WO2020081486A1

WO2020081486A1 - Control of insect infestation

Info

Publication number: WO2020081486A1
Application number: PCT/US2019/056187
Authority: WO
Inventors: Thais BARROS RODRIGUES; Suresh Desai
Original assignee: Greenlight Biosciences, Inc.
Priority date: 2018-10-15
Filing date: 2019-10-15
Publication date: 2020-04-23

Abstract

Provided herein are methods for using RNAi molecules targeting a ras opposite (ROP) gene for controlling Coleopteran insects, methods for producing RNAi molecules targeting ROP, and compositions comprising RNAi molecules targeting ROP.

Description

CONTROU OF INSECT INFESTATION

REUATED APPUICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/745,891 filed October 15, 2018, which is incorporated by reference herein in its entirety.

BACKGROUND

Crops are often the target of insect attacks. Globally, farmers lose 30 to 40 of their crops due to pests and diseases, according to the UN Food and Agricultural Organization. Crop maintenance and crop health is essential for yield and quality of produce, which ultimately require long-term strategies for the minimization of pest and disease occurrence. The annual costs of controlling crop pests ( e.g ., Colorado potato beetle) are estimated to be in the tens of millions of dollars, with projected annual costs of crop loss reaching billions of dollars if left uncontrolled.

Progress has been made towards developing more efficient methods and compositions for controlling pest infestations. While chemical pesticides have been one solution for eradicating pest infestations, alternative, more environmentally safe, solutions are needed. Chemical pesticides are harmful to the environment and may lack specificity or selectivity which ultimately results in non-target effects. Additionally, given the slow metabolism of chemical pesticides and the likelihood of chemical pesticides to accumulate, resistance is likely to occur. Thus, there has been a long felt need for more environmentally friendly methods for controlling or eradicating insect infestations which are more selective, environmentally safe, and

biodegradable.

SUMMARY

The present disclosure provides, in some aspects, compositions, genetic constructs, and methods for controlling infestation of pests (e.g., insects of the order of Coleoptera, Lepidoptera, Hemiptera and/or Diptera) that cause damage to crop plants. For example, aspects of the present disclosure provide compositions that include interfering RNA molecules (e.g., double-stranded RNA) for controlling crop infestation by these pests. To reduce our dependence on broad- spectrum chemical insecticides and their related problems, reduced-risk pesticides are required.

A new technology that offers the promise of a reduced risk approach to insect pest control is RNA interference (RNAi). In some embodiments, the present disclosure provides RNAi-based technologies that can mitigate insect (e.g., Colorado potato beetle damage by delivering ribonucleic acid (RNA) interference (RNAi) molecules that target (e.g., bind to) and interfere with the messenger RNA (mRNA) of an insect (e.g., Colorado potato beetle ras opposite (ROP) gene.

ROP primarily effects vesicle docking involved in exocytosis. ROP may be a component of one of the vesicle trafficking pathways or interact functionally with the Ras2 proteins, such as Seel -like protein, Seel -like superfamily, and Seel -like, domain 2. Its molecular function is described by SNARE binding.

It should be understood that while various aspects and embodiments provided herein are directed to controlling infestation of Leptinotarsa decemlineata (Colorado potato beetle), this disclosure is not intended to be limited to this particular genus/species. One skilled in the art appreciates that the methods and compositions provided herein can be used to target any insect expressing a-SNAP (e.g., an a-SNAP homolog, orthologs, or paralog). Non-limiting examples of such insects include Coleoptera, Lepidoptera, Hemiptera and Diptera.

Laboratory studies have confirmed that oral delivery of RNA molecules whose mode of action is through the RNAi process (e.g., double-stranded RNA (dsRNA)) are effective for many insect species and hence, topical dsRNA is considered a suitable form of delivery. However, spray-on dsRNA insect pest control technology does not exist today. The cost of production of dsRNA at relatively low price is a major challenge for the Ag-Bio industry. For agricultural pests, transgenic plants that can express insecticidal dsRNA may protect the plants from insect herbivory. However, not all countries are receptive to genetically-modified crops, and spray-on application of dsRNA is being considered as an alternative delivery method of protection.

To identify targets for RNAi knockdown, whole genome information was used to identify the appropriate gene sequence for ROP in the target species (e.g., Leptinotarsa decemlineata), which when silenced selectively, controls these key pests, without adversely affecting non-target species in the potato agriculture ecosystem. Given a DNA sequence of interest and a rule set of design criteria for the output sequences, a propriety computational algorithm was combined with publicly available RNAi design tools, to create output sequences that meet these criteria. The original/initial region selected to design the dsRNA was identified by searching comprehensive sequence databases for Tribolium and Drosophila genomes (e.g., Flybase, SnapDragon,

Beetlebase, etc.). The publicly available E-RNAi tool, that can be used to design dsRNA using a predicted siRNA-based approach, was combined with proprietary algorithms to create the design workflow. This design workflow was then used to create specific long dsRNA sequences of a (a) desired length (b) desired percent identity to original sequence (by introducing random mutations), and (c) by sectioning the initial ROP gene sequence into multiple fragments.. In some embodiments, the RNAi molecules comprise single-stranded RNA (ssRNA), and in some embodiments, the RNAi molecules comprise double- stranded RNA (dsRNA) or partially dsRNA. In still other embodiments, the RNAi molecules may be single-stranded RNA molecules with secondary structure containing significant double- stranded character, such as, but not limited to, hairpin RNA. The present disclosure provides RNA, for example single stranded RNA (ssRNA), small interfering (siRNA), micro RNA (miRNA), messenger RNA (mRNA), short hairpin (shRNA) or double stranded RNA (dsRNA) for targeting ROP mRNA.

ROP RNA, in some embodiments, is effective for reducing ROP expression in an insect, stunting of larvae, inhibiting growth, reproduction ( e.g ., fertility and/or fecundity) and/or repair of the insect, killing of the larvae or the insect, and decreasing feeding of the insect. Accordingly, one aspect of the present disclosure provides a method for controlling an insect comprising delivering (e.g., contacting) an effective amount of an ROP-targeting RNA with a plant and/or an insect. ROP RNA is particularly useful for controlling a Coleopteran insect (e.g., Colorado potato beetle), thereby reducing and/or preventing infestation of certain plants (e.g., a potato) that are a major food source for humans.

Some aspects of the present disclosure also provide cell-free methods of producing ROP- targeting RNA, the method comprising: (a) incubating in a reaction mixture cellular RNA, and a ribonuclease under conditions appropriate for the production of 5' nucleoside monophosphates (5' NMPs); (b) eliminating the ribonuclease; and (c) incubating the reaction mixture, or in a second reaction mixture, the 5’ NMPs, a polyphophospate kinase, a polyphosphate, a

polymerase, and a DNA (also referred to a DNA template) under conditions appropriate for the production of the ROP-targeting RNA from the DNA.

Also provided herein are compositions comprising an ROP-targeting RNA. In some embodiments, the composition comprising an ROP-targeting RNA further comprises an additive, for example, a chemical, a pesticide, a surfactant, a biological, or other non-pesticidal ingredient. In some embodiments, ROP-targeting RNA is provided in an expression vector. In some embodiments, an ROP-targeting RNA is provided in a plant or a plant cell.

It should be understood that an“RNAi molecule targeting ROP” encompasses“RNAi molecules targeting mRNA encoded by ROP.” A RNAi molecule is considered to target a gene of interest if the RNAi molecule binds to (e.g., transiently binds to) and inhibits (reduces or blocks) translation of the mRNA, e.g., due to the mRNA being degraded. In some embodiments, if there are epigenetic changes, a RNAi molecule may inhibit expression of the mRNA encoded by the gene of interest. It should also be understood that in some embodiments, the

polynucleotide is a double-stranded RNA (e.g., dsRNA GS3) that inhibits expression of a coding region of the gene (e.g., ROP). In other embodiments, the polynucleotide is a DNA sequence that encodes a dsRNA. In yet other embodiments, the polynucleotide is an antisense RNA. It should be understood that the sequences disclosed herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine with a uracil.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B include graphs showing the percent mortality of Colorado potato beetles (CPBs) (FIG. 1A) and percent leaf disc consumption by CPBs (FIG. IB) following a nine-day exposure of the CPBs to either a ROP RNAi (GS 65) composition of the present disclosure or to a control RNAi (GS 4) composition (10 pg/cm² concentration of RNAi).

DETAILED DESCRIPTION

According to some aspects of the present disclosure, RNAi molecules ( e.g ., dsRNAs) targeting ROP are effective at interfering with the mRNA encoded by an ROP gene in insect (e.g. Coleopteran ) cells, thereby reducing or eliminating translation of the mRNA (e.g., into its corresponding protein). Accordingly, in some aspects, the present disclosure provides compositions and methods for controlling insect (e.g. Coleopteran ) infestations by contacting any portion of a plant (e.g., roots, tubers, stem, branches, leaves, flower, etc.), ground (e.g., soil, dirt, grass, etc.), insect (e.g., Coleopteran) and/or diet (e.g., food and/or water ingested by) of the insect with an RNAi molecule as provided herein. Also provided herein are cell-free methods of synthesizing RNAi molecules that target ROP gene products (mRNA).

An insect, as used herein, refers to an insect in any stage of development. In some embodiments, the insect is an insect egg. In some embodiments, the insect is an insect larva. In some embodiments, the insect is an insect pupa. In some embodiments, the insect is an adult insect.

A Lepidopteran insect may be any Lepidopteran insect of order Lepidoptera. Examples of insects of the order Lepidoptera include, but are not limited to, Nxmphalidae (brushfooted butterflies), Danaidae (milkweed butterflies), Pieridae (whites and sulfurs) Papilionidae

(swallowtails), Lycaenidae (blues, coppers, and hairstreaks), Hesperiidae (skippers),

Tineidae (clothes moths), Sesiidae (clearwing moths), Pyralidae (snout moths),

Lasiocampidae (lappet moths), Saturniidae (giant silk moths), Sphinsidae (hawk moths),

Arctiidae (tiger moths), Lymantriidae (tussock moths), Noctuidae (loopers, owlet moths, and underwings).

A Dipteran insect may be any Dipteran insect of order Diptera. Examples of insects of the order Diptera include, but are not limited to, Culicidae (mosquitoes), Tabanidae (horse flies/ deer flies), Simulidae (black flies), Psychodidae (moth flies), Ceratopogonidae (punkies, no-see- urns), Muscidae (House flies), Cecidomyiidae (gall midges), Tephritidae (fruit flies), Agromyzidae (leaf miners), Anthomyiidae (maggots), Drosophilidae (pomace flies),

Tipulidae (crane flies), Calliphoridae (blow flies), Chironomidae (midges), and

Sarcophagidae (flesh flies)

A Hemipteran insect may be a Hemipteran insect of order Hemiptera. Examples of insects of the order of Hemiptera include, but are not limited to Miridae (Plant Bugs),

Lygaeidae (Seed Bugs), Tingidae (lace bugs), Coreidae (squash bugs and leaffooted bugs), Alydidae (broadheaded bugs), Rhopalidae (scentless plant bugs), Berytidae (stilt bugs),

Reduviidae (assassin bugs), Phymatidae (ambush bugs), Nabidae (damsel bugs),

Anthocoridae (minute pirate bugs), Corixidae (water boatmen), Gerridae (water striders), Nepidae (water scorpions), Belostomatidae (giant water bugs), Naucoridae (creeping water bugs), Notonectidae (backswimmers), Cicadidae (cicadas), Cicadellidae (leafhoppers),

Membracidae (treehoppers), Cercopidae (spittlebugs or froghoppers),

Fulgoridae (planthoppers), Psyllidae (psyllids or jumping plant lice), Aleyrodidae (whiteflies), Aphididae (aphids, plantlice), and Coccidae (soft scale insects).

A Coleopteran insect may be any Coleopteran insect of order Coleoptera. Examples of insects of the order Coleoptera include, but are not limited to, Chrysomelidae (leaf beetle), Curculionidae (snout beetle), Meloidae (blister beetle), Tenebrionidae (darkling beetle),

Scarabaeidae (scarab beetle), Cerambycidae (Japanese pine sawyer), Curculionidae (Chinese white pine beetle), Nitidulidae (small hive beetle), Chrysomelidae (broad-shouldered leaf beetle), Cerambycidae (mulberry longhorn beetle), Phyllotreta (flea beetle), Diabrotica (corn rootworm) Chrysomela (cottonwood leaf beetle), Hypothenemus (coffee berry borer), Sitophilus (maize weevil), Epitrix (tobacco flea beetle), E. cucumeris (potato flea beetle), P. pusilla (western black flea beetle); Anthonomus (pepper weevil), Hemicrepidus (wireworms), Melanotus (wireworm), Ceutorhychus (cabbage seedpod weevil), Aeolus (wireworm), Horistonotus (sand wireworm), Sphenophorus (maize billbug), S. zea (timothy billbug), S. parvulus (bluegrass billbug), S.

callosus (southern corn billbug); Phyllophaga (white grubs), Chaetocnema (corn flea beetle), Popillia (Japanese beetle), Epilachna (Mexican bean beetle), Cerotoma (bean leaf beetle), Epicauta (blister beetle), Chrysomelidae (alligator weed flea beetle) and any combination thereof.

