Patent Grant
11091770
Disclosed herein are methods for controlling invertebrate pest infestations, particularly in plants, and compositions and polynucleotides useful in such methods. More specifically, this invention is related to polynucleotides and methods of use thereof for modifying the expression of genes in an invertebrate pest, particularly through RNA interference. Pest species of interest include insects that infest crop plants, e. g., Plutella xylostella (diamondback moth), Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), and Euschistus heros (neotropical brown stink bug).
Commercial crops are often the targets of attack by invertebrate pests such as insects. Compositions for controlling insect infestations in plants have typically been in the form of chemical insecticides. However, there are several disadvantages to using chemical insecticides. For example, chemical insecticides are generally not selective, and applications of chemical insecticides intended to control insect pests in crop plants can exert their effects on non-target insects and other invertebrates as well. Chemical insecticides often persist in the environment and can be slow to degrade, thus potentially accumulating in the food chain. Furthermore the use of persistent chemical insecticides can result in the development of resistance in the target insect species. Thus there has been a long felt need for more environmentally friendly methods for controlling or eradicating insect infestation on or in plants, i. e., methods which are species-selective, environmentally inert, non-persistent, and biodegradable, and that fit well into pest resistance management schemes.
Insecticidal compositions that include Bacillus thuringiensis (“Bt”) bacteria have been commercially available and used as environmentally safe and acceptable insecticides for more than thirty years. The effectiveness of these compositions is due to insecticidal proteins that are produced exclusively by Bt bacteria. The insecticidal Bt proteins do not persist in the environment, are highly selective as to the target species affected, exert their effects only upon ingestion by a target insect, and have been shown to be harmless to plants and other non-targeted organisms, including humans and other vertebrates. Transgenic plants containing one or more recombinant genes encoding insecticidal Bt proteins are also available in the art and are resistant to insect pest infestation. One positive environmental result of the use of transgenic plants expressing Bt proteins is a decrease in the amount of chemical insecticides that are applied to control pest infestation in such transgenic crop fields, resulting in decreased contamination of soil and waters by non-degraded or excess chemical insecticides. In addition, there has been a noticeable increase in the numbers of beneficial insects in fields in which Bt protein-expressing transgenic crop plants are grown because of the decrease in the use of non-selective chemical insecticides.
RNA interference (RNAi, RNA-mediated gene suppression) is another approach used for pest control. In invertebrates RNAi-based gene suppression was first demonstrated in nematodes (Fire et al., (1998) Nature, 391:806-811; Timmons & Fire (1998) Nature, 395:854). Subsequently, RNAi-based suppression of invertebrate genes using recombinant nucleic acid techniques has been reported in a number of species, including agriculturally or economically important pests from various insect and nematode taxa, such as: root-knot nematodes (Meloidogyne spp.), see Huang et al. (2006) Proc. Natl. Acad. Sci. USA, 103:14302-14306, doi:10.1073/pnas.0604698103); cotton bollworm (Helicoverpa armigera), see Mao et al. (2007) Nature Biotechnol., 25:1307-1313, doi:10.1038/nbt1352; Western corn rootworm (Diabrotica virgifera LeConte), see Baum et al. (2007) Nature Biotechnol., 25:1322-1326, doi:10.1038/nbt1359; sugar beet cyst nematode (Heterodera schachtii), see Sindhu et al. (2008) J. Exp. Botany, 60:315-324, doi:10.1093/jxb/ern289; mosquito (Aedes aegypti), see Pridgeon et al. (2008) J. Med. Entomol., 45:414-420, doi: full/10.1603/0022-2585%282008%2945%5B414%3ATAADRK %5D2.0.CO %3B2 fruit flies (Drosophila melanogaster), flour beetles (Tribolium castaneum), pea aphids (Acyrthosiphon pisum), and tobacco hornworms (Manduca sexta), see Whyard et al. (2009) Insect Biochem. Mol. Biol., 39:824-832, doi:10.1016/j.ibmb.2009.09.00; diamondback moth (Plutella xylostella), see Gong et al. (2011) Pest Manag. Sci., 67: 514-520, doi:10.1002/ps.2086; green peach aphid (Myzus persicae), see Pitino et al. (2011) PLoS ONE, 6:e25709, doi: 10.1371/journal.pone.0025709; brown planthopper (Nilaparvata lugens), see Li et al. (2011) Pest Manag. Sci., 67:852-859, doi:10.1002/ps.2124; and whitefly (Bemisia tabaci), see Upadhyay et al. (2011) J. Biosci., 36:153-161, doi:10.1007/s12038-011-9009-1.
This invention is related to methods of controlling insect pests, in particular insects which infest crop plants and have previously been found to be recalcitrant to RNA-mediated gene suppression methods, e. g., Plutella xylostella (diamondback moth), Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), and Euschistus heros (neotropical brown stink bug). Double-stranded RNA (dsRNA) trigger sequences have been identified for testing on the recalcitrant insect species Plutella xylostella (diamondback moth, DBM), Spodoptera frugiperda (fall armyworm, FAW), Lygus hesperus (western tarnished plant bug, WTPB), and Euschistus heros (neotropical brown stink bug, NBSB). These triggers are designed to suppress novel target genes that are putative orthologues of genes previously demonstrated to be efficacious targets for RNAi-mediated mortality in Western corn rootworm (Diabrotica virgifera).
This invention is further related to polynucleotides and recombinant DNA molecules and constructs useful in methods of controlling insect pests. This invention is further related to insecticidal compositions, as well as to transgenic plants resistant to infestation by insect pests. This invention is also related to methods of identifying efficacious double-stranded RNA triggers for controlling insect pests, and methods for identifying target genes that are likely to represent essential functions, making these genes preferred targets for RNAi-mediated silencing and control of insect pests.
This invention is related to control of insect species, especially those that are economically or agriculturally important pests. The compositions and methods of this invention include recombinant polynucleotide molecules, such as recombinant DNA constructs for making transgenic plants resistant to infestation by insect species and RNA “triggers” that are useful, e. g., as topically applied agents for causing RNAi-mediated suppression of a target gene in a insect species and thus controlling or preventing infestation by that insect species. Another utility of this invention is a polynucleotide-containing composition (e. g., a composition containing a dsRNA trigger for suppressing a target gene) that is topically applied to an insect species or to a plant, plant part, or seed to be protected from infestation by an insect species. This invention is further related to methods for selecting preferred insect target genes that are more likely to be effective targets for RNAi-mediated control of an insect species.
In one aspect, this invention provides a method for controlling an insect infestation of a plant including contacting with a dsRNA an insect that infests a plant, wherein the dsRNA includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of the insect, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In embodiments, the dsRNA includes an RNA strand with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the dsRNA includes an RNA strand with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the dsRNA trigger suppresses a gene in the insect and stunts or kills the insect.
In another aspect, this invention provides a method of causing mortality or stunting in an insect, including providing in the diet of an insect at least one recombinant RNA including at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the recombinant RNA by the insect results in mortality or stunting in the insect. In embodiments, the silencing element includes an RNA strand with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the silencing element includes an RNA strand with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
In another aspect, this invention provides an insecticidal composition including an insecticidally effective amount of a recombinant RNA molecule, wherein the recombinant RNA molecule includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of an insect that infests a plant, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In embodiments, the recombinant RNA molecule is dsRNA. In embodiments, the recombinant RNA molecule includes at least one segment (e. g., an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the recombinant RNA molecule includes at least one segment (e. g., an RNA strand or segment of an RNA strand) with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
In another aspect, this invention provides a method of providing a plant having improved resistance to an insect, including expressing in the plant a recombinant DNA construct including DNA encoding at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the recombinant RNA by the insect results in mortality or stunting in the insect. In embodiments, the silencing element is ssRNA. In other embodiments, the silencing element is dsRNA. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
In another aspect, this invention provides a recombinant DNA construct including a heterologous promoter, such as a heterologous promoter functional in a bacterial cell or in a eukaryotic cell (e. g., a plant cell or an insect cell), operably linked to DNA encoding an RNA transcript including a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the RNA transcript is ssRNA. In other embodiments, the RNA transcript is dsRNA. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
In related aspects, this invention provides man-made compositions including the polynucleotide or trigger of this invention, such as dsRNA formulations useful for topical application to a plant or substance in need of protection from an insect infestation, recombinant constructs and vectors useful for making transgenic plant cells and transgenic plants, formulations and coatings useful for treating plants (including plant seeds or propagatable parts such as tubers), plant seeds or propagatable parts such as tubers treated with or containing a polynucleotide of this invention as well as commodity products and foodstuffs produced from such plants, seeds, or propagatable parts (especially commodity products and foodstuffs having a detectable amount of a polynucleotide of this invention). A further aspect of this invention are polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44; another aspect of this invention are polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 or the complement thereof. Such antibodies are made by routine methods as known to one of ordinary skill in the art.
Other aspects and specific embodiments of this invention are disclosed in the following detailed description and working Examples.
Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used and the manufacturing or laboratory procedures described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Heritage® Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only.
Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. One of skill in the art would be aware that a given DNA sequence is understood to define a corresponding RNA sequence which is identical to the DNA sequence except for replacement of the thymine (T) nucleotides of the DNA with uracil (U) nucleotides. Thus, providing a specific DNA sequence is understood to define the exact RNA equivalent. A given first polynucleotide sequence, whether DNA or RNA, further defines the sequence of its exact complement (which can be DNA or RNA), i. e., a second polynucleotide that hybridizes perfectly to the first polynucleotide by forming Watson-Crick base-pairs. By “essentially identical” or “essentially complementary” to a target gene or a fragment of a target gene is meant that a polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize (generally under physiological conditions such as those found in a living plant or animal cell) to a target gene or to a fragment of a target gene or to the transcript of the target gene or the fragment of a target gene; one of skill in the art would understand that such hybridization does not necessarily require 100% sequence identity or complementarity. A first nucleic acid sequence is “operably” connected or “linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter sequence is “operably linked” to DNA if the promoter provides for transcription or expression of the DNA. Generally, operably linked DNA sequences are contiguous.
The term “polynucleotide” commonly refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Polynucleotides also include molecules containing multiple nucleotides including non-canonical nucleotides or chemically modified nucleotides as commonly practiced in the art; see, e. g., chemical modifications disclosed in the technical manual “RNA Interference (RNAi) and DsiRNAs”, 2011 (Integrated DNA Technologies Coralville, Iowa). Generally, polynucleotides or triggers of this invention, whether DNA or RNA or both, and whether single- or double-stranded, include at least one segment of 18 or more contiguous nucleotides (or, in the case of double-stranded polynucleotides, at least 18 contiguous base-pairs) that are essentially identical or complementary to a fragment of equivalent size of the DNA of a target gene or the target gene's RNA transcript. Throughout this disclosure, “at least 18 contiguous” means “from about 18 to about 10,000, including every whole number point in between”. Thus, embodiments of this invention include compositions including oligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e. g., polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target gene including coding or non-coding or both coding and non-coding portions of the target gene). Where a polynucleotide is double-stranded, such as the dsRNA triggers described in the working Examples, its length can be similarly described in terms of base pairs. Double-stranded polynucleotides, such as the dsRNA triggers described in the working examples, can further be described in terms of one or more of the single-stranded components.
The polynucleotides or triggers of this invention are generally designed to suppress or silence one or more genes (“target genes”). The term “gene” refers to any portion of a nucleic acid that provides for expression of a transcript or encodes a transcript. A “gene” can include, but is not limited to, a promoter region, 5′ untranslated regions, transcript encoding regions that can include intronic regions, 3′ untranslated regions, or combinations of these regions. In embodiments, the target genes can include coding or non-coding sequence or both. In other embodiments, the target gene has a sequence identical to or complementary to a messenger RNA, e. g., in embodiments the target gene is a cDNA.
Controlling Insect Infestations of a Plant by Contacting with a dsRNA
A first aspect of this invention provides a method for controlling an insect infestation of a plant including contacting with a double-stranded RNA (dsRNA) an insect that infests a plant, wherein the dsRNA includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a target gene of the insect, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In this context “controlling” includes inducement of a physiological or behavioural change in an insect (adult or larvae or nymphs) such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. “Double-stranded” refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions.
In various embodiments, the insect is selected from the group consisting of Spodoptera spp., Lygus spp., Euschistus spp., and Plutella spp. Insects of particular interest include Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), Euschistus heros (neotropical brown stink bug), and Plutella xylostella (diamondback moth).
