarose (LONZA, Allendale, N.J.) gel in NORTH assays to detect a segment of the AAD-1 herbicide tolerance ERNMAX 10x glyoxal running buffer (AMBION/INVIT gene (SEQ ID NO:57: GAAD1 oligonucleotides in Table 9) ROGEN). RNAs are separated by electrophoresis at 65 are designed using PRIMER EXPRESS software (AP volts/30 mA for 2 hours and 15 minutes. PLIED BIOSYSTEMS). Table 9 shows the sequences of the primers and probes. Assays are multiplexed with reagents 0301 Following electrophoresis, the gel is rinsed in for an endogenous maize chromosomal gene (Invertase 2xSSC for 5 min and imaged on a GEL DOC station (SEQ ID NO:58: GENBANK Accession No: U16123; (BIORAD, Hercules, Calif.), then the RNA is passively referred to herein as IVR1), which serves as an internal transferred to a nylon membrane (MILLIPORE) overnight at reference sequence to ensure g|NA is present in each assay. RT, using 10xSSC as the transfer buffer (20xSSC consists of For amplification, LIGHTCYCLERR480 PROBES MAS 3 sodium chloride and 300 mM trisodium citrate, pH 7.0). TER mix (ROCHE APPLIED SCIENCE) is prepared at 1X Following the transfer, the membrane is rinsed in 2xSSC for final concentration in a 10 LL Volume multiplex reaction 5 minutes, the RNA is UV-crosslinked to the membrane containing 0.4 uM of each primer and 0.2 M of each probe (AGILENT/STRATAGENE), and the membrane is allowed (Table 10). A two-step amplification reaction is performed as to dry at room temperature for up to 2 days. outlined in Table 11. Fluorophore activation and emission 0302) The membrane is pre-hybridized in ULTRA for the FAM- and HEX-labeled probes are as described HYBTM buffer (AMBION/INVITROGEN) for 1 to 2 hr. The above: CY5 conjugates are excited maximally at 650 nm and probe consists of a PCR amplified product containing the fluoresce maximally at 670 nm. sequence of interest, (for example, the antisense sequence 0307 Cp scores (the point at which the fluorescence portion of SEQ ID NOS:3-5, as appropriate) labeled with signal crosses the background threshold) are determined digoxigenin by means of a ROCHE APPLIED SCIENCE from the real time PCR data using the fit points algorithm DIG procedure. Hybridization in recommended buffer is (LIGHTCYCLER(R) SOFTWARE release 1.5) and the Rela overnight at a temperature of 60° C. in hybridization tubes. tive Quant module (based on the AACt method). Data are Following hybridization, the blot is subjected to DIG handled as described previously (above; qPCR). TABLE 9 Sequences of primers and probes (with fluorescent conjugate) used for gene copy number determinations and binary vector plasmid backbone detection.
Name Sequence
GAAD1-F TGTTCGGTTCCCTCTACCAA (SEQ ID NO. 59)
GAAD1-R CAACATCCATCACCTTGACTGA (SEO ID NO : 6O) GAAD1-P (FAM) CACAGAACCGTCGCTTCAGCAACA (SEO ID NO : 61) TGGCGGACGACGACTTGT (SEO ID NO: 62) AAAGTTTGGAGGCTGCCGT (SEO ID NO : 63) US 2017/0218391 A1 Aug. 3, 2017 37
TABLE 9- continued Sequences of primers and probes (with fluorescent conjugate) used for gene copy number determinations and binary vector plasmid backbone detection. Name Sequence IVR1-P (HEX) CGAGCAGACCGCCGTGTACTTCTACC (SEO ID NO : 64) SPC1A CTTAGCTGGATAACGCCAC (SEO ID NO : 65) SPC1S GACCGTAAGGCTTGATGAA (SEQ ID NO: 66) TOSPEC (CY5 k) CGAGATTCTCCGCGCTGTAGA (SEO ID NO : 67) ST-LS1-F GTATGTTTCTGCTTCTACCTTTGAT (SEQ ID NO : ST-LS1-R CCATGTTTTGGTCATATATTAGAAAAGTT (SEO ID NO: 69) ST-LS1-P (FAM) AGTAATATAGTATTTCAAGTATTTTTTTCAAAAT (SEO ID NO : 7 O) CY5 = Cyanine-5
TABLE 10 assays are compared to similarly conducted bioassays that employ appropriate control tissues from host plants that do Reaction components for gene copy number not produce an insecticidal dsRNA, or to other control analyses and plasmid backbone detection. samples. Growth and Survival of target insects on the test Component Amt. (IL) Stock Final Concin diet is reduced compared to that of the control group. 0310. Insect Bioassays with Transgenic Maize Events. 2x Buffer S.O 2x 1X Appropriate Forward Primer 0.4 10 M 0.4 0311. Two western corn rootworm larvae (1 to 3 days old) Appropriate Reverse Primer 0.4 10 M 0.4 hatched from washed eggs are selected and placed into each Appropriate Probe 0.4 5 M 0.2 well of the bioassay tray. The wells are then covered with a IVR1-Forward Primer 0.4 10 M 0.4 IVR1-Reverse Primer 0.4 10 M 0.4 “PULL NPEEL tab cover (BIO-CV-16, BIO-SERV) and IVR1-Probe 0.4 5 M O.2 placed in a 28°C. incubator with an 18 hr/6 hr light/dark HO O6 NA* NA cycle. Nine days after the initial infestation, the larvae are gDNA 2.0 ND* * ND assessed for mortality, which is calculated as the percentage of dead insects out of the total number of insects in each Total 1O.O treatment. The insect samples are frozen at -20°C. for two *NA = Not Applicable days then the insect larvae from each treatment are pooled NTD = Not Determined and weighed. The percent of growth inhibition is calculated as the mean weight of the experimental treatments divided by the mean of the average weight of two control well TABLE 11 treatments. The data are expressed as a Percent Growth Thermocycler conditions for DNA qPCR. Inhibition (of the negative controls). Mean weights that Genomic copy number analyses exceed the control mean weight are normalized to Zero. 0312 Insect Bioassays in the Greenhouse. Process Temp. Time No. Cycles 0313 Western corn rootworm (WCR, Diabrotica vir Target Activation 95° C. 10 min 1 gifera virgifera LeConte) eggs are received in Soil from Denature 95° C. 10 sec 40 Extend & Acquire 60° C. 40 sec CROP CHARACTERISTICS (Farmington, Minn.). WCR FAM, HEX, or CY5 eggs are incubated at 28° C. for 10 to 11 days. Eggs are Cool 40° C. 10 sec 1 washed from the soil, placed into a 0.15% agar solution, and the concentration is adjusted to approximately 75 to 100 eggs per 0.25 mL aliquot. A hatch plate is set up in a Petri dish with an aliquot of egg Suspension to monitor hatch Example 8: Bioassay of Transgenic Maize rates. 0308 Insect Bioassays. 0314. The soil around the maize plants growing in 0309 Bioactivity of dsRNA of the subject invention ROOTRANERSR) is infested with 150 to 200 WCR eggs. produced in plant cells is demonstrated by bioassay meth The insects are allowed to feed for 2 weeks, after which time ods. See, e.g., Baum et al. (2007) Nat. Biotechnol. 25(11): a “Root Rating is given to each plant. A Node-Injury Scale 1322-1326. One is able to demonstrate efficacy, for example, is utilized for grading, essentially according to Oleson et al. by feeding various plant tissues or tissue pieces derived from (2005) J. Econ. Entomol. 98: 1-8. Plants passing this bioas a plant producing an insecticidal dsRNA to target insects in say, showing reduced injury, are transplanted to 5-gallon a controlled feeding environment. Alternatively, extracts are pots for seed production. Transplants are treated with insec prepared from various plant tissues derived from a plant ticide to prevent further rootworm damage and insect release producing the insecticidal dsRNA, and the extracted nucleic in the greenhouses. Plants are hand pollinated for seed acids are dispensed on top of artificial diets for bioassays as production. Seeds produced by these plants are saved for previously described herein. The results of such feeding evaluation at the T and Subsequent generations of plants. US 2017/0218391 A1 Aug. 3, 2017
0315 Transgenic negative control plants are generated by of the target genes in the coleopteran pest through RNA transformation with vectors harboring genes designed to mediated gene silencing. When the function of a target gene produce a yellow fluorescent protein (YFP). Bioassays are is important at one or more stages of development, the conducted with negative controls included in each set of growth and/or development of the coleopteran pest is plant materials. affected, and in the case of at least one of WCR, NCR, SCR, Example 9: Transgenic Zea mays Comprising MCR, D. balteata LeConte, D. speciosa Germar, D. u. Coleopteran Pest Sequences tenella, and D. u. undecimpunctata Mannerheim, leads to failure to successfully infest, feed, and/or develop, or leads 0316 10-20 transgenic To Zea mays plants are generated to death of the coleopteran pest. The choice of target genes as described in EXAMPLE 6. A further 10-20 T Zea mays and the successful application of RNAi are then used to independent lines expressing hairpin dsRNA for an RNAi control coleopteran pests. construct are obtained for corn rootworm challenge. Hairpin 0320 Phenotypic Comparison of Transgenic RNAi Lines dsRNA comprise a portion of SEQ ID NO:1. Additional hairpin dsRNAs are derived, for example, from coleopteran and Nontransformed Zea mays. pest sequences such as, for example, Caf1-180 (U.S. Patent 0321 Target coleopteran pest genes or sequences Application Publication No. 2012/0174258), VatpaseC (U.S. selected for creating hairpin dsRNA have no similarity to Patent Application Publication No. 2012/0174259), Rhol any known plant gene sequence. Hence, it is not expected (U.S. Patent Application Publication No. 2012/0174260), that the production or the activation of (systemic) RNAi by VatpaseH (U.S. Patent Application Publication No. 2012/ constructs targeting these coleopteran pest genes or 0198586), PPI-87B (U.S. Patent Application Publication sequences will have any deleterious effect on transgenic No. 2013/009 1600), RPA70 (U.S. Patent Application Pub plants. However, development and morphological charac lication No. 2013/009 1601), RPS6 (U.S. Patent Application teristics of transgenic lines are compared with non-trans Publication No. 2013/0097730), ROP (U.S. patent applica formed plants, as well as those of transgenic lines trans tion Ser. No. 14/577,811), RNA polymerase II140 (U.S. formed with an “empty' vector having no hairpin patent application Ser. No. 14/577,854), RNA polymerase I1 expressing gene. Plant root, shoot, foliage and reproduction (U.S. Patent Application No. 62/133,214), RNA polymerase characteristics are compared. Plant shoot characteristics II-215 (U.S. Patent Application No. 62/133.202), RNA Such as height, leaf numbers and sizes, time of flowering, polymerase 33 (U.S. Patent Application No. 62/133,210), floral size and appearance are recorded. In general, there are incm (U.S. Patent Application No. 62/095,487), Drea. (U.S. no observable morphological differences between transgenic patent application Ser. No. 14/705,807), transcription elon lines and those without expression of target iRNA molecules gation factor spts (U.S. Patent Application No. 62/168,613), when cultured in vitro and in soil in the glasshouse. and sptó (U.S. Patent Application No. 62/168,606). These Example 10: Transgenic Zea mays Comprising a are confirmed through RT-PCR or other molecular analysis Coleopteran Pest Sequence and Additional RNAi methods. Constructs 0317 Total RNA preparations from selected independent T lines are optionally used for RT-PCR with primers 0322. A transgenic Zea mays plant comprising a heter designed to bind in the linker of the hairpin expression ologous coding sequence in its genome that is transcribed cassette in each of the RNAi constructs. In addition, specific into an iRNA molecule that targets an organism other than primers for each target gene in an RNAi construct are a coleopteran pest is secondarily transformed via Agrobac optionally used to amplify and confirm the production of the terium or WHISKERSTM methodologies (see Petolino and pre-processed mRNA required for siRNA production in Arnold (2009) Methods Mol. Biol. 526:59-67) to produce planta. The amplification