Further, the Coleopteran insect may be any species of Leptinotarsa. Leptinotarsa species include, but are not limited to, Leptinotarsa decemlineata (Colorado potato beetle), Leptinotarsa behrensi, Leptinotarsa collinsi, Leptinotarsa defecta, Leptinotarsa haldemani (Haldeman’s green potato beetle), Leptinotarsa heydeni, Leptinotarsa juncta (false potato beetle), Leptinotarsa lineolata (burrobrush leaf beetle), Leptinotarsa peninsularis, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsa tlascalana, Leptinotarsa tumamoca, and Leptinotarsa typographica.

RNAi Molecule Targeting Ras Opposite (ROP)

RNAi molecules targeting ROP have been identified through examination of ROP mRNA and in vivo (e.g., plant/field) testing. Such RNAi molecules targeting ROP are useful for controlling Coleopteran insects (e.g., Colorado potato beetles), for example, by inhibiting or reducing expression of ROP, and consequently, by increasing insect mortality, as well as decreasing growth, reproduction (e.g., fertility and/or fecundity), and/or feeding (e.g., eating and/or drinking) of Coleopteran insects.

Expression of a gene in a cell (e.g., insect cell), for example, is considered to be inhibited or reduced through contact with an RNAi molecule if the level of mRNA and/or protein encoded by the gene is reduced in the cell by at least 10% relative to a control cell that has not been contacted with the RNAi molecule. For example, delivering to a cell (e.g., contacting a cell) with an RNAi molecule (e.g., dsRNA) targeting ROP may result in a reduction (e.g., by at least 10%) in the amount of RNA transcript and/or protein (e.g., encoded by the ROP gene) compared to a cell that is not contacted with RNAi molecular targeting ROP.

In some embodiments, RNAi molecules of the present disclosure specifically inhibit expression of an ROP gene without biologically relevant or biologically significant off-target effects (no relevant or significant change in the expression of non-ROP genes). In some embodiments, an RNAi molecule specifically inhibits (reduces or blocks) translation of an ROP protein by specifically inhibiting expression of (e.g., degrading) an ROP mRNA (e.g., ROP mRNA of SEQ ID NO: 2) that encodes the ROP protein. Specific inhibition of an ROP gene includes a measurable reduction in ROP gene expression (e.g., ROP mRNA expression, and/or ROP protein expression) or a complete lack of detectable gene expression (e.g., ROP mRNA expression, and/or ROP protein expression).

In some embodiments, RNAi molecules of the present disclosure specifically inhibit expression of an ROP gene without biologically relevant or biologically significant off-target effects (no relevant or significant change in the expression of non-ROP genes). In some embodiments, an RNAi molecule specifically inhibits the expression of an ROP protein by specifically inhibiting an mRNA that encodes an ROP protein (e.g., ROP mRNA of SEQ ID NO: 2). Specific inhibition of an ROP gene involves a measurable reduction in ROP gene expression (e.g., ROP mRNA expression, and/or ROP protein expression) or a complete lack of detectable gene expression (e.g., ROP mRNA expression, and/or ROP protein expression). RNAi molecules targeting ROP provided herein, in some embodiments, are designed to have complementarity to ROP mRNA of a Coleopteran insect, e.g., a Colorado potato beetle. An example of a DNA sequence encoding Colorado potato beetle ROP is provided in the sequence of SEQ ID NO: 1. An example of an mRNA sequence encoding Colorado potato beetle ROP is provided in the sequence of SEQ ID NO: 2. Examples of a RNA molecules targeting ROP are provided in the sequences of SEQ ID NO: 3 or 5.

In some embodiments, the RNAi molecule targeting ROP provided herein is designed to have complementarity to ROP mRNA of a Coleopteran insect, e.g., a Chrysomelidae (a leaf beetle), a Curculionidae (a snout beetle), a Meloidae (a blister beetle), Tenebrionidae (a darkling beetle), a Scarabaeidae (a scarab beetle), a Cerambycidae (a japanese pine sawyer), a

Curculionidae (a Chinese white pine beetle), a Nitidulidae (a small hive beetle), a

Chrysomelidae (a broad-shouldered leaf beetle), a Cerambycidae (a mulberry longhorn beetle),

C. scripta (cottonwood leaf beetle), H. hampei (coffee berry borer), S. Zeamais (maize weevil),/ hirtipennis (tobacco flea beetle), F. cucumeris (potato flea beetle), P. cruciferae (crucifer flea beetle) and P. pusilla (western black flea beetle), A. eugenii (pepper weevil), H. memnonius (wireworms), M. communis (wireworm), C. assimilis (cabbage seedpod weevil), P. striolata (striped flea beetle), A. mellillus (wireworm), A. mancus (wheat wireworm), H. uhlerii (sand wireworm), S. maidis (maize billbug), S. zeae (timothy billbug), S. parvulus (bluegrass billbug), and S. callosus (southern corn billbug), Phyllophaga spp. (White grubs), C. pulicaria (corn flea beetle), P. japonica (Japanese beetle), F. varivestis (Mexican bean beetle), C. trifurcate (Bean leaf beetle), F. pestifera and F. lemniscata (Blister beetles), Oulema melanapus (Cereal leaf beetle), Hypera postica (Alfalfa weevil), Dendroctonus (Mountain Pine beetle), Agrilus (Emarald Ash Borer), Hylurgopinus (native elm bark beetle), Scolytus (European elm bark beetle) and/or a Chrysomelidae (an alligator weed flea beetle).

In some embodiments, the RNAi molecule targeting ROP provided herein is designed to have complementarity to ROP mRNA of a Leptinotarsa insect, e.g., a Leptinotarsa decemlineata (a Colorado potato beetle), a Leptinotarsa behrensi, a Leptinotarsa collinsi, a Leptinotarsa defecta, a Leptinotarsa haldemani (a Haldeman’s green potato beetle), a Leptinotarsa hey deni, a Leptinotarsa juncta (a false potato beetle), a Leptinotarsa lineolata (a burrobrush leaf beetle), a Leptinotarsa peninsularis, a Leptinotarsa rubiginosa, a Leptinotarsa texana, a Leptinotarsa tlascalana, a Leptinotarsa tumamoca, and/or a Leptinotarsa typographica.

A double- stranded RNA (dsRNA) of the present disclosure, in some embodiments, comprises a first strand that binds to (e.g., is at least partially complementary to or is wholly complementary to) a messenger RNA (mRNA) encoded by a Coleopteran ROP gene, and a second strand that is complementary to the first strand. dsRNA may comprise RNA strands that are the same length or different lengths. In some embodiments, a dsRNA comprises a first strand (e.g., an antisense strand) that is the same length as a second strand (e.g., a sense strand). In some embodiments, a dsRNA comprises a first strand (e.g., an antisense strand) that is a different length than a second strand (e.g., a sense strand). A first strand may be about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more than 20% longer than a second strand. A first strand may be 1-5, 2-5, 2-10, 5-10, 5-15, 10-20, 15-20, or more than 20 nucleotides longer than a second strand.

dsRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the RNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single-stranded RNA having two or more loop structures and a stem comprising self

complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active RNAi molecule capable of mediating RNAi. An RNAi molecule may comprise a 3' overhang at one end of the molecule, The other end may be blunt- ended or have also an overhang (5' or 3')· When the RNAi molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different.

A single- stranded RNA of the present disclosure, in some embodiments, comprises a strand that binds to a mRNA encoded by a Coleopteran ROP gene.

RNAi molecules targeting ROP as provided herein may vary in length. It should be understood that, in some embodiments, while a long RNA (e.g., dsRNA or ssRNA) molecule is applied (e.g., to a plant) as the insecticide, after entering cells this dsRNA is cleaved by the Dicer enzyme into shorter double- stranded RNA fragments having a length of, for example, 15 to 25 nucleotides. Thus, RNAi molecules of the present disclosure may be delivered as 15 to 25 nucleotide fragments, for example, or they may be delivered as longer double-stranded nucleic acids (e.g., at least 100 nucleotides).

Thus, in some embodiments, RNAi molecules targeting ROP comprise 15-3913 nucleotides (ssRNA) or nucleotide base pairs (dsRNA). For example, an RNAi molecule of the present disclosure may comprise 15-1000, 15-950, 15-900, 15-850, 15-800, 15-750, 15-700, 15- 650, 15-600, 15-500, 15-450, 15-400, 15-350, 15-300, 15-250, 15-200, 15-150, 15-100, 15-50, 19-1000, 18-950, 18-900, 18-850, 18-800, 18-750, 18-700, 18-650, 18-600, 18-500, 18-450, 18- 400, 18-350, 18-300, 18-250, 18-200, 18-180, 18-100, 18-50, 19-1000, 19-950, 19-900, 19-850,

19-800, 19-750, 19-700, 19-650, 19-600, 19-500, 19-450, 19-400, 19-350, 19-300, 19-250, 19- 200, 19-190, 19-100, 19-50, 20-1000, 20-950, 20-900, 20-850, 20-800, 20-750, 20-700, 20-650,

20-600, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-200, 20-100, 20-50, 15211000, 21-950, 21-900, 21-850, 21-800, 21-750, 21-700, 21-650, 21-600, 21-500, 21-450, 21-400, 21-350, 21-300, 21-250, 21-210, 21-210, 21-100, 21-50, 22-1000, 22-950, 22-900, 22- 850, 22-800, 22-750, 22-700, 22-650, 22-600, 22-500, 22-450, 22-400, 22-350, 22-300, 22-250,

22-220, 22-220, 22-100, 22-50, 23-1000, 23-950, 23-900, 23-850, 23-800, 23-750, 23-700, 23- 650, 23-600, 23-500, 23-450, 23-400, 23-350, 23-300, 23-250, 23-230, 23-230, 23-100, 23-50,

24-1000, 24-950, 24-900, 24-850, 24-800, 24-750, 24-700, 24-650, 24-600, 24-500, 24-450, 24- 400, 24-350, 24-300, 24-250, 24-240, 24-240, 24-100, 24-50, 25-1000, 25-950, 25-900, 25-850,

25-800, 25-750, 25-700, 25-650, 25-600, 25-500, 25-450, 25-400, 25-350, 25-300, 25-250, 25- 250, 25-250, 25-100, or 25-50 nucleotides or nucleotide base pairs. In some embodiments, RNAi molecules targeting ROP comprise or consist of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 50, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleotides or nucleotide base pairs.

In some embodiments, an RNAi molecule targeting ROP comprises or consists of a sequence that is complementary to an mRNA or a segment of an mRNA encoded by a

Coleopteran ROP gene. In some embodiments, an RNAi molecule targeting ROP comprises or consists of a sequence that is complementary to an mRNA or a segment of an mRNA encoded by a DNA sequence of SEQ ID NO: 1. In some embodiments, an RNAi molecule targeting ROP comprises or consists of a sequence that is complementary to an mRNA encoded by a DNA sequence of SEQ ID NO: 1.

In some embodiments, an RNAi molecule targeting ROP comprises or consists of a sequence that is complementary to an mRNA encoded by a region or segment of a Coleopteran ROP DNA. In some embodiments, an RNAi molecule targets an mRNA encoded by a 5’ region or segment of a Coleopteran ROP DNA. A 5’ region of a Coleopteran ROP DNA may comprise or consist of any sequence encompassed by nucleotides 1 to 1500, nucleotides 10 to 1500, nucleotides 25 to 1500, nucleotides 50 to 1500, nucleotides 100 to 1500, nucleotides 150 to 1500, nucleotides 200 to 1500, nucleotides 250 to 1500, nucleotides 300 to 1500, nucleotides 350 to 1500, nucleotides 400 to 1500, nucleotides 450 to 1500, nucleotides 500 to 1500, nucleotides 550 to 1500, nucleotides 600 to 1500, nucleotides 650 to 1500, nucleotides 700 to 1500, nucleotides 750 to 1500, nucleotides 800 to 1500, nucleotides 850 to 1500, nucleotides 900 to 1500, nucleotides 950 to 1500, or nucleotides 1000 to 1500 of the ROP DNA (e.g., nucleotides 1-1500 of SEQ ID NO: 1 or 2). In some embodiments, an RNAi molecule targets an mRNA encoded by a central region or segment of a Coleopteran ROP DNA. A central region of a Coleopteran ROP DNA may comprise or consist of any sequence encompassed by nucleotides 1000 to 3000, nucleotides 1050 to 3000, nucleotides 1150 to 3000, nucleotides 1200 to 3000, nucleotides 1250 to 3000, nucleotides 1300 to 3000, nucleotides 1350 to 3000, nucleotides 1400 to 3000, nucleotides 1450 to 3000, nucleotides 1500 to 3000, nucleotides 1550 to 3000, nucleotides 1600 to 3000, nucleotides 1650 to 3000, nucleotides 1700 to 3000, nucleotides 1750 to 3000, nucleotides 1800 to 3000, nucleotides 1850 to 3000, nucleotides 1900 to 3000, nucleotides 1950 to 3000, or nucleotides 2000 to 3000 of the ROP DNA ( e.g ., nucleotides 1000- 3000 of SEQ ID NO: 1 or 2). In some embodiments, an RNAi molecule targets an mRNA encoded by a 3’ region or segment of a Coleopteran ROP DNA. A 3’ region of a Coleopteran ROP DNA may comprise or consist of any sequence encompassed by nucleotides 2500 to 3913, nucleotides 2550 to 3913, nucleotides 2600 to 3913, nucleotides 2650 to 3913, nucleotides 2700 to 3913, nucleotides 2750 to 3913, nucleotides 2800 to 3913, nucleotides 2850 to 3913, nucleotides 2900 to 3913, nucleotides 2950 to 3913, nucleotides 3000 to 3913, nucleotides 3050 to 3913, nucleotides 3100 to 3913, nucleotides 3150 to 3913, nucleotides 3200 to 3913, nucleotides 3250 to 3913, nucleotides 3300 to 3913, nucleotides 3350 to 3913, nucleotides 3400 to 3913, nucleotides 3450 to 3913, or nucleotides 3500 to 3913 of the ROP DNA (e.g., nucleotides 2500-3913 of SEQ ID NO: 1 or 2).