Various embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; wherein the insect is Lygus hesperus (western tarnished plant bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; wherein the insect is Euschistus heros (neotropical brown stink bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; and wherein the insect is Plutella xylostella (diamondback moth) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:10-12. Other embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Other embodiments include those where the dsRNA has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.
The plant can be any plant that is subject to infestation by an insect that can be controlled by the polynucleotides disclosed herein. Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading “Plants”.
In embodiments of the method, the dsRNA includes multiple segments each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a target gene of the insect. For example, the dsRNA can include segments corresponding to different regions of the target gene, or can include multiple copies of a segment. In other embodiments of the method, the dsRNA includes multiple segments, each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a different target gene; in this way multiple target genes, or multiple insect species, can be suppressed.
In embodiments of the method, the dsRNA is blunt-ended. In other embodiments, the dsRNA has an overhang at one or both ends (termini); the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5′ end or on the 3′ end of a strand. The dsRNA can be chemically synthesized, or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. The dsRNA can be chemically modified, e. g., to improve stability or efficacy.
In some embodiments of the method, the contacting includes application of a composition including the dsRNA to a surface of the insect or to a surface of the plant infested by the insect. The composition can include or be in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, or seed treatment. In embodiments, the composition can be applied to a seed, e. g., by soaking the seed in a liquid composition including the dsRNA, wherein the seed imbibes or takes up the dsRNA into the seed interior or seed endosperm in an effective amount to provide improved resistance to the insect pest by the seed or a plant or seedling grown from the seed. In embodiments, the contacting includes providing the dsRNA in a composition that further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. In embodiments, the contacting includes providing the dsRNA in a composition that further includes at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.
In some embodiments of the method, the contacting includes providing the dsRNA in a composition that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. Such compositions can further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. Such compositions can further include at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In embodiments, the combination of the dsRNA and the pesticidal agent provides a level of insect control that is synergistic, i. e., greater than the sum of the effects of the dsRNA and the pesticidal agent components if tested separately.
Methods of Causing Mortality or Stunting in an Insect
Another aspect of this invention provides a method of causing mortality or stunting in an insect, including providing in the diet of an insect at least one recombinant RNA including at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the recombinant RNA by the insect results in mortality or stunting in the insect. The method is applicable to insects at various life stages. In embodiments, the method causes mortality or stunting in an insect larva or nymph. In other embodiments, the method causes mortality in adult insects.
In embodiments of the method the recombinant RNA includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. Embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; wherein the insect is Lygus hesperus (western tarnished plant bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; wherein the insect is Euschistus heros (neotropical brown stink bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; and wherein the insect is Plutella xylostella (diamondback moth) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:10-12. Other embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Other embodiments include those where the recombinant RNA has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.
In embodiments of the method, the recombinant RNA is dsRNA. In these embodiments, the dsRNA can be blunt-ended dsRNA, or can be dsRNA with an overhang at one or both ends (termini); the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5′ end or on the 3′ end of a strand. The dsRNA can be chemically synthesized, or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. The dsRNA can be chemically modified, e. g., to improve stability or efficacy. In embodiments of the method where the recombinant RNA is dsRNA, the dsRNA includes at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
In some embodiments of the method, the recombinant RNA is provided in the insect's diet in the form of an ingestible composition, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. Such ingestible compositions can further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. Such ingestible compositions can further include at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In embodiments, the combination of the recombinant RNA and the pesticidal agent provides a level of insect stunting or mortality that is synergistic, i. e., greater than the sum of the effects of the recombinant RNA and the pesticidal agent components if tested separately.
Insecticidal Compositions
Another aspect of the invention provides an insecticidal composition including an insecticidally effective amount of a recombinant RNA molecule, wherein the recombinant RNA molecule includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a target gene of an insect that infests a plant, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. By “insecticidally effective” is meant effective in inducing a physiological or behavioural change in an insect (adult or larvae or nymphs) that infests a plant such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development; in embodiments, application of an insecticidally effective amount of the recombinant RNA molecule to a plant improves the plant's resistance to infestation by the insect.
In embodiments of the insecticidal composition, the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In specific embodiments, the recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the recombinant RNA molecule is a dsRNA of at least 50 base pairs in length. In embodiments of the method, the recombinant RNA molecule is dsRNA. In embodiments, the recombinant RNA molecule is a dsRNA which can be blunt-ended dsRNA, or can be dsRNA with an overhang at one or both ends (termini); the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5′ end or on the 3′ end of a strand. In embodiments, the recombinant RNA molecule is a dsRNA which can be chemically synthesized, or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. In embodiments, the recombinant RNA molecule is a dsRNA of a length greater than that which is typical of naturally occurring regulatory small RNAs (such as endogenously produced siRNAs and mature miRNAs), i. e., the polynucleotide is double-stranded RNA of at least about 30 contiguous base-pairs in length. In embodiments, the recombinant RNA molecule is a dsRNA with a length of between about 50 to about 500 base-pairs.
Embodiments of the insecticidal composition include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; wherein the insect is Lygus hesperus (western tarnished plant bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; wherein the insect is Euschistus heros (neotropical brown stink bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; and wherein the insect is Plutella xylostella (diamondback moth) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:10-12. Other embodiments of the insecticidal composition include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Specific embodiments of the insecticidal composition include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Other embodiments of the insecticidal composition include those where the recombinant RNA molecule has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.
In various embodiments the insecticidal composition includes an insecticidally effective amount of a recombinant RNA molecule that consists of naturally occurring ribonucleotides, such as those found in naturally occurring RNA. In certain embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S. Patent Publication 2011/0152353, U.S. Patent Publication 2011/0152346, and U.S. Patent Publication 2011/0160082, which are herein incorporated by reference. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide which can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (e. g., fluorescein or rhodamine) or other label (e. g., biotin).
The recombinant RNA molecule of is provided by suitable means known to one in the art. Embodiments include those wherein the recombinant RNA molecule is chemically synthesized (e. g., by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture (such as plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.
In embodiments the recombinant RNA molecule of use in this method is provided as an isolated RNA fragment (not part of an expression construct, i. e., lacking additional elements such as a promoter or terminator sequences). Such recombinant RNA molecules can be relatively short, such as single- or double-stranded RNA molecules of between about 18 to about 300 or between about 50 to about 500 nucleotides (for single-stranded polynucleotides) or between about 18 to about 300 or between about 50 to about 500 base-pairs (for double-stranded polynucleotides). Embodiments include those in which the polynucleotide is a dsRNA including a segment having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
In embodiments, the insecticidal composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment. In embodiments, the insecticidal composition can be applied to a seed, e. g., by soaking the seed in a liquid insecticidal composition including the dsRNA, wherein the seed imbibes or takes up the dsRNA into the seed interior or seed endosperm in an effective amount to provide improved resistance to the insect pest by the seed or a plant or seedling grown from the seed. In some embodiments, the insecticidal composition is provided in a form that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. The insecticidal compositions can further include one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. The insecticidal compositions can further include at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In embodiments, the combination of the recombinant RNA molecule and the pesticidal agent provides a level of insect control that is synergistic, i. e., greater than the sum of the effects of the recombinant RNA molecule and the pesticidal agent components if tested separately.
A related aspect of the invention is a plant treated with an insecticidal composition as described herein, or a seed of the treated plant, wherein the plant exhibits improved resistance to the insect. In embodiments, the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not treated with the insecticidal composition.
Methods of Providing Plants with Improved Insect Resistance
Another aspect of the invention provides a method of providing a plant having improved resistance to an insect, including expressing in the plant a recombinant DNA construct including DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the RNA by the insect results in mortality or stunting in the insect.
In embodiments of the method, the silencing element has a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 45, and 46. In specific embodiments, the silencing element is RNA that forms double-stranded RNA from two separate, essentially complementary strands, wherein at least one RNA strand includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 45, and 46. In other embodiments, the silencing element is RNA that forms double-stranded RNA from a single self-hybridizing hairpin transcript, wherein one “arm” of the hairpin includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 45, and 46. Other embodiments include those where the silencing element has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.
In embodiments of the method, the recombinant DNA construct further includes a heterologous promoter operably linked to the DNA encoding RNA that includes at least one silencing element, wherein the heterologous promoter is functional in a plant cell. Promoters functional in a plant cell include those listed under the heading “Promoters”.
In embodiments of the method, the recombinant DNA construct is expressed in the plant by means of transgenic expression or transient expression. In some embodiments, the method further includes expression in the plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. The pesticidal agent can be expressed from the same recombinant DNA construct that includes the DNA encoding at least one silencing element, or from a different recombinant DNA construct.
A related aspect of the invention is a plant having improved resistance to an insect, or the seed of such a plant, wherein the plant is provided by the method including expressing in the plant a recombinant DNA construct including DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the RNA by the insect results in mortality or stunting in the insect. In embodiments, the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not treated with the insecticidal composition. Also encompassed by the invention are fruit, seed, or propagatable parts of the plant provided by this method and exhibiting improved resistance to the insect.
A related aspect of the invention is a plant or seedling having improved resistance to an insect, wherein the plant or seedling is grown from a seed treated with a recombinant DNA construct including DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44; alternatively the plant is grown from a seed directly treated with the RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In embodiments, the recombinant DNA construct (or the encoded RNA that includes at least one silencing element) is applied by soaking the seed in a liquid composition including the recombinant DNA construct (or the encoded RNA that includes at least one silencing element), wherein the seed imbibes or takes up the DNA or encoded RNA into the seed interior or seed endosperm in an effective amount to provide improved resistance to the insect pest by a plant or seedling grown from the seed.
Recombinant DNA Constructs Encoding RNA for Insect Control
Another aspect of the invention provides a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript including a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
In specific embodiments, the RNA transcript forms double-stranded RNA from two separate, essentially complementary strands (e. g., where one strand is encoded on a separate DNA construct or where the two strands are encoded on separate sections of the DNA encoding an RNA transcript, and which are separately transcribed or made separate, for example, by the action of a recombinase or nuclease), wherein at least one RNA strand includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In other embodiments, the RNA transcript forms double-stranded RNA from a single self-hybridizing hairpin transcript, wherein one “arm” of the hairpin includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.
Embodiments of the recombinant DNA construct include those wherein the heterologous promoter is functional for expression of the RNA transcript in a bacterium. In embodiments where the recombinant DNA construct is to be expressed in a bacterium, the bacterium is selected from the group consisting of Escherichia coli, Bacillus species, Pseudomonas species, Xenorhabdus species, or Photorhabdus species. In other embodiments, the recombinant DNA construct includes a heterologous promoter that is functional in a plant cell. In embodiments, the recombinant DNA construct is contained in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In embodiments, the recombinant DNA construct is integrated into a plant chromosome or plastid, e. g., by stable transformation.
Related aspects of the invention include a transgenic plant cell having in its genome the recombinant DNA construct, and a transgenic plant including such a transgenic plant cell. Transgenic plant cells and plants are made by methods known in the art, such as those described under the heading “Making and Using Transgenic Plant Cells and Transgenic Plants”. Further aspects of the invention include a commodity product produced from such a transgenic plant, and transgenic progeny seed or propagatable plant part of the transgenic plant.
Related Information and Techniques
Plants
The methods and compositions described herein for treating and protecting plants from insect infestations are useful across a broad range of plants. Suitable plants in which the methods and compositions disclosed herein can be used include, but are not limited to, cereals and forage grasses (rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilseed crops (soybean, oilseed brassicas including canola and oilseed rape, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (common bean, cowpea, pea, faba bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupine, alfalfa, and clovers), temperate fruits and nuts (apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut), tropical and subtropical fruits and nuts (citrus including limes, oranges, and grapefruit; banana and plantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit, and persimmon), vegetable crops (solanaceous plants including tomato, eggplant, and peppers; vegetable brassicas; radish, carrot, cucurbits, alliums, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turf grasses), and forest tree species.