of the desired bands for each target one or more insecticidal dsRNA molecules (for example, at gene confirms the expression of the hairpin RNA in each least one dsRNA molecule including a dsRNA molecule transgenic Zea mays plant. Processing of the dsRNA hairpin targeting a gene comprising SEQID NO:1). Plant transfor of the target genes into siRNA is Subsequently optionally mation plasmid vectors prepared essentially as described in confirmed in independent transgenic lines using RNA blot EXAMPLE 4 are delivered via Agrobacterium or WHIS hybridizations. KERSTM-mediated transformation methods into maize sus 0318 Moreover, RNAi molecules having mismatch pension cells or immature maize embryos obtained from a sequences with more than 80% sequence identity to target transgenic Hi II or B104 Zea mays plant comprising a genes affect corn rootworms in a way similar to that seen heterologous coding sequence in its genome that is tran with RNAi molecules having 100% sequence identity to the scribed into an iRNA molecule that targets an organism target genes. The pairing of mismatch sequence with native other than a coleopteran pest. sequences to form a hairpin dsRNA in the same RNAi construct delivers plant-processed siRNAs capable of affect Example 11: Transgenic Zea mays Comprising an ing the growth, development, and viability of feeding cole RNAi Construct and Additional Coleopteran Pest opteran pests. Control Sequences 0319. In planta delivery of dsRNA, siRNA, or miRNA 0323 A transgenic Zea mays plant comprising a heter corresponding to target genes and the Subsequent uptake by ologous coding sequence in its genome that is transcribed coleopteran pests through feeding results in down-regulation into an iRNA molecule that targets a coleopteran pest US 2017/0218391 A1 Aug. 3, 2017 39 organism (for example, at least one dsRNA molecule includ BION: INVITROGEN) according to the manufacturers ing a dsRNA molecule targeting a gene comprising SEQID protocol. RNA sequencing using an Illumina R. HiSegTM NO: 1) is secondarily transformed via Agrobacterium or system (San Diego, Calif.) provided candidate target gene WHISKERSTM methodologies (see Petolino and Arnold sequences for use in RNAi insect control technology. HiSeq TM generated a total of about 378 million reads for the (2009) Methods Mol. Biol. 526:59-67) to produce one or six samples. The reads were assembled individually for each more insecticidal protein molecules, for example, Cry3, sample using TRINITYTM assembler software (Grabherr et Cry34 and Cry35 insecticidal proteins. Plant transformation al. (2011) Nature Biotech. 29:644-652). The assembled plasmid vectors prepared essentially as described in transcripts were combined to generate a pooled transcrip EXAMPLE 4 are delivered via Agrobacterium or WHIS tome. This BSB pooled transcriptome contained 378,457 KERSTM-mediated transformation methods into maize sus Sequences. pension cells or immature maize embryos obtained from a 0330 BSB Gw Ortholog Identification. transgenic B104 Zea mays plant comprising a heterologous 0331. A tBLASTn search of the BSB pooled transcrip coding sequence in its genome that is transcribed into an tome was performed using as query, Drosophila gw protein iRNA molecule that targets a coleopteran pest organism. isoforms A, B, E, F, I, and J (GENBANK Accession No. Doubly-transformed plants are obtained that produce iRNA NP 726600, NP 72.6597, NP 726601, NP 726596, molecules and insecticidal proteins for control of coleop NP 726599, respectively). BSB gw-1 (SEQID NO:71), was teran pests. identified as an Euschistus heros candidate target gw gene, the product of which has the predicted amino acid sequence Example 12: Screening of Candidate Target Genes of SEQ ID NO:72. in Neotropical Brown Stink Bug (Euschistus heros) 0332 Template Preparation and dsRNA Synthesis. 0324 Neotropical Brown Stink Bug (BSB, Euschistus 0333 cDNA was prepared from total BSB RNA extracted heros) Colony. from a single young adult insect (about 90 mg) using 0325 BSB were reared in a 27°C. incubator, at 65% TRIZolR Reagent (LIFE TECHNOLOGIES). The insect relative humidity, with 16: 8 hour light: dark cycle. One was homogenized at room temperature in a 1.5 mL micro gram of eggs collected over 2-3 days were seeded in 5 L centrifuge tube with 200 uL TRIZolR) using a pellet pestle containers with filter paper discs at the bottom, and the (FISHERBRAND Catalog No. 12-141-363) and Pestle containers were covered with #18 mesh for ventilation. Each Motor Mixer (COLE-PARMER, Vernon Hills, Ill.). Follow rearing container yielded approximately 300-400 adult BSB. ing homogenization, an additional 800 uL TRIZolR) was At all stages, the insects were fed fresh green beans three added, the homogenate was Vortexed, and then incubated at times per week, a Sachet of seed mixture that contained room temperature for five minutes. Cell debris was removed Sunflower seeds, soybeans, and peanuts (3:1:1 by weight by centrifugation, and the Supernatant was transferred to a ratio) was replaced once a week. Water was Supplemented in new tube. Following manufacturer-recommended TRIZolR) vials with cotton plugs as wicks. After the initial two weeks, extraction protocol for 1 mL TRIZol R, the RNA pellet was insects were transferred into a new container once a week. dried at room temperature and resuspended in 200 uL Tris 0326 BSB Artificial Diet. Buffer from a GFX PCR DNA and GEL, EXTRACTION 0327 A BSB artificial diet was prepared as follows. KIT (IllustraTM; GE HEALTHCARE LIFE SCIENCES) Lyophilized green beans were blended to a fine powder in a using Elution Buffer Type 4 (i.e., 10 mM Tris-HCl; pH8.0). MAGIC BULLETR) blender, while raw (organic) peanuts The RNA concentration was determined using a NANO were blended in a separate MAGIC BULLETR) blender. DROPTM 8000 spectrophotometer (THERMO SCIEN Blended dry ingredients were combined (weight percent TIFIC, Wilmington, Del.). ages: green beans, 35%; peanuts, 35%; sucrose, 5%; Vitamin 0334 cDNA Amplification. complex (e.g., VanderZant Vitamin Mixture for insects, SIGMA-ALDRICH, Catalog No. V1007), 0.9%); in a large 0335 cDNA was reverse-transcribed from 5ug BSB total MAGIC BULLETR) blender, which was capped and shaken RNA template and oligo dT primer, using a SUPERSCRIPT well to mix the ingredients. The mixed dry ingredients were III FIRST-STRAND SYNTHESIS SYSTEMTM for RT-PCR then added to a mixing bowl. In a separate container, water (INVITROGEN), following the supplier's recommended and benomyl anti-fungal agent (50 ppm; 25 uL of a 20,000 protocol. The final volume of the transcription reaction was ppm solution/50 mL diet solution) were mixed well, and brought to 100 uL with nuclease-free water. then added to the dry ingredient mixture. All ingredients 0336 Primers as shown in Table 12 were used to amplify were mixed by hand until the solution was fully blended. BSB gw-1 reg1. The DNA template was amplified by The diet was shaped into desired sizes, wrapped loosely in touch-down PCR (annealing temperature lowered from 60° aluminum foil, heated for 4 hours at 60° C., and then cooled C. to 50° C., in a 1° C./cycle decrease) with 1 uL cDNA and stored at 4°C. The artificial diet was used within two (above) as the template. A fragment comprising a 493 bp weeks of preparation. segment of BSB gw-1 reg1 (SEQ ID NO:73), was gener 0328 BSB Transcriptome Assembly. ated during 35 cycles of PCR. The above procedure was also 0329 Six stages of BSB development were selected for used to amplify a 301 bp negative control template YFPv2 mRNA library preparation. Total RNA was extracted from (SEQ ID NO:76), using YFPv2-F (SEQ ID NO:77) and insects frozen at -70° C., and homogenized in 10 volumes YFPv2-R (SEQ ID NO:78) primers. The BSB gw-1 reg1 of Lysis/Binding buffer in Lysing MATRIX A 2 mL tubes and YFPv2 primers contained a T7 phage promoter (MP BIOMEDICALS, Santa Ana, Calif.) on a FastPrep R-24 sequence (SEQ ID NO:6) at their 5' ends, and thus enabled Instrument (MP BIOMEDICALS). Total mRNA was the use of YFPv2 and BSB gw DNA fragments for dsRNA extracted using a mirVanaTM miRNA Isolation Kit (AM transcription. US 2017/0218391 A1 Aug. 3, 2017 40
TABL E 12 Primers and Primer Pairs used to amplify portions of coding regions of exemplary gw target genes and a YFP negative control gene. Gene ID Primer ID Sequence Pair gw- 1 BSB gw- TTAATACGACTCACTATAGGGAGAGTATGTTAAGCATCAAC 19 reg1 1. For AGCATACAC (SEO ID NO : 74) BSB gw- TTAATACGACTCACTATAGGGAGACACAACCGAACCAGGTG 1 Rev TAG (SEO ID NO: 75)
Pair YFP YFPW2 - F TTAATACGACT CACTATAGGGAGAGCATCTGGAGCACTTCT 2O CTTTCA (SEQ ID No. 77) YFPW2-R TTAATACGACT CACTATAGGGAGACCATCTCCTTCAAAGGT GATTG (SEO ID NO : 78)
0337 dsRNA Synthesis. TABLE 13 0338 dsRNA was synthesized using 2 uL PCR product Results of BSB gw dsRNA injection into the hemocoel of * instar (above) as the template with a MEGAScript TM T7 RNAi kit Neotropical Brown Stink Bug nymphs seven days after injection. (AMBION) used according to the manufacturers instruc N Mean 96 p value tions. See FIG. 1. dsRNA was quantified on a NANO Treatment Trials Mortality + SEM** t-test DROPTM 8000 spectrophotometer, and diluted to 500 ng/uL BSB gw-1 reg1 3 67 - 8.8 0.031i in nuclease-free 0.1x TE buffer (1 mM Tris HCL, 0.1 mM Not injected 1 7 O.35 EDTA, pH 7.4). YFPv2 3 19 - 11.6 *Ten insects injected per trial for each dsRNA. 0339. Injection of dsRNA into BSB Hemocoel. Standard error of the mean indicates significant difference from the YFPv2 dsRNA control using a Student's t-test p 0340 BSB were reared on a green bean and seed diet, as a 0.05. the colony, in a 27°C. incubator at 65% relative humidity and 16:8 hour light: dark photoperiod. Second instar nymphs Example 13: Transgenic Zea mays Comprising (each weighing 1 to 1.5 mg) were gently handled with a Hemipteran Pest Sequences small brush to prevent injury, and were placed in a Petri dish on ice to chill and immobilize the insects. Each insect was 0343 Ten to 20 transgenic To Zea mays plants harboring injected with 55.2 nL 500 ng/uL dsRNA solution (i.e., 27.6 expression vectors for nucleic acids comprising any portion ng dsRNA; dosage of 18.4 to 27.6 g/g body weight). of SEQ ID NO:71 (e.g., SEQ ID NO:73) are generated as Injections were performed using a NANOJECTTM II injector described in EXAMPLE 4. A further 10-20 T Zea mays (DRUMMOND SCIENTIFIC, Broomhall, Pa.), equipped independent lines expressing hairpin dsRNA for an RNAi with an injection needle pulled from a Drummond 3.5 inch construct are obtained for BSB challenge. Hairpin dsRNA #3-000-203-G/X glass capillary. The needle tip was broken, are derived comprising a portion of SEQ ID NO:71 or and the capillary was backfilled with light mineral oil and segments thereof (e.g., SEQ ID NO:73). These are con then filled with 2 to 3 uI dsRNA. dsRNA was injected into firmed through RT-PCR or other molecular analysis meth the abdomen of the nymphs (10 insects injected per dsRNA ods. Total RNA preparations from selected independent T. per trial), and the trials were repeated on three different days. lines are optionally used for RT-PCR with primers designed to bind in the linker intron of the hairpin expression cassette Injected insects (5 per well) were transferred into 32-well in each of the RNAi constructs. In addition, specific primers trays (Bio-RT-32 Rearing Tray; BIO-SERV, Frenchtown, for each target gene in an RNAi construct are optionally N.J.) containing a pellet of artificial BSB diet, and covered used to amplify and confirm the production of the pre with Pull-N-PeelTM tabs (BIO-CV-4: BIO-SERV). Moisture processed mRNA required for siRNA production in planta. was supplied by means of 1.25 mL water in a 1.5 mL The amplification of the desired bands for each target gene microcentrifuge tube with a cotton wick. The trays were confirms the expression of the hairpin RNA in each trans incubated at 26.5°C., 60% humidity, and 16: 8 hour light: genic Zea mays plant. Processing of the dsRNA hairpin of dark photoperiod. Viability counts and weights were taken the target genes into siRNA is Subsequently optionally on day 7 after the injections. confirmed in independent transgenic lines using RNA blot (0341 BSB Gw is a Lethal dsRNA Target. hybridizations. 0344) Moreover, RNAi molecules having mismatch 0342. As summarized in Table 13, in each replicate, at sequences with more than 80% sequence identity to target least ten 2" instar BSB nymphs (1-1.5 mg each) were genes affect hemipterans in a way similar to that seen with injected into the hemocoel with 55.2 nL BSB gw-1 reg1 RNAi molecules having 100% sequence identity to the dsRNA (500 ng/uL), for an approximate final concentration target genes. The pairing of mismatch sequence with native of 18.4-27.6 ug dsRNA/g insect. The mortality determined sequences to form a hairpin dsRNA in the same RNAi for BSB gw-1 reg1 dsRNA was higher than that observed construct delivers plant-processed siRNAs capable of affect with the same amount of injected YFPv2 dsRNA (negative ing the growth, development, and viability of feeding control). hemipteran pests. US 2017/0218391 A1 Aug. 3, 2017