It should be understood that the term gene encompasses coding and non-coding nucleic acid. Thus, in some embodiments, an ROP gene encodes an mRNA that comprises a 5’ untranslated region, an open reading frame, and a 3’ untranslated region. Thus, an RNAi molecule herein, in some embodiments, binds to a 5’ untranslated region, an open reading frame, and/or a 3’ untranslated region of an mRNA.

In some embodiments, an RNAi molecule targeting ROP comprises or consists of an RNA sequence of SEQ ID NO: 3 or 5. In some embodiments, an RNAi molecule targeting ROP comprises or consists of an RNA sequence of SEQ ID NO: 3 or 5.

In some embodiments, an RNAi molecule targeting ROP comprises or consists of a sequence that is complementary to a RNA sequence of ID NO: 2. In some embodiments, an RNAi molecule targeting ROP comprises or consists of a sequence that is complementary to a RNA sequence of SEQ ID NO: 2.

In some embodiments, RNAi molecules targeting ROP comprise or consist of a (at least one) contiguous sequence that has 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence encoded by a Coleopteran ROP gene. In some embodiments, the ROP gene comprises a DNA sequence of SEQ ID NO: 1. In some embodiments, RNAi molecules targeting ROP comprise or consist of a (at least one) contiguous sequence that has 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence encoded by a DNA sequence of SEQ ID NO:

1.

In some embodiments, RNAi molecules targeting ROP comprise or consist of a (at least one) contiguous sequence that is 70% to 100% complementary ( e.g ., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%

complementary) to an RNA sequence encoded by a Coleopteran ROP gene. In some

embodiments, the ROP gene comprises a DNA sequence of SEQ ID NO: 1. In some

embodiments, RNAi molecules targeting ROP comprise or consist of a (at least one) contiguous sequence that is 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence encoded by a DNA sequence of SEQ ID NO: 1.

In some embodiments, RNAi molecules targeting ROP comprise or consist of a (at least one) contiguous sequence that has 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence of SEQ ID NOS: 3 or 5. In some embodiments, RNAi molecules targeting ROP comprise or consist of a contiguous sequence that has 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence of SEQ ID NO: 3 or 5.

In some embodiments, RNAi molecules targeting ROP comprise or consist of a (at least one) contiguous sequence is 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to an RNA sequence of SEQ ID NO: 2 or 4. In some embodiments, RNAi molecules targeting ROP comprise or consist of a contiguous sequence is 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to an RNA sequence of SEQ ID NO: 2 or 4.

In some embodiments, RNAi molecules targeting ROP comprise or consist of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs having 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence or segment of an RNA sequence of SEQ ID NOS: 3 or 5. In some embodiments, RNAi molecules targeting ROP comprise or consist of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs having 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence or segment of an RNA sequence of SEQ ID NO: 3 or 5.

In some embodiments, RNAi molecules targeting ROP comprise or consist of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs having 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to an RNA sequence or segment of an RNA sequence of SEQ ID NO: 2 or 4. In some embodiments, RNAi molecules targeting ROP comprise or consist of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs having 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to an RNA sequence or segment of an RNA sequence of SEQ ID NO: 2 or 4.

In some embodiments, RNAi molecules targeting ROP comprise or consist of 10 to 25,

10 to 24, 10 to 23, 10 to 22, 10 to 21, 10 to 20, 11 to 25, 11 to 24, 11 to 23, 11 to 22, 11 to 21, 11 to 20, 12 to 25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 13 to 25, 13 to 24, 13 to 23, 13 to 22, 13 to 21, 13 to 20, 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, 14 to 20, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 16 to 25, 16 to 24, 16 to 23, 16 to 22, 16 to 21, 16 to 20, 17 to 25, 17 to 24, 17 to 23, 17 to 22, 17 to 21, 17 to 20, 18 to 25, 18 to 24, 18 to 23, 18 to

22, 18 to 21, or 18 to 20 contiguous nucleotides having 70% to 100% identity (e.g., 70% to

100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence or segment of an RNA sequence of SEQ ID NOS: 3 or 5. In some embodiments, RNAi molecules targeting ROP comprise or consist of 10 to 25, 10 to 24, 10 to 23, 10 to 22, 10 to 21, 10 to 20, 11 to 25, 11 to 24, 11 to 23, 11 to 22, 11 to 21, 11 to 20, 12 to

25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 13 to 25, 13 to 24, 13 to 23, 13 to 22, 13 to

21, 13 to 20, 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, 14 to 20, 15 to 25, 15 to 24, 15 to

23, 15 to 22, 15 to 21, 15 to 20, 16 to 25, 16 to 24, 16 to 23, 16 to 22, 16 to 21, 16 to 20, 17 to

25, 17 to 24, 17 to 23, 17 to 22, 17 to 21, 17 to 20, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to

21, or 18 to 20 contiguous nucleotides having 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an RNA sequence or segment of an RNA sequence of SEQ ID NO: 3 or 5.

10 to 24, 10 to 23, 10 to 22, 10 to 21, 10 to 20, 11 to 25, 11 to 24, 11 to 23, 11 to 22, 11 to 21, 11 to 20, 12 to 25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 13 to 25, 13 to 24, 13 to 23, 13 to

22, 13 to 21, 13 to 20, 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, 14 to 20, 15 to 25, 15 to

24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 16 to 25, 16 to 24, 16 to 23, 16 to 22, 16 to 21, 16 to

20, 17 to 25, 17 to 24, 17 to 23, 17 to 22, 17 to 21, 17 to 20, 18 to 25, 18 to 24, 18 to 23, 18 to

22, 18 to 21, or 18 to 20 contiguous nucleotides having 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,

99%, or 100% complementary) to an RNA sequence or segment of an RNA sequence of SEQ ID NO: 2 or 4. In some embodiments, RNAi molecules targeting ROP comprise or consist of 10 to

25, 10 to 24, 10 to 23, 10 to 22, 10 to 21, 10 to 20, 11 to 25, 11 to 24, 11 to 23, 11 to 22, 11 to

21, 11 to 20, 12 to 25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 13 to 25, 13 to 24, 13 to

23, 13 to 22, 13 to 21, 13 to 20, 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, 14 to 20, 15 to

25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 16 to 25, 16 to 24, 16 to 23, 16 to 22, 16 to

21, 16 to 20, 17 to 25, 17 to 24, 17 to 23, 17 to 22, 17 to 21, 17 to 20, 18 to 25, 18 to 24, 18 to

23, 18 to 22, 18 to 21, or 18 to 20 contiguous nucleotides having 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to an RNA sequence or segment of an RNA sequence of SEQ ID NO: 2 or 4.

The“percent identity” of two nucleic acid sequences (e.g., RNAi molecules targeting ROP provided herein and any one of, for example, SEQ ID NOS: 1-5) may be determined by any method known in the art. The variants provided herein, in some embodiments, contain randomly placed mutations with the four nucleotides (A, U, G, C) selected at an approximately equal probability for a given mutation. In some embodiments, these mutations might be distributed either over a small region of the sequence, or widely distributed across the length of the sequence. In some embodiments, the percent identity of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=l00, wordlength-l2 to obtain guide sequences homologous to a target nucleic acid. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25(l7):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

The polynucleotides provided herein, such as RNAi molecules targeting ROP, in some embodiments, are designed to have at least one silencing element complementary (e.g., wholly (100%) or partially (less than 100%, e.g., 90% to 99%) complementary) to a segment of a sequence of ROP mRNA of a Coleopteran insect, e.g., a Colorado potato beetle. In some embodiments, polynucleotides comprise at least one silencing element that is essentially identical or essentially complementary to ROP mRNA of a Coleopteran insect. In some embodiments, the polynucleotides comprise 2 to 5, to 10, 2 to 20, 2 to 20, 2 to 40, or 2 to 50 silencing elements. In some embodiments, the polynucleotides comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 silencing elements.

RNAi molecules targeting ROP provided herein may be of any form of RNA, including single-stranded RNA (ssRNA) and double- stranded RNA (dsRNA). Non-limiting examples of single-stranded RNA include mRNA, micro RNA (miRNA) (e.g., artificial miRNA (amiRNA)), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), and antisense RNA. Double- stranded RNA includes wholly double-stranded molecules that do not contain a single- stranded region (e.g., a loop or overhang), as well as partially double-stranded molecules that contain a double-stranded region and a single- stranded region (e.g., a loop or overhang). Lurther, the RNAi molecules may be single- stranded RNA molecules with secondary structure containing significant double- stranded character, such as, but not limited to, hairpin RNA. Thus, RNAi molecules targeting ROP, in some embodiments, may be short hairpin RNA (shRNA).

In some embodiments, RNAi molecules targeting ROP comprise dsRNA, ssRNA, siRNA, miRNA (e.g., amirRNA), piRNA, mRNA, or shRNA. In some embodiments, RNAi molecules targeting ROP comprise more than one form of RNA. For example, the RNAi molecules targeting ROP may comprise ssRNA and dsRNA. In some embodiments, RNAi molecules targeting ROP comprise a hybrid with RNA and DNA. In some embodiments, RNAi molecules targeting ROP comprise amiRNAs processed from a long precursor transcript of nonprotein-coding RNA, that is partially self-complementary to mediate silencing of target mRNAs. amiRNAs are designed, in some embodiments, by replacing the mature 21 nucleotide miRNA sequences within pre-miRNA with 21 nucleotide long fragments derived from the target gene ( Frontiers in Plant Science , Sebastian et al., 2017). An amiRNA may have a length of, for example, at least 18 to 500 nucleotides, at least 21 to 500 nucleotides, at least 50 to 500 nucleotides, at least 100 to 500 nucleotides, or at least 200 to 500 nucleotides.

RNAi molecules targeting ROP may be provided as a mixture of RNAi molecules targeting ROP, for example, a mixture of RNAi molecules targeting ROP having different sequences. Any number of distinct RNAi molecules targeting ROP may be provided in a mixture of RNAi molecules targeting ROP. In some embodiments, the mixture of RNAi molecules targeting ROP comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 distinct (having different sequences/nucleotide compositions) RNAi molecules targeting ROP.

In some embodiment, RNAi molecules targeting ROP are provided as a mixture of RNAi molecules that are complementary (wholly or partially) to different segments of an mRNA encoded by an ROP gene (e.g., comprising a sequence of SEQ ID NO: 1). In some embodiment, RNAi molecules targeting ROP are provided as a mixture of RNAi molecules that are complementary (wholly or partially) to different segments of an RNA sequence of SEQ ID NO:

2. Any number of RNAi molecules targeting ROP that are complementary to different segments of an mRNA (e.g., comprising a sequence of SEQ ID NO: 2) encoded by an ROP gene (e.g., comprising a sequence of SEQ ID NO: 1) may be provided in a mixture of RNAi molecules targeting ROP. In some embodiments, the mixture of RNAi molecules targeting ROP comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 RNAi molecules targeting ROP. In some embodiments, the mixture of RNAi molecules targeting ROP comprises 2 to 5, or 2 to 10 RNAi molecules targeting ROP. In some embodiments, RNAi molecules targeting ROP provided herein may have one or more mismatches compared with the corresponding sequence of ROP mRNA (e.g., SEQ ID NO: 2). A region of complementarity on RNAi molecule targeting ROP may have up to 1, up to 2, up to 3, up to 4, etc. mismatches provided that it maintains the ability to form complementary base pairs with ROP mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on RNAi molecules targeting ROP may have no more than 1, no more than 2, no more than 3, or no more than 4 mismatches provided that it maintains the ability to form complementary base pairs with ROP mRNA under appropriate hybridization conditions. In some embodiments, if there is more than one mismatch in a region of complementarity, they may be positioned consecutively (e.g., 2, 3, 4, or more in a row), or interspersed throughout the region of complementarity provided that the RNAi molecule targeting ROP maintains the ability to form complementary base pairs with ROP mRNA under appropriate hybridization conditions.

RNAi molecules targeting ROP may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, degradation, resistance to nuclease degradation, base-pairing properties, RNA distribution, and cellular uptake, and other features relevant to its use. See, e.g., Bramsen et al, Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen and Kjems, Frontiers in Genetics, 3 (2012): 1-22. Accordingly, in some embodiments, RNAi molecules targeting ROP may include one or more (at least one) suitable modifications. In some

embodiments, a modified RNAi molecule targeting ROP has a modification in its base, sugar (e.g., ribose, deoxyribose), or phosphate group.

