US 2016O194658A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0194658 A1 Narva et al. (43) Pub. Date: Jul. 7, 2016 (54) NUCAMPHOLIN NUCLECACID Publication Classification MOLECULES TO CONTROL COLEOPTERAN INSECT PESTS (51) Int. Cl. (71) Applicants: Dow AgroSciences LLC, Indianapolis, CI2N 5/82 (2006.01) IN (US); Fraunhofer-Gesellschaft zur AOIN57/6 (2006.01) Forderung der angewandten Forschung E. V., Munchen (DE) CI2N IS/II3 (2006.01) (52) U.S. Cl. (72) Inventors: Kenneth E. Narva, Zionsville, IN (US); CPC .......... CI2N 15/8286 (2013.01); C12N 15/I 13 Sarah Worden, Indianapolis, IN (US); Meghan Frey, Greenwood, IN (US); (2013.01); A0IN 57/16 (2013.01); C12N Chaoxian Geng, Zionsville, IN (US); 23 10/14 (2013.01) Murugesan Rangasamy, Zionsville, IN (US); Kanika Arora, Indianapolis, IN (US); Balaji Veeramani, Indianapolis, (57) ABSTRACT IN (US); Premchand Gandra, Indianapolis, IN (US); Andreas This disclosure concerns nucleic acid molecules and methods Vilcinskas, Giessen (DE); Eileen of use thereof for control of insect pests through RNA inter Knorr, Giessen (DE) ference-mediated inhibition of target coding and transcribed (21) Appl. No.: 14/979,181 non-coding sequences in insect pests, including coleopteran (22) Filed: Dec. 22, 2015 pests. The disclosure also concerns methods for making Related U.S. Application Data transgenic plants that express nucleic acid molecules useful (60) Provisional application No. 62/095,487, filed on Dec. for the control of insect pests, and the plant cells and plants 22, 2014. obtained thereby. Patent Application Publication Jul. 7, 2016 Sheet 1 of 2 US 2016/O194658 A1 FIG. 1. Generation of dsRNA from a single transcription template with a single pair of primers ----------------------------------- polymerase g x8:a: %ao Hill: *** Patent Application Publication Jul. 7, 2016 Sheet 2 of 2 US 2016/O194658 A1 FIG. 2. Generation of dsRNA from two transcription templates. RNA polyherase & as&ssexxxxxxxxxxxxxxxxxxxsexex : engiate 3 M wanooooooo ti -- assa a w %OH: US 2016/0 194658 A1 Jul. 7, 2016 NUCAMPHOLIN NUCLECACID adults also feed on reproductive tissues of the corn plant, but MOLECULES TO CONTROL COLEOPTERAN in contrast rarely feed on corn leaves. INSECT PESTS 0006 Most of the rootworm damage in corn is caused by larval feeding. Newly hatched rootworms initially feed on PRIORITY CLAIM fine corn root hairs and burrow into root tips. As the larvae grow larger, they feed on and burrow into primary roots. 0001. This application claims the benefit of the filing date When corn rootworms are abundant, larval feeding often of U.S. Provisional Patent Application Ser. No. 62/095,487, results in the pruning of roots all the way to the base of the filed Dec. 22, 2014, for “NUCAMPHOLIN NUCLEICACID corn stalk. Severe root injury interferes with the roots’ ability MOLECULES TO CONTROL INSECT PESTS which is to transport water and nutrients into the plant, reduces plant incorporated herein in its entirety. growth, and results in reduced grain production, thereby often drastically reducing overall yield. Severe root injury also TECHNICAL FIELD OF THE DISCLOSURE often results in lodging of corn plants, which makes harvest 0002 The present invention relates generally to genetic more difficult and further decreases yield. Furthermore, feed control of plant damage caused by insect pests (e.g., ing by adults on the corn reproductive tissues can result in coleopteran pests). In particular embodiments, the present pruning of silks at the ear tip. If this “silk clipping is severe invention relates to identification of target coding and non enough during pollen shed, pollination may be disrupted. coding polynucleotides, and the use of RNAi technologies for 0007 Control of corn rootworms may be attempted by post-transcriptionally repressing or inhibiting expression of crop rotation, chemical insecticides, biopesticides (e.g., the target coding and non-coding polynucleotides in the cells of spore-forming gram-positive bacterium, Bacillus thuringien an insect pest to provide a plant protective effect. sis), or a combination thereof. Crop rotation suffers from the significant disadvantage of placing unwanted restrictions BACKGROUND upon the use of farmland. Moreover, oviposition of some rootworm species may occur in Soybean fields, thereby miti 0003. The western cornrootworm (WCR), Diabrotica vir gating the effectiveness of crop rotation practiced with corn gifera virgifera LeConte, is one of the most devastating corn and soybean. rootworm species in North America and is a particular con 0008 Chemical insecticides are the most heavily relied cern in corn-growing areas of the Midwestern United States. upon strategy for achieving corn rootworm control. Chemical The northern corn rootworm (NCR), Diabrotica barberi insecticide use, though, is an imperfect corn rootWorm con Smith and Lawrence, is a closely-related species that co trol strategy; over S1 billion may be lost in the United States inhabits much of the same range as WCR. There