(0345. In planta delivery of dsRNA, siRNA, shRNA, remove the embryonic axis, wherein about /2-/3 of the hpRNA, or miRNA corresponding to target genes and the embryo axis remains attached to the nodal end of the Subsequent uptake by hemipteran pests through feeding cotyledon. results in down-regulation of the target genes in the Inoculation. hemipteran pest through RNA-mediated gene silencing. 0351 When the function of a target gene is important at one or 0352. The split soybean seeds comprising a partial por more stages of development, the growth, development, tion of the embryonic axis are then immersed for about 30 and/or survival of the hemipteran pest is affected, and in the minutes in a solution of Agrobacterium tumefaciens (e.g., case of at least one of Euschistus heros, E. serous, Nezara strain EHA 101 or EHA 105) containing a binary plasmid viridula, Piezodorus guildinii, Halyomorpha haly's, China comprising SEQ ID NO:71 and/or segments thereof (e.g., via hilare, C. marginiatum, Dichelops melacanthus, D. fur SEQID NO:73). The A. tumefaciens solution is diluted to a catus, Edessa meditabunda, Thyanta perditor; Horcias final concentration of w=0.6 ODs before immersing the nobilellus, Taedia Stigmosa, Dysdercus peruvianus, Neo cotyledons comprising the embryo axis. megalotomus parvus, Leptoglossus zonatus, Niesthrea 0353 Co-Cultivation. sidae, Lygus hesperus, and L. lineolaris leads to failure to 0354 Following inoculation, the split soybean seed is successfully infest, feed, develop, and/or leads to death of allowed to co-cultivate with the Agrobacterium tumefaciens the hemipteran pest. The choice of target genes and the strain for 5 days on co-cultivation medium (Agrobacterium successful application of RNAi is then used to control Protocols, vol. 2, 2" Ed., Wang, K. (Ed.) Humana Press, hemipteran pests. New Jersey, 2006) in a Petri dish covered with a piece of 0346 Phenotypic Comparison of Transgenic RNAi Lines filter paper. and Non-Transformed Zea mays. 0355 Shoot Induction. 0347 Target hemipteran pest genes or sequences selected 0356. After 5 days of co-cultivation, the split soybean for creating hairpin dsRNA have no similarity to any known seeds are washed in liquid Shoot Induction (SI) media plant gene sequence. Hence it is not expected that the consisting of B5 salts, B5 vitamins, 28 mg/L Ferrous, 38 production or the activation of (systemic) RNAi by con mg/L NaEDTA, 30 g/L sucrose, 0.6 g/L MES, 1.11 mg/L structs targeting these hemipteran pest genes or sequences BAP 100 mg/L TIMENTINTM, 200 mg/L cefotaxime, and will have any deleterious effect on transgenic plants. How 50 mg/L. Vancomycin (pH 5.7). The split soybean seeds are ever, development and morphological characteristics of then cultured on Shoot Induction I (SII) medium consisting transgenic lines are compared with non-transformed plants, of B5 salts, B5 vitamins, 7 g/L Noble agar, 28 mg/L Ferrous, as well as those of transgenic lines transformed with an 38 mg/L Na-EDTA, 30 g/L sucrose, 0.6 g/L MES, 1.11 mg/L 'empty vector having no hairpin-expressing gene. Plant BAP, 50 mg/L TIMENTINTM, 200 mg/L cefotaxime, and 50 root, shoot, foliage and reproduction characteristics are mg/L. Vancomycin (pH 5.7), with the flat side of the coty compared. There is no observable difference in root length ledon facing up and the nodal end of the cotyledon imbedded and growth patterns of transgenic and non-transformed into the medium. After 2 weeks of culture, the explants from plants. Plant shoot characteristics Such as height, leaf num the transformed split soybean seed are transferred to the bers and sizes, time of flowering, floral size and appearance Shoot Induction II (SI II) medium containing SII medium are similar. In general, there are no observable morphologi supplemented with 6 mg/L glufosinate)(LIBERTYR). cal differences between transgenic lines and those without 0357 Shoot Elongation. expression of target iRNA molecules when cultured in vitro 0358. After 2 weeks of culture on SI II medium, the and in soil in the glasshouse. cotyledons are removed from the explants and a flush shoot pad containing the embryonic axis are excised by making a Example 14: Transgenic Glycine max Comprising cut at the base of the cotyledon. The isolated shoot pad from Hemipteran Pest Sequences the cotyledon is transferred to Shoot Elongation (SE) medium. The SE medium consists of MS salts, 28 mg/L 0348 Ten to 20 transgenic To Glycine max plants har Ferrous, 38 mg/L NaEDTA, 30 g/L sucrose and 0.6 g/L boring expression vectors for nucleic acids comprising a MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid, portion of SEQID NO:71 or segments thereof (e.g., SEQID 0.1 mg/L IAA, 0.5 mg/L GA3, 1 mg/L Zeatin riboside, 50 NO:73) are generated as is known in the art, including for mg/L TIMENTINTM, 200 mg/L cefotaxime, 50 mg/L van example by Agrobacterium-mediated transformation, as fol comycin, 6 mg/L glufosinate, and 7 g/L Noble agar, (pH lows. Mature soybean (Glycine max) seeds are sterilized 5.7). The cultures are transferred to fresh SE medium every overnight with chlorine gas for sixteen hours. Following 2 weeks. The cultures are grown in a CONVIRONTM growth sterilization with chlorine gas, the seeds are placed in an chamber at 24° C. with an 18 h photoperiod at a light open container in a LAMINARTM flow hood to dispel the intensity of 80-90 Lumol/m sec. chlorine gas. Next, the sterilized seeds are imbibed with Rooting. sterile HO for sixteen hours in the dark using a black box 0359 at 24° C. 0360 Elongated shoots which developed from the coty ledon shoot pad are isolated by cutting the elongated shoot 0349 Preparation of Split-Seed Soybeans. at the base of the cotyledon shoot pad, and dipping the 0350. The split soybean seed comprising a portion of an elongated shoot in 1 mg/L IBA (Indole 3-butyric acid) for embryonic axis protocol requires preparation of soybean 1-3 minutes to promote rooting. Next, the elongated shoots seed material which is cut longitudinally, using a #10 blade are transferred to rooting medium (MS salts, B5 vitamins, 28 affixed to a scalpel, along the hilum of the seed to separate mg/L Ferrous, 38 mg/L Na-EDTA, 20 g/L Sucrose and 0.59 and remove the seed coat, and to split the seed into two g/L MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic cotyledon sections. Careful attention is made to partially acid 7 g/L Noble agar, pH 5.6) in phyta trays. US 2017/0218391 A1 Aug. 3, 2017 42
0361 Cultivation. production or the activation of (systemic) RNAi by con 0362 Following culture in a CONVIRONTM growth structs targeting these hemipteran pest genes or sequences chamber at 24° C., 18 h photoperiod, for 1-2 weeks, the will have any deleterious effect on transgenic plants. How shoots which have developed roots are transferred to a soil ever, development and morphological characteristics of mix in a covered sundae cup and placed in a CONVIRONTM transgenic lines are compared with non-transformed plants, growth chamber (models CMP4030 and CMP3244, Con as well as those of transgenic lines transformed with an trolled Environments Limited, Winnipeg, Manitoba, 'empty' vector having no hairpin-expressing gene. Plant Canada) under long day conditions (16 hours light/8 hours root, shoot, foliage, and reproduction characteristics are dark) at a light intensity of 120-150 umol/m sec under compared. There is no observable difference in root length constant temperature (22°C.) and humidity (40-50%) for and growth patterns of transgenic and non-transformed acclimatization of plantlets. The rooted plantlets are accli plants. Plant shoot characteristics Such as height, leaf num mated in Sundae cups for several weeks before they are bers and sizes, time of flowering, floral size and appearance transferred to the greenhouse for further acclimatization and are similar. In general, there are no observable morphologi establishment of robust transgenic soybean plants. cal differences between transgenic lines and those without 0363 A further 10-20 T. Glycine max independent lines expression of target iRNA molecules when cultured in vitro expressing hairpin dsRNA for an RNAi construct are and in soil in the glasshouse. obtained for BSB challenge. Hairpin dsRNA may be derived comprising SEQ ID NO:71 or segments thereof (e.g., SEQ Example 15: E. heros Bioassays on Artificial Diet ID NO:73). These are confirmed through RT-PCR or other 0368. In dsRNA feeding assays on artificial diet, 32-well molecular analysis methods as known in the art. Total RNA trays are set up with an ~18 mg pellet of artificial diet and preparations from selected independent T lines are option water, as for injection experiments (See EXAMPLE 12). ally used for RT-PCR with primers designed to bind in the dsRNA at a concentration of 200 ng/uL is added to the food linker intron of the hairpin expression cassette in each of the pellet and water sample; 100 L to each of two wells. Five RNAi constructs. In addition, specific primers for each 2" instar E. heros nymphs are introduced into each well. target gene in an RNAi construct are optionally used to Water samples and dsRNA that targets a YFP transcript are amplify and confirm the production of the pre-processed used as negative controls. The experiments are repeated on mRNA required for siRNA production in planta. The ampli three different days. Surviving insects are weighed, and the fication of the desired bands for each target gene confirms the expression of the hairpin RNA in each transgenic Gly mortality rates are determined after 8 days of treatment. cine max plant. Processing of the dsRNA hairpin of the Mortality and/or growth inhibition is observed in the wells target genes into siRNA is Subsequently optionally con provided with BSB gw dsRNA, compared to the control firmed in independent transgenic lines using RNA blot wells. hybridizations. Example 16: Transgenic Arabidopsis thaliana 0364 RNAi molecules having mismatch sequences with Comprising Hemipteran Pest