Additional Construct Elements
Embodiments of the polynucleotides and nucleic acid molecules of this invention can include additional elements, such as promoters, small RNA recognition sites, aptamers or ribozymes, additional and additional expression cassettes for expressing coding sequences (e. g., to express a transgene such as an insecticidal protein or selectable marker) or non-coding sequences (e. g., to express additional suppression elements). For example, an aspect of this invention provides a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript that includes a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In another embodiment, a recombinant DNA construct including a promoter operably linked to DNA encoding: (a) an RNA transcript that includes a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46, and (b) an aptamer, is stably integrated into the plant's genome from where RNA transcripts including the RNA aptamer and the RNA silencing element are expressed in cells of the plant; the aptamer serves to guide the RNA silencing element to a desired location in the cell. In another embodiment, inclusion of one or more recognition sites for binding and cleavage by a small RNA (e. g., by a miRNA or an siRNA that is expressed only in a particular cell or tissue) allows for more precise expression patterns in a plant, wherein the expression of the recombinant DNA construct is suppressed where the small RNA is expressed. Such additional elements are described below.
Promoters
Promoters of use in the invention are functional in the cell in which the construct is intended to be transcribed. Generally these promoters are heterologous promoters, as used in recombinant constructs, i. e., they are not in nature found to be operably linked to the other nucleic elements used in the constructs of this invention. In various embodiments, the promoter is selected from the group consisting of a constitutive promoter, a spatially specific promoter, a temporally specific promoter, a developmentally specific promoter, and an inducible promoter. In many embodiments the promoter is a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
Non-constitutive promoters suitable for use with the recombinant DNA constructs of this invention include spatially specific promoters, temporally specific promoters, and inducible promoters. Spatially specific promoters can include organelle-, cell-, tissue-, or organ-specific promoters (e. g., a plastid-specific, a root-specific, a pollen-specific, or a seed-specific promoter for expression in plastids, roots, pollen, or seeds, respectively). In many cases a seed-specific, embryo-specific, aleurone-specific, or endosperm-specific promoter is especially useful. Temporally specific promoters can include promoters that tend to promote expression during certain developmental stages in a plant's growth cycle, or during different times of day or night, or at different seasons in a year. Inducible promoters include promoters induced by chemicals or by environmental conditions such as, but not limited to, biotic or abiotic stress (e. g., water deficit or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection). MicroRNA promoters are useful, especially those having a temporally specific, spatially specific, or inducible expression pattern; examples of miRNA promoters, as well as methods for identifying miRNA promoters having specific expression patterns, are provided in U.S. Patent Application Publications 2006/0200878, 2007/0199095, and 2007/0300329, which are specifically incorporated herein by reference. An expression-specific promoter can also include promoters that are generally constitutively expressed but at differing degrees or “strengths” of expression, including promoters commonly regarded as “strong promoters” or as “weak promoters”.
Promoters of particular interest include the following examples: an opaline synthase promoter isolated from T-DNA of Agrobacterium; a cauliflower mosaic virus 35S promoter; enhanced promoter elements or chimeric promoter elements such as an enhanced cauliflower mosaic virus (CaMV) 35S promoter linked to an enhancer element (an intron from heat shock protein 70 of Zea mays); root specific promoters such as those disclosed in U.S. Pat. Nos. 5,837,848; 6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in U.S. Pat. No. 6,433,252; a promoter for a plant nuclear gene encoding a plastid-localized aldolase disclosed in U.S. Patent Application Publication 2004/0216189; cold-inducible promoters disclosed in U.S. Pat. No. 6,084,089; salt-inducible promoters disclosed in U.S. Pat. No. 6,140,078; light-inducible promoters disclosed in U.S. Pat. No. 6,294,714; pathogen-inducible promoters disclosed in U.S. Pat. No. 6,252,138; and water deficit-inducible promoters disclosed in U.S. Patent Application Publication 2004/0123347 A1. All of the above-described patents and patent publications disclosing promoters and their use, especially in recombinant DNA constructs functional in plants are incorporated herein by reference.
Plant vascular- or phloem-specific promoters of interest include a rolC or rolA promoter of Agrobacterium rhizogenes, a promoter of a Agrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RSs1 gene promoter, a Commelina yellow mottle badnavirus promoter, a coconut foliar decay virus promoter, a rice tungro bacilliform virus promoter, the promoter of a pea glutamine synthase GS3A gene, a invCD111 and invCD141 promoters of a potato invertase genes, a promoter isolated from Arabidopsis shown to have phloem-specific expression in tobacco by Kertbundit et al. (1991) Proc. Natl. Acad. Sci. US A., 88:5212-5216, a VAHOX1 promoter region, a pea cell wall invertase gene promoter, an acid invertase gene promoter from carrot, a promoter of a sulfate transporter gene Sultr1;3, a promoter of a plant sucrose synthase gene, and a promoter of a plant sucrose transporter gene.
Promoters suitable for use with a recombinant DNA construct or polynucleotide of this invention include polymerase II (“pol II”) promoters and polymerase III (“pol III”) promoters. RNA polymerase II transcribes structural or catalytic RNAs that are usually shorter than 400 nucleotides in length, and recognizes a simple run of T residues as a termination signal; it has been used to transcribe siRNA duplexes (see, e. g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Pol II promoters are therefore preferred in certain embodiments where a short RNA transcript is to be produced from a recombinant DNA construct of this invention. In one embodiment, the recombinant DNA construct includes a pol II promoter to express an RNA transcript flanked by self-cleaving ribozyme sequences (e. g., self-cleaving hammerhead ribozymes), resulting in a processed RNA, such as a single-stranded RNA that binds to the transcript of the Leptinotarsa target gene, with defined 5′ and 3′ ends, free of potentially interfering flanking sequences. An alternative approach uses pol III promoters to generate transcripts with relatively defined 5′ and 3′ ends, i. e., to transcribe an RNA with minimal 5′ and 3′ flanking sequences. In some embodiments, Pol III promoters (e. g., U6 or H1 promoters) are preferred for adding a short AT-rich transcription termination site that results in 2 base-pair overhangs (UU) in the transcribed RNA; this is useful, e. g., for expression of siRNA-type constructs. Use of pol III promoters for driving expression of siRNA constructs has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnol., 20: 446-448. Baculovirus promoters such as baculovirus polyhedrin and p10 promoters are known in the art and commercially available; see, e. g., Invitrogen's “Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques”, 2002 (Life Technologies, Carlsbad, Calif.) and F. J. Haines et al. “Baculovirus Expression Vectors”, undated (Oxford Expression Technologies, Oxford, UK).
The promoter element can include nucleic acid sequences that are not naturally occurring promoters or promoter elements or homologues thereof but that can regulate expression of a gene. Examples of such “gene independent” regulatory sequences include naturally occurring or artificially designed RNA sequences that include a ligand-binding region or aptamer (see “Aptamers”, below) and a regulatory region (which can be cis-acting). See, for example, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such “riboregulators” could be selected or designed for specific spatial or temporal specificity, for example, to regulate translation of DNA that encodes a silencing element for suppressing a target gene only in the presence (or absence) of a given concentration of the appropriate ligand. One example is a riboregulator that is responsive to an endogenous ligand (e. g., jasmonic acid or salicylic acid) produced by the plant when under stress (e. g., abiotic stress such as water, temperature, or nutrient stress, or biotic stress such as attach by pests or pathogens); under stress, the level of endogenous ligand increases to a level sufficient for the riboregulator to begin transcription of the DNA that encodes a silencing element for suppressing a target gene.
Recombinase Sites
In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA construct includes at least a pair of loxP sites, wherein site-specific recombination of DNA between the loxP sites is mediated by a Cre recombinase. The position and relative orientation of the loxP sites is selected to achieve the desired recombination; for example, when the loxP sites are in the same orientation, the DNA between the loxP sites is excised in circular form. In another embodiment, the recombinant DNA construct includes DNA encoding one loxP site; in the presence of Cre recombinase and another DNA with a loxP site, the two DNAs are recombined.
Aptamers
In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA that is processed to an RNA aptamer, that is, an RNA that binds to a ligand through binding mechanism that is not primarily based on Watson-Crick base-pairing (in contrast, for example, to the base-pairing that occurs between complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure). See, for example, Ellington and Szostak (1990) Nature, 346:818-822. Examples of aptamers can be found, for example, in the public Aptamer Database, available on line at aptamer.icmb.utexas.edu (Lee et al. (2004) Nucleic Acids Res., 32(1):D95-100). Aptamers useful in the invention can, however, be monovalent (binding a single ligand) or multivalent (binding more than one individual ligand, e. g., binding one unit of two or more different ligands).
Ligands useful in the invention include any molecule (or part of a molecule) that can be recognized and be bound by a nucleic acid secondary structure by a mechanism not primarily based on Watson-Crick base pairing. In this way, the recognition and binding of ligand and aptamer is analogous to that of antigen and antibody, or of biological effector and receptor. Ligands can include single molecules (or part of a molecule), or a combination of two or more molecules (or parts of a molecule), and can include one or more macromolecular complexes (e. g., polymers, lipid bilayers, liposomes, cellular membranes or other cellular structures, or cell surfaces). Examples of specific ligands include vitamins such as coenzyme B12 and thiamine pyrophosphate, flavin mononucleotide, guanine, adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine, glucosamine-6-phosphate, caffeine, theophylline, antibiotics such as chloramphenicol and neomycin, herbicides such as glyphosate and dicamba, proteins including viral or phage coat proteins and invertebrate epidermal or digestive tract surface proteins, and RNAs including viral RNA, transfer-RNAs (t-RNAs), ribosomal RNA (rRNA), and RNA polymerases such as RNA-dependent RNA polymerase (RdRP). One class of RNA aptamers useful in the invention are “thermoswitches” that do not bind a ligand but are thermally responsive, that is to say, the aptamer's conformation is determined by temperature; see, for example, Box 3 in Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463.
Transgene Transcription Units
In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes a transgene transcription unit. A transgene transcription unit includes DNA sequence encoding a gene of interest, e. g., a natural protein or a heterologous protein. A gene of interest can be any coding or non-coding sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi, protists, plants, invertebrates, and vertebrates. Particular genes of interest are genes encoding at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. The transgene transcription unit can further include 5′ or 3′ sequence or both as required for transcription of the transgene.
Introns
In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding a spliceable intron. By “intron” is generally meant a segment of DNA (or the RNA transcribed from such a segment) that is located between exons (protein-encoding segments of the DNA or corresponding transcribed RNA), wherein, during maturation of the messenger RNA, the intron present is enzymatically “spliced out” or removed from the RNA strand by a cleavage/ligation process that occurs in the nucleus in eukaryotes. The term “intron” is also applied to non-coding DNA sequences that are transcribed to RNA segments that can be spliced out of a maturing RNA transcript, but are not introns found between protein-coding exons. One example of these are spliceable sequences that that have the ability to enhance expression in plants (in some cases, especially in monocots) of a downstream coding sequence; these spliceable sequences are naturally located in the 5′ untranslated region of some plant genes, as well as in some viral genes (e. g., the tobacco mosaic virus 5′ leader sequence or “omega” leader described as enhancing expression in plant genes by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638). These spliceable sequences or “expression-enhancing introns” can be artificially inserted in the 5′ untranslated region of a plant gene between the promoter and any protein-coding exons. Examples of such expression-enhancing introns include, but are not limited to, a maize alcohol dehydrogenase (Zm-Adh1), a maize Bronze-1 expression-enhancing intron, a rice actin 1 (Os-Act1) intron, a Shrunken-1 (Sh-1) intron, a maize sucrose synthase intron, a heat shock protein 18 (hsp18) intron, and an 82 kilodalton heat shock protein (hsp82) intron. U.S. Pat. Nos. 5,593,874 and 5,859,347, specifically incorporated by reference herein, describe methods of improving recombinant DNA constructs for use in plants by inclusion of an expression-enhancing intron derived from the 70 kilodalton maize heat shock protein (hsp70) in the non-translated leader positioned 3′ from the gene promoter and 5′ from the first protein-coding exon.
Ribozymes
In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding one or more ribozymes. Ribozymes of particular interest include a self-cleaving ribozyme, a hammerhead ribozyme, or a hairpin ribozyme. In one embodiment, the recombinant DNA construct includes DNA encoding one or more ribozymes that serve to cleave the transcribed RNA to provide defined segments of RNA, such as silencing elements for suppressing a Leptinotarsa target gene.
Gene Suppression Elements
In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding additional gene suppression element for suppressing a target gene other than a Leptinotarsa target gene. The target gene to be suppressed can include coding or non-coding sequence or both.