RNAi molecules targeting ROP produced by the methods provided herein may be modified as described herein. In some embodiments, RNAi molecules targeting ROP is produced according to a method described herein and subsequently modified. In some embodiments, RNAi molecules targeting ROP are produced according to a method described herein using a modified starting material. In some embodiments, the modified starting material is a modified nucleobase. In some embodiments, the modified starting material is a modified nucleoside. In some embodiments, the modified starting material is a modified nucleotide.

In some embodiments, modified RNAi molecules targeting ROP comprise a backbone modification. In some embodiments, backbone modification results in a longer half-life for the RNA due to reduced degradation (e.g., nuclease-mediated degradation). This in turn results in a longer half-life. Examples of suitable backbone modifications include, but are not limited to, phosphorothioate modifications, phosphorodithioate modifications, p-ethoxy modifications, methylphosphonate modifications, methylphosphorothioate modifications, alkyl- and aryl- phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), alkylphosphotriesters (in which the charged oxvsen moiety is alkylated), peptide nucleic acid (PNA) backbone modifications, and locked nucleic acid (LNA) backbone modifications. These modifications may be used in combination with each other and/or in combination with phosphodiester backbone linkages.

Alternatively or additionally, RNAi molecules targeting ROP may comprise other modifications, including modifications at the base or sugar moiety. Examples include RNA having sugars that are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3 ' position and other than a phosphate group at the 5 ' position (e.g., a 2'-0- alkylated ribose), or RNA having sugars such as arabinose instead of ribose. RNA also embraces substituted purines and pyrimidines such as C-5 propyne modified bases (Wagner el al, Nature Biotechnology 14:840-844, 1996). Other purines and pyrimidines include, but are not limited to, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, and

hypoxanthine. Other such modifications are well known to those of skill in the art.

RNAi molecules that comprise a nucleotide sequence complementary to all or a segment of the target sequence can be designed and prepared using any suitable methods. In some embodiments, an RNAi molecule may be designed with assistance from comprehensive sequence databases, such as those known for Tribolium and Drosophila genetics (e.g., Flybase, SnapDragon, Beetlebase, etc.). In some embodiments, a sequence database is utilized to determine off-target effects of a designed RNAi molecule (e.g., as in Arziman, Z., Horn, T., & Boutros, M. (2005). E-RNAi: a web application to design optimized RNAi constructs. Nucleic Acids Research, 33 (Web Server issue), W582-W588. doi:l0.l093/nar/gki468.)

Methods of Use

Aspects of the present disclosure, in some embodiments, provide methods for controlling a Coleopteran insect infestation comprising delivering to a plant or Coleopteran insect (e.g., Colorado potato beetle) an effective amount of an RNAi molecule targeting ROP (or a composition comprising an RNAi molecule targeting ROP). In some embodiments, the method of delivery comprises applying to a surface of a plant or Coleopteran insect, a composition comprising the RNAi molecule. In some embodiments, a composition comprising an RNAi molecule targeting ROP is a solid or liquid (e.g., solution, suspension, or emulsions). Non limiting examples include emulsifiable concentrates, concentrate solutions, low concentrate solutions, ultra-low volume concentrate solutions, water soluble concentrate solutions, water soluble liquid solutions, baits (paste, gel, liquid, solid or injectable), smoke, fog, invert emulsions, flowables, aerosols, homogenous and non-homogenous mixtures, suspensions (water and oil based), dust, powders (wettable or soluble), granules (water-dispersible or dry flowables), pellets, capsules, fumigants, encapsulated or micro-encapsulation formulations, or any combinations thereof.

In some embodiments, a compositing comprising an RNAi molecule targeting ROP may be applied as a concentrate, spray (after dilution or concentrate), fog, in furrow, seed treatment, drench, drip, insect diet, bait, or any other forms suited for applying to a furrow. The RNAi molecule targeting ROP described herein may be delivered to any portion of a plant, including, but are not limited to, leaf, stem, flower, fruit, shoot, root, seed, tuber, anther, stamen, and/or pollen. In some embodiments, RNAi is delivered mechanically, through high pressure spray or sand blasting. In some embodiments, a composition comprises an RNAi molecules and at least one additive selected from adjuvants, attractants, sterilizing agents, growth-regulating substances, carriers or diluents, stabilizers, and/or pesticidal agent(s) ( e.g ., insecticides, fungicides, and/or herbicides). Pesticidal agents include, for example, other dsRNA targeting genes distinct from ROP, patatins, plant lectins, phytoecdy steroids, cry proteins, vegetative insecticidal proteins (vip), cytolytic proteins (cyt), biotin-binding proteins, protease inhibitors, chitinases, organic compounds, or any combination thereof. Non-pesticidal agents may also be used (e.g. adjuvants, such as antifoaming agents, buffers, compatibility agents, drift control additives, emulsifiers, extenders, invert emulsifiers, plant penetrants, safeners, spreaders, stickers, surfactants, thickeners, and wetting agents).

A composition, in some embodiments, include a mixture of an RNAi molecule targeting ROP and at least one of a variety of agricultural chemicals, insecticides, miticides, fungicides, pesticidal agents and/or biopesticidal (e.g., microbial, PIP, and/or biochemical) agents, such as Spiromesifen, Spirodiclofen, Spirotetramat, Pyridaben, Tebufenpyrad, Tolfenpyrad,

Fenpyroximate, Flufenerim, Pyrimidifen, Fenazaquin, Rotenone, Cyenopyrafen,

Hydramethylnon, Acequinocyl, Fluacrypyrim, Aluminium phosphide, Calcium phosphide, Phosphine, Zinc phosphide, Cyanide, Diafenthiuron, Azocyclotin, Cyhexatin, Fenbutatin oxide, Propargite, Tetradifon, Bensultap, Thiocyclam, Thiosultap-sodium, Flonicamid, Etoxazole, Clofentezine, Diflovidazin, Hexythiazox, Chlorfluazuron, Bistrifluron, Diflubenzuron,

Flucycloxuron, Flufenoxuron, Hexaflumuron, Lufenuron, Novaluron, Noviflumuron,

Teflubenzuron, Triflumuron, Buprofezin, Cyromazine, Hydroprene, Kinoprene, Methoprene, Fenoxycarb, Pyriproxyfen, Pymetrozine, Pyrifluquinazon, Chlorfenapyr, Tralopyril, methyl bromide and/or other alkyl halides, Chloropicrin, Sulfuryl fluoride, Benclothiaz,

Chinomethionat, Cryolite, Methylneodecanamide, Benzoximate, Cymiazole, Fluensulfone, Azadirachtin, Bifenazate, Amidoflumet, Dicofol, Plifenate, Cyflumetofen, Pyridalyl, Beauveria bassiana GHA, Sulfoxaflor, Spinetoram, Spinosad, Spinosad, Emamectin benzoate, Lepimectin, Milbemectin, Abamectin, Methoxyfenozide, Chromafenozide, Halofenozide, Tebufenozide, Amitraz, Chlorantraniliprole, Cyantraniliprole, Flubendiamide, alpha-endosulfan, Chlordane, Endosulfan, Fipronil, Acetoprole, Ethiprole, Pyrafluprole, Pyriprole, Indoxacarb,

Metaflumizone, Acrinathrin, Allethrin, Allethrin-cis-trans, Allethrin-trans, beta-Cyfluthrin, beta- Cypermethrin, Bifenthrin, Bioallethrin, Bioallethrin S-cyclopentenyl, Bioresmethrin,

Cycloprothrin, Cyfluthrin, Cyhalothrin, Cypermethrin, Cyphenothrin [(lR)-trans-isomers], Dimefluthrin, Empenthrin [(EZ)-(lR)-isomers], Esfenvalerate, Etofenprox, Fenpropathrin, Fenvalerate, Flucythrinate, Flumethrin, Gamma-cyhalothryn, lambda-Cyhalothrin,

Meperfluthrin, Metofluthrin, Permethrin, Phenothrin [(lR)-trans-isomer], Prallethrin,

Profluthrin, Protrifenbute, Resmethrin, Silafluofen, tau-Fluvalinate, Tefluthrin, Tetramethrin, Tetramethrin [(lR)-isomers], Tetramethylfluthrin, theta-Cypermethrin, Tralomethrin,

Transfluthrin, zeta-Cypermethrin, alpha-Cypermethrin, Deltamethrin, DDT, Methoxychlor, Thiodicarb, Alanycarb, Aldicarb, Bendiocarb, Benfuracarb, Butoxycarboxim, Carbaryl, Carbofuran, Carbosulfan, Ethiofencarb, Fenobucarb, Formetanate, Furathiocarb, Isoprocarb, Methiocarb, Methomyl, Metolcarb, Oxamyl, Pirimicarb, Propoxur, Thiofanox, Triazamate, Trimethacarb, XMC, Xylylcarb, Chlorpyrifos, Malathion, Acephate, Azamethiphos, Azinphos- ethyl, Azinphos-methyl, Cadusafos, Chlorethoxyfos, Chlorfenvinphos, Chlormephos,

Chlorpyrifos-methyl, Coumaphos, Cyanophos, Demeton-S -methyl, Diazinon,

Dichlorvos/DDVP, Dicrotophos, Dimethoate, Dimethylvinphos, Disulfoton, EPN, Ethion, Ethoprophos, Famphur, Fenamiphos, Fenitrothion, Fenthion, Fonofos, Fosthiazate, Imicyafos, Isofenphos-methyl, Mecarbam, Methamidophos, Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate, Oxydemeton-methyl, Parathion, Parathion-methyl, Phenthoate, Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim, Pirimiphos-ethyl, Profenofos, Propaphos, Propetamphos, Prothiofos, Pyraclofos, Pyridaphenthion, Quinalphos, Sulfotep, Tebupirimfos, Temephos, Terbufos, Tetrachlorvinphos, Thiometon, Triazophos, Trichlorfon, Vamidothion Imidacloprid, Thiamethoxam, Acetamiprid, Clothianidin, Dinotefuran, Nitenpyram, Nithiozine, Nicotine, Thiacloprid, cyantraniliprole, carbamates, organophosphates, cyclodiene

organochlorines, phenylpyrazoles (fiproles), pyrethroids, pyrethins, DDT Methoxychlor, Neonicotinoids, Nicotine, Sulfoximines, Butenolides, Mesoionics, Spinosyns, Avermectins, Milbernycins, Juvenile hormone analogues, Fenoxycarb, Pyriproxyfen, Alkyl halides,

Chloropicrin, Fluorides, Borates, Tarter emetic, Methyl isothiocyanate generators, Pyridine azomethine derivatives, Pyropenes, Clofentezine, Diflovidazin, Hexythiazox, Etoxazole, Diafenthiuron, Organotin miticides, Propargite, Tetradifon, Pyrroles, Dinitrophenols,

Sulfuramid, Nereistoxin analogues, Benzoylureas, Buprofezin, Cyromazine, Diacylhydrazines, Amitraz, Hydramethylnon, Acequinocyl, Fluacrypyrim, Bifenazate, METI acaricides and insecticides, Rotenone, Oxadiazines, Semicarbazones, Tetronic and Tetramic acid derivatives, Phosphides, Cyanides, Beta-ketonitrile derivatives, Carboxanilides, Diamides, Flonicamid, Meta-diamides Isoxazolines, Granuloviruses (GVs), Nucleopolyhedroviruses (NPVs), GS- omega/kappa HXTX-Hvla peptide, Azadirachtin, Benzoximate, Bromopropylate,

Chinomethionat, Dicofol, Lime sulfur, Mancozeb, Pyridalyl, Sulfur, Benzimidazoles,

Dicarboximides, Pyridines, Pyrimidines, Triazoles, Acylalanines, Pyridine carboxamides, Anilino-pyrimidines, Quinone outside Inhibitors (Qol- fungicides), Phenylpyrroles, Quinolines, Hydroxyanilides, Toluamides, Cyanoacetamide-oximes, Dinitrophenyl crotonates,

Phosphonates, Carboxylic Acid Amides (CAA-fungicides), Ml inorganic, M2 inorganic, M3 dithiocarbamates, M4 phthalimides, paraffinic oil, petroleum-based horticultural oils, palmitic oil, steric oil, linoleic oil, oleic oils, canola oil, soybean oil, oregano oil, tagetes oil, balsam fir oil, thyme oil, black pepper oil, mint oil, cedarwood oil, fish oil, jojoba oil, lavadin oil, castor oil, eucalyptus oil, ocimum oil, patchouli oil, citrus oil, artemisia oil, camphor oil, wintergreen oil, methyl eugenol oil, thymol oil, geranium oil, sesame oil, linseed oil, cottonseed oil, lemongrass oil, bergamot oil, mustard oil, orange oil, citronella oil, tea tree oil, neem oil, garlic oil, Bacillus sphaericus, Bacillus thuringiensis (e.g., Bacillus thuringiensis var. aizawai, Bacillus

thuringiensis var. israelensis, Bacillus thuringiensis var. kurstaki, Bacillus thuringiensis var. sphaericus , Bacillus thuringiensis var. tenebrionensis) and the insecticidal proteins they produce (e.g., CrylAb, Cry 1 Ac, CrylFa, Cry 1 A.105, Cry2Ab, Vip3A, mCry3A, Cry3Ab, Cry3Bb, Cry34Abl/Cr35Abl), Paenibacillus popilliae, Serratia entomophila, nuclear polyhedrosis viruses, granulosis viruses, non-occluded baculoviruses, Beauveria spp,