Sequences more than 80% sequence identity to target genes affect BSB in a way similar to that seen with RNAi molecules having 0369 Arabidopsis transformation vectors containing a 100% sequence identity to the target genes. The pairing of target gene construct for hairpin formation comprising seg mismatch sequence with native sequences to form a hairpin ments of gw (SEQ ID NO:71) are generated using standard dsRNA in the same RNAi construct delivers plant-processed molecular methods similar to EXAMPLE 4. Arabidopsis siRNAs capable of affecting the growth, development, and transformation is performed using standard Agrobacterium viability of feeding hemipteran pests. based procedure. T seeds are selected with glufosinate 0365. In planta delivery of dsRNA, siRNA, shRNA, or tolerance selectable marker. Transgenic T Arabidopsis miRNA corresponding to target genes and the Subsequent plants are generated and homozygous simple-copy T2 trans uptake by hemipteran pests through feeding results in down genic plants are generated for insect studies. Bioassays are regulation of the target genes in the hemipteran pest through performed on growing Arabidopsis plants with inflores RNA-mediated gene silencing. When the function of a target cences. Five to ten insects are placed on each plant and gene is important at one or more stages of development, the monitored for survival within 14 days. growth, development, and viability of feeding of the 0370 Construction of Arabidopsis Transformation Vec hemipteran pest is affected, and in the case of at least one of tOrS. Euschistus heros, Piezodorus guildinii, Halyomorpha haly's, 0371 Entry clones based on an entry vector harboring a Nezara viridula, Chinavia hilare, Euschistus serous, Dich target gene construct for hairpin formation comprising a elops melacanthus, Dichelops furcatus, Edessa medit segment of BSB gw (SEQ ID NO:71) are assembled using abunda, Thyanta perditor, Chinavia marginiatum, Horcias a combination of chemically synthesized fragments (DNA2. nobilellus, Taedia Stigmosa, Dysdercus peruvianus, Neo 0, Menlo Park, Calif.) and standard molecular cloning megalotomus parvus, Leptoglossus zonatus, Niesthrea methods. Intramolecular hairpin formation by RNA primary sidae, and Lygus lineolaris leads to failure to Successfully transcripts is facilitated by arranging (within a single tran infest, feed, develop, and/or leads to death of the hemipteran Scription unit) two copies of a target gene segment in pest. The choice of target genes and the Successful applica opposite orientations, the two segments being separated by tion of RNAi is then used to control hemipteran pests. a linker sequence (e.g. a loop or an ST-LS1 intron) (Van 0366 Phenotypic Comparison of Transgenic RNAi Lines canneyt et al. (1990) Mol. Gen. Genet. 220(2):245-50). and Non-Transformed Glycine max. Thus, the primary mRNA transcript contains the two gw 0367 Target hemipteran pest genes or sequences selected gene segment sequences as large inverted repeats of one for creating hairpin dsRNA have no similarity to any known another, separated by the linker sequence. A copy of a plant gene sequence. Hence it is not expected that the promoter (e.g. Arabidopsis thaliana ubiquitin 10 promoter US 2017/0218391 A1 Aug. 3, 2017
(Callis et al. (1990).J. Biological Chem. 265:12486-12493)) Example 17: Growth and Bioassays of Transgenic is used to drive production of the primary mRNA hairpin Arabidopsis transcript, and a fragment comprising a 3' untranslated 0379 Selection of T Arabidopsis Transformed with region from Open Reading Frame 23 of Agrobacterium dsRNA Constructs. tumefaciens (AtuORF23 3' UTR v1; U.S. Pat. No. 5,428, 0380. Up to 200 mg of T seeds from each transformation 147) is used to terminate transcription of the hairpin-RNA are stratified in 0.1% agarose solution. The seeds are planted expressing gene. in germination trays (10.5"x21"x1"; T.O. Plastics Inc., 0372. The hairpin clones within entry vectors are used in Clearwater, Minn.) with #5 sunshine media. Transformants standard GATEWAY(R) recombination reactions with a typi are selected for tolerance to Ignite R (glufosinate) at 280 g/ha cal binary destination vector to produce hairpin RNA expres at 6 and 9 days post planting. Selected events are trans sion transformation vectors for Agrobacterium-mediated planted into 4" diameter pots. Insertion copy analysis is performed within a week of transplanting via hydrolysis Arabidopsis transformation. quantitative Real-Time PCR (qPCR) using Roche LightCy 0373) A binary destination vector comprises a herbicide cler480TM. The PCR primers and hydrolysis probes are tolerance gene, DSM-2v2 (U.S. Patent Publication No. designed against DSM2V2 selectable marker using Light 2011/0107455), under the regulation of a Cassava vein CyclerTM Probe Design Software 2.0 (Roche). Plants are mosaic virus promoter (CsVMV Promoter v2. U.S. Pat. No. maintained at 24°C., with a 16:8 hour light: dark photope 7,601,885; Verdaguer et al. (1996) Plant Mol. Biol. 31:1129 riod under fluorescent and incandescent lights at intensity of 39). A fragment comprising a 3' untranslated region from 100-150 mE/ms. Open Reading Frame 1 of Agrobacterium tumefaciens (At 0381 E. heros Plant Feeding Bioassay. uORF1 3' UTR V6; Huang et al. (1990) J. Bacteriol. 172: 0382. At least four low copy (1-2 insertions), four 1814-22) is used to terminate transcription of the DSM2V2 medium copy (2-3 insertions), and four high copy (>4 mRNA. insertions) events are selected for each construct. Plants are grown to a reproductive stage (plants containing flowers and 0374. A negative control binary construct which com siliques). The surface of soil is covered with ~50 mL volume prises a gene that expresses a YFP hairpin RNA, is con of white sand for easy insect identification. Five to ten 2" structed by means of standard GATEWAYOR) recombination instar E. heros nymphs are introduced onto each plant. The reactions with a typical binary destination vector and entry plants are covered with plastic tubes that are 3" in diameter, vector. The entry construct comprises a YFP hairpin 16" tall, and with wall thickness of 0.03" (Item No. 484485, sequence under the expression control of an Arabidopsis Visipack Fenton Mo.); the tubes are covered with nylon Ubiquitin 10 promoter (as above) and a fragment comprising mesh to isolate the insects. The plants are kept under normal an ORF23 3' untranslated region from Agrobacterium tume temperature, light, and watering conditions in a conviron. In faciens (as above). 14 days, the insects are collected and weighed; percent mortality as well as growth inhibition (1-weight treatment/ 0375 Production of Transgenic Arabidopsis Comprising weight control) are calculated. YFP hairpin-expressing Insecticidal RNAs: Agrobacterium-Mediated Transforma plants are used as controls. Significant mortality and/or tion. growth inhibition is observed in nymphs feeding on trans 0376 Binary plasmids containing hairpin dsRNA genic BSB gw dsRNA plants, compared to that of nymphs sequences are electroporated into Agrobacterium strain on control plants. GV3101 (pMP90RK). The recombinant Agrobacterium 0383 T2 Arabidopsis Seed Generation and T2 Bioassays. clones are confirmed by restriction analysis of plasmids 0384 T2 seed is produced from selected low copy (1-2 preparations of the recombinant Agrobacterium colonies. A insertions) events for each construct. Plants (homozygous Qiagen Plasmid Max Kit (Qiagen, Catil 12162) is used to and/or heterozygous) are subjected to E. heros feeding extract plasmids from Agrobacterium cultures following the bioassay, as described above. T3 seed is harvested from manufacture recommended protocol. homozygotes and stored for future analysis. 0377 Arabidopsis Transformation and T. Selection. Example 18: Transformation of Additional Crop 0378 Twelve to fifteen Arabidopsis plants (c.V. Colum Species bia) are grown in 4" pots in the green house with light 0385 Cotton is transformed with a gw dsRNA transgene intensity of 250 umol/m, 25°C., and 18:6 hours of light: to provide control of hemipteran insects by utilizing a dark conditions. Primary flower stems are trimmed one method known to those of skill in the art, for example, week before transformation. Agrobacterium inoculums are Substantially the same techniques previously described in prepared by incubating 10 LL recombinant Agrobacterium EXAMPLE 14 of U.S. Pat. No. 7,838,733, or Example 12 of glycerol stock in 100 mL LB broth (Sigma L3022)+100 PCT International Patent Publication No. WO 2007/053482. mg/L Spectinomycin-50 mg/L. Kanamycin at 28° C. and shaking at 225 rpm for 72 hours. Agrobacterium cells are Example 19: Gw dsRNA in Insect Management harvested and suspended into 5% sucrose+0.04% Silwet L77 (Lehle Seeds Cat if VIS-02)+10 ug/L benzamino purine 0386 Gw dsRNA transgenes are combined with other (BA) solution to ODoo 0.8-1.0 before floral dipping. The dsRNA molecules in transgenic plants to provide redundant above-ground parts of the plant are dipped into the Agro RNAi targeting and synergistic RNAi effects. Transgenic bacterium solution for 5-10 minutes, with gentle agitation. plants including, for example and without limitation, corn, The plants are then transferred to the greenhouse for normal Soybean, and cotton expressing dsRNA that targets gw are growth with regular watering and fertilizing until seed set. useful for preventing feeding damage by coleopteran and US 2017/0218391 A1 Aug. 3, 2017 44 hemipteran insects. Gw dsRNA transgenes are also com When combined with other dsRNA molecules that target bined in plants with Bacillus thuringiensis, PIP-1, and/or insect pests and/or with insecticidal proteins in transgenic AflP insecticidal protein technology to represent new modes plants, a synergistic insecticidal effect is observed that also of action in Insect Resistance Management gene pyramids. mitigates the development of resistant insect populations.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 84
<21 Os SEQ ID NO 1 &211s LENGTH: 45.31 &212s. TYPE: DNA <213> ORGANISM: Diabrotica virgifera
<4 OOs SEQUENCE: 1 gcaccattat caaagaacta tdggtgaatc. cacaattitta caaacataac atttgaccaa 60 aatgttatcc aaaagttaaa tttgt attat tctggaattt ttct tact.cc agtaatatac 12O attggagat c aaactgtata aataaattgt at aaataaat ctaaatcaag ataattattt 18O
cacacat ct c totacatatic cagttacctt coat ct ctitt ct catgttgg aaacaatgga 24 O gtatic tittct ctdtgaagct cogcc cactt tdatat citaa ccaattitatic ct gcacccaa 3 OO gttgttgattt gtgatggtgt gttct tatt c to attct ct c catattaatt tat cqacgta 360 aagccaatgt gatttitt tag tdatatt cog titttaatcgc at cacatttic gaggatatag 42O atctotggct ggc.ca.gactg at atggagca ct attgaaga tigcgc.gc.ccc taccCcct Co 48O
gag.ccgaagt ct acatttcc tacctaccala gtgc ct caaa agt cagc.cat gaggggcagc 54 O
gcaccCC cag tacaagttgc agggc catct toggggggt c gagc.cgatcc cc caagtagt 6 OO accc.gttgcg cc gatgaagg cqctctgtct gtgatat cog gct Caagttg cc.gttcaatc 660
gacaacticta at attagaat goaatctgtg accoaaaatt gtc.ttctgaa citctgttacc 72O gtaccaaata togcaacgttt agaccatggc atgg to accc acaataatag ctittaagtta 78O
gttagtaagt ttggtgctitt acticcc.cgga cdagacatt c ccaatcaaaa gtctgatgac 84 O
citcgaactac tacgcgatga cct caatgta citgaattcaa ctaaatacga tactaaaa.ca 9 OO citctg.cgata acaacgatga aaaagacgac catgatgcat accalaatgtc. galacattgaa 96.O
act catacct gcacaaataa tdacaac agc tat caagagc tigtacaagcc tittgagacitt O2O agagggggag gCaaagttc cct cagc act gg tacttctg gatgggggiac gocaccitt ct O8O
caatctggta acaacaatgc aaataagagc aatggc.cagc aaccacctac ct cocaatca 14 O aacaa.cactg gttggggtca acctggaacg aaaactgcaa at aacaatgc aatgccacct 2 OO