Suitable gene suppression elements are described in detail in U.S. Patent Application Publication 2006/0200878, which disclosure is specifically incorporated herein by reference, and include one or more of:
In some embodiments, an intron is used to deliver a gene suppression element in the absence of any protein-coding exons (coding sequence). In one example, an intron, such as an expression-enhancing intron (preferred in certain embodiments), is interrupted by embedding within the intron a gene suppression element, wherein, upon transcription, the gene suppression element is excised from the intron. Thus, protein-coding exons are not required to provide the gene suppressing function of the recombinant DNA constructs disclosed herein.
Transcription Regulatory Elements
In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding a transcription regulatory element. Transcription regulatory elements include elements that regulate the expression level of the recombinant DNA construct of this invention (relative to its expression in the absence of such regulatory elements). Examples of suitable transcription regulatory elements include riboswitches (cis- or trans-acting), transcript stabilizing sequences, and miRNA recognition sites, as described in detail in U.S. Patent Application Publication 2006/0200878, specifically incorporated herein by reference.
Making and Using Transgenic Plant Cells and Transgenic Plants
Transformation of a plant can include any of several well-known methods and compositions. Suitable methods for plant transformation include virtually any method by which DNA can be introduced into a cell. One method of plant transformation is microprojectile bombardment, for example, as illustrated in U.S. Pat. No. 5,015,580 (soybean), U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No. 5,914,451 (soybean), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice), U.S. Pat. No. 6,365,807 (rice), and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize), all of which are incorporated by reference for enabling the production of transgenic plants.
Another useful method of plant transformation is Agrobacterium-mediated transformation by means of Agrobacterium containing a binary Ti plasmid system, wherein the Agrobacterium carries a first Ti plasmid and a second, chimeric plasmid containing at least one T-DNA border of a wild-type Ti plasmid, a promoter functional in the transformed plant cell and operably linked to a polynucleotide or recombinant DNA construct of this invention. See, for example, the binary system described in U.S. Pat. No. 5,159,135, incorporated by reference. Also see De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such a binary system, the smaller plasmid, containing the T-DNA border or borders, can be conveniently constructed and manipulated in a suitable alternative host, such as E. coli, and then transferred into Agrobacterium.
Detailed procedures for Agrobacterium-mediated transformation of plants, especially crop plants, include procedures disclosed in U.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soybean); U.S. Pat. Nos. 5,591,616 and 5,981,840 (maize); U.S. Pat. No. 5,463,174 (brassicas including canola), U.S. Pat. No. 7,026,528 (wheat), and U.S. Pat. No. 6,329,571 (rice), and in U.S. Patent Application Publications 2004/0244075 (maize) and 2001/0042257 A1 (sugar beet), all of which are specifically incorporated by reference for enabling the production of transgenic plants. U.S. Patent Application Publication 2011/0296555 discloses in Example 5 the transformation vectors (including the vector sequences) and detailed protocols for transforming maize, soybean, canola, cotton, and sugarcane) and is specifically incorporated by reference for enabling the production of transgenic plants. Similar methods have been reported for many plant species, both dicots and monocots, including, among others, peanut (Cheng et al. (1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37); rice (Toriyama et al. (1988) Bio/Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology, 10:667; Becker et al. (1994) Plant J., 5:299), alfalfa (Masoud et al. (1996) Transgen. Res., 5:313); and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431). See also a description of vectors, transformation methods, and production of transformed Arabidopsis thaliana plants where transcription factors are constitutively expressed by a CaMV35S promoter, in U.S. Patent Application Publication 2003/0167537 A1, incorporated by reference. Transformation methods specifically useful for solanaceous plants are well known in the art. See, for example, publicly described transformation methods for tomato (Sharma et al. (2009), J. Biosci., 34:423-433), eggplant (Arpaia et al. (1997) Theor. Appl. Genet., 95:329-334), potato (Bannerjee et al. (2006) Plant Sci., 170:732-738; Chakravarty et al. (2007) Amer. J. Potato Res., 84:301-311; S. Millam “Agrobacterium-mediated transformation of potato.” Chapter 19 (pp. 257-270), “Transgenic Crops of the World: Essential Protocols”, Ian S. Curtis (editor), Springer, 2004), and peppers (Li et al. (2003) Plant Cell Reports, 21: 785-788). Stably transgenic potato, tomato, and eggplant have been commercially introduced in various regions; see, e. g., K. Redenbaugh et al. “Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the FLAVR SAVR™ Tomato”, CRC Press, Boca Raton, 1992, and the extensive publicly available documentation of commercial genetically modified crops in the GM Crop Database; see: CERA. (2012). GM Crop Database. Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C., available electronically at www.cera-gmc.org/?action=gm_crop_database. Various methods of transformation of other plant species are well known in the art, see, for example, the encyclopedic reference, “Compendium of Transgenic Crop Plants”, edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.interscience.wiley.com/emrw/9781405181099/hpt/toc), which describes transformation procedures for cereals and forage grasses (rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilseed crops (soybean, oilseed brassicas, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (common bean, cowpea, pea, faba bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupine, alfalfa, and clovers), temperate fruits and nuts (apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut), tropical and subtropical fruits and nuts (citrus, grapefruit, banana and plantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit, and persimmon), vegetable crops (tomato, eggplant, peppers, vegetable brassicas, radish, carrot, cucurbits, alliums, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turf grasses), and forest tree species.
Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos or parts of embryos, and gametic cells such as microspores, pollen, sperm, and egg cells. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of this invention. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention (e. g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004/0216189, which are specifically incorporated by reference.
In general transformation practice, DNA is introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are generally used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell is resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the recombinant DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin or paromomycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of useful selective marker genes and selection agents are illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all of which are specifically incorporated by reference. Screenable markers or reporters, such as markers that provide an ability to visually identify transformants can also be employed. Examples of useful screenable markers include, for example, a gene expressing a protein that produces a detectable color by acting on a chromogenic substrate (e. g., beta glucuronidase (GUS) (uidA) or luciferase (luc)) or that itself is detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those of skill in the art will recognize that many other useful markers or reporters are available for use.
Detecting or measuring transcription of a recombinant DNA construct in a transgenic plant cell can be achieved by any suitable method, including protein detection methods (e. g., western blots, ELISAs, and other immunochemical methods), measurements of enzymatic activity, or nucleic acid detection methods (e. g., Southern blots, northern blots, PCR, RT-PCR, fluorescent in situ hybridization).
Other suitable methods for detecting or measuring transcription in a plant cell of a recombinant polynucleotide of this invention targetting an insect target gene include measurement of any other trait that is a direct or proxy indication of the level of expression of the target gene in the insect, relative to the level of expression observed in the absence of the recombinant polynucleotide, e. g., growth rates, mortality rates, or reproductive or recruitment rates of the insect, or measurements of injury (e. g., root injury) or yield loss in a plant or field of plants infested by the insect. In general, suitable methods for detecting or measuring transcription in a plant cell of a recombinant polynucleotide of interest include, e. g., gross or microscopic morphological traits, growth rates, yield, reproductive or recruitment rates, resistance to pests or pathogens, or resistance to biotic or abiotic stress (e. g., water deficit stress, salt stress, nutrient stress, heat or cold stress). Such methods can use direct measurements of a phenotypic trait or proxy assays (e. g., in plants, these assays include plant part assays such as leaf or root assays to determine tolerance of abiotic stress). Such methods include direct measurements of resistance to an invertebrate pest or pathogen (e. g., damage to plant tissues) or proxy assays (e. g., plant yield assays, or invertebrate pest bioassays such as the Western corn rootworm (Diabrotica virgifera virgifera LeConte) larval bioassay described in International Patent Application Publication WO2005/110068 A2 and U.S. Patent Application Publication US 2006/0021087 A1, specifically incorporated by reference, or the soybean cyst nematode bioassay described by Steeves et al. (2006) Funct. Plant Biol., 33:991-999, wherein cysts per plant, cysts per gram root, eggs per plant, eggs per gram root, and eggs per cyst are measured, or the bioassays described herein in the working Examples.
The recombinant DNA constructs of this invention can be stacked with other recombinant DNA for imparting additional traits (e. g., in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, and the like) for example, by expressing or suppressing other genes. Constructs for coordinated decrease and increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 A1, specifically incorporated by reference.
Seeds of fertile transgenic plants can be harvested and used to grow progeny generations, including hybrid generations, of transgenic plants of this invention that include the recombinant DNA construct in their genome. Thus, in addition to direct transformation of a plant with a recombinant DNA construct of this invention, transgenic plants of this invention can be prepared by crossing a first plant having the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. A transgenic plant of this invention can be crossed with a plant line having other recombinant DNA that confers one or more additional trait(s) (such as, but not limited to, herbicide resistance, pest or disease resistance, environmental stress resistance, modified nutrient content, and yield improvement) to produce progeny plants having recombinant DNA that confers both the desired target sequence expression behavior and the additional trait(s).
In such breeding for combining traits the transgenic plant donating the additional trait can be a male line (pollinator) and the transgenic plant carrying the base traits can be the female line. The progeny of this cross segregate such that some of the plant will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, e. g., usually 6 to 8 generations, to produce a homozygous progeny plant with substantially the same genotype as one original transgenic parental line as well as the recombinant DNA of the other transgenic parental line.
Yet another aspect of this invention is a transgenic plant grown from the transgenic seed of this invention. This invention contemplates transgenic plants grown directly from transgenic seed containing the recombinant DNA as well as progeny generations of plants, including inbred or hybrid plant lines, made by crossing a transgenic plant grown directly from transgenic seed to a second plant not grown from the same transgenic seed. Crossing can include, for example, the following steps:
It is often desirable to introgress recombinant DNA into elite varieties, e. g., by backcrossing, to transfer a specific desirable trait from one source to an inbred or other plant that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention. The progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent “B”, and then the selected progeny are mated back to the superior recurrent parent “A”. After five or more backcross generations with selection for the desired trait, the progeny can be essentially hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes. The last backcross generation would be selfed to give progeny which are pure breeding for the gene(s) being transferred, i. e., one or more transformation events.
Through a series of breeding manipulations, a selected DNA construct can be moved from one line into an entirely different line without the need for further recombinant manipulation. One can thus produce inbred plants which are true breeding for one or more DNA constructs. By crossing different inbred plants, one can produce a large number of different hybrids with different combinations of DNA constructs. In this way, plants can be produced which have the desirable agronomic properties frequently associated with hybrids (“hybrid vigor”), as well as the desirable characteristics imparted by one or more DNA constructs.
In certain transgenic plant cells and transgenic plants of this invention, it is sometimes desirable to concurrently express a gene of interest while also modulating expression of a Leptinotarsa target gene. Thus, in some embodiments, the transgenic plant contains recombinant DNA further including a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct of this invention is preferably effected with concurrent transcription of the gene expression element.
In some embodiments, the recombinant DNA constructs of this invention can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage. Transgenic plants can be derived from any monocot or dicot plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood- or pulp-producing trees, vegetable plants, fruit plants, and ornamental plants. Examples of plants of interest include grain crop plants (such as wheat, oat, barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicots including alfalfa, vetch, clover, and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee; wood- or pulp-producing trees; vegetable crop plants such as legumes (for example, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, the brassicas (for example, cabbages, kales, mustards, and other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example, cucumbers, melons, summer squashes, winter squashes), edible alliums (for example, onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers, groundcherries), and edible members of the Chenopodiaceae (for example, beet, chard, spinach, quinoa, amaranth); fruit crop plants such as apple, pear, citrus fruits (for example, orange, lime, lemon, grapefruit, and others), stone fruits (for example, apricot, peach, plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado, and berries; plants grown for biomass or biofuel (for example, Miscanthus grasses, switchgrass, jatropha, oil palm, eukaryotic microalgae such as Botryococcus braunii, Chlorella spp., and Dunaliella spp., and eukaryotic macroalgae such as Gracilaria spp., and Sargassum spp.); and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental groundcovers, and ornamental grasses.
This invention also provides commodity products produced from a transgenic plant cell, plant, or seed of this invention, including, but not limited to, harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts of a plant, meals, oils, extracts, fermentation or digestion products, crushed or whole grains or seeds of a plant, or any food or non-food product including such commodity products produced from a transgenic plant cell, plant, or seed of this invention. The detection of one or more of nucleic acid sequences of the recombinant DNA constructs of this invention in one or more commodity or commodity products contemplated herein is de facto evidence that the commodity or commodity product contains or is derived from a transgenic plant cell, plant, or seed of this invention.