Metarhizium, Entomophaga, Zoopthora, Paecilomyces fumosoroseus, Normuraea, Lecanicillium lecanii, Nosema, Thelohania, Vairimorpha, Steinernema spp, Heterorhabditis spp or any combination thereof, which may further comprise an active ingredient selected from the group consisting of azinphos-methyl, acephate, isoxathion, isofenphos, ethion, etrimfos, oxydemeton- methyl, oxydeprofos, quinalphos, chlorpyrifos, chlorpyrifos-methyl, chlorfenvin phos, cyanophos, dioxabenzofos, dichlorvos, disulfoton, dimethylvinphos, dimethoate, sulprofos, diazinon, thiometon, tetrachlorvinphos, temephos, tebupirimfos, terbufos, naled, vamidothion, pyraclofos, pyridafen thion, pirimiphos-methyl, fenitrothion, fenthion, phenthoate,

flupyrazophos, prothiofos, propaphos, profenofos, phoxime, phosalone, phosmet, formothion, phorate, malathion, mecarbam, mesulfenfos, methamidophos, methidathion, parathion, methyl parathion, monocrotophos, trichlorphon, EPN, isazophos, isamidofos, cadusafos, diamidaphos, dichlofenthion, thionazin, fenamiphos, fosthiazate, fosthietan, phosphocarb, DSP, ethoprophos, alanycarb, aldicarb, isoprocarb, ethiofen carb, carbaryl, carbosulfan, xylylcarb, thiodicarb, pirimicarb, fenobucarb, furathiocarb, propoxur, ben diocarb, benfuracarb, methomyl, metolcarb, XMC, carbofuran, aldoxycarb, oxamyl, acrin athrin, allethrin, esfenvalerate, empenthrin, cycloprothrin, cyhalothrin, gamma-cyhalothrin, lambda-cyhalothrin, cyfluthrin, beta-cyfluthrin, cypermethrin, alpha-cypermethrin, zeta-cyper-methrin, silafluofen, tetramethrin, tefluthrin, deltamethrin, tralomethrin, bifenthrin, phenothrin, fenvalerate, fenpropathrin, furamethrin, prallethrin, flucythrinate, fluvalinate, flubrocythrinate, permethrin, resmethrin, ethofenprox, cartap, thiocyclam, ben sultap, acetamiprid, imidacloprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, nitenpyram, chlorfluazuron, difluben zuron, teflubenzuron, triflumuron, novaluron, noviflumuron, bistrifluoron, fluazuron, flucy-cloxuron, flufenoxuron, hexaflumuron, lufenuron, chromafen ozide, tebufenozide, halofen ozide, methoxyfen ozide, diofen olan, cyromazin e, pyriproxyfen, buprofezin, methop-rene, hydroprene, kinoprene, triazamate, endosulfan, chlorfenson, chlorobenzilate, dicofol, bromopropylate, acetoprole, flpronil, ethiprole, pyrethrin, rotenone, nicotinesulphate, spinosad, , finpronil, spirotetramat abamectin, acequinocyl, amidoflumet, amitraz, etoxazole, chinomethionat, clofentezine, fenbutatin oxide, dienochlor, cyhexatin, spirodiclofen, spiromesifen, tetradifon, tebufenpyrad, binapacryl, bifenazate, pyridaben, pyrimidifen, fenazaquin, fenothiocarb,fenpyroximate, fluacrypyrim,flu- azinam, flufenzin, hexythiazox, propargite, polynactin complex, milbemectin, lufenuron, mecarbam, methiocarb,mevinphos,halfenprox,azadirachtin,diafenthiuron, indoxacarb, emamectin benzoate, potassium oleate, sodium oleate, chlorfenapyr, tolfenpyrad, pymetrozine,

fenoxycarb,hydramethylnon, hydroxy propyl starch, pyridalyl, flufenerim, flubendiamide, flonicamid, metaflumizole, lepimectin, TPIC, albendazole, oxibendazole, oxfendazole, trichlamide,fensulfothion,fenbendazole,levamisole hydrochloride, morantel tartrate, dazomet, metam-sodium, tri- adimefon, hexaconazole, propiconazole, ipconazole, prochloraz, triflumizole, tebuconazole, epoxiconazole, difenoconazole, flusilazole, triadimenol, cyproconazole, metconazole,fluquinconazole,bitertanol,tetraconazole,triti- conazole, flutriafol,penconazole, diniconazole, fenbuconazole, bromuconazole, imibenconazole, simeconazole, myclobutanil, hymexazole, imazalil, furametpyr, thifluzamide, etridiazole, oxpoconazole, oxpoconazole fumarate, pefurazoate, prothioconazole, pyrifenox, fenarimol, nuari- mol, bupirimate, mepanipyrim, cyprodinil, pyrimethanil, metalaxyl, mefenoxam, oxadixyl, benalaxyl,

thiophanate, thiophanate-methyl, benomyl, carbendazim, fuberidazole, thiabendazole, manzeb, propineb, zineb, metiram, maneb, ziram, thiuram, chlorothalonil, ethaboxam, oxycarboxin, carboxin, flutolanil, silthiofam, mepronil, dimethomorph, fenpropidin, fenpropimorph, spiroxamine, tridemorph, dodemorph, flumorph, azoxystrobin, kresoxim-methyl,

metominostrobin, orysastrobin, fluoxastrobin, trifloxystrobin, dimoxystrobin, pyraclostrobin, picoxystrobin, iprodione, procymidone, vinclozolin, chlozolinate, flusulfamide, dazomet, methyl isothiocyanate, chloropicrin, methasulfocarb, hydroxyisoxazole, potassium hydroxyisoxazole, echlomezol, D-D, carbarn, basic copper chloride, basic copper sulfate, copper nonylphenolsulfonate, oxine copper, DBEDC, anhydrous copper sulfate, copper sulfate pentahydrate, cupric hydroxide, inorganic sulfur, wettable sulfur, lime sulfur, zinc sulfate, fentin, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hypochlorite, silver, edifenphos, tolclofos-methyl, fosetyl, iprobenfos, dinocap, pyrazophos, carpropamid, fthalide, tricyclazole, pyroquilon, diclocymet, fenoxanil, kasugamycin, validamycin, polyoxins, blasticiden S, oxytetracycline, mildiomycin, streptomycin, rape seed oil, machine oil, benthiavalicarbisopropyl, iprovalicarb, propamocarb, diethofencarb, fluoroimide, fludioxanil, fenpiclonil, quinoxyfen, oxolinic acid, chlorothalonil, captan, folpet, probenazole, acibenzolar-S- methyl, tia-dinil, cyflufenamid, fenhexamid, diflumetorim, metrafenone, picobenzamide, proquinazid, famoxadone, cyazofamid, fenamidone, zoxamide, boscalid, cymoxanil, dithianon, fluazinam, dichlofluanide, triforine, isoprothiolane, ferimzone, diclomezine, tecloftalam, pencycuron, chinomethionat, iminoctadine acetate, iminoctadine albesilate, ambam,

polycarbamate, thiadiazine, chloroneb, nickel dimethyldithiocarbamate, guazatine,

dodecylguanidine acetate, quintozene, tolylfluanid, anilazine, nitrothalisopropyl, fenitropan, dimethirimol, benthiazole, flumetover, mandipropamide, and penthiopyrad, or any combinations thereof.

In some embodiments, an RNAi molecule targeting ROP is supplied in the diet of a Coleopteran insect. For example, an RNAi molecule targeting ROP may be applied topically to a plant, or seeds (e.g. via soaking, coating, dusting or spraying), or cells of a plant may be engineered to express the RNAi molecule. RNAi molecules may also be supplied in another food or water source.

The plant may be any plant that is subject to infestation by a Coleopteran insect. In some embodiments, the plant is a Solanaceous plant (e.g., family Solanaceae). Examples of

Solanaceous plants include, but are not limited to, potato plants (Solanum tuberosum), buffalo bur plants (Solanum rostratum), eggplant plants (Solanum melongena ), tomato plants (Solanum lycopersicum), tobacco plants (Nicotiana tabacum), pepper plants (Capsicum annum) and woody nightshade plants (Solanum dulcamara).

Thus, in some embodiments, the methods comprise delivering to a plant (e.g., a potato plant) with an RNAi molecule targeting ROP, for example, in an effective amount to suppress infestation of the plant by a Coleopteran insect (e.g., Colorado potato beetle). In other embodiments, the methods comprise delivering to a buffalo bur plant with an RNAi molecule targeting ROP, for example, in an effective amount to suppress infestation of the plant by a Coleopteran insect (e.g., Colorado potato beetle). In yet other embodiments, the methods comprise delivering to an eggplant plant with an RNAi molecule targeting ROP, for example, in an effective amount to suppress infestation of the plant by a Coleopteran insect (e.g., Colorado potato beetle). In still other embodiments, the methods comprise delivering to a tomato plant with an RNAi molecule targeting ROP, for example, in an effective amount to suppress infestation of the plant by a Coleopteran insect ( e.g ., Colorado potato beetle). In further embodiments, the methods comprise delivering to a tobacco plant with an RNAi molecule targeting ROP, for example, in an effective amount to suppress infestation of the plant by a Coleopteran insect (e.g., Colorado potato beetle). In additional embodiments, the methods comprise delivering to a pepper plant with an RNAi molecule targeting ROP, for example, in an effective amount to suppress infestation of the plant by a Coleopteran insect (e.g., Colorado potato beetle).

Delivering to a plant (e.g., a part of a plant) and/or Coleopteran insect an RNAi molecule targeting ROP may include, for example, applying (e.g., soaking, coating, or dusting) the RNAi molecule or a composition comprising the RNAi molecule topically to any portion of a plant (e.g., roots, tubers, stem, branches, leaves, flower, etc), ground (e.g., soil, dirt, grass, etc.), insect and/or diet of the insect. A delivering step may also include genetically engineering cells of a plant to express the RNAi molecule. A delivering step may also include exposing a plant or Coleopteran insect to an organism (e.g., virus, bacteria, fungus, etc.) that has been genetically engineered to express and/or deliver the RNAi molecule to the plant or Coleopteran insect.

An effective amount is the amount of an RNAi molecule targeting ROP required to confer a beneficial effect on infestation (e.g. death, cessation of feeding, inhibition of growth, development or reproduction) by a Coleopteran insect, either alone or in combination with one or more other additives. Beneficial effects include a reduction in infestation, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, relative to a control. In some embodiments, the control is the absence of an insecticide and/or pesticide. In some embodiments, an effective amount of an RNAi molecule targeting ROP completely eliminates Coleopteran insect (e.g., Colorado potato beetle) infestation of a plant.

Effective amounts vary, as recognized by those skilled in the art, depending on the particular plant, the severity of the infestation, the duration of the infestation, previous exposure to insecticides and like factors within the knowledge and expertise of a practitioner. These factors are well known to those of ordinary skill in that art and can be addressed with no more than routine experimentation. It is generally preferred that lower effective concentrations be used, that is, the lowest concentration that provides control of an insect, to increase efficiency and decrease cost.

An effective amount of an RNAi molecule targeting ROP may also vary depending on the method of delivery. In some embodiments, an effective amount of an RNAi molecule targeting ROP is expressed as micrograms (m g) of RNAi molecule targeting ROP per centimeter squared (cm²) of a surface of a plant or ground ( e.g ., soil, dirt, grass, etc.), i.e., pg/cm². Thus, in some

embodiments, an effective amount of an RNAi molecule targeting ROP comprises 0.001 pg/cm² to 10 pg/cm². In some embodiments, an effective amount of an RNAi molecule targeting ROP comprises 0.001 pg/cm² to 9 pg/cm², 0.001 pg/cm² to 8 pg/cm², 0.001 pg/cm² to 7 pg/cm²,

0.001 pg/cm² to 6 pg/cm², 0.001 pg/cm² to 5 pg/cm², 0.001 pg/cm² to 4 pg/cm², 0.001 pg/cm² to 3 pg/cm², 0.001 pg/cm² to 2 pg/cm², 0.001 pg/cm² to 1 pg/cm², 0.001 pg/cm² to 0.1 pg/cm², or 0.001 pg/cm² to 0.01 pg/cm². In some embodiments, an effective amount of an RNAi molecule targeting ROP comprises 0.01 pg/cm² to 10 pg/cm², 0.1 pg/cm² to 10 pg/cm², 1 pg/cm² to 10 pg/cm², 2 pg/cm² to 10 pg/cm², 3 pg/cm² to 10 pg/cm², 4 pg/cm² to 10 pg/cm², 5 pg/cm² to 10 pg/cm², 6 pg/cm² to 10 pg/cm², 7 pg/cm² to 10 pg/cm², 8 pg/cm² to 10 pg/cm², or 9 pg/cm² to 10 pg/cm².