aatagt caac ct cottacct c tactgctaat tct cagaaca acaatggacc aagcaacaat 26 O accaaacaac aattggalaca acticaac agt atgagagaag cc atttittag cc aggatggc 32O tggggcggac aa catgtcaa tdaagataca aattgggaca tt CC cagtag ccc.cgagc ct 38O
cc cattaaaa tigatggitt C cqgaggit coa ccaccatgga aaccggctgt gaataatggit 4 4 O
accga attat gggaagcgaa tott.cgaaac ggtggacaac ct cotccaca acct Caacag 5 OO aaaac cc ctt ggggt cacac accct ctacg aa cataggcg gtacctgggg caagacgat 560
gacgctgaca Cttctaatgt ttggaccggc gtaccatcca at Caacctica atggggtggit 62O
gcaggtggala at acgaataa tigagc.catg tdgggcggcc ct aagaaaga aaacgattgg 68O
ggtacaggtg caa.gcaatac C9gtggctgg ggtgat CCaC gtgcagctga t c cacgtcaa 740
actgg tatgg accct Caga aatcc.gc.cca galactgagag atatgcgggc aggtaataca 8 OO
US 2017/0218391 A1 Aug. 3, 2017 46
- Continued cgtgaggaag citat caaagc ticagaccacc ct caacaact gtgtact cqg taacacaa.ca 414 O atactago cq aaaatccaac cqattgggat gcaaacactt togctic caa.ca agtagcaagt 42OO
Calacagagcg gct Ctt Cogg cqcatggcga ggttcaa.gca aacaa.cccac tdgggcagac 426 O acctggagta CC9gctggcc caacaattica agcagcacca gtttgttgggc agctic ct caa 432O citcgacaact cagat.ccc.gc ticgtggaacc ccatc tagt c taaattctitt tottcctaac 438 O gacct cittag gtggtgagtic catgitaagtt aaggatgaaa ccaaaataat tccatct tag 4 44 O ttacaagtgt tdatat ct ct citctg.cgcta titt cactata aaagtttitat tdaatgttitt 4500 taatgttitta taat attaaa tittaacaatt g 4531
<210s, SEQ ID NO 2 &211s LENGTH: 1315 212. TYPE: PRT <213> ORGANISM: Diabrotica virgifera <4 OOs, SEQUENCE: 2 Met Arg Ala Pro Thr Pro Ser Glu Pro Llys Ser Thr Phe Pro Thr Tyr 1. 5 1O 15 Glin Val Pro Gln Lys Ser Ala Met Arg Gly Ser Ala Pro Pro Val Glin 2O 25 3O Val Ala Gly Pro Ser Trp Gly Gly Arg Ala Asp Pro Pro Ser Ser Thr 35 4 O 45 Arg Cys Ala Asp Glu Gly Ala Lieu Ser Val Ile Ser Gly Ser Ser Cys SO 55 6 O Arg Ser Ile Asp Asn. Ser Asn. Ile Arg Met Glin Ser Val Thr Glu Asn 65 70 7s 8O Cys Lieu. Lieu. Asn. Ser Val Thr Val Pro Asn Met Glin Arg Lieu. Asp His 85 90 95 Gly Met Val Thr His Asn Asn Ser Phe Llys Lieu Val Ser Llys Phe Gly 1OO 105 11 O Ala Lieu. Lieu Pro Gly Arg Asp Ile Pro Asn Glin Llys Ser Asp Asp Lieu 115 12 O 125 Glu Lieu. Lieu. Arg Asp Asp Lieu. Asn Val Lieu. Asn. Ser Thr Lys Tyr Asp 13 O 135 14 O Thir Lys Thr Lieu. Cys Asp Asn. Asn Asp Glu Lys Asp Asp His Asp Ala 145 150 155 160 Tyr Gln Met Ser Asn Ile Glu Thr His Thr Cys Thr Asn Asn Asp Asn 1.65 17O 17s Ser Tyr Glin Glu Lieu. Tyr Llys Pro Lieu. Arg Lieu. Arg Gly Gly Gly Glu 18O 185 19 O Ser Ser Leu Ser Thr Gly. Thir Ser Gly Trp Gly Thr Pro Pro Ser Glin 195 2OO 2O5
Ser Gly Asn. Asn. Asn Ala Asn Llys Ser Asn Gly Glin Gln Pro Pro Thr 21 O 215 22O
Ser Glin Ser Asn Asn Thr Gly Trp Gly Glin Pro Gly Thr Lys Thr Ala 225 23 O 235 24 O
Asn. Asn. Asn Ala Met Pro Pro Asn. Ser Glin Pro Pro Thir Ser Thir Ala 245 250 255
Asn Ser Glin Asn. Asn. Asn Gly Pro Ser Asn. Asn. Thir Lys Glin Glin Lieu 26 O 265 27 O
Glu Gln Lieu. Asn. Ser Met Arg Glu Ala Ile Phe Ser Glin Asp Gly Trp US 2017/0218391 A1 Aug. 3, 2017 47
- Continued
27s 28O 285 Gly Gly Gln His Val Asn Glin Asp Thr Asn Trp Asp Ile Pro Ser Ser 29 O 295 3 OO Pro Glu Pro Pro Ile Llys Met Asp Gly Ser Gly Gly Pro Pro Pro Trp 3. OS 310 315 32O Llys Pro Ala Val Asn. Asn Gly Thr Glu Lieu. Trp Glu Ala Asn Lieu. Arg 3.25 330 335 Asn Gly Gly Glin Pro Pro Pro Gln Pro Glin Gln Lys Thr Pro Trp Gly 34 O 345 35. O His Thr Pro Ser Thr Asn Ile Gly Gly. Thir Trp Gly Glu Asp Asp Asp 355 360 365 Ala Asp Thir Ser Asn Val Trp Thr Gly Val Pro Ser Asn Gln Pro Glin 37 O 375 38O Trp Gly Gly Ala Gly Gly Asn. Thir Asn. Asn Gly Ala Met Trp Gly Gly 385 390 395 4 OO Pro Llys Lys Glu Asn Asp Trp Gly Thr Gly Ala Ser Asn Thr Gly Gly 4 OS 41O 415 Trp Gly Asp Pro Arg Ala Ala Asp Pro Arg Glin Thr Gly Met Asp Pro 42O 425 43 O Arg Glu Ile Arg Pro Glu Lieu. Arg Asp Met Arg Ala Gly Asn Thr Glu 435 44 O 445 Thr Met Arg Ile Met Asp Pro Arg Glu Thr Met Arg Gln Met Ser Asn 450 45.5 460 Ser Asp Met Arg Gly Asp Pro Arg Gly Ile Thr Gly Arg Lieu. Asn Gly 465 470 47s 48O Ala Gly Ala Glu Ala Phe Trp Gly Glin Gly Thr Pro His Ala Ala Ser 485 490 495 Gln Pro Ile His His His Asn Llys Met Pro Val Pro Pro Gly Asin Gly SOO 505 51O Thr Gly Gly Trp Glu Glu Pro Ser Pro Pro Ser Glin Arg Arg Asn Met 515 52O 525 Pro Asn Tyr Asp Asp Gly. Thir Ser Leu Trp Gly Asn Pro Glin Glin Gly 53 O 535 54 O Ser His Trp Lys Asp Leu Pro Thr Gly Gly Ser Met Gly Arg Gly Gly 5.45 550 555 560 Asn Pro Ala Gly Pro Pro Gly Met Asn Glin Ala Arg Gly Met Lys Glin 565 st O sts Pro Glu Gly Ser Met Trp Gly Gly His Gly Arg Asn Gly Ser Trp Asp 58O 585 59 O Glu Thr Gly Pro Gly Ala Ala Trp Asp Glu Pro Asn. Ser Trp Ala Lys 595 6OO 605 Glin Llys Met Pro Asp Pro Lieu. Trp Asp Glu Ser Glu Trp Gly His Lys 610 615 62O
Gln Glin Ser Llys Pro Glin Lieu. Thir Lys Glu Met Val Trp Asn Ser Lys 625 630 635 64 O Glin Phe Arg Met Lieu Val Asp Met Gly. His Llys Lys Glu Asp Val Glu 645 650 655
Asn Ala Lieu. Arg Lieu. Arg Ala Met Asn Val Glu Glu Ala Lieu. Asp Lieu 660 665 67 O
Lieu. Ser Pro Met Arg Asn. Asn Arg Ala Asn Asp Gly Trp Asn. Thir Arg 675 68O 685 US 2017/0218391 A1 Aug. 3, 2017 48
- Continued
His Asp Asp His Tyr Glu. His Pro Pro Phe Cys Glin Arg Gly Phe Ser 69 O. 695 7 OO Thr Gly Pro Gly Gly Glin Lieu. Thr Gly Phe Gln Pro Gly Asn Asn Ala 7 Os 71O 71s 72O Pro Asn Lieu. Lieu. Asn. Asn Met Ser Asn Pro Gly Thr Asn. Asn. Ser Lieu. 72 73 O 73 Ile Asin Asn. Ile Ala Pro Ala Val Val Glin Llys Lieu. Lieu. Thr Glin Glin 740 74. 7 O Glin Gly Gly Gly Ser Glin Gly Phe Gly Gly Ser Ser Ala Asn Ala Gly 7ss 760 765 Arg Asn Ile Glin Pro Glin Ser Glin Pro Ser Thr Glin Gln Lieu. Arg Met 770 775 78O Lieu Val Glin Glin Ile Glin Met Ala Val Glin Ala Gly Tyr Lieu. Asn His 78s 79 O 79. 8OO
Glin Ile Lieu. Asn. Glin Pro Leu Ala Pro Glin. Thir Lieu Wall Lieu. Lieu. Asn 805 810 815 Glin Lieu. Lieu. Glin Glin Ile Lys Asn Lieu. Glin Glin Lieu. Ile Ser Glin Glin 82O 825 83 O Ser Ile Thr Gly Thr Pro Ile Asin Gly Lys Glin Asn Asn Ala Tyr Met 835 84 O 845 Glin Phe Ser Val Lieu. Ile Thr Lys Thr Lys Glin Ser Ile Ala Asn Lieu. 850 855 860 Glin Asn Glin Ile Ala Ala Glin Glin Ala Thir Tyr Val Lys Glin Glin Glin 865 87O 87s 88O His Glin Ser Ser Met Gly Ala Tyr Asp Ser Phe Lys Thr Asn Pro Met 885 890 895 His Asp Ser Ile Asn Ala Lieu. Glin Thr Asn. Phe Gly Asp Lieu. Gly Ile 9 OO 905 91 O Asn Lys Glu Pro Gln Met Asn Pro Glin Glin Ser Arg Lieu. Thr Glin Trp 915 92 O 925 Ile Ser Lys Asp Lys Asp Asp Gly Gly Glu Phe Ser Arg Ala Pro Gly 93 O 935 94 O Ser Ser Ser Llys Pro Pro Asn Thr Ser Pro Asn Met Asn Pro Leu Val 945 950 955 96.O Lieu. Asn Pro Ser Asp Gly Pro Trp Ser Thr Gly Arg Thr Gly Asp Thr 965 97O 97. Gly Trp Pro Asp Ser Ser Ala Asn Asp Asn. Ser Asn Asp Wall Lys Asp 98O 985 99 O Ala Glin Trp Ser Thr Thr Thr Glin Pro Ser Lieu. Thir Asp Lieu Val Pro 995 1OOO 1005 Glu Phe Glu Pro Gly Llys Pro Trp Llys Gly Asn Glin Ile Lys Ile 1010 1015 1 O2O
Glu Asp Asp Pro Ser Ile Thr Pro Gly Ser Val Val Arg Ser Pro 1025 1O3 O 1035
Lieu. Ser Ile Ala Thir Ile Lys Asp Asn. Glu Lieu. Phe Asn Met Asn 104 O 1045 1 OSO
Pro Ser Lys Ser Pro Pro Ala Thr Asp Gly Ile Glin Ser Leu Ser 105.5 106 O 1065
Lieu. Ser Ser Ser Thr Trp Ser Phe Asin Pro Ser Gly Thr Ser Thr 1070 1075 108 O US 2017/0218391 A1 Aug. 3, 2017 49
- Continued
Ser Ser Ala Phe Thr Ser Pro Pro Gly Lys Lieu Pro Thr Ser Lys O85 O9 O O95 Ala Lieu. Gly Asp Lieu. Asn Pro Ser Thr Ala Val Thir Ser Glu Lieu. OO O5 10 Trp Gly Ala Pro Llys Ser Ser Arg Gly Pro Pro Pro Gly Lieu Ser 15 2O 25 Ala Lys Gly Ser Gly Ala e Ser Asn Gly Trp Ser Ala Val Asn 3O 35 4 O Thr Met Pro Trp Gly Pro Gly Gly Glin Arg Thr Ser Gly Asn Trp 45 SO 55 Gly Gly Ser Ser Glin Trp Lieu. Lieu. Lieu. Arg Asn Lieu. Thir Ala Glin 60 65 70 Ile Asp Gly Ser Thr Lieu. Arg Thr Lieu. Cys Lieu Gln His Gly Pro
Lieu. Lieu. Ser Phe His Lieu. Tyr Lieu. His Glin Gly Phe Ala Lieu Ala 90 95 2OO Tyr Ser Ser Arg Glu Glu Ala Ile Lys Ala Glin Thir Thr Lieu. 2O5 21 O 215 Asn Asn. CyS Val Lieu. Gly Asn. Thir Thir Ile Lieu Ala Glu ASn Pro 22O 225 23 O Thir Asp Trp Asp Ala Asn. Thir Lieu. Lieu. Glin Glin Val Ala Ser Glin 235 24 O 245 Gln Ser Gly Ser Ser Gly Ala Trp Arg Gly Ser Ser Lys Glin Pro 250 255 26 O Thr Gly Ala Asp Thir Trp Ser Thr Gly Trp Pro Asn Asn Ser Ser 265 27 O 27s Ser Thir Ser Lieu. Trp Ala Ala Pro Glin Lieu. Asp Asn. Ser Asp Pro 28O 285 29 O Ala Arg Gly Thr Pro Ser Ser Lieu. Asn. Ser Phe Lieu Pro Asn Asp 295 3OO 305 Lieu. Lieu. Gly Gly Glu Ser Met 310 315
<210s, SEQ ID NO 3 &211s LENGTH: 469 &212s. TYPE: DNA <213> ORGANISM: Diabrotica virgifera
<4 OOs, SEQUENCE: 3 acgcaacaac tacggatgtt ggtgcaacaa atacagatgg cagttcaggc agggitat ct c 6 O aatcaccaga ttcttaatca acctittggcg ccacaaacgt toggttcttct aaatcaactg 12 O ttgcaacaga t caagaattt acago agctic at at cacaac aat caataac togg tacgc.ct 18O atcaacggaa alacagaataa cqcttatatg cagttitt cag tact cat cac aaaaacaaaa 24 O caat caattig cca atttaca gaatcaaatc gctgctcaac aagcdactta cqttaa.gcaa. 3OO caacaacacic aaag.ca.gcat giggtgccitat gact cattta aaacgaatcc catgcatgat 360 tcqataaacg ctittacaaac caattittggit gacittaggca ttaacaaaga gcct caaatg 42O alacc cacaac aat cacgact cacccagtgg ataagtaaag ataaggatg 469
<210s, SEQ ID NO 4 &211s LENGTH: 130 &212s. TYPE: DNA
US 2017/0218391 A1 Aug. 3, 2017 51
- Continued
&211s LENGTH: 50 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Dvv-gw-1 Rev <4 OOs, SEQUENCE: 9 ttaatacgac toactatagg gagacatcct tat ctitt act tat coactgg SO
<210s, SEQ ID NO 10 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Dvv- g1-1 v1. For <4 OOs, SEQUENCE: 10 ttaatacgac toactatagg gagaaaaacg aatcc catgc atgattic 47
<210s, SEQ ID NO 11 &211s LENGTH: 50 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Dvv-gw-1 v1 Rev <4 OOs, SEQUENCE: 11 ttaatacgac toactatagg gagacatcct tat ctitt act tat coactgg SO
<210s, SEQ ID NO 12 &211s LENGTH: 49 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Dvv-gw-1 v2 For <4 OOs, SEQUENCE: 12 ttaatacgac toactatagg gagat caatc accagattct taatcaacc 49
<210s, SEQ ID NO 13 &211s LENGTH: 50 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Dvv-gw-1 v2. Rev <4 OOs, SEQUENCE: 13 ttaatacgac toactatagg gagagittatt gattgttgttgatatgagctg SO
<210s, SEQ ID NO 14 &211s LENGTH: 705 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: YFP gene
<4 OOs, SEQUENCE: 14 atgtcatctg gag cacttct Cttt catggg aagattic ctt acgttgttgga gatggaaggg 6 O aatgttgatg gcc acaccitt tag catacgt gggaaaggct acggagatgc ct cagtggga 12 O alaggttgatg cacagttcat Ctgcacaact ggtgatgttc Ctgtgccttg gag cacactt 18O gtcaccactic ticacct atgg agcacagtgc tittgccaagt atggit coaga gttgaaggac 24 O
US 2017/0218391 A1 Aug. 3, 2017 54
- Continued ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer YFP-F T7
SEQUENCE: 21 ttaatacgac toactatagg gagacaccat gggct coagc ggcgc cc 47
SEQ ID NO 22 LENGTH: 23 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer YFP-R
SEQUENCE: 22 agat Cttgaa ggcgct Cttic agg 23
SEQ ID NO 23 LENGTH: 23 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer YFP-F
SEQUENCE: 23
Caccatgggc tccagcggcg C cc 23
SEQ ID NO 24 LENGTH: 47 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer YFP-RT7
SEQUENCE: 24 ttaatacgac toactatagg gagaagat.ct taaggcgct Ctt Cagg 47
SEO ID NO 25 LENGTH: 46 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer Ann-F1. T7
SEQUENCE: 25 ttaatacgac toactatagg gagagcticca acagtggttc ctitatic 46
SEQ ID NO 26 LENGTH: 29 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer Ann-R1
SEQUENCE: 26 ctaataattic titttittaatgttcct gagg 29
SEO ID NO 27 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer Ann-F1 US 2017/0218391 A1 Aug. 3, 2017 55
- Continued
<4 OOs, SEQUENCE: 27 gctic caacag tdgttcctta t c 22