Generally a transgenic plant having in its genome a recombinant DNA construct of this invention exhibits increased resistance to an insect infestation. In various embodiments, for example, where the transgenic plant expresses a recombinant DNA construct of this invention that is stacked with other recombinant DNA for imparting additional traits, the transgenic plant has at least one additional altered trait, relative to a plant lacking the recombinant DNA construct, selected from the group of traits consisting of:
(a) improved abiotic stress tolerance;
(b) improved biotic stress tolerance;
(c) modified primary metabolite composition;
(d) modified secondary metabolite composition;
(e) modified trace element, carotenoid, or vitamin composition;
(f) improved yield;
(g) improved ability to use nitrogen, phosphate, or other nutrients;
(h) modified agronomic characteristics;
(i) modified growth or reproductive characteristics; and
(j) improved harvest, storage, or processing quality.
In some embodiments, the transgenic plant is characterized by: improved tolerance of abiotic stress (e. g., tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or of biotic stress (e. g., crowding, allelopathy, or wounding); by a modified primary metabolite (e. g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition; a modified secondary metabolite (e. g., alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition; a modified trace element (e. g., iron, zinc), carotenoid (e. g., beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e. g., tocopherols) composition; improved yield (e. g., improved yield under non-stress conditions or improved yield under biotic or abiotic stress); improved ability to use nitrogen, phosphate, or other nutrients; modified agronomic characteristics (e. g., delayed ripening; delayed senescence; earlier or later maturity; improved shade tolerance; improved resistance to root or stalk lodging; improved resistance to “green snap” of stems; modified photoperiod response); modified growth or reproductive characteristics (e. g., intentional dwarfing; intentional male sterility, useful, e. g., in improved hybridization procedures; improved vegetative growth rate; improved germination; improved male or female fertility); improved harvest, storage, or processing quality (e. g., improved resistance to pests during storage, improved resistance to breakage, improved appeal to consumers); or any combination of these traits.
In another embodiment, transgenic seed, or seed produced by the transgenic plant, has modified primary metabolite (e. g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition, a modified secondary metabolite composition, a modified trace element, carotenoid, or vitamin composition, an improved harvest, storage, or processing quality, or a combination of these. In another embodiment, it can be desirable to change levels of native components of the transgenic plant or seed of a transgenic plant, for example, to decrease levels of an allergenic protein or glycoprotein or of a toxic metabolite.
Generally, screening a population of transgenic plants each regenerated from a transgenic plant cell is performed to identify transgenic plant cells that develop into transgenic plants having the desired trait. The transgenic plants are assayed to detect an enhanced trait, e. g., enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein, and enhanced seed oil. Screening methods include direct screening for the trait in a greenhouse or field trial or screening for a surrogate trait. Such analyses are directed to detecting changes in the chemical composition, biomass, physiological properties, or morphology of the plant. Changes in chemical compositions such as nutritional composition of grain are detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch, tocopherols, or other nutrients. Changes in growth or biomass characteristics are detected by measuring plant height, stem diameter, internode length, root and shoot dry weights, and (for grain-producing plants such as maize, rice, or wheat) ear or seed head length and diameter. Changes in physiological properties are identified by evaluating responses to stress conditions, e. g., assays under imposed stress conditions such as water deficit, nitrogen or phosphate deficiency, cold or hot growing conditions, pathogen or insect attack, light deficiency, or increased plant density. Other selection properties include days to flowering, days to pollen shed, days to fruit maturation, fruit or tuber quality or amount produced, days to silking in maize, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, staying green, stalk lodging, root lodging, plant health, fertility, green snap, and pest resistance. In addition, phenotypic characteristics of harvested fruit, seeds, or tubers can be evaluated.
This example illustrates non-limiting embodiments of coding DNA sequences useful as target genes for controlling insect species and for making compositions and plants of this invention, and identifies dsRNA trigger sequences useful for controlling insect species. Orthologues to genes previously demonstrated to be efficacious targets for RNAi-mediated mortality in western corn rootworm were identified from insect species that have not previously been reported to be susceptible to orally delivered RNA. These orthologous target genes and examples of dsRNA trigger sequences are provided in Table 1.
Spodoptera frugiperda
Spodoptera frugiperda
Spodoptera frugiperda
Lygus hesperus
Lygus hesperus
Lygus hesperus
Lygus hesperus
Lygus hesperus
Euschistus heros
Euschistus heros
Euschistus heros
Plutella xylostella
Plutella xylostella
Plutella xylostella
Plutella xylostella
Plutella xylostella
Lygus hesperus
Lygus hesperus
The dsRNA trigger sequences that are confirmed to be effective in suppressing a target gene in a sequence-specific manner are useful for identifying efficacious RNA delivery agents and formulations. The insecticidal activity of formulations containing the dsRNA triggers can be optimized by various techniques, such as modifying the chemical entities in the formulation or modifying the ratio of the chemical components in the formulation. Non-limiting examples of delivery agents and formulations are provided in Examples 6 and 8.
This example illustrates non-limiting embodiments of dsRNA trigger sequences useful for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect, and methods for validating dsRNA trigger efficacy or for causing stunting or mortality in an insect. More specifically this example illustrates use of dsRNA triggers for sequence-specific reduction of target gene mRNA transcript level in insect cells.
Cultured Spodoptera frugiperda (fall armyworm, FAW) SF9 cells were incubated with the dsRNA triggers T34640 (SEQ ID NO:17, targetting V-ATPase A subunit), T34642 (SEQ ID NO:18, targetting COPI coatomer B (beta) subunit), or T34644 (SEQ ID NO:19, targetting COPI coatomer B′ (beta prime) subunit), or with a control trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent protein); see Table 1. The dsRNA triggers were formulated with the commercial transfection agent Cellfectin® II (Life Technologies, Inc., Grand Island, N.Y. 14072).
Similar experiments were carried out with Plutella xylostella (diamondback moth, DBM) cells. The results demonstrate a similar target-gene-specific cytotoxic response to the DBM triggers but not to a non-specific trigger targetting green fluorescent protein. Cultured Plutella xylostella (diamondback moth) PxE-PO#5A3 cells were incubated with the dsRNA triggers T42017 (SEQ ID NO:21, targetting V-ATPase subunit A), T33310 (SEQ ID NO:29, targetting V-ATPase subunit A), T32938 (SEQ ID NO:28, targetting COPI coatomer beta subunit), and T32937 (SEQ ID NO:13, targetting COPI coatomer beta prime subunit), or with a control trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent protein); see Table 1. The dsRNA triggers were formulated with the commercial transfection agent Cellfectin® II (Life Technologies, Inc., Grand Island, N.Y. 14072).
This example illustrates non-limiting embodiments of dsRNA trigger sequences useful for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect, and methods for validating dsRNA trigger efficacy or for causing stunting or mortality in an insect. More specifically this example illustrates oral delivery of dsRNA triggers for causing stunting or mortality in insects.
An assay using Euschistus heros (neotropical brown stink bug, NBSB) nymphs fed on an artificial diet was used for testing the efficacy of dsRNA triggers designed specifically to target Euschistus heros genes. Using this assay, a ˜500 base pair dsRNA trigger, T33199, which targets the E. heros ubiquitin C gene, was observed to effect a dose-dependent stunting and mortality response in E. heros nymphs. A shorter (302 bp) dsRNA trigger, T44042 (SEQ ID NO:26) was designed, based on the sequence of T33199, for formulating for oral or topical delivery.
This example illustrates non-limiting embodiments of dsRNA trigger sequences useful for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect, and methods for validating dsRNA trigger efficacy or for causing stunting or mortality in an insect. More specifically this example illustrates embodiments of dsRNA triggers for causing stunting or mortality in insects, and demonstrates systemic RNAi efficacy of these triggers.
Table 2 provides dsRNA triggers tested by microinjection delivery in Lygus hesperus (Western tarnished plant bug) nymphs. The non-Lygus-specific trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent protein) was used as a control.
Lygus hesperus Target Gene
Table 3 presents mortality results for dsRNA triggers, T34617 (SEQ ID NO:15, targetting Lygus hesperus V-ATPase subunit A), and T34622 (SEQ ID NO:16, targetting Lygus hesperus COPI coatomer beta prime subunit) tested by microinjection delivery in Lygus hesperus (Western tarnished plant bug) nymphs. Control nymphs were microinjected with T35763 (targetting green fluorescent protein) or with deionized water. Increased percent mortality by day was observed for Lygus-target-gene-specific treatment groups (T34617 and T34622) compared to negative control treatment groups over the 5-day observation period.
Lygus
Lygus
hesperus
hesperus
Table 4 presents mortality results for dsRNA triggers, T34616 (SEQ ID NO:14, targetting Lygus hesperus ribosomal protein rpL19), T34622 (SEQ ID NO:16, targetting Lygus hesperus COPI coatomer beta prime subunit), and T42772 (SEQ ID NO:22, targetting Lygus hesperus ubiquitin C), tested by microinjection delivery in Lygus hesperus (Western tarnished plant bug) nymphs. Control nymphs were microinjected with T35763 (targetting green fluorescent protein). Increased percent mortality by day was observed for Lygus-target-gene-specific treatment groups (T34616, T34622, and T42772) compared to the negative control (T35763) treatment group over the 6-day observation period. Repeat activity of T34622 confirmed activity of this trigger observed in the earlier trial (Table 3).
Lygus
Lygus
hesperus
hesperus
This example discloses embodiments related to polynucleotide molecules having a nucleotide sequence containing specific modifications such as nucleotide substitutions. Embodiments of such modifications include modified dsRNA triggers that provide improved sequence discrimination between the intended target gene of the insect pest of interest, and genetic sequences of other, non-target species.
Table 5 identifies examples of matches between the sequence of a target gene provided in Table 1 and a sequence identified in a non-target organism (NTO), where the match is a segment of at least 19 contiguous nucleotides. Table 5 further provides examples of sequence modifications (e. g., nucleotide changes at a specified location in the original target gene sequence) to eliminate the sequence match to a non-target organism.
Spodoptera
Danaus plexippus
frugiperda
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Apis mellifera
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Spodoptera
Danaus plexippus
frugiperda
Danaus plexippus
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Apis mellifera
Homo sapiens
Apis mellifera
Homo sapiens
Apis mellifera
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Apis mellifera
Spodoptera
Danaus plexippus
frugiperda
Homo sapiens
Homo sapiens
Bombus impatiens
Bombus terrestris
Danaus plexippus
Danaus plexippus
Homo sapiens
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Lygus
Danaus plexippus
hesperus
Bombus impatiens
Bombus terrestris
Danaus plexippus
Homo sapiens
Bombus impatiens
Bombus terrestris
Homo sapiens
Apis mellifera
Apis mellifera
Lygus
Homo sapiens
hesperus
Homo sapiens
Homo sapiens
Lygus
Bombus terrestris
hesperus
Danaus plexippus
Homo sapiens
Danaus plexippus
Danaus plexippus
Apis mellifera
Danaus plexippus
Homo sapiens
Homo sapiens
Plutella
Danaus plexippus
xylostella
Danaus plexippus
Danaus plexippus
Danaus plexippus
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Plutella
Homo sapiens
xylostella
Danaus plexippus
Homo sapiens
Apis mellifera
Danaus plexippus
Danaus plexippus
Homo sapiens
Danaus plexippus
Plutella
Danaus plexippus
xylostella
Danaus plexippus
Danaus plexippus
Danaus plexippus
Homo sapiens
Homo sapiens
Homo sapiens
Danaus plexippus
Danaus plexippus
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Homo sapiens
Homo sapiens
Homo sapiens
Bombus terrestris
Homo sapiens
Homo sapiens
Danaus plexippus
Danaus plexippus
Homo sapiens
Danaus plexippus
Homo sapiens
Danaus plexippus
Table 6 identifies examples of matches between the sequence of a dsRNA trigger provided in Table 1 and a sequence identified in a non-target organism (NTO), where the match is a segment of at least 19 contiguous nucleotides. Table 6 provides examples of sequence modifications (e. g., nucleotide changes at a specified location in the original dsRNA trigger sequence) which eliminate a specific sequence match of at least 19 contiguous nucleotides to a non-target organism. Table 6 further provides non-limiting embodiments of a modified trigger sequence in which all of the nucleotide changes recited in Table 6 for a given original trigger sequence have been made to eliminate the all of the recited match(es) of at least 19 contiguous nucleotides to a non-target organism sequence. For example, the modified dsRNA trigger having SEQ ID NO:31 has one nucleotide change (A→U) at position 213, when compared to the original dsRNA trigger having SEQ ID NO:15; this single change eliminates matches of at least 19 contiguous nucleotides to two non-target organisms, Bombus impatiens and Bombus terrestris. In another example, the modified dsRNA trigger having SEQ ID NO:35 has four nucleotide changes (at positions 42, 79, 124, and 194), when compared to the original dsRNA trigger having SEQ ID NO:20; these changes eliminate four matches of at least 19 contiguous nucleotides to the NTO Danaus plexippus. In another example, the modified dsRNA trigger having SEQ ID NO:37 has four nucleotide changes (at positions 64, 72, 131, and 280), when compared to the original dsRNA trigger having SEQ ID NO:20; these changes eliminate four matches of at least 19 contiguous nucleotides to the NTOs Bombus impatiens, Bombus terrestris, Homo sapiens, and Danaus plexippus.