In some embodiments, an effective amount of an RNAi molecule targeting ROP is expressed as grams (g) of RNAi molecule targeting ROP per acre (ac.) of a surface of a plant or ground (e.g., soil, dirt, grass, etc.), i.e., g/ac. Thus, in some embodiments, an effective amount of an RNAi molecule targeting ROP comprises 0.01 g/ac. to 100 g/ac. In some embodiments, an effective amount of an RNAi molecule targeting ROP comprises 0.01 g/ac. to 90 g/ac., 0.01 g/ac. to 80 g/ac., 0.01 g/ac. to 70 g/ac., 0.01 g/ac. to 60 g/ac., 0.01 g/ac. to 50 g/ac., 0.01 g/ac. to 40 g/ac., 0.01 g/ac. to 30 g/ac., 0.01 g/ac. to 20 g/ac., 0.01 g/ac. to 10 g/ac., 0.01 g/ac. to 1 g/ac., or 0.01 g/ac. to 0.1 g/ac. In some embodiments, an effective amount of an RNAi molecule targeting ROP comprises 0.1 g/ac. to 100 g/ac., 1 g/ac. to 100 g/ac., 10 g/ac. to 100 g/ac., 20 g/ac. to 100 g/ac., 30 g/ac. to 100 g/ac., 40 g/ac. to 100 g/ac., 50 g/ac. to 100 g/ac., 60 g/ac. to 100 g/ac., 70 g/ac. to 100 g/ac., 80 g/ac. to 100 g/ac., or 90 g/ac. to 100 g/ac.

In some embodiments, the effectiveness of an RNAi molecule to control Coleopteran insects can be determined using the ability of the RNAi molecule to kill or cause death of an insect or population of insects. The rate of death in a population of insects may be determined by percent mortality (e.g., percent mortality over time). Generally, percent mortality of a population of insects reflects the percentage of insects in said population that have died as a result of the RNAi molecule (e.g., 75% mortality indicates that an RNAi molecule has killed 75% of the total insect population). In some embodiments, percent mortality is measured over time (e.g., over the course of a multi-day exposure of insects to an RNAi molecule). In some embodiments, percent mortality is measured after at least 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 days of exposure. In some embodiments, an RNAi molecule causes a percent mortality of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of a Coleopteran insect population. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of a Coleopteran insect population are killed by an RNAi molecule that targets ROP. In some embodiments, percent mortality of an RNAi molecule is compared to a control ( e.g ., a control molecule or untreated conditions). In some embodiments, percent mortality of an RNAi molecule is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200% higher than a control (e.g., a control molecule or untreated conditions).

In some embodiments, the effectiveness of an RNAi molecule to control Coleopteran insects can be determined using the ability of the RNAi molecule to limit the leaf disc consumption of a Coleopteran insect or an insect population. Leaf disc consumption refers to the amount (e.g., percentage) of plant material (e.g., an eggplant leaf) that is consumed or eaten by an insect or population of insects. In some embodiments, an RNAi molecule causes at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in the leaf disc consumption by an insect or population of insects. In some embodiments, the ability of an RNAi molecule to decrease leaf disc consumption is compared relative to a control (e.g., a control molecule or untreated conditions). In some embodiments, leaf disc consumption is measured over time (e.g., over the course of a multi-day exposure of insects to an RNAi molecule). In some embodiments, leaf disc consumption is measured after 3, 4, 5, 6, 7, 8, 9, 10, or more days of exposure.

In some embodiments, the effectiveness of an RNAi molecule to control Coleopteran insects can be determined using the ability of the RNAi molecule to decrease percent plant defoliation by a Coleopteran insect or an insect population. Percent plant defoliation refers to the percentage of plant material (e.g., an eggplant leaf) that is destroyed (e.g., consumed) by an insect or population of insects. In some embodiments, an RNAi molecule causes at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in the percent plant defoliation by an insect or population of insects. In some embodiments, an RNAi molecule causes percent plant defoliation to decrease below 40%, 30, 25%, 20%, 15%, 10%, 5%, 3%, or 1%. In some embodiments, percent plant defoliation remains below 40%, 30, 25%, 20%, 15%, 10%, 5%, 3%, or 1% for at least 5, 6, 7, 8, 9, 10, 15, or 20 days following exposure of insects to an RNAi molecule. In some embodiments, the ability of an RNAi molecule to decrease percent plant defoliation is compared relative to a control (e.g., a control molecule or untreated conditions). In some embodiments, percent plant defoliation is measured over time (e.g., over the course of a multi-day exposure of insects to an RNAi molecule). In some embodiments, percent plant defoliation is measured after 3, 4, 5, 6, 7, 8, 9, 10, or more days of exposure.

In some embodiments, an RNAi molecule targeting ROP may be formulated in a solution (e.g., that is applied to a surface of the Coleopteran insect and/or diet (e.g., food and/or water ingested), a plant or ground ( e.g ., soil, dirt, grass, etc.)). In some embodiments, the effective amount of the RNAi molecule targeting ROP in the solution is expressed as nanograms (ng) or micrograms (pg) of RNAi molecule targeting ROP per milliliter (ml) of the solution, i.e., ng/ml. Thus, in some embodiments, a solution comprises an RNAi molecule targeting ROP at a concentration of 10 ng/ml to 100 pg/ml. In some embodiments, a solution comprises an RNAi molecule targeting ROP at a concentration of 10 ng/ml to 100 pg/ml, 100 ng/ml to 100 pg/ml, 250 ng/ml to 100 pg/ml, 750 ng/ml to 100 pg/ml, 1000 ng/ml to 100 pg/ml, 10 pg/ml to 100 pg/ml, 25 pg/ml to 100 pg/ml, 50 pg/ml to 100 pg/ml, or 75 pg/ml to 100 pg/ml. In some embodiments, a solution comprises an RNAi molecule targeting ROP at a concentration of 10 ng/ml to 100 pg/ml, 10 ng/ml to 75 pg/ml, 10 ng/ml to 50 pg/ml, 10 ng/ml to 25 pg/ml, 10 ng/ml to 10 pg/ml, 10 ng/ml to 1000 ng/ml, 10 ng/ml to 1000 ng/ml, 10 ng/ml to 750 ng/ml, 10 ng/ml to 500 ng/ml, 10 ng/ml to 250 ng/ml, 10 ng/ml to 100 ng/ml, 10 ng/ml to 75 ng/ml, 10 ng/ml to 50 ng/ml, or 10 ng/ml to 25 ng/ml.

A solution, in some embodiments, comprises an RNAi molecule targeting ROP and at least one additional additive (e.g., a pesticide, surfactant or other non-pesticidal agent). In some embodiments, such a mixture comprises an RNAi molecule targeting ROP at a concentration of 0.0001 pg/ml to 10 pg/ml (e.g., that is applied to a surface of a plant and/or ground (e.g., soil, dirt, grass, etc.)). In some embodiments, such a mixture comprises an RNAi molecule targeting ROP at a concentration of 0.001 pg/ml to 10 pg/ml, 0.01 pg/ml to 10 pg/ml, 0.1 pg/ml to 10 pg/ml, 1 pg/ml to 10 pg/ml, 2 pg/ml to 10 pg/ml, 3 pg/ml to 10 pg/ml, 4 pg/ml to 10 pg/ml, 5 pg/ml to 10 pg/ml, 6 pg/ml to 10 pg/ml, 7 pg/ml to 10 pg/ml, 8 pg/ml to 10 pg/ml, or 9 pg/ml to 10 pg/ml. In some embodiments, such a mixture comprises an RNAi molecule targeting ROP at a concentration of 0.0001 pg/ml to 9 pg/ml, 0.0001 pg/ml to 8 pg/ml, 0.0001 pg/ml to 7 pg/ml, 0.0001 pg/ml to 6 pg/ml, 0.0001 pg/ml to 5 pg/ml, 0.0001 pg/ml to 4 pg/ml, 0.0001 pg/ml to 3 pg/ml, 0.0001 pg/ml to 2 pg/ml, 0.0001 pg/ml to 1 pg/ml, 0.0001 pg/ml to 0.1 pg/ml, 0.0001 pg/ml to 0.01 pg/ml, or 0.0001 pg/ml to 0.001 pg/ml.

In some embodiments, an RNAi molecule targeting ROP is provided in a diet of an insect. Thus, in some embodiments, an effective amount of an RNAi molecule targeting ROP is expressed as micrograms (pg) of RNAi molecule targeting ROP per milliliter (ml) of the diet of the insect, i.e., pg/ml. In some embodiments, the diet of an insect comprises an RNAi molecule targeting ROP at a concentration of 0.001 pg/ml to 10 pg/ml. In some embodiments, the diet of an insect comprises an RNAi molecule targeting ROP at a concentration of 0.001 pg/ml to 9 pg/ml, 0.001 pg/ml to 8 pg/ml, 0.001 pg/ml to 7 pg/ml, 0.001 pg/ml to 6 pg/ml, 0.001 pg/ml to 5 pg/ml, 0.001 pg/ml to 4 pg/ml, 0.001 pg/ml to 3 pg/ml, 0.001 pg/ml to 2 pg/ml, 0.001 pg/ml to 1 pg/ml, 0.001 pg/ml to 0.1 pg/ml, or 0.001 us/ml to 0.01 pg/ml. In some embodiments, the diet of an insect comprises an RNAi molecule targeting ROP at a concentration of 0.01 pg/ml to 10 pg/ml, 0.1 pg/ml to 10 pg/ml, 1 pg/ml to 10 pg/ml, 2 pg/ml to 10 pg/ml, 3 pg/ml to 10 pg/ml, 4 pg/ml to 10 pg/ml, 5 pg/ml to 10 pg/ml, 6 pg/ml to 10 pg/ml, 7 pg/ml to 10 pg/ml, 8 pg/ml to 10 pg/ml, or 9 pg/ml to 10 pg/ml.

The step of delivering to any portion of a plant ( e.g ., roots, tubers, stem, branches, leaves, flower, etc), ground (e.g., soil, dirt, grass, etc.), insect and/or diet of the insect with an RNAi molecule targeting ROP may include a single application (single contact) or multiple

applications (multiple contacts) of the RNAi molecule targeting ROP to the plant, ground (e.g., soil, dirt, grass, etc.), insect and/or diet of the insect. Delivery to a portion of a plant, insect and/or diet of the insect may be in the form of a spray (e.g., pressurized/aerosolized spray, pump) solid, (e.g. powder, pellet, bait), or liquid (e.g., homogeneous mixtures such as solutions and non-homogeneous mixtures such as suspensions (water and oil based), colloids, micelles, and emulsions). The period of time of contact may vary. In some embodiments, delivering comprises an exposure of an RNAi molecule targeting ROP with a portion of a plant and/or Coleopteran insect for a suitable period sufficient for reduction of growth, reproduction (e.g., fertility and/or fecundity), and/or feeding of the Coleopteran insect and/or death of the Coleopteran insect, if any.

In some embodiments, delivery of an RNAi molecule targeting ROP with a plant and/or Coleopteran insect is followed by ingestion and/or absorption of the RNAi molecule targeting ROP by the plant and/or Coleopteran insect. In some embodiments, ingestion of the RNAi molecule targeting ROP by the Coleopteran insect alters a biological function of the Coleopteran insect, thereby controlling infestation by the Coleopteran insect. Examples of altered biological function of the Coleopteran insect include, but are not limited to, reduced growth, reduced reproduction (e.g., fertility and/or fecundity), reduced feeding, decreased movement, decreased development, decreased cellular repair, and/or increased mortality.

In some embodiments, delivering comprises applying an RNAi molecule targeting ROP to a portion of the surface of a plant and/or a surface contacted by a Coleopteran insect (e.g., ground (e.g., soil, dirt, grass, etc.)). In some embodiments, applying an RNAi molecule targeting ROP to a portion of a surface comprises spraying, coating, and/or dusting the surface or portion thereof. In some embodiments, applying an RNAi molecule targeting ROP RNA to a portion of a surface comprises ground drenching or applying the RNAi molecule as a granulated or powdered formulation to the soil adjacent to the roots of the plant .

A RNAi molecule targeting ROP may be applied to any portion of a plant (e.g., roots, tubers, stem, branches, leaves, flower, etc). In some embodiments, the RNAi molecule targeting ROP is contacted with an above-ground portion of a plant (e.g., a leaf) and/or with a below- ground portion of a plant ( e.g ., a root), which may include at least one in furrow formulation selected from the group consisting of a powder, granule, pellet, capsule, soluble liquid concentrate, spray(after dilution or concentrate), fog, in furrow, seed treatment, insect diet, bait, drench, drip irrigation, or any other forms suited for applying to a furrow. Portions of a plant that may be contacted with the RNAi molecule targeting ROP described herein include, but are not limited to, leaf, stem, flower, fruit, shoot, root, seed, tuber, anther, stamen, or pollen. In some embodiments, RNAi is delivered mechanically, through high pressure spray or sand blasting.

In some embodiments, delivering comprises providing an RNAi molecule targeting ROP for dietary uptake by the Coleopteran insect. In some embodiments, contacting comprises providing an RNAi molecule targeting ROP that can be ingested or otherwise absorbed internally by the Coleopteran insect. In some embodiments, the RNAi molecule targeting ROP is provided in a diet for dietary uptake by the Coleopteran insect. In some embodiments, the RNAi molecule targeting ROP is provided in/on a plant or plant part, or topically applied to a plant or plant part (e.g., soaking, coating, dusting). In some embodiments, the RNAi molecule targeting ROP is expressed in a plant or plant part.

In some embodiments, delivering an RNAi molecule targeting ROP to a Coleopteran insect inhibits expression of (reduces or inhibits expression of) an endogenous complementary nucleotide sequence (e.g. , RNA sequence) in the Coleopteran insect. In some embodiments, the endogenous complementary nucleotide sequence is an endogenous ROP sequence.