<210s, SEQ ID NO 28 &211s LENGTH: 53 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Ann-R1. T7
<4 OOs, SEQUENCE: 28 ttaatacgac toactatagg gagactaata attcttttitt aatgttcct g agg 53
<210s, SEQ ID NO 29 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Ann-F2 T7
<4 OOs, SEQUENCE: 29 ttaatacgac toactatagg gagattgtta caa.gctggag aacttctic 48
<210s, SEQ ID NO 3 O &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Primer Ann-R2
<4 OOs, SEQUENCE: 30 cittalaccaac aacggctaat aagg 24
<210s, SEQ ID NO 31 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer Ann-F2
<4 OOs, SEQUENCE: 31 ttgttacaag ctggagaact tctic 24
<210s, SEQ ID NO 32 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer Ann-R2Tf
<4 OOs, SEQUENCE: 32 ttaatacgac toactatagg gagacittaac caacaacggc taataagg 48
<210s, SEQ ID NO 33 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F1. T7
<4 OOs, SEQUENCE: 33 ttaatacgac toactatagg gagaagatgt totgcatc tagagaa 47 US 2017/0218391 A1 Aug. 3, 2017 56
- Continued
<210s, SEQ ID NO 34 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R1 <4 OOs, SEQUENCE: 34 gtcc attcgt. c catccactg. ca. 22
<210s, SEQ ID NO 35 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F1 <4 OOs, SEQUENCE: 35 agatgttggc tigcatctaga gaa 23
<210s, SEQ ID NO 36 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R1. T7 <4 OOs, SEQUENCE: 36 ttaatacgac toactatagg gagagtcc at t cqtc catcc actgca 46
<210s, SEQ ID NO 37 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F2 T7 <4 OO > SEQUENCE: 37 ttaatacgac toactatagg gaga.gcagat galacaccagc gagaaa 46
<210s, SEQ ID NO 38 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R2 <4 OOs, SEQUENCE: 38 ctgggcagct tcttgtttico to 22
<210s, SEQ ID NO 39 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F2
<4 OOs, SEQUENCE: 39 gCagatgaac accago.gaga aa 22
<210s, SEQ ID NO 4 O &211s LENGTH: 46 &212s. TYPE: DNA US 2017/0218391 A1 Aug. 3, 2017 57
- Continued ORGANISM: Artificial Sequence FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R2 T7 SEQUENCE: 4 O ttaatacgac toactatagg gag actgggc agctt cittgt titcctic 46
SEQ ID NO 41 LENGTH: 51 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer L4-F1. T7
SEQUENCE: 41 ttaatacgac toactatagg gagaagtgaa atgttagcaa atata acatc c 51
SEQ ID NO 42 LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer L4 -R1
SEQUENCE: 42 acct ct cact tcaaatcttg actittg 26
SEQ ID NO 43 LENGTH: 27 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer L4 - F1
SEQUENCE: 43 agtgaaatgt tagcaaatat aac atcc 27
SEQ ID NO 44 LENGTH: SO TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer L4-R1. T7
SEQUENCE: 44 ttaatacgac toactatagg gagaacct ct cacttcaaat cittgactittg SO
SEO ID NO 45 LENGTH: SO TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer L4-F2 T7
SEQUENCE: 45 ttaatacgac toactatagg gagacaaagt caagatttga agtgagaggit SO
SEQ ID NO 46 LENGTH: 25 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer L4 -R2
US 2017/0218391 A1 Aug. 3, 2017 59
- Continued
SEO ID NO 5 O LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Oligonucleotide T2 OVN FEATURE: NAMEAKEY: misc feature LOCATION: (22) ... (22) OTHER INFORMATION: n is a, c, g, or t SEQUENCE: 5 O ttitt tttitt t t t t t t t t t t t win 22
SEO ID NO 51 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer P5U76S (F)
SEQUENCE: 51 ttgttgatgtt ggtggcgitat
SEO ID NO 52 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Primer P5U76A (R)
SEQUENCE: 52 tgttaaataa aaccc.caaag atcg 24
SEO ID NO 53 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer TIPmxF
SEQUENCE: 53 tgaggg taat gccaactggt t 21
SEO ID NO 54 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer TIPmxR
SEQUENCE: 54 gcaatgtaac coagtgtc.tc. tcaa 24
SEO ID NO 55 LENGTH: 32 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe HXTIP
SEQUENCE: 55 tttittggctt agagttgatg gtgtactgat ga 32
US 2017/0218391 A1 Aug. 3, 2017 63
- Continued
&211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer IWR1-R
<4 OOs, SEQUENCE: 63 aaagtttgga ggctgc.cgt. 19
<210s, SEQ ID NO 64 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe IVR1-P (HEX)
<4 OOs, SEQUENCE: 64 cgagcagacic gcc.gtgtact tct acc 26
<210s, SEQ ID NO 65 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer SPC1A
<4 OOs, SEQUENCE: 65 cittagotgga taacgc.cac 19
<210s, SEQ ID NO 66 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer SPC1S
<4 OOs, SEQUENCE: 66 gaccgtaagg Cttgatgaa 19
<210s, SEQ ID NO 67 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe TOSPEC (CY5*)
<4 OO > SEQUENCE: 67 cgagattct c cqc.gctgtag a 21
<210s, SEQ ID NO 68 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer ST-LS1-F
<4 OOs, SEQUENCE: 68 gtatgtttct gcttctacct ttgat 25
<210s, SEQ ID NO 69 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
US 2017/0218391 A1 Aug. 3, 2017 66
- Continued ggtcttctgt aaatcgtgaa tictagttggc CtgattcatC cqgtgatgat gct tctggca 384 O aggattggcc aaattic cagt caacct coat citcaa.gcatt citctgat citt gttcc tigagt 3900 ttgaac Cagg aaa.gc.cttgg aagggaalacc Cactaaaaag catcgaggat gatccaa.gc.c 396 O ttacacctgg titcggttgtg aggtotcct c titt citctgcc ttcaataaag gatacacata 4 O2O tatt at caac tag tactggit gctggcaaag citt caccitac taccagttct tctittagata 4 O8O titat cocatc ticttggcttg tdatcatcta cittggagctt taatccacca cct tctt cat 414 O Ctalacactag titgaagctgaattictagtg gagctgctgg aggtgg tagt gggtcaiacat 42OO caaataatgg taggcaaa aatag tactt Caacttggga aactaatticg tctgaattgt 426 O gggctic ccaa aagagggcct cct coaggitt taccagctaa acctagtggt ggttcaagtg 432O gtggacaggc tigcaaatggit toggacctt ttct agtag togcc.gttgg tdaactgggc 438 O alaggttggcc tigaccgaat Caggcggctg. Caact cagoc aggttct act tdgttgttat 4 44 O tgcgaaatct tacticct cag attgacggitt caactittaaa aactic tatgt ttaca acatg 4500 ggcc attatc aaattt coat citctacctta accatgg cat cqct cittgct aaatatgcat 456 O Ctcgggaaga agccaataag gcc calaggtg Ctttaaacaa ttgttgttctt ggtaiacact a 462O caat atttgc tigagagtic cc agtgagaccg atgtgttgtc attacttaat catcttggtg 468O gacaaggagg gaccgc.cagt ggcagct cag gatggcgtgg taaggaagct tdggcaatt 474. O cc.ca.gctttggggagccaat ggagcaa.gct cagctgctgc titctttgttgg gCaggagata 48OO gtgat cagca togtaacact c catcct caa taaattctta tittgc.caggit gacct tcttg 486 O gtggtgagtic tatttaggca aat citt catt cittct ct caa acctt cacca aattcttctic 492 O gatctataaa tacgtcaatc aaaac tattgaacaaaaaaa atacaaaaaa accaaaaaaa 498O aacaagtact ttgat citcag aaacaccaca tacctttitt attataaata tatatgatat 5040 gaagtatatgcaattaatta tttgtaccag gaacgtatat cittatt atta 509 O
<210s, SEQ ID NO 72 &211s LENGTH: 1453 212. TYPE: PRT <213> ORGANISM; Euschistus heros
<4 OOs, SEQUENCE: 72 Met Phe Arg Asn Asn Ser Ser Ser Asn Glu Ile Ser Ser Lys Thr Asn 1. 5 1O 15 Ala Phe Val Glin Asn Lys Asp Glu Glu Asp Llys Ser Glu Ser Lieu. Lieu 2O 25 3O Arg Gly Met Ala Gln Pro Pro Llys Pro Thr Ser Pro Thr His Glin Val 35 4 O 45 Pro Glu Lys Arg Asp Val Met Val Val Asp Ile Gly Val Arg Glu Asp SO 55 6 O Asp Gly Pro Val Lieu. Thr Val Ile Thr Asn His Pro Ser Glin Ala Pro 65 70 7s 8O
Ala Lys Ile Phe Ser Ser Glu Ile Gly Glu Ser Glu Ser Asp Gly Ser 85 90 95
Ser Lys Met Pro Lieu Ala Glu Thr Glin Gly Thr Gly Ala Lieu. Cys Lieu. 1OO 105 11 O
Asp Ser Ile Llys Ser Ile Ser Val Asn. Glu Ser Phe Ser Val Lys Asp 115 12 O 125 US 2017/0218391 A1 Aug. 3, 2017 67
- Continued
Llys Phe Ile Cys Pro Ser Lys Ser Lieu. Ile Leu Pro Pro Asn Ile Pro 13 O 135 14 O Asn. Thir Asn Asp Asp Thr Asp Glin Asp Wall Lys Ser Phe Lys Ile Cys 145 150 155 160 Asp Tyr Tyr Thr Arg Trp Gly Ile Pro Arg Asn Lieu Lys Lieu. Lieu. Gly 1.65 17O 17s Gly Gly Glu Ser Ser Lieu. Thir Thr Gly Thr Thr Gly Trp Gly Ser Pro 18O 185 19 O Pro Ser Asn Glin Gly Gly Ser Thr Gly Trp Asn Ser Ala Asn. Thir Thr 195 2OO 2O5 Ser Gly Ser Asn Ser Ser Ser Gly Glin Gly Glin Ala Gly Thr Gly Glin 21 O 215 22O Ser Pro Ala Pro Ala Ser Ala Gly Glin Thr Trp Gly Ser Ser Glin Asn 225 23 O 235 24 O Asn. Thir Asn. Asn. Ser Asn. Ser Asn. Ser Asn. Asn. Asn. Asn Gly Ser Arg 245 250 255 Ser Ser Val Ser Glin Glin Gly Gly Gly Ser Thr Glin Glin Glin Pro Gly 26 O 265 27 O Gly Gly Pro Pro Ser Glin Ser Thr Ala Pro Pro Val Ala Thr Val Ser 27s 28O 285
Thir Ser Thir Wall. Thir Thir Ala Pro Ala Ser Ser Ala Thir Asn. Thir Ser 29 O 295 3 OO ASn Ile ASn Thr Ala Thr Thr Ser Ala Ser Glin Glin ASn Gly Ser Ala 3. OS 310 315 32O Ser Gly Asn Glin Val Val Gly Ser Gly Ser Thr Trp Ala Thr Ala Val 3.25 330 335 Gly Lys Gly Lieu Pro Pro Thir Ser Thr Val Ser Thr Pro Thr Ser Ser 34 O 345 35. O Gly Ser Thr Ser Thr Lys Glin Gln Met Glu Gln Lieu. Asn Thr Met Arg 355 360 365 Glu Ala Lieu. Tyr Ser Glin Asp Gly Trp Gly Gly Glin Asn. Wall Asn Glin 37 O 375 38O Asp Ser Asn Trp Asp Ile Pro Gly Ser Pro Glu Pro Gly. Thir Lys Asp 385 390 395 4 OO Ser Asn. Asn Ala Ala Pro Val Pro Lieu. Trp Llys Lieu Pro Ile Asn. Asn 4 OS 41O 415 Gly. Thir Asp Lieu. Trp Glu Ala Asn Lieu. Arg Asn Gly Gly Val Pro Pro 42O 425 43 O Pro Val Ser Glin Glin Ser Gln Lys Thr Pro Trp Val His Thr Pro Ser 435 44 O 445 Thir Asn. Ile Gly Gly. Thir Trp Gly Glu Asp Asp Glu Gly Asp Ala Ser 450 45.5 460
Asn Val Trp Thr Gly Val Pro Glin Ala Glin Thr Gly Cys Gly Pro Glin 465 470 47s 48O
Trp Pro Ala Glin Pro Pro Pro Ile Trp Pro Ala Thr Lys Lys Glu Gly 485 490 495
Asp Trp Gly Gly Pro Asn Trp Asn Asp Glin Arg Asp Thr Arg Asp Lieu SOO 505 51O Arg His Ser Asp Met Arg Glin Met Met Asp Ala Arg Asp His Met Arg 515 52O 525 Pro Thir Ser Ile Asp His Arg Ser Met Gly Gly Asn Asp Val Ile Met US 2017/0218391 A1 Aug. 3, 2017 68
- Continued
53 O 535 54 O Arg Gly Asp Pro Arg Gly Ile Ser Gly Arg Lieu. Asn Gly Val Thir Ser 5.45 550 555 560 Glu Ala Met Trp Pro Gly Pro Gly Pro His His His Ile Pro His His 565 st O sts Gln Gly Lys Lieu Pro Ser Gln Pro Asn Glin Pro Val Asn Glin Trp Ser 58O 585 59 O Ser Ser Gly Pro Pro Met Lys Asp Met Thr Gly Lieu. Gly Gly Lys Ser 595 6OO 605 Thr Gly Trp Glu Glu Pro Ser Pro Pro Ala Glin Arg Arg Asn Met Pro 610 615 62O Asn Tyr Asp Asp Gly Thr Ser Leu Trp Gly Pro Gln His Pro Arg Pro 625 630 635 64 O Thir Ile Glin Gly Glin Asn Llys Val Ser His Trp Lys Glu Met Pro Ala 645 650 655 Pro Gly Ile Gly Arg Gly Gly Lieu. Glin Cys Pro Pro Gly Arg Ala Asn 660 665 67 O Pro Thr Met Llys Pro Asp Gln Pro Leu Trp Pro His His Pro Arg Asn 675 68O 685 Glu Arg Gly Trp Glu Gly Gly Met Asp Ser Gly Pro Trp Gly Asp Glu 69 O. 695 7 OO Llys Pro Thr Pro Ala Ala Ala Pro Trp Met Asp Glin Gly Lieu Ala Pro 705 710 715 72O Ser Ser Trp Glin Gly Gly Pro Llys His Llys Pro Ala Trp Asp Gly Ser 72 73 O 73 Asp Lieu. Asp Pro Thr Ser Trp Val His Ser Lys Gln Pro Ser Lys Ser 740 74. 7 O Val Ser Lys Glu Phe Ile Trp Thr Ser Lys Glin Phe Arg Ile Leu Ser 7ss 760 765 Glu Met Gly Phe Llys Lys Glu Asp Ile Glu Ser Ala Lieu. Arg Ser Ser 770 775 78O Gly Met Ser Lieu. Glu Asp Ala Lieu. Asp Gln Lieu. Asn. Thir Asn Arg Gly 78s 79 O 79. 8OO Lieu. Ser Ala Gly Gly Gly Ser Glu Arg Trp Pro Arg His Gly Asp Lieu. 805 810 815 Asp Ser Glu. His Ala Ala Ile Met Asn Thr Phe Pro Ser Pro Glin Glin 82O 825 83 O Thir Ile Cys Lieu Ala Pro Phe Pro Glin Gly Gly Gly Gly Gly Gly Ser 835 84 O 845 Gly Ser Gly Pro Gly Gly Gly Pro Thr Lieu Ala Thr Ile Thr Pro Ala 850 855 860
Val Met Gln Lys Lieu. Leu Ala Glin Gln Pro Pro Glin Glin Gln Pro Phe 865 87O 87s 88O
Ala Glin Glin Ser Ser Arg Thr Glin Gln Thr Glin Gln Pro Ser Ala Glin 885 890 895
Gln Leu Arg Met Leu Val Glin Glin Ile Glin Met Ala Val Glin Thr Gly 9 OO 905 91 O
Tyr Lieu Ser Pro Glin Ile Lieu. Asn Gln Pro Leu Ala Pro Glin Thr Lieu. 915 92 O 925
Ile Lieu. Lieu. Asn Gln Lieu. Lieu. Glin Glin Ile Lys Asn Lieu. Glin Glin Lieu. 93 O 935 94 O US 2017/0218391 A1 Aug. 3, 2017 69
- Continued
Met Glin His His Thir Wall Met Glin Val Asn Pro Lieu. Gly Llys Pro Ser 945 950 955 96.O
Ser Asn His Luell Lieu. Glin Lieu. Ser Val Glin Ile Thr Lys Thr Lys Glin 965 97O 97.