Homo sapiens
Bombus impatiens
Bombus terrestris
Bombus terrestris
Danaus plexippus
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Danaus plexippus
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Danaus plexippus
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Apis mellifera
Bombus terrestris
Danaus plexippus
Apis mellifera
Apis mellifera
Apis mellifera
Bombus impatiens
Bombus terrestris
Homo sapiens
Danaus plexippus
Bombus impatiens
Danaus plexippus
Bombus impatiens
Apis mellifera
Apis mellifera
Danaus plexippus
Apis mellifera
Bombus impatiens
Apis mellifera
Apis mellifera
Bombus impatiens
Bombus terrestris
Danaus plexippus
Apis mellifera
Apis mellifera
Bombus impatiens
Homo sapiens
Apis mellifera
Bombus impatiens
Bombus impatiens
Bombus impatiens
Bombus terrestris
Danaus plexippus
Bombus impatiens
Bombus impatiens
Bombus terrestris
Bombus impatiens
Apis mellifera
Apis mellifera
Apis mellifera
Apis mellifera
Bombus impatiens
Apis mellifera
Apis mellifera
Bombus impatiens
Apis mellifera
Bombus impatiens
Bombus impatiens
Bombus terrestris
Danaus plexippus
Bombus impatiens
Bombus impatiens
Bombus terrestris
Bombus impatiens
Apis mellifera
Apis mellifera
Bombus impatiens
Danaus plexippus
Bombus impatiens
Apis mellifera
Apis mellifera
Danaus plexippus
Homo sapiens
Danaus plexippus
Homo sapiens
Homo sapiens
Bombus terrestris
Danaus plexippus
Danaus plexippus
Homo sapiens
Danaus plexippus
Danaus plexippus
Danaus plexippus
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Bombus terrestris
This example discloses embodiments related to polynucleotide molecules, such as dsRNA triggers designed to silence or suppress a target gene of an insect pest of interest, and techniques for determining efficacy of such molecules in suppressing a target gene or in controlling an insect pest. This example discloses a method of providing to an insect a polynucleotide, such as a recombinant RNA molecule or dsRNA trigger, in the form of an ingestible composition.
One method for determining efficacy of polynucleotide molecules in suppressing a target gene or in controlling a pest Lygus species is a sucrose feeding assay. In brief, this assay involves contacting western tarnished plant bug (Lygus hesperus) nymphs with dsRNA triggers in an ingestible composition (a sucrose solution), followed by maintenance on an artificial diet and monitoring of the nymphs' condition.
Parafilm sachets containing 2 milliliters of a 15% sucrose solution were prepared for the feeding experiments, with the treatment sachets containing dsRNA triggers (see Table 1) at either 500 micrograms/milliliter (or parts per million, ppm) or 1000 micrograms/milliliter (or parts per million, ppm). In some experiments the sucrose solution further included 5 milligrams/milliliter yeast tRNA to inhibit potential nuclease activity. Artificial diet sachets were also prepared using parafilm sachets containing 2 milliliters of a western tarnished plant bug (Lygus hesperus) artificial diet prepared by combining autoclaved, boiling water (518 milliliters) with 156.3 grams of Bio-Serv® diet F9644B (Bio-Serv, Frenchtown, N.J.) in a surface-sterilized blender.
Third-instar Lygus hesperus nymphs were anesthetized under carbon dioxide vapor and distributed among 4-ounce glass jars (40-75 individuals per treatment). A piece of tissue paper was added to each jar to absorb honeydew. Each experiment was carried out over 7 days. The nymphs were allowed to feed for a 72-hour period (3 days) on the sucrose sachets, after which the sucrose sachets were removed and replaced with artificial diet sachets for the remaining 4 days of the 7-day observation period. All insects for a single treatment group were observed in a single arena, incubated at 27 degrees Celsius, 60% relative humidity. Initial mortality at day 0 (start of the 72-hour sucrose-feeding stage) was 0 in all cases. Mortality was recorded from 3 days (i. e., the end of the 72-hour sucrose-feeding stage) up to 7 days from the start of the experiment. For gene expression studies, immediately following the end of the 72-hour sucrose-feeding stage, 12 living nymphs were removed from each jar and immediately frozen on dry ice in individual matrix tubes to serve as samples for RNA measurement assays.
A series of feeding experiments using the above protocol was performed to assess activity of the dsRNA triggers T34616 (SEQ ID NO:14), T34617 (SEQ ID NO:15), T34619 (SEQ ID NO:45), T34622 (SEQ ID NO:16), T42772 (SEQ ID NO:22), T42768 (SEQ ID NO:46), and T44045 (SEQ ID NO:26) against Lygus hesperus. The dsRNA triggers T42772 (SEQ ID NO:22) and T44045 (SEQ ID NO:26) were also tested on Lygus lineolaris. A control trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent protein) was used in the assays. At 500 micrograms/milliliter (500 ppm), enhanced mortality was observed with all Lygus-specific dsRNA triggers compared to control treatments in the presence (Table 7) or absence of yeast tRNA supplement (Table 8). T34622 (SEQ ID NO:16) (targetting the putative COPI coatomer beta prime subunit gene with SEQ ID NO:6) and T42772 (SEQ ID NO:22) (targetting the putative ubiquitin C gene with SEQ ID NO:7) were consistently the best performing triggers. Triggers T42772 (SEQ ID NO:22) and T44045 (SEQ ID NO:26), both targetting a putative ubiquitin C gene (SEQ ID NO:7), exhibited similar activity in the Western tarnished plant bug Lygus hesperus (Table 9A); both triggers also appeared active (although slower to act) in a related pest species, the tarnished plant bug, Lygus lineolaris (Table 9B).
A three-replicate experiment tested the ubiquitin C triggers T44045 (SEQ ID NO:26) and T42768 (SEQ ID NO:46) on Lygus hesperus at 1000 micrograms/milliliter (1000 ppm); the results demonstrated statistically significant differences in the means for insect mortality at days 3, 4, 5, 6, and 7 due to ubiquitin C trigger treatments when compared to control treatments with 15% sucrose alone or 15% sucrose plus the control trigger T35763 (SEQ ID NO:27) at 1000 micrograms/milliliter (1000 ppm) (Table 10).
Lygus hesperus mortality (as percent of total insects in treatment),
Lygus lineolaris mortality (as percent of total insects in treatment),
Lygus hesperus mortality (as percent of total insects in treatment),
Target gene suppression was assessed in Lygus hesperus by Quantigene analysis of three target genes (COPI coatomer beta prime subunit, V-ATPase subunit A, and COPI coatomer beta subunit). Sucrose feeding assays were carried out using the dsRNA triggers T34616 (SEQ ID NO:14), T34617 (SEQ ID NO:15), T34619 (SEQ ID NO:45), T34622 (SEQ ID NO:16), T42772 (SEQ ID NO:22), and T44045 (SEQ ID NO:26) tested at 500 or 100 ppm in 15% sucrose as described above. Immediately following the end of the 72-hour sucrose-feeding stage, the nymphs were individually frozen and subjected to Quantigene analysis. All values were normalized against the expression levels of two reference genes (EF1alpha and actin). The results (Tables 11A-C) demonstrated that each of the three tested target genes was specifically suppressed by the corresponding dsRNA trigger, coincident with observed increased mortality when compared to treatment with sucrose only or with the control GFP trigger T35763 (SEQ ID NO:27). The observed target gene suppression (reduction in target gene expression) was significant (p=0.05) compared to sucrose controls for five of six trigger experiments; in the sixth trigger experiment (T34622 at 1000 ppm, see Table 11A) visual suppression was observed just below the significance level. These results support the conclusion that the mortality observed in 15% sucrose feeding assays is mediated by RNAi.
The polynucleotides of this invention are generally designed to modulate expression by inducing regulation or suppression of an insect target gene and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence an insect target gene or cDNA (e. g., SEQ ID NOs:1-12 and 43-44) or to the sequence of RNA transcribed from an insect target gene, which can be coding sequence or non-coding sequence. These effective polynucleotide molecules that modulate expression are referred to herein as a “trigger”, or “triggers”. This example describes non-limiting techniques useful in the design and selection of polynucleotides as “triggers” to modulate expression of an insect target gene.
Selection of Polynucleotide Triggers by “Tiling”
Polynucleotides of use in the invention need not be of the full length of a target gene, and in many embodiments are of much shorter length in comparison to the target gene. An example of a technique that is useful for selecting effective triggers is “tiling”, or evaluation of polynucleotides corresponding to adjacent or partially overlapping segments of a target gene.
Effective polynucleotide “triggers” can be identified by “tiling” gene targets in selected length fragments, e. g., fragments of 200-300 nucleotides in length, with partially overlapping regions, e. g., of about 25 nucleotides, along the length of the target gene. To suppress a single gene, trigger sequences are designed to correspond to (have a nucleotide identity or complementarity with) regions that are unique to the target gene; the selected region of the target gene can include coding sequence or non-coding sequence (e. g., promoter regions, 3′ untranslated regions, introns and the like) or a combination of both.
Where it is of interest to design a target effective in suppressing multiple target genes, the multiple target gene sequences are aligned and polynucleotide triggers designed to correspond to regions with high sequence homology in common among the multiple targets. Conversely, where it is of interest to design a target effective in selectively suppressing one among multiple target sequences, the multiple target gene sequences are aligned and polynucleotide triggers designed to correspond to regions with no or low sequence homology in common among the multiple targets.
In a non-limiting example, anti-sense single-stranded RNA triggers are designed for each of the target genes listed in Table 1 as follows. Multiple anti-sense single-stranded RNA triggers, each of 200-300 nucleotides in length and with a sequence corresponding to (i. e., for anti-sense triggers, complementary to) a fragment of a target gene having a sequence selected from SEQ ID NOs:1-12 and 43-44 are designed so that each trigger's sequence overlaps about 25 nucleotides of the next adjacent trigger's sequence, in such a way that the multiple triggers in combination cover the full length of the target gene. (Sense triggers are designed in an analogous fashion, where the trigger sequence is identical to a fragment of the target gene. Similarly, double-stranded triggers can be designed by providing pairs of sense and anti-sense triggers, each pair of triggers overlapping the next adjacent pair of triggers.)
The polynucleotide triggers are tested by any convenient means for efficacy in silencing the insect target gene. Examples of a suitable test include the bioassays described herein in the working Examples. Another test involves the topical application of the polynucleotide triggers either directly to individual insects or to the surface of a plant to be protected from an insect infestation. One desired result of treatment with a polynucleotide of this invention is prevention or control of an insect infestation, e. g., by inducing in an insect a physiological or behavioural change such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. Another desired result of treatment with a polynucleotide of this invention is provision of a plant that exhibits improved resistance to an insect infestation.
The tiling procedure can be repeated, if desired. A polynucleotide trigger found to provide desired activity can itself be subjected to a tiling procedure. For example, multiple overlapping anti-sense single-stranded RNA triggers are designed, each of 50-60 nucleotides in length and with a sequence corresponding to (i. e., for anti-sense triggers, complementary to) the fragment of a target gene having a sequence selected from SEQ ID NOs:1-12 and 43-44 for which a single polynucleotide trigger of 300 nucleotides was found to be effective. Additional rounds of tiling analysis can be carried out, where triggers as short as 18 or 19 nucleotides are tested.