are several each year due to corn rootworm when the costs of the chemi other related subspecies of Diabrotica that are significant cal insecticides are added to the costs of the rootworm dam pests in North America: the Mexican corn rootworm (MCR), age that may occur despite the use of the insecticides. High D. virgifera zeae Krysan and Smith; the Southern corn root populations of larvae, heavy rains, and improper application worm (SCR), D. undecimpunctata howardi Barber: D. bal of the insecticide(s) may all result in inadequate corn root teata LeConte; D. undecimpunctata tenella, D. speciosa Ger worm control. Furthermore, the continual use of insecticides mar; and D. u. undecimpunctata Mannerheim. The United may select for insecticide-resistant rootworm strains, as well States Department of Agriculture has estimated that corn as raise significant environmental concerns due to the toxicity rootworms cause S1 billion in lost revenue each year, includ of many of them to non-target species. ing S800 million in yield loss and S200 million in treatment 0009 European pollen beetles (PB) are serious pests in COStS. oilseed rape, both the larvae and adults feed on flowers and 0004 Both WCR and NCR are deposited in the soil as pollen. Pollen beetle damage to the crop can cause 20-40% eggs during the Summer. The insects remain in the egg stage yield loss. The primary pest species is Melligethes aeneus throughout the winter. The eggs are oblong, white, and less Fabricius. Currently, pollen beetle control in oilseed rape than 0.004 inches in length. The larvae hatch in late May or relies mainly on pyrethroids which are expected to be phased early June, with the precise timing of egg hatching varying out soon because of their environmental and regulatory pro from year to year due to temperature differences and location. file. Moreover, pollen beetle resistance to existing chemical The newly hatched larvae are white worms that are less than insecticides has been reported. Therefore, urgently needed 0.125 inches in length. Once hatched, the larvae begin to feed are environmentally friendly pollen beetle control solutions on corn roots. Cornrootworms go through three larval instars. with novel modes of action. After feeding for several weeks, the larvae molt into the pupal 0010. In nature, pollen beetles overwinter as adults in the stage. They pupate in the Soil, and then they emerge from the soil or under leaf litter. In spring the adults emerge from soil as adults in July and August. Adult rootworms are about hibernation and start feeding on flowers of weeds, and 0.25 inches in length. migrate onto flowering oilseed rape plants. The eggs are laid 0005 Corn rootworm larvae complete development on in oilseed rape flower buds. The larvae feed and develop in the corn and several other species of grasses. Larvae reared on buds and flowers. Late stage larvae find a pupation site in the yellow foxtail emerge later and have a smaller head capsule soil. The second generation adults emerge in July and August size as adults than larvae reared on corn. Ellsbury et al. (2005) and feed on various flowering plants before finding sites for Environ. Entomol. 34:627-34. WCRadults feed on corn silk, overwintering. pollen, and kernels on exposed ear tips. If WCRadults emerge 0011 RNA interference (RNAi) is a process utilizing before corn reproductive tissues are present, they may feed on endogenous cellular pathways, whereby an interfering RNA leaf tissue, thereby slowing plant growth and occasionally (iRNA) molecule (e.g., a dsRNA molecule) that is specific for killing the host plant. However, the adults will quickly shift to all, or any portion of adequate size, of a target gene results in preferred silks and pollen when they become available. NCR the degradation of the mRNA encoded thereby. In recent US 2016/0 194658 A1 Jul. 7, 2016 years, RNAi has been used to perform gene “knockdown” in sand sequences provided would be lethal, or even otherwise a number of species and experimental systems; for example, useful, in species of corn rootworm when used as dsRNA or Caenorhabditis elegans, plants, insect embryos, and cells in siRNA.U.S. Pat. No. 7,943,819 provides no suggestion to use tissue culture. See, e.g., Fire et al. (1998) Nature 391:806-11; any particular sequence of the more than nine hundred Martinez et al. (2002) Cell 110:563-74; McManus and Sharp sequences listed therein for RNA interference, other than the (2002) Nature Rev. Genetics 3:737-47. particular partial sequence of a charged multivesicular body 0012 RNAi accomplishes degradation of mRNA through protein 4b gene. Furthermore, U.S. Pat. No. 7,943,819 pro an endogenous pathway including the DICER protein com vides no guidance as to which other of the over nine hundred plex. DICER cleaves long dsRNA molecules into short frag sequences provided would be lethal, or even otherwise useful, ments of approximately 20 nucleotides, termed Small inter in species of corn rootworm when used as dsRNA or siRNA.