Glin Ile Thir Asn Lieu. Glin Asn Glin Ile Ala Ala Glin Glin Ala Wall Tyr 9 85 99 O
Wall His Glin Gln His Thir Pro Pro Thir Ser G ul Phe Phe Llys Ser 995 1OOO 1005
Ser Luell His Glu Pro Ile Ser Ala Luell His Pro Phe Ser Asp O1O O15
Lell Ser Lell Asp Pro Pro Thir Ser Gly Thir Glin Glin Ser O25 O3 O
Arg Luell Asn Glin Trp Luell Pro Ala Luell Glu Asp Ser Asp O4 O O45
Ile Thir Gly Glu Phe Ser Arg Ala Pro Gly Thir Ala Lys O6 O
Ser Glin Gly Ser Ser Ser Pro Asn Thir Asn Luell Lell Gly O7
Glin Asp Gly Thir Trp Ser Ser Wall ASn Arg Ser Ser Trp O9 O
Pro Ser Ser Gly Asp Asp Ala Ser Gly Trp Pro Asn O5
Ser Glin Pro Pro Ser Ala Phe Ser Asp Wall Pro Glu
Phe Pro Gly Pro Gly ASn Pro Ser Ile
Glu Asp Pro Ser Luell Pro Gly Ser Wall Arg Ser Pro
Lell Lell Pro Ser Ile Asp Thir His Ile Ser Thir Ser
Thir Ala Gly Ala Pro Thir Thir Ser Ser Lell Asp
Ile Pro Ser Luell Gly Ser Ser Ser Thir Ser Phe Asn 2OO
Pro Pro Ser Ser Ser Asn Thir Ser Wall Lell ASn Ser Ser 21 O 215
Gly Ala Gly Gly Gly Ser Gly Ser Thir Ser Asn ASn Gly Gly 225 23 O
Gly Asn Ser Thir Ser Trp Glu Thir Asn Ser Glu Lell 24 O
Trp Pro Arg Gly Pro Pro Pro Gly Lell Ala Pro 255
Ser Gly Ser Ser Gly Glin Ala Ala Asn Trp Gly Pro 27 O
Lell Ser Ser Ser Gly Arg Ser Thir Gly Glin Trp Pro Gly 28O 285
Pro Asn Glin Ala Ala Ala Glin Pro Gly Ser Trp Lell Lell 295 305
Lell Asn Lell Thir Pro Ile Asp Gly Ser Luell Thir 310 315 US 2017/0218391 A1 Aug. 3, 2017 70
- Continued Lieu. Cys Lieu. Glin His Gly Pro Lieu. Ser Asn. Phe His Lieu. Tyr Lieu. 3.25 33 O 335 Asn His Gly Ile Ala Lieu Ala Lys Tyr Ala Ser Arg Glu Glu Ala 34 O 345 350 Asn Lys Ala Glin Gly Ala Lieu. Asn. Asn. Cys Val Lieu. Gly Asn Thr 355 360 365 Thir Ile Phe Ala Glu Ser Pro Ser Glu Thir Asp Val Lieu Ser Lieu. 37O 375 38O Lieu. Asn His Lieu. Gly Gly Glin Gly Gly Thr Ala Ser Gly Ser Ser 385 390 395 Gly Trp Arg Gly Lys Glu Ala Trp Gly Asn. Ser Glin Lieu. Trp Gly 4 OO 405 41 O Ala Asn Gly Ala Ser Ser Ala Ala Ala Ser Lieu. Trp Ala Gly Asp 415 42O 425 Ser Asp Gln His Arg Asn Thr Pro Ser Ser Ile Asn Ser Tyr Lieu. 43 O 435 44 O Pro Gly Asp Lieu. Lieu. Gly Gly Glu Ser Ile
<210s, SEQ ID NO 73 &211s LENGTH: 493 &212s. TYPE: DNA <213> ORGANISM; Euschistus heros
< 4 OO SEQUENCE: 73 gtatgttaag catcaa.ca.gc atacaccacc tacttctgag tttittcaaga gttcattaca 6 O tgaaccaatt totgcactitc atcctaattt ttctgat citt tot cittaaag atc.ccc.cgac 12 O Cagtggaact agc.ca.gcaat cacgattaaa t cagtggaag ttacctg.ccc tdgaaaaaga 18O
Ctcagatatt gggacaggtgaattittctag agctic Caggit acaac agcta agt cagctica 24 O aggct cittct t cacctaata caaatttatt acttgggcag gctgatggta cittggtc.ttic 3OO tgtaaatcgt gaatct agtt ggcctgattic atc.cggtgat gatgcttctg gcaaggattg 360 gccaaatticc agt caacctic catct caa.gc attct ctdat cittgttcct g agtttgaacc 42O aggaaagcct tdaagggaa accCactaaa aag catcgag gatgatccala gcctt acacc 48O tggttcggitt gtg 493
<210s, SEQ ID NO 74 &211s LENGTH: 50 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer BSB gw-1 For <4 OOs, SEQUENCE: 74 ttaatacgac toactatagg gagag tatgt taa.gcatcaa cagcatacac SO
<210s, SEQ ID NO 75 &211s LENGTH: 45 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer BSB gw-1 Rev
<4 OO > SEQUENCE: 75 ttaatacgac toactatagg gagacacaac Caac Caggit gtaag 45
US 2017/0218391 A1 Aug. 3, 2017 77
- Continued aggaaagccu. luggaagggaa acc calculaala aag caucgag gaugaluccala gccuulacacc 48O lugglulu.cggull glug 493
What may be claimed is: v. Zeae, D. balteata LeConte; D. u. tenella, D. speciosa, D. 1. An isolated nucleic acid comprising at least one poly u. undecimpunctata Mannerheim; Euschistus heros (Fabr.) nucleotide operably linked to a heterologous promoter, (Neotropical Brown Stink Bug), Nezara viridula (L.) wherein the polynucleotide is selected from the group con (Southern Green Stink Bug), Piezodorus guildinii (West sisting of: wood) (Red-banded Stink Bug), Halyomorpha halys (Stal) SEQID NO:1; the complement or reverse complement of (Brown Marmorated Stink Bug), Chinavia hilare (Say) SEQ ID NO:1; a fragment of at least 15 contiguous (Green Stink Bug), Euschistus servus (Say) (Brown Stink nucleotides of SEQ ID NO:1; the complement or Bug), Dichelops melacanthus (Dallas), Dichelops furcatus reverse complement of a fragment of at least 15 con (F.), Edessa meditabunda (F.), Thyanta perditor (F) (Neo tiguous nucleotides of SEQ ID NO:1; a native coding tropical Red Shouldered Stink Bug), Chinavia marginatum sequence of a Diabrotica organism comprising SEQID (Palisot de Beauvois), Horcias nobilellus (Berg) (Cotton NOs:3-5; the complement or reverse complement of a Bug), Taedia Stigmosa (Berg), Dysdercus peruvianus native coding sequence of a Diabrotica organism com (Guérin-Méneville), Neomegalotomus parvus (Westwood), prising SEQ ID NOS:3-5; a fragment of at least 15 Leptoglossus Zonatus (Dallas), Niesthreasidae (F.), Lygus contiguous nucleotides of a native coding sequence of hesperus (Knight) (Western Tarnished Plant Bug), and Lygus a Diabrotica organism comprising SEQ ID NOS:3-5; lineolaris (Palisot de Beauvois). the complement or reverse complement of a fragment 5. A plant transformation vector comprising the poly of at least 15 contiguous nucleotides of a native coding nucleotide of claim 1. sequence of a Diabrotica organism comprising SEQID 6. A ribonucleic acid (RNA) molecule transcribed from NOs:3-5; the polynucleotide of claim 1. SEQ ID NO:71; the complement or reverse complement 7. A double-stranded ribonucleic acid molecule produced of SEQID NO:71; a fragment of at least 15 contiguous from the expression of the polynucleotide of claim 1. nucleotides of SEQ ID NO:71; the complement or 8. The double-stranded ribonucleic acid molecule of reverse complement of a fragment of at least 15 con claim 7, wherein contacting the polynucleotide sequence tiguous nucleotides of SEQID NO:71; a native coding with a coleopteran or hemipteran insect inhibits the expres sequence of a Euschistus organism comprising SEQID sion of an endogenous nucleotide sequence specifically NO:73; the complement or reverse complement of a complementary to the polynucleotide. native coding sequence of a Euschistus organism com 9. The double-stranded ribonucleic acid molecule of prising SEQ ID NO:73; a fragment of at least 15 claim 8, wherein contacting said ribonucleotide molecule contiguous nucleotides of a native coding sequence of with a coleopteran or hemipteran insect kills or inhibits the a Euschistus organism comprising SEQID NO:73; and growth, viability, and/or feeding of the insect. the complement or reverse complement of a fragment 10. The double stranded RNA of claim 7, comprising a of at least 15 contiguous nucleotides of a native coding first, a second and a third RNA segment, wherein the first sequence of a Euschistus organism comprising SEQID RNA segment comprises the polynucleotide, wherein the NO:73. third RNA segment is linked to the first RNA segment by the 2. The polynucleotide of claim 1, wherein the polynucle second polynucleotide sequence, and wherein the third RNA otide is selected from the group consisting of SEQID NO:1; segment is substantially the reverse complement of the first the complement of SEQ ID NO:1; a fragment of at least 15 RNA segment, such that the first and the third RNA seg contiguous nucleotides of SEQID NO:1; the complement of ments hybridize when transcribed into a ribonucleic acid to a fragment of at least 15 contiguous nucleotides of SEQ ID form the double-stranded RNA. NO:1; a native coding sequence of a Diabrotica organism 11. The RNA of claim 6, selected from the group con comprising any of SEQ ID NOS:3-5; the complement of a sisting of a double-stranded ribonucleic acid molecule and a native coding sequence of a Diabrotica organism compris single-stranded ribonucleic acid molecule of between about ing any of SEQ ID NOS:3-5; a fragment of at least 15 15 and about 30 nucleotides in length. contiguous nucleotides of a native coding sequence of a 12. A plant transformation vector comprising the poly Diabrotica organism comprising any of SEQ ID NOS:3-5; nucleotide of claim 1, wherein the heterologous promoter is and the complement of a fragment of at least 15 contiguous functional in a plant cell. nucleotides of a native coding sequence of a Diabrotica 13. A cell transformed with the polynucleotide of claim 1. organism comprising any of SEQ ID NOS:3-5. 14. The cell of claim 13, wherein the cell is a prokaryotic 3. The polynucleotide of claim 1, wherein the polynucle cell. otide is selected from the group consisting of SEQID NO:1, 15. The cell of claim 13, wherein the cell is a eukaryotic SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID cell. NO:71, SEQ ID NO:73, and the complement or reverse 16. The cell of claim 15, wherein the cell is a plant cell. complement of any of the foregoing. 17. A plant transformed with the polynucleotide of claim 4. The polynucleotide of claim 3, wherein the organism is 1. selected from the group consisting of D. v. virgifera 18. A seed of the plant of claim 17, wherein the seed LeConte; D. barberi Smith and Lawrence; D. u. howardi. D. comprises the polynucleotide. US 2017/0218391 A1 Aug. 3, 2017
19. A commodity product produced from the plant of providing an agent comprising a first and a second poly claim 17, wherein the commodity product comprises a nucleotide sequence that functions upon contact with detectable amount of the polynucleotide, the commodity the coleopteran pest to inhibit a biological function product being preferably a food or oil. within the coleopteran pest, wherein the first polynucle 20. The plant of claim 17, wherein the at least one otide sequence comprises a region that exhibits from polynucleotide is expressed in the plantas a double-stranded about 90% to about 100% sequence identity to from ribonucleic acid molecule. about 15 to about 30 contiguous nucleotides of any of SEQ ID NOS:79-82, and wherein the first polynucle 21. The cell of claim 16, wherein the cell is a corn, otide sequence is specifically hybridized to the second Soybean, or cotton cell. polynucleotide sequence. 22. The plant of claim 17, wherein the plant is corn, 30. A method for controlling a hemipteran pest popula Soybean, or cotton. tion, the method comprising: 23. The plant of claim 17, wherein the at least one providing an agent comprising a first and a second poly polynucleotide is expressed in the plant as a ribonucleic acid nucleotide sequence that functions upon contact with molecule, and the ribonucleic acid molecule inhibits the the hemipteran pest to inhibit a biological function expression of an endogenous polynucleotide that is specifi within the hemipteran pest, wherein the first polynucle cally complementary to the at least one polynucleotide when otide sequence comprises a region that exhibits from a coleopteran or hemipteran insectingests a part of the plant. about 90% to about 100% sequence identity to from 24. The polynucleotide of claim 1, further comprising at about 15 to about 30 contiguous nucleotides of either of least one additional polynucleotide that encodes an RNA SEQ ID NO:83 and SEQ ID NO:84, and wherein the molecule that inhibits the expression of an endogenous first polynucleotide sequence is specifically hybridized insect gene. to the second polynucleotide sequence. 