Effective polynucleotide triggers of any size can be used, alone or in combination, in the various methods of this invention. In some embodiments, a single polynucleotide trigger is used to make a composition of this invention (e. g., a composition for topical application, or a recombinant DNA construct useful for making a transgenic plant). In other embodiments, a mixture or pool of different polynucleotide triggers is used; in such cases the polynucleotide triggers can be for a single target gene or for multiple target genes. In some embodiments, a polynucleotide trigger is designed to target different regions of the target gene, e. g., a trigger can include multiple segments that correspond to different exon regions of the target gene, and “spacer” nucleotides which do not correspond to a target gene can optionally be used in between or adjacent to the segments.
Thermodynamic Considerations in Selecting Polynucleotide Triggers
Polynucleotide triggers can be designed or their sequence optimised using thermodynamic considerations. For example, polynucleotide triggers can be selected based on the thermodynamics controlling hybridization between one nucleic acid strand (e. g., a polynucleotide trigger or an individual siRNA) and another (e. g., a target gene transcript)
Methods and algorithms to predict nucleotide sequences that are likely to be effective at RNAi-mediated silencing of a target gene are known in the art. Non-limiting examples of such methods and algorithms include “i-score”, described by Ichihara et al. (2007) Nucleic Acids Res., 35(18): 123e; “Oligowalk”, publicly available at rna.urmc.rochester.edu/servers/oligowalk and described by Lu et al. (2008) Nucleic Acids Res., 36:W104-108; and “Reynolds score”, described by Khovorova et al. (2004) Nature Biotechnol., 22:326-330.
Permitted Mismatches
By “essentially identical” or “essentially complementary” is meant that the trigger polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to the target gene or to the RNA transcribed from a target gene (e. g., the transcript) to suppress expression of a target gene (e. g., to effect a reduction in levels or activity of the target gene transcript and/or encoded protein). Polynucleotides of this invention need not have 100 percent identity or complementarity to a target gene or to the RNA transcribed from a target gene to suppress expression of the target gene (e. g., to effect a reduction in levels or activity of the target gene transcript or encoded protein, or to provide control of an insect species). In some embodiments, the polynucleotide or a portion thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In certain embodiments, an “essentially identical” polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides in either the endogenous target gene or to an RNA transcribed from the target gene. In certain embodiments, an “essentially complementary” polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to the sequence of 18 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene.
Polynucleotides containing mismatches to the target gene or transcript can be used in certain embodiments of the compositions and methods of this invention. In some embodiments, the polynucleotide includes at least 18 or at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript. In certain embodiments, a polynucleotide of 19 contiguous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript (i. e., 1 or 2 mismatches between the polynucleotide's 19 contiguous nucleotides and the segment of equivalent length in the target gene or target gene's transcript). In certain embodiments, a polynucleotide of 20 or more nucleotides that contains a contiguous 19 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 21 continuous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 22 or more nucleotides that contains a contiguous 21 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript.
In designing polynucleotides with mismatches to an endogenous target gene or to an RNA transcribed from the target gene, mismatches of certain types and at certain positions that are more likely to be tolerated can be used. In certain exemplary embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used as described by Du et al. (2005) Nucleic Acids Res., 33:1671-1677. In some embodiments, mismatches in 19 base-pair overlap regions are located at the low tolerance positions 5, 7, 8 or 11 (from the 5′ end of a 19-nucleotide target), at medium tolerance positions 3, 4, and 12-17(from the 5′ end of a 19-nucleotide target), and/or at the high tolerance positions at either end of the region of complementarity, i. e., positions 1, 2, 18, and 19 (from the 5′ end of a 19-nucleotide target) as described by Du et al. (2005) Nucleic Acids Res., 33:1671-1677. Tolerated mismatches can be empirically determined in routine assays such as those described herein in the working Examples.
In some embodiments, the polynucleotides include additional nucleotides for reasons of stability or for convenience in cloning or synthesis. In one embodiment, the polynucleotide is a dsRNA including an RNA strand with a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 and further including an additional 5′ G or an additional 3′ C or both, adjacent to the segment. In another embodiment, the polynucleotide is a double-stranded RNA including additional nucleotides to form an overhang, for example, a dsRNA including 2 deoxyribonucleotides to form a 3′ overhang.
Embedding Active Triggers in Neutral Sequence
In an embodiment, a bioactive trigger (i. e., a polynucleotide with a sequence corresponding to the target gene and which is responsible for an observed suppression of the target gene) is embedded in “neutral” sequence, i. e., inserted into additional nucleotides that have no sequence identity or complementarity to the target gene. Neutral sequence can be desirable, e. g., to increase the overall length of a polynucleotide. For example, it can be desirable for a polynucleotide to be of a particular size for reasons of stability, cost-effectiveness in manufacturing, or biological activity.
It has been reported that in another coleopteran species, Diabrotica virgifera, dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays; see Bolognesi et al. (2012) PLoS ONE 7(10): e47534. doi:10.1371/journal.pone.0047534. Thus, in one embodiment, a 21-base-pair dsRNA trigger corresponding to a target gene in Table 1 and found to provide control of an insect infestation is embedded in neutral sequence of an additional 39 base pairs, thus forming a polynucleotide of about 60 base pairs. In another embodiment, a single 21-base-pair trigger is found to be efficacious when embedded in larger sections of neutral sequence, e. g., where the total polynucleotide length is from about 60 to about 300 base pairs. In another embodiment, at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 is embedded in larger sections of neutral sequence to provide an efficacious trigger. In another embodiment, segments from multiple sequences selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 are embedded in larger sections of neutral sequence to provide an efficacious trigger.
It is anticipated that the combination of certain recombinant RNAs of this invention (e. g., the dsRNA triggers having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46, or active fragments of these triggers) with one or more non-polynucleotide pesticidal agents will result in a synergetic improvement in prevention or control of insect infestations, when compared to the effect obtained with the recombinant RNA alone or the non-polynucleotide pesticidal agent alone. Routine insect bioassays such as the bioassays described herein in the working Examples are useful for defining dose-responses for larval mortality or growth inhibition using combinations of the polynucleotides of this invention and one or more non-polynucleotide pesticidal agents (e. g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein). One of skill in the art can test combinations of polynucleotides and non-polynucleotide pesticidal agents in routine bioassays to identify combinations of bioactives that are synergistic and desirable for use in protecting plants from insect infestations.
This example illustrates non-limiting embodiments of the use of polynucleotides of this invention in topically applied compositions for preventing or controlling insect infestations.
Compositions containing one or more polynucleotides of this invention are useful as topical treatments of plants, animals, or environments wherein prevention or control of a Leptinotarsa species infestation is desired. In embodiments, a polynucleotide trigger for a target gene with a sequence selected from SEQ ID NOs:1-12 and 43-44, i. e., the target genes identified in Table 1, as described in the preceding examples, is included in an effective amount in a composition designed to be provided directly (e. g., by contact or ingestion) to an insect species, or a plant or environment wherein prevention or control of infestation by that insect is desired. In embodiments, a polynucleotide trigger for a target gene with a sequence selected from SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 is included in an effective amount in such compositions. In embodiments, a dsRNA trigger with a strand having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46, or active fragments of these triggers, is included in an effective amount in such compositions. Such compositions are formulated and manufactured according to the art and can be in any convenient form, e. g., a solution or mixture of solutions, an emulsion, a suspension, a dispersible powder, a solid or liquid bait, a seed coating, or a soil drench. Embodiments of such compositions include those where the polynucleotide of this invention is provided in a living or dead microorganism such as a bacterium or fungal or yeast cell, or provided as a microbial fermentation product, or provided in a living or dead plant cell, or provided as a synthetic recombinant polynucleotide. In an embodiment the composition includes a non-pathogenic strain of a microorganism that contains a polynucleotide of this invention; ingestion or intake of the microorganism results in stunting or mortality of the insect pest; non-limiting examples of suitable microorganisms include E. coli, B. thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia entomophila and related Serratia sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae, Clostridium bifermentans and other Clostridium species, or other spore-forming gram-positive bacteria. In an embodiment, the composition includes a plant virus vector including a polynucleotide of this invention; feeding by an insect on a plant treated with the plant virus vector results in stunting or mortality of the insect. In an embodiment, the composition includes a baculovirus vector including a polynucleotide of this invention; ingestion or intake of the vector results in stunting or mortality of the insect. In an embodiment, a polynucleotide of this invention is encapsulated in a synthetic matrix such as a polymer or attached to particulates and topically applied to the surface of a plant; feeding by an insect on the topically treated plant results in stunting or mortality of the insect. In an embodiment, a polynucleotide of this invention is provided in the form of a plant cell (e. g., a transgenic plant cell of this invention) expressing the polynucleotide; ingestion of the plant cell or contents of the plant cell by an insect results in stunting or mortality of the insect.
Embodiments of the compositions optionally include the appropriate stickers and wetters required for efficient foliar coverage as well as UV protectants to protect polynucleotides such as dsRNAs from UV damage. Such additives are commonly used in the bioinsecticide industry and are known to those skilled in the art. Compositions for soil application can include granular formulations that serve as bait for insect larvae. Embodiments include a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. In embodiments, the composition further includes at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.
Such compositions are applied in any convenient manner, e. g., by spraying or dusting the insect directly, or spraying or dusting a plant or environment wherein prevention or control of infestation by that insect is desired, or by applying a coating to a surface of a plant, or by applying a coating to a seed (or seed potato) in preparation for the seed's planting, or by applying a soil drench around roots of a plant for which prevention or control of infestation by that insect is desired.
An effective amount of a polynucleotide of this invention is an amount sufficient to provide control of the insect, or to prevent infestation by the insect; determination of effective amounts of a polynucleotide of this invention are made using routine assays such as those described in the working Examples herein. While there is no upper limit on the concentrations and dosages of a polynucleotide of this invention that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency and economy. Non-limiting embodiments of effective amounts of a polynucleotide include a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of a polynucleotide in a liquid form sprayed on a plant, or from about 10 milligrams per acre to about 100 grams per acre of polynucleotide applied to a field of plants, or from about 0.001 to about 0.1 microgram per milliliter of polynucleotide in an artificial diet for feeding the insect. Where compositions of this invention are topically applied to a plant, the concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using 25-mer polynucleotides of this invention is about 1 nanomole (nmol) of polynucleotides per plant, for example, from about 0.05 to 1 nmol polynucleotides per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per plant. In certain embodiments, about 40 to about 50 nmol of a ssDNA polynucleotide of this invention are applied. In certain embodiments, about 0.5 nmol to about 2 nmol of a dsRNA of this invention is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of a dsRNA or an ssDNA (21-mer) of this invention is applied. In certain embodiments, a composition of about 0.5 to about 1.5 milligrams per milliliter of a dsRNA polynucleotide of this invention of about 50 to about 200 or more nucleotides is applied. In certain embodiments, about 1 nmol to about 5 nmol of a dsRNA of this invention is applied to a plant. In certain embodiments, the polynucleotide composition as topically applied to the plant contains at least one polynucleotide of this invention at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines can require correspondingly larger amounts of polynucleotides. When using long dsRNA molecules of this invention that can be processed into multiple oligonucleotides (e. g., multiple triggers encoded by a single recombinant DNA molecule of this invention), lower concentrations can be used. Non-limiting examples of effective polynucleotide treatment regimes include a treatment of between about 0.1 to about 1 nmol of polynucleotide molecule per plant, or between about 1 nmol to about 10 nmol of polynucleotide molecule per plant, or between about 10 nmol to about 100 nmol of polynucleotide molecule per plant.
Embodiments of compositions of this invention include a “transfer agent”, i. e., an agent that, when combined with a composition including a polynucleotide of this invention that is topically applied to the surface of an organism, enables the polynucleotide to enter the cells of that organism. Such transfer agents can be incorporated as part of the composition including a polynucleotide of this invention, or can be applied prior to, contemporaneously with, or following application of the composition including a polynucleotide of this invention. In embodiments, a transfer agent is an agent that improves the uptake of a polynucleotide of this invention by an insect. In embodiments, a transfer agent is an agent that conditions the surface of plant tissue, e. g., seeds, leaves, stems, roots, flowers, or fruits, to permeation by a polynucleotide of this invention into plant cells. In embodiments, the transfer agent enables a pathway for a polynucleotide of this invention through cuticle wax barriers, stomata, and/or cell wall or membrane barriers into plant cells.