Consequences of inhibition can be confirmed by any appropriate assay to evaluate one or more properties of an insect, or by biochemical techniques that evaluate molecules indicative of ROP expression (e.g., RNA, protein). In some embodiments, the extent to which an RNAi molecule targeting ROP provided herein reduces levels of expression of ROP is evaluated by comparing expression levels (e.g., mRNA or protein levels of ROP to an appropriate control (e.g., a level of ROP expression in a cell or population of cells to which an RNAi molecule targeting ROP has not been delivered or to which a negative control has been delivered). In some embodiments, an appropriate control level of ROP expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

In some embodiments, delivering an RNAi molecule targeting ROP as described herein results in a reduction in the level of ROP expression in a cell of an insect. In some embodiments, the reduction in levels of ROP expression may be a reduction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% relative to a control level. In some embodiments, the control level is a level of ROP expression in a similar insect cell (or average level among a population of cells) not contacted with the RNAi molecule. In some embodiments, the control level is a level of ROP expression in a similar insect cell (or average level among a population of cells) contacted with an RNAi molecule targeting a gene not expressed by the insect cell, e.g., green fluorescent protein (GFP).

In some embodiments, the effect of delivering to a cell or insect an RNAi molecule targeting ROP is assessed after a finite period of time. For example, levels of ROP may be determined in a cell or insect at least 4 hours, 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after delivering to the cell or insect the RNAi molecule targeting ROP.

In some embodiments, delivery of an RNAi molecule targeting ROP as described herein results in a reduction in the level of growth, reproduction (e.g., fertility and/or fecundity), and/or feeding of an insect. In some embodiments, the reduction in levels of growth, reproduction (e.g., fertility and/or fecundity), and/or feeding may be a reduction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% relative to a control level. In some embodiments, the control level is a level of growth, reproduction (e.g., fertility and/or fecundity), and/or feeding of a similar insect not contacted with the RNAi molecule. In some embodiments, the control level is a level of growth, reproduction (e.g., fertility and/or fecundity), and/or feeding of a similar insect contacted with an RNAi molecule targeting a gene not expressed by the insect cell, e.g., green fluorescent protein (GFP).

In some embodiments, delivery of an RNAi molecule targeting ROP as described herein results in an increase in mortality among a population of insects. In some embodiments, the increase in level of mortality may be an increase by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% relative to a control. In some embodiments, the control is mortality among a population of insects not contacted with the RNAi molecule. In some embodiments, the control is among a population of insects contacted with an RNAi molecule targeting a gene not expressed by the insect cell, e.g., green fluorescent protein (GFP).

Aspects of the present disclosure provide plants that expresses an RNAi molecule targeting ROP as described herein. In some embodiments, DNA encoding an RNAi molecule targeting ROP provided herein is provided to a plant (seed or cells of a plant) such that the plant expresses the RNAi molecule targeting ROP. In some embodiments, DNA encoding an RNAi molecule targeting ROP is expressed in a plant by transgenic expression, e.g., by stably integrating DNA encoding an RNAi molecule targeting ROP into a genome of a plant such that the plant expresses the RNAi molecule targeting ROP.

Methods of Producing RNAi Molecules Targeting ROP

RNAi molecules targeting ROP as provided herein may be produced by any suitable method known in the art. Examples of methods for producing an RNAi molecule targeting ROP include, but are not limited to, in vitro transcription (IVT), chemical synthesis, expression in an organism ( e.g ., a plant), or expression in cell culture (e.g., a plant cell culture), and microbial fermentation.

RNAi molecules targeting ROP may be produced, in some embodiments, according to cell-free production methods described in International Application Publication WO

2017/176963 Al, published October 12, 2017, entitled“Cell-Free Production of Ribonucleic Acid”; U.S. Provisional Application U.S.S.N. 62/571,071 filed October 11, 2017, entitled “Methods and Compositions for Nucleoside Triphosphate and Ribonucleic Acid Production”; and International Application Publication WO 2019/075167 Al, published April 18, 2019, entitled“Methods and Compositions for Nucleoside Triphosphate and Ribonucleic Acid

Production”; each of which is incorporated herein by reference.

Any suitable DNA encoding RNAi molecules targeting ROP described herein may be used in the methods described herein. A DNA may be a single- stranded DNA (ssDNA) or a double-stranded DNA (dsDNA). In some embodiments, a DNA comprises one or more DNA expression cassette(s) that when transcribed produces a single- stranded RNA (ssRNA) molecule (e.g., that remains single stranded or folds into an RNA hairpin) or complementary ssRNA molecules that anneal to produce the double-stranded RNA (dsRNA) molecule.

In some embodiments, a DNA comprises a promoter (e.g., an inducible promoter) operably linked to a nucleotide sequence encoding RNA that is complementary to a segment of ROP, and optionally a terminator. In other embodiments, a DNA comprises a first promoter (e.g., an inducible promoter) operably linked to a nucleotide sequence encoding RNA that is complementary to a segment of ROP, and optionally a terminator, and a second promoter (e.g., an inducible promoter) operably linked to a nucleotide sequence encoding a second RNA that is complementary to the first RNA, and optionally a terminator. In yet other embodiments, a DNA comprises a promoter (e.g., an inducible promoter) operably linked to a nucleotide sequence encoding a first region of an RNA, followed by one or more nucleotides of a loop region, followed by a second region of the RNA, and optionally followed by a terminator, wherein the first region of the RNA is complementary to a segment of ROP and the second region is complementary to the first region. In still other embodiments, a DNA comprises a first strand comprising a first promoter (e.g., an inducible promoter) operably linked to a nucleotide sequence encoding a first RNA that is complementary to a segment of ROP, and optionally a terminator, and a second strand comprising a second promoter (e.g., an inducible promoter) operably linked to a nucleotide sequence encoding a second RNA that is complementary to the first RNA, and optionally a terminator wherein the first and second promoters are operably linked to the nucleotide sequence encoding a desired ROP-targeting RNA and wherein the bidirectional transcription of the nucleotide sequence encoding the desired ROP-targeting RNA results in complementary RNA molecules which anneal to form the dsRNA molecule.

A DNA is typically provided on a vector, such as a plasmid, although other template formats may be used (e.g., linear DNA generated by polymerase chain reaction (PCR), chemical synthesis, or other means known in the art). In some embodiments, more than one DNA is used in a reaction mixture. In some embodiments, 2, 3, 4, 5, or more different DNAs are used in a reaction mixture.

A promoter or terminator may be a naturally-occurring sequence or an engineered (e.g., synthetic) sequence. In some embodiments, an engineered sequence is modified to enhance transcriptional activity. In some embodiments, the promoter is a naturally-occurring sequence. In other embodiments, the promoter is an engineered sequence. In some embodiments, the terminator is a naturally-occurring sequence. In other embodiments, the terminator is an engineered sequence.

EXAMPLES

In order that the invention described herein may be more fully understood, the following examples are set forth. The Examples described in this Application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

Example 1: ROP RNAi composition kills Colorado potato beetles

To evaluate the effect of the ROP RNAi polynucleotide (SEQ ID NO: 4) on Colorado potato beetles (CPBs), a composition ( e.g ., comprising water) comprising the ROP RNAi polynucleotide (hereafter,“GL 64”) was applied (at a concentration of 10 pg/cm²) onto the leaves of potato plants and allowed to dry. Up to 80% of CPBs died following a 8 day exposure to the GL 64-covered potato plant leaves, compared with less than 10% of CPBs that die following exposure to the GL 32 control (GL 32) leaves (FIG. 1A). This increased mortality in response to exposure to GL 64 also results in a decrease of potato leaf consumption to nearly 20% (FIG. IB). Percent potato leaf consumption refers to the percentage of potato leaf discs (punched out of potato leaves) following treatment of the discs with the RNAi composition and subsequent exposure of the discs to Colorado potato beetle, for example.

Additional Embodiments

Additional embodiments of the present disclosure are encompassed by the following numbered paragraphs.

1. A polynucleotide molecule targeting a Coleopteran ras opposite (ROP) gene, wherein the polynucleotide molecule is selected from the group consisting of:

a polynucleotide molecule that binds to and inhibits expression of a messenger RNA (mRNA) encoded by a deoxynucleic acid (DNA) comprising a sequence of SEQ ID NO: 1;

a polynucleotide molecule that binds to and inhibits expression of a mRNA comprising a sequence of SEQ ID NO: 2 or 4;

a polynucleotide molecule that comprises a sequence having at least 80% identity to a sequence of SEQ ID NO: 3 or 5; and

a polynucleotide molecule that comprises a segment that comprises at least 18 contiguous nucleotides, wherein the segment has at least 90% identity to a segment of a sequence of SEQ ID NO: 3 or 5.

2. The polynucleotide molecule of paragraph 1, wherein the polynucleotide molecule binds to a sequence of SEQ ID NO: 2.

3. The polynucleotide molecule of paragraph 1 or 2, wherein the polynucleotide molecule comprises a sequence that has at least 85%, at least 90%, at least 95%, or at least 98% identity to a sequence of SEQ ID NO: 3 or 5.

4. The polynucleotide molecule of paragraph 1 or 2, wherein the polynucleotide molecule comprises a segment that comprises at least 18 contiguous nucleotides, wherein the segment shares at least 95% or at least 98% identity with a sequence of SEQ ID NO: 3 or 5.

5. The polynucleotide molecule of paragraph 3 or 4, wherein the polynucleotide molecule comprises the sequence of SEQ ID NO: 3 or 5.

6. The polynucleotide molecule of any one of paragraphs 1-5, wherein the polynucleotide molecule is a single-stranded RNA (ssRNA) molecule, optionally comprising the sequence of SEQ ID NO: 3 or 5.

7. The polynucleotide molecule of paragraph 6, wherein the ssRNA molecule is selected from the group consisting of small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), and antisense RNAs. 8. The polynucleotide molecule of any one of paragraphs 1-5, wherein the polynucleotide molecule is a double-stranded RNA (dsRNA) molecule, optionally comprising the sequence of SEQ ID NO: 3 or 5, or a segment of SEQ ID NO: 3 or 5.

9. A composition comprising the polynucleotide molecule of any one of paragraphs

1-8.

10. The composition of paragraph 9, wherein the composition further comprises an additive selected from the group consisting of insect feed, insect attractants, pheromones, proteins, carbohydrates, polymers, and pesticides.

11. A method for controlling Coleopteran infestation, the method comprising contacting a plant, ground, a Coleopteran insect, or a diet of a Coleopteran insect with the polynucleotide molecule of any one of paragraphs 1-8 or the composition of paragraph 9 or 10.

12. The method of paragraph 11, wherein the Coleopteran insect is of a species selected from the group consisting of: Leptinotarsa spp., Phyllotreta spp., Cerotoma spp., Diabrotica spp., tribolium spp., Anthonomus spp. and Alticini spp.

13. The method of paragraph 11 or 12, wherein the Coleopteran insect is a

Leptinotarsa spp. insect.

14. The method of paragraph 13, wherein the Leptinotarsa spp. insect is a Colorado potato beetle.

15. The method of any one of paragraphs 11-14, wherein the plant is selected from the group consisting of Solanaceae plants, Brassicaceae plants, Poaceae plants, Cucurbitaceae plants, Fobaceae plants, Apiaceae plants, Amaranthaceae plants, and Malvaceae plants.

16. The method of any one of paragraphs 11-15, wherein the method impairs growth, reproduction, and/or feeding of the Coleopteran insect.

17. The method of any one of paragraphs 11-15, wherein the method results in death of the Coleopteran insect.

18. A method for producing a polynucleotide for use in insect control, the method comprising:

(a) incubating in a reaction mixture cellular ribonucleic acid (RNA) and a

ribonuclease and producing 5' nucleoside monophosphates (5' NMPs);

(b) eliminating the ribonuclease; and

(c) incubating in the reaction mixture, or in a second reaction mixture, the 5' NMPs, a polyphosphate kinase, a polyphosphate, a polymerase, and a deoxyribonucleic acid (DNA) template having at least 80% identity to SEQ ID NO: 1, or encoding a RNA sequence that comprises a segment that comprises at least 18 contiguous nucleotides, wherein the segment has at least 90% identity to a segment of a scq ^nr^ ^nl TD NO: 2, and producing the RNA of interest, optionally wherein the reaction mixture of step (c) further comprises a nucleoside kinase, a NMP kinase, and/or a NDP kinase.

19. The method of paragraph 18, wherein the cellular RNA comprises ribosomal RNA, messenger RNA, and/or transfer RNA.

20. The method of paragraph 18 or 19, wherein the polyphosphate kinase is selected from PPK1 family enzymes or PPK2 family enzymes, and optionally wherein the polyphosphate kinase comprises a Class III polyphosphate kinase 2 from Deinococcus geothermalis.

21. The method of any one of paragraphs 18-20, wherein the polyphosphate comprises hexametaphosphate.

22. The method according to paragraph 18, wherein the DNA template is a promotor operably linked to a nucleotide sequence encoding a desired ROP-targeting RNA, and optionally, a transcriptional terminator.

23. The method according to paragraph 22, wherein the DNA template further comprises a second template comprising a promoter operably linked to the reverse complement of the nucleotide sequence encoding a desired ROP-targeting RNA, wherein the two individual RNA molecules anneal to form a dsRNA molecule.