25. A plant transformation vector comprising the poly 31. A method for controlling a coleopteran pest popula nucleotide of claim 24, wherein the additional polynucle tion, the method comprising: otide(s) are each operably linked to a heterologous promoter providing in a host plant of a coleopteran pest a trans functional in a plant cell. formed plant cell comprising the polynucleotide of 26. A method for controlling a coleopteran or hemipteran claim 2, wherein the polynucleotide is expressed to pest population, the method comprising providing an agent produce a ribonucleic acid molecule that functions comprising a ribonucleic acid (RNA) molecule that func upon contact with a coleopteran pest belonging to the tions upon contact with the pest to inhibit a biological population to inhibit the expression of a target sequence function within the pest, wherein the RNA is specifically within the coleopteran pest and results in decreased hybridizable with a polynucleotide selected from the group growth and/or Survival of the coleopteran pest or pest consisting of SEQ ID NO:79; the complement or reverse population, relative to reproduction of the same pest complement of SEQID NO:79; SEQID NO:83; the comple species on a plant of the same host plant species that ment or reverse complement of SEQID NO:83; a fragment does not comprise the polynucleotide. of at least 15 contiguous nucleotides of either of SEQ ID 32. The method according to claim 31, wherein the NOs:79 and 83; the complement or reverse complement of ribonucleic acid molecule is a double-stranded ribonucleic a fragment of at least 15 contiguous nucleotides of either of acid molecule. SEQ ID NOS:79 and 83; a transcript of either of SEQ ID 33. The method according to claim 31, wherein the NOs: 1 and 71; the complement or reverse complement of a coleopteran pest population is reduced relative to a popula transcript of either of SEQID NOs: 1 and 71; a fragment of tion of the same pest species infesting a host plant of the at least 15 contiguous nucleotides of a transcript of either of same host plant species lacking the transformed plant cell. SEQ ID NOs: 1 and 71; and the complement or reverse 34. The method according to claim 32, wherein the complement of a fragment of at least 15 contiguous nucleo coleopteran pest population is reduced relative to a coleop tides of a transcript of either of SEQ ID NOS:1 and 71. teran pest population infesting a host plant of the same 27. The method according to claim 26, wherein the RNA species lacking the transformed plant cell. of the agent is specifically hybridizable with a polynucle 35. A method of controlling insect pest infestation in a otide selected from the group consisting of any of SEQ ID plant, the method comprising providing in the diet of a NOs:80-82 and 84; the complement or reverse complement coleopteran pest a ribonucleic acid (RNA) that is specifically of any of SEQ ID NOS:80-82 and 84; a fragment of at least hybridizable with a polynucleotide selected from the group 15 contiguous nucleotides of any of SEQID NOS:80-82 and consisting of: 84; the complement or reverse complement of a fragment of SEQ ID NO:79 and SEQ ID NO:83; at least 15 contiguous nucleotides of any of SEQ ID NOs: the complement or reverse complement of either of SEQ 80-82 and 84; a transcript of any of SEQID NOS:3-5 and 73: ID NOs: 79 and 83; the complement or reverse complement of a transcript of any a fragment of at least 15 contiguous nucleotides of either of SEQ ID NOS:3-5 and 73; a fragment of at least 15 of SEQ ID NOs:79 and 83; contiguous nucleotides of a transcript of any of SEQ ID the complement or reverse complement of a fragment of NOs:3-5 and 73; and the complement or reverse comple at least 15 contiguous nucleotides of either of SEQ ID ment of a fragment of at least 15 contiguous nucleotides of NOs:79 and 83; a transcript of any of SEQ ID NOS:3-5 and 73. a transcript of either of SEQ ID NOS:1 and 71; 28. The method according to claim 26, wherein the agent the complement or reverse complement of a transcript of is a double-stranded RNA molecule. either of SEQ ID NOS:1 and 71; 29. A method for controlling a coleopteran pest popula a fragment of at least 15 contiguous nucleotides of a tion, the method comprising: transcript of either of SEQ ID NOS:1 and 71; and US 2017/0218391 A1 Aug. 3, 2017 79
the complement or reverse complement of a fragment of screening the transformed plant cells for expression of a at least 15 contiguous nucleotides of a transcript of ribonucleic acid (RNA) molecule encoded by the at either of SEQ ID NOS:1 and 71. least one polynucleotide; and 36. The method according to claim 35, wherein the diet selecting a plant cell that expresses the RNA. comprises a plant cell transformed to express the polynucle 46. The method according to claim 45, wherein the vector otide or an RNAi bait comprising the RNA. comprises a polynucleotide selected from the group consist 37. The method according to claim 35, wherein the ing of: SEQ ID NO:1; the complement or reverse comple specifically hybridizable RNA is comprised in a double ment of SEQID NO:1; a fragment of at least 15 contiguous stranded RNA molecule. nucleotides of SEQ ID NO:1; the complement or reverse 38. A method of controlling insect pest infestation in a complement of a fragment of at least 15 contiguous nucleo plant, the method comprising contacting an insect pest with tides of SEQ ID NO:1; a native coding sequence of a a ribonucleic acid (RNA) that is specifically hybridizable Diabrotica organism comprising any of SEQ ID NOS:3-5; with a polynucleotide selected from the group consisting of the complement or reverse complement of a native coding SEQ ID NO:79 and SEQ ID NO:83; sequence of a Diabrotica organism comprising any of SEQ the complement or reverse complement of either of SEQ ID NOS:3-5; a fragment of at least 15 contiguous nucleotides ID NOs: 79 and 83; of a native coding sequence of a Diabrotica organism a fragment of at least 15 contiguous nucleotides of either comprising any of SEQID NOS:3-5; and the complement or of SEQ ID NOs:79 and 83; reverse complement of a fragment of at least 15 contiguous the complement or reverse complement of a fragment of nucleotides of a native coding sequence of a Diabrotica at least 15 contiguous nucleotides of either of SEQ ID organism comprising any of SEQ ID NOS:3-5. NOs:79 and 83; 47. The method according to claim 45, wherein the RNA a transcript of either of SEQ ID NOS:1 and 71; molecule is a double-stranded RNA molecule. the complement or reverse complement of a transcript of 48. A method for producing transgenic plant protected either of SEQ ID NOS:1 and 71; against a coleopteran pest, the method comprising: a fragment of at least 15 contiguous nucleotides of a providing the transgenic plant cell produced by the transcript of either of SEQ ID NOS:1 and 71; and the complement or reverse complement of a fragment of method of claim 46; and at least 15 contiguous nucleotides of a transcript of regenerating a transgenic plant from the transgenic plant either of SEQ ID NOS:1 and 71. cell, wherein expression of the ribonucleic acid mol 39. The method according to claim 38, wherein contacting ecule encoded by the at least one polynucleotide is the insect pest with the RNA comprises spraying the plant Sufficient to modulate the expression of a target gene in with a composition comprising the RNA. a coleopteran pest that contacts the transformed plant. 40. The method according to claim 38, wherein the 49. A method for producing a transgenic plant cell, the specifically hybridizable RNA is comprised in a double method comprising: stranded RNA molecule. transforming a plant cell with a vector comprising a gw 41. A method for improving the yield of a plant crop, the means for providing coleopteran pest protection to a method comprising: plant; introducing the nucleic acid of claim 1 into a plant to culturing the transformed plant cell under conditions produce a transgenic plant; and sufficient to allow for development of a plant cell cultivating the plant to allow the expression of the at least culture comprising a plurality of transformed plant one polynucleotide; wherein expression of the at least cells; one polynucleotide inhibits insect pest reproduction or selecting for transformed plant cells that have integrated growth and loss of yield due to insect pest infection. the gw means for providing coleopteran pest protection 42. The method according to claim 41, wherein expres to a plant into their genomes; sion of the at least one polynucleotide produces an RNA screening the transformed plant cells for expression of a molecule that Suppresses at least a first target gene in an gw means for inhibiting expression of an essential gene insect pest that has contacted a portion of the corn plant. in a coleopteran pest; and 43. The method according to claim 41, wherein the selecting a plant cell that expresses the gw means for polynucleotide is selected from the group consisting of SEQ inhibiting expression of an essential gene in a coleop ID NO:1, SEQID NO:3, SEQID NO:4, SEQID NO:5, SEQ teran pest. ID NO:71, SEQ ID NO:73, and the complement or reverse 50. A method for producing a transgenic plant protected complement of any of the foregoing. against a coleopteran pest, the method comprising: 44. The method according to claim 41, wherein the plant providing the transgenic plant cell produced by the is a corn, soybean, or cotton plant. method of claim 49; and 45. A method for producing a transgenic plant cell, the regenerating a transgenic plant from the transgenic plant method comprising: cell, wherein expression of the gw means for inhibiting transforming a plant cell with a vector comprising the expression of an essential gene in a coleopteran pest is nucleic acid of claim 1: Sufficient to modulate the expression of a target gene in culturing the transformed plant cell under conditions a coleopteran pest that contacts the transformed plant. sufficient to allow for development of a plant cell 51. A method for producing a transgenic plant cell, the culture comprising a plurality of transformed plant method comprising: cells; transforming a plant cell with a vector comprising a gw Selecting for transformed plant cells that have integrated means for providing hemipteran pest protection to a the at least one polynucleotide into their genomes; plant; US 2017/0218391 A1 Aug. 3, 2017
culturing the transformed plant cell under conditions 55. The cell of claim 16, wherein the cell comprises a sufficient to allow for development of a plant cell polynucleotide encoding a polypeptide from Bacillus culture comprising a plurality of transformed plant thuringiensis, a PIP-1 polypeptide, or an AflP-1A polypep cells; tide. selecting for transformed plant cells that have integrated the gw means for providing hemipteran pest protection 56. The cell of claim 55, wherein the polynucleotide to a plant into their genomes: encodes a polypeptide from B. thuringiensis that is selected screening the transformed plant cells for expression of a from a group comprising Cry 1B, Cry1 I, Cry2A, Cry3, gW means for inhibiting expression of an essential gene Cry7A, Cry8, Cry9D, Cry 14, Cry 18, Cry22, Cry23, Cry34, in a hemipteran pest; and Cry35, Cry36, Cry37, Cry43, Crys5, Cyt1A, and Cyt2C. Selecting a plant cell that expresses the gw means for 57. The plant of claim 17, wherein the plant comprises a inhibiting expression of an essential gene in a polynucleotide encoding a polypeptide from Bacillus hemipteran pest. thuringiensis, a PIP-1 polypeptide, or an AflP-1A polypep 52. A method for producing a transgenic plant protected tide. against a hemipteran pest, the method comprising: providing the transgenic plant cell produced by the 58. The plant of claim 57, wherein the polynucleotide method of claim 51; and encodes a polypeptide from B. thuringiensis that is selected regenerating a transgenic plant from the transgenic plant from a group comprising Cry 1B, Cry1 I, Cry2A, Cry3, cell, wherein expression of the gw means for inhibiting Cry7A, Cry8, Cry9D, Cry 14, Cry 18, Cry22, Cry23, Cry34, expression of an essential gene in a hemipteran pest is Cry35, Cry36, Cry37, Cry43, Crys5, Cyt1A, and Cyt2C. Sufficient to modulate the expression of a target gene in 59. The method according to claim 45, wherein the a hemipteran pest that contacts the transformed plant. transformed plant cell comprises a polynucleotide encoding 53. The nucleic acid of claim 1, further comprising a a polypeptide from Bacillus thuringiensis, a PIP-1 polypep polynucleotide encoding a polypeptide from Bacillus tide, or an AflP-1A polypeptide. thuringiensis, a PIP-1 polypeptide, or an AflP-1A polypep tide. 60. The method according to claim 59, wherein the 54. The nucleic acid of claim 53, wherein the polynucle polynucleotide encodes a polypeptide from B. thuringiensis otide encodes a polypeptide from B. thuringiensis that is that is selected from a group comprising Cry 1B, Cry 1I, selected from a group comprising Cry 1B, Cry1 I, Cry2A, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry 14, Cry 18, Cry22, Cry3, Cry7A, Cry8, Cry9D, Cry 14, Cry 18, Cry22, Cry23, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, CryS5, Cyt1A, Cry34, Cry35, Cry36, Cry37, Cry43, Crys5, Cyt1A, and and Cyt2C. Cyt2C.