24. The method according to paragraph 18, wherein the DNA template is a promoter operably linked to a nucleotide sequence encoding: (a) a desired ROP RNA, (b) one or more nucleotides of a loop region of an RNA transcript, (c) the reverse compliment of the nucleotide sequence encoding the desired ROP-targeting RNA and optionally, a transcriptional terminator.

25. The method according to paragraph 18 wherein the DNA template comprises: a. a first promoter,

b. a nucleotide sequence encoding a desired ROP-targeting RNA,

c. a second promoter, and

d. optionally, one or more transcriptional terminators,

wherein the first and second promoters are operably linked to the nucleotide sequence encoding a desired ROP-targeting RNA and wherein the bidirectional transcription of the nucleotide sequence encoding the desired ROP-targeting RNA results in complementary RNA molecules which anneal to form the dsRNA molecule

26. The method of paragraph 18, wherein the ribonuclease, the polyphosphate kinase, the DNA template, and/or the polymerase is prepared from cells that express the ribonuclease, the polyphosphate kinase, the DNA template, and/or the polymerase.

27. The method of paragraph 18, wherein the reaction mixture of (a) comprises a cell lysate prepared from cells that express the ribonuclease, the polyphosphate kinase, the DNA template, and/or the polymerase. 28. The method of paragraph 18, wherein step (b) comprises eliminating the ribonuclease and native enzymatic activities in the cell lysate via temperature, pH, salt, detergent, alcohol, and/or chemical inhibitors.

29. The method of paragraph 18, wherein step (b) comprises eliminating native enzymatic activity of enzymes in the cell lysate via separation, precipitation, filtration, capture, and/or chromatography.

30. The method of paragraph 18, wherein step (b) comprises eliminating native enzymatic activity of enzymes in the cell lysate via genetic modification, enzyme secretion from a cell, and/or protease targeting.

31. The method of any one of paragraphs 28-30, wherein the native enzymatic activities are selected from phosphatases, nucleases, proteases, deaminases, and hydrolases.

32. The method of any one of paragraphs 28-31, wherein the polyphosphate kinase, and/or the polymerase can withstand elimination conditions.

33. The method of paragraph 18, wherein the polymerase comprises at least one RNA polymerase.

34. A double- stranded ribonucleic acid (dsRNA) comprising a sequence with at least 80% identity to the sequence of SEQ ID NO: 3 or 5.

35. The dsRNA of paragraph 34 comprising a sequence with at least 90% or at least 95% identity to the sequence of SEQ ID NO: 3 or 5.

36. The dsRNA of paragraph 34 comprising a sequence of SEQ ID NO: 3 or 5.

37. A composition comprising the dsRNA of any one of paragraphs 34-36, optionally formulated at a concentration of 0.0001 pg/cm²to 10 pg/cm².

38. The method of paragraph 11, wherein the contacting step comprises applying the polynucleotide to the surface of the plant, ground, Coleopteran insect, or diet of a Coleopteran insect at a concentration of at least 0.0001 pg/cm².

39. The method of paragraph 38, wherein the contacting step comprises applying the polynucleotide to the surface of the plant, ground, Coleopteran insect, or diet of a Coleopteran insect at a concentration of 0.0001 pg/cm² to 10 pg/cm².

40. The method of paragraph 39, wherein the contacting step comprises applying the polynucleotide to the surface of the plant, ground, Coleopteran insect, or diet of a Coleopteran insect at a concentration of 0.0001 pg/cm² to 0.1 pg/cm².

41. The method of any one of paragraphs 38-40, wherein percent mortality of Coleopteran insects increase to at least 30% following fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, or fewer than 5 days of exposure of the Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions. 42. The method of paragraph 41, wherein percent mortality of Coleopteran insects increase to at least 40% following fewer than 10, fewer than 9, fewer than 8, fewer than 7, or fewer than 6 days of exposure of the Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

43. The method of paragraph 42, wherein percent mortality of Coleopteran insects increase to at least 50% following fewer than 10, fewer than 9, fewer than 8, or fewer than 7 days of exposure of the Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

44. The method of paragraph 43, wherein percent mortality of Coleopteran insects increase to at least 60% or at least 70% following fewer than 10, fewer than 9, or fewer than 8 days of exposure of the Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

45. The method of paragraph 43, wherein percent mortality of Coleopteran insects increase to at least 90% following fewer than 10 days or fewer than 9 days of exposure of the Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

46. The method of any one of paragraphs 38-45, wherein leaf disc consumption decrease to less than 20% following fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, or fewer than 5 days of exposure of Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

47. The method of paragraph 46, wherein leaf disc consumption decrease to less than 10% following fewer than 10% following fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, or fewer than 5 days of exposure of Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

48. The method of any one of paragraphs 38-47, wherein percent plant defoliation decreases to less than 10% following fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, or fewer than 4 days of exposure of Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

49. The method of any one of paragraphs 38-48, wherein percent plant defoliation remains less than 10% following at least 10, at least 15, or at least 20 days following exposure of Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

Table 1. Sequences,

EQUIVALENTS AND SCOPE

In the claims articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain

embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

It is also noted that the terms“comprising” and“containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the

specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

What is claimed is: CLAIMS

1. A polynucleotide that specifically inhibits expression of a Coleopteran a ras opposite (ROP) gene.

2. The polynucleotide of claim 1, wherein the Coleopteran ROP gene comprises a deoxynucleic acid (DNA) sequence of SEQ ID NO: 1.

3. The polynucleotide of claim 1 or 2, wherein the polynucleotide is a ribonucleic acid (RNA).

4. The polynucleotide of claim 3, wherein the RNA is a double-stranded RNA (dsRNA) comprising a first strand that is complementary to a messenger RNA (mRNA) encoded by the Coleopteran ROP gene, and a second strand is complementary to the first strand.

5. The polynucleotide of claim 4, wherein the mRNA comprises the RNA sequence of SEQ ID NO: 2.

6. The polynucleotide of claim 4 or 5, wherein the first strand of the dsRNA comprises an RNA sequence 70% to 100% complementary to the mRNA or a segment of the mRNA encoded by the Coleopteran ROP gene.

7. The polynucleotide of claim 6, wherein the first strand of the dsRNA comprises an RNA sequence 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100%

complementary to the mRNA or the segment of the mRNA encoded by the Coleopteran ROP gene.

8. The polynucleotide of claim 7, wherein the segment of the mRNA has a length of at least 18 to 500 nucleotides, at least 21 to 500 nucleotides, at least 50 to 500 nucleotides, at least 100 to 500 nucleotides, or at least 200 to 500 nucleotides.

9. The polynucleotide of any one of claims 4-8, wherein the first strand of the dsRNA comprises at least 18 to 21 contiguous nucleotides at least 90% to 100%, 95% to 100%, or at least 98% to 100%, complementary to the mRNA or the segment of the mRNA encoded by the Coleopteran ROP gene.

10. The polynucleotide of any one of claims 4-9, wherein the first strand of the dsRNA comprises an RNA sequence that has 70% to 100% identity to the RNA sequence or a segment of the RNA sequence of SEQ ID NO: 3.

11. The polynucleotide of claim 10, wherein the first strand of the dsRNA comprises an RNA sequence that has 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identity to the RNA sequence or the segment of the RNA sequence of SEQ ID NO: 3.

12. The polynucleotide of claim 10 or 11, wherein the segment of the RNA sequence of SEQ ID NO: 3 has a length of at least 18 to 500 nucleotides, at least 21 to 500 nucleotides, at least 50 to 500 nucleotides, at least 100 to 500 nucleotides, or at least 200 to 500 nucleotides.

13. The polynucleotide of any one of claims 10-12, wherein the first strand of the dsRNA comprises at least 18 to 21 contiguous nucleotides that have at least 90% to 100%, 95% to 100%, or at least 98% to 100%, identity to the RNA sequence of SEQ ID NO: 3.

14. The polynucleotide of claim 2 or 3, wherein the RNA is a single-stranded RNA (ssRNA) that binds to an mRNA encoded by the Coleopteran ROP gene.

15. The polynucleotide of claim 14, wherein the mRNA comprises the RNA sequence of SEQ ID NO: 2.

16. The polynucleotide of claim 14 or 15, wherein the ssRNA comprises an RNA sequence 70% to 100% complementary to the mRNA or a segment of the mRNA encoded by the

Coleopteran ROP gene.

17. The polynucleotide of claim 16, wherein the ssRNA comprises an RNA sequence 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100%

18. The polynucleotide of claim 17, wherein the segment of the mRNA has a length of at least 18 to 500 nucleotides, at least 21 to 500 nucleotides, at least 50 to 500 nucleotides, at least 100 to 500 nucleotides, or at least 200 to 500 nucleotides.

19. The polynucleotide of any one of claims 14-18, wherein the ssRNA comprises at least 18 to 21 contiguous nucleotides at least 90% to 100%, 95% to 100%, or at least 98% to 100%, complementary to the mRNA.

20. The polynucleotide of any one of claims 14-19, wherein the ssRNA comprises an RNA sequence that has 70% to 100% identity to the RNA sequence of SEQ ID NO: 3.

21. The polynucleotide of claim 20, wherein the ssRNA comprises an RNA sequence that has 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, or 98% to 100% identity to the RNA sequence or the segment of the RNA sequence of SEQ ID NO: 3.

22. The polynucleotide of claim 21, wherein the segment of the RNA sequence of SEQ ID NO: 3 has a length of at least 18 to 500 nucleotides, at least 21 to 500 nucleotides, at least 50 to 500 nucleotides, at least 100 to 500 nucleotides, or at least 200 to 500 nucleotides.

23. The polynucleotide of any one of claims 20-22, wherein the ssRNA comprises at least 18 to 21 contiguous nucleotides that have at least 90% to 100%, 95% to 100%, or at least 98% to 100%, identity to the RNA sequence of SEQ ID NO: 3.

24. A composition comprising the polynucleotide of any one of claims 1-23.

25. The composition of claim 24, wherein the composition

(a) further comprises at least one additive selected from the group consisting of:

adjuvants, attractants, growth-regulating substances, insect feed, pheromones, proteins, carbohydrates, polymers, organic compounds, biologies, and pesticidal agents;

(b) is formulated as a liquid, a solution, a suspension, an emulsion, an emulsifiable concentrate, a concentrate solution, a low concentrate solution, an ultra-low volume concentrate solution, a water soluble concentrate solutions bait, an invert emulsion, a flowable, an aerosol, a smoke, a fog, a flowable, a homogenous mixture, a non-homogenous mixture, a solid, a dust, a powder, a granule, a pellet, a capsule, a fumigant, an encapsulated formulation, or a micro encapsulation formulation; or

(c) is delivered as a spray, fog, seed treatment, drench, drip irrigation, in furrow, insect diet, or bait.

26. The composition of any one of claims 24-25 formulated at a concentration of 0.001 pg/cm² to 10 pg/cm².

27. A deoxyribonucleic acid (DNA) encoding the RNA of any one of claims 3-26.

28. A method for controlling Coleopteran insect infestation, the method comprising delivering to a plant, ground, a Coleopteran insect, or a diet of a Coleopteran insect with the polynucleotide molecule of any one of claims 1-23 or the composition of any one of claims 24- 27.

29. A method for controlling Coleopteran insect infestation, the method comprising delivering to a plant, ground, a Coleopteran insect, or a diet of a Coleopteran insect with a polynucleotide or a composition comprising a polynucleotide that specifically inhibits expression of a Coleopteran ras opposite (ROP) gene.

30. The method of claim 28 or 29, wherein the composition is delivered to a leaf, stem, seed, root, or soil of the plant.

31. The method of any one of claims 28-30, wherein the plant is selected from the group consisting of Solanaceae plants, Brassicaceae plants, Poaceae plants, Cucurbitaceae plants, Fobaceae plants, Apiaceae plants, Amaranthaceae plants, and Malvaceae plants.

32. The method of any one of claims 28-31, wherein the polynucleotide or composition is delivered in an amount sufficient to cause stunting, mortality, decreased feeding, or inhibited reproduction of a Coleopteran insect.

33. The method of any one of claims 28-32, wherein the Coleopteran insect is of a species selected from the group consisting of: Leptinotarsa spp., Phyllotreta spp., Cerotoma spp., Diabrotica spp., tribolium spp., Anthonomus spp. and Alticini spp.

34. The method of claim 33, wherein the Coleopteran insect is a Leptinotarsa spp. insect.

35. The method of claim 34, wherein the Leptinotarsa spp. insect is a Colorado potato beetle.

36. The method of any one of claims 28-35, wherein the delivering step comprises applying the polynucleotide to the surface of the plant, to the ground, to the Coleopteran insect, or to the diet of a Coleopteran insect at a concentration of at least 0.001 pg/cm², of 0.001 pg/cm² to 10 pg/cm², or 0.001 pg/cm² to 0.1 pg/cm².

37. The method of any one of claims 29-36, wherein percent mortality of Coleopteran insects increases to at least 30% following fewer than 10 days of exposure of the Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.

38. The method of any one of claims 28-37, wherein percent plant defoliation decreases to less than 15% following fewer than 10 days of exposure of Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions; or remains less than 15% following at least 10 days following exposure of Coleopteran insects to the polynucleotide, relative to a control, optionally under untreated conditions.