Suitable transfer agents include agents that increase permeability of the exterior of the organism or that increase permeability of cells of the organism to polynucleotides of this invention. Suitable transfer agents include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. In embodiments, application of a composition of this invention and a transfer agent optionally includes an incubation step, a neutralization step (e. g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. Suitable transfer agents can be in the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition, or can cause the polynucleotide composition to take the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition. Embodiments of transfer agents include counter-ions or other molecules that are known to associate with nucleic acid molecules, e. g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Embodiments of transfer agents include organic solvents such as DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents miscible with water or that dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Embodiments of transfer agents include naturally derived or synthetic oils with or without surfactants or emulsifiers, e. g., plant-sourced oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available on-line at herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.
Embodiments of transfer agents include organosilicone preparations. For example, a suitable transfer agent is an organosilicone preparation that is commercially available as SILWET L-77® brand surfactant having CAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currently available from Momentive Performance Materials, Albany, N.Y. In embodiments where a SILWET L-77® brand surfactant organosilicone preparation is used as transfer agent in the form of a spray treatment (applied prior to, contemporaneously with, or following application of the composition including a polynucleotide of this invention) of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of a polynucleotide of this invention into plant cells from a topical application on the surface. One embodiment includes a composition that comprises a polynucleotide of this invention and a transfer agent including an organosilicone preparation such as Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent). One embodiment includes a composition that comprises a polynucleotide of this invention and a transfer agent including SILWET L-77® brand surfactant in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1%, by weight (wt percent).
Organosilicone compounds useful as transfer agents for use in this invention include, but are not limited to, compounds that include: (a) a trisiloxane head group that is covalently linked to, (b) an alkyl linker including, but not limited to, an n-propyl linker, that is covalently linked to, (c) a polyglycol chain, that is covalently linked to, (d) a terminal group. Trisiloxane head groups of such organosilicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an n-propyl linker. Polyglycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Polyglycol chains can comprise a mixture that provides an average chain length “n” of about “7.5”. In certain embodiments, the average chain length “n” can vary from about 5 to about 14. Terminal groups can include, but are not limited to, alkyl groups such as a methyl group. Organosilicone compounds useful as transfer agents for use in this invention include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane. An example of a transfer agent for use in this invention is Compound I:
Organosilicone compounds useful as transfer agents for use in this invention are used, e. g., as freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent).
Embodiments of transfer agents include one or more salts such as ammonium chloride, tetrabutylphosphonium bromide, and ammonium sulfate, provided in or used with a composition including a polynucleotide of this invention. In embodiments, ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate are used at a concentration of about 0.5% to about 5% (w/v), or about 1% to about 3% (w/v), or about 2% (w/v). In certain embodiments, the composition including a polynucleotide of this invention includes an ammonium salt at a concentration greater or equal to 300 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes an organosilicone transfer agent in a concentration of about 0.015 to about 2 percent by weight (wt percent) as well as ammonium sulfate at concentrations from about 80 to about 1200 mM or about 150 mM to about 600 mM.
Embodiments of transfer agents include a phosphate salt. Phosphate salts useful in a composition including a polynucleotide of this invention include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts. In certain embodiments, the composition including a polynucleotide of this invention includes a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes a phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the composition including a polynucleotide of this invention includes sodium phosphate at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes sodium phosphate at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes a sodium phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the composition including a polynucleotide of this invention includes a sodium phosphate salt in a range of about 10 mM to about 160 mM or in a range of about 20 mM to about 40 mM. In certain embodiments, the composition including a polynucleotide of this invention includes a sodium phosphate buffer at a pH of about 6.8.
Embodiments of transfer agents include surfactants and/or effective molecules contained therein. Surfactants and/or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organosilicone surfactants. In certain embodiments, the composition including a polynucleotide of this invention is formulated with counter-ions or other molecules that are known to associate with nucleic acid molecules. Non-limiting examples include, tetraalkyl ammonium ions, trialkyl ammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine. In certain embodiments, the composition including a polynucleotide of this invention is formulated with a non-polynucleotide herbicide e. g., glyphosate, auxin-like benzoic acid herbicides including dicamba, chloramben, and TBA, glufosinate, auxin-like herbicides including phenoxy carboxylic acid herbicide, pyridine carboxylic acid herbicide, quinoline carboxylic acid herbicide, pyrimidine carboxylic acid herbicide, and benazolin-ethyl herbicide, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides. In certain embodiments, the composition including a polynucleotide of this invention is formulated with a non-polynucleotide pesticide, e. g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.
All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure. Although the materials and methods of this invention have been described in terms of preferred embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of this invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of this invention as defined by the appended claims.
This application is a U.S. National Entry of International Application No. PCT/US2015/022985, filed on Mar. 27, 2015 which claims priority to U.S. Provisional Patent Application No. 61/973,484 filed Apr. 1, 2014, which is incorporated by reference in its entirety herein. A sequence listing contained in the file named “P34352US01_SEQ.txt” which is 71,047 bytes in size (measured in MS-Windows®) and created on Sep. 29, 2016 is filed electronically herewith and incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/022985 | 3/27/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/153339 | 10/8/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 3687808 | Merigan et al. | Aug 1972 | A |
| 3791932 | Schuurs et al. | Feb 1974 | A |
| 3839153 | Schuurs et al. | Oct 1974 | A |
| 3850578 | McConnell | Nov 1974 | A |
| 3850752 | Schuurs et al. | Nov 1974 | A |
| 3853987 | Dreyer | Dec 1974 | A |
| 3867517 | Ling | Feb 1975 | A |
| 3879262 | Schuurs et al. | Apr 1975 | A |
| 3901654 | Gross | Aug 1975 | A |
| 3935074 | Rubenstein et al. | Jan 1976 | A |
| 3984533 | Uzgiris | Oct 1976 | A |
| 3996345 | Ullman et al. | Dec 1976 | A |
| 4034074 | Miles | Jul 1977 | A |
| 4098876 | Piasio et al. | Jul 1978 | A |
| 4469863 | Ts'o et al. | Sep 1984 | A |
| 4476301 | Imbach et al. | Oct 1984 | A |
| 4535060 | Comai | Aug 1985 | A |
| 4581847 | Hibberd et al. | Apr 1986 | A |
| 4666828 | Gusella | May 1987 | A |
| 4683202 | Mullis | Jul 1987 | A |
| 4732250 | Maucher et al. | Mar 1988 | A |
| 4761373 | Anderson et al. | Aug 1988 | A |
| 4769061 | Comai | Sep 1988 | A |
| 4801531 | Frossard | Jan 1989 | A |
| 4810648 | Stalker | Mar 1989 | A |
| 4879219 | Wands et al. | Nov 1989 | A |
| 4940835 | Shah et al. | Jul 1990 | A |
| 4971908 | Kishore et al. | Nov 1990 | A |
| 5004863 | Umbeck | Apr 1991 | A |
| 5011771 | Bellet et al. | Apr 1991 | A |
| 5013659 | Bedbrook et al. | May 1991 | A |
| 5015580 | Christou et al. | May 1991 | A |
| 5023243 | Tullis | Jun 1991 | A |
| 5034506 | Summerton et al. | Jul 1991 | A |
| 5094945 | Comai | Mar 1992 | A |
| 5141870 | Bedbrook et al. | Aug 1992 | A |
| 5145783 | Kishore et al. | Sep 1992 | A |
| 5159135 | Umbeck | Oct 1992 | A |
| 5166315 | Summerton et al. | Nov 1992 | A |
| 5177196 | Meyer, Jr. et al. | Jan 1993 | A |
| 5185444 | Summerton et al. | Feb 1993 | A |
| 5188642 | Shah et al. | Feb 1993 | A |
| 5188897 | Suhadolnik et al. | Feb 1993 | A |
| 5192659 | Simons | Mar 1993 | A |
| 5214134 | Weis et al. | May 1993 | A |
| 5216141 | Benner | Jun 1993 | A |
| 5235033 | Summerton et al. | Aug 1993 | A |
| 5264423 | Cohen et al. | Nov 1993 | A |
| 5264562 | Matteucci | Nov 1993 | A |
| 5264564 | Matteucci | Nov 1993 | A |
| 5272057 | Smulson et al. | Dec 1993 | A |
| 5276019 | Cohen et al. | Jan 1994 | A |
| 5278302 | Caruthers et al. | Jan 1994 | A |
| 5281521 | Trojanowski et al. | Jan 1994 | A |
| 5286634 | Stadler et al. | Feb 1994 | A |
| 5286717 | Cohen et al. | Feb 1994 | A |
| 5304732 | Anderson et al. | Apr 1994 | A |
| 5310667 | Eichholtz et al. | May 1994 | A |
| 5312910 | Kishore et al. | May 1994 | A |
| 5321131 | Agrawal et al. | Jun 1994 | A |
| 5331107 | Anderson et al. | Jul 1994 | A |
| 5339107 | Henry et al. | Aug 1994 | A |
| 5346107 | Bouix et al. | Sep 1994 | A |
| 5378824 | Bedbrook et al. | Jan 1995 | A |
| 5390667 | Kumakura et al. | Feb 1995 | A |
| 5392910 | Bell et al. | Feb 1995 | A |
| 5393175 | Courville | Feb 1995 | A |
| 5399676 | Froehler | Mar 1995 | A |
| 5405938 | Summerton et al. | Apr 1995 | A |
| 5405939 | Suhadolnik et al. | Apr 1995 | A |
| 5416011 | Hinchee et al. | May 1995 | A |
| 5453496 | Caruthers et al. | Sep 1995 | A |
| 5455233 | Spielvogel et al. | Oct 1995 | A |
| 5459127 | Felgner et al. | Oct 1995 | A |
| 5460667 | Moriyuki et al. | Oct 1995 | A |
| 5462910 | Ito et al. | Oct 1995 | A |
| 5463174 | Moloney et al. | Oct 1995 | A |
| 5463175 | Barry et al. | Oct 1995 | A |
| 5466677 | Baxter et al. | Nov 1995 | A |
| 5470967 | Huie et al. | Nov 1995 | A |
| 5476925 | Letsinger et al. | Dec 1995 | A |
| 5489520 | Adams et al. | Feb 1996 | A |
| 5489677 | Sanghvi et al. | Feb 1996 | A |
| 5491288 | Chaubet et al. | Feb 1996 | A |
| 5510471 | Lebrun et al. | Apr 1996 | A |
| 5518908 | Corbin et al. | May 1996 | A |
| 5519126 | Hecht | May 1996 | A |
| 5536821 | Agrawal et al. | Jul 1996 | A |
| 5538880 | Lundquist et al. | Jul 1996 | A |
| 5539082 | Nielsen et al. | Jul 1996 | A |
| 5541306 | Agrawal et al. | Jul 1996 | A |
| 5541307 | Cook et al. | Jul 1996 | A |
| 5550111 | Suhadolnik et al. | Aug 1996 | A |
| 5550318 | Adams et al. | Aug 1996 | A |
| 5550398 | Kocian et al. | Aug 1996 | A |
| 5550468 | Haberlein et al. | Aug 1996 | A |
| 5558071 | Ward et al. | Sep 1996 | A |
| 5561225 | Maddry et al. | Oct 1996 | A |
| 5561236 | Leemans et al. | Oct 1996 | A |
| 5563253 | Agrawal et al. | Oct 1996 | A |
| 5569834 | Hinchee et al. | Oct 1996 | A |
| 5571799 | Tkachuk et al. | Nov 1996 | A |
| 5587361 | Cook et al. | Dec 1996 | A |
| 5591616 | Hiei et al. | Jan 1997 | A |
| 5593874 | Brown et al. | Jan 1997 | A |
| 5596086 | Matteucci et al. | Jan 1997 | A |
| 5597717 | Guerineau et al. | Jan 1997 | A |
| 5602240 | De Mesmaeker et al. | Feb 1997 | A |
| 5605011 | Bedbrook et al. | Feb 1997 | A |
| 5608046 | Cook et al. | Mar 1997 | A |
| 5610289 | Cook et al. | Mar 1997 | A |
| 5618704 | Sanghvi et al. | Apr 1997 | A |
| 5623070 | Cook et al. | Apr 1997 | A |
| 5625050 | Beaton et al. | Apr 1997 | A |
| 5627061 | Barry et al. | May 1997 | A |
| 5633360 | Bischofberger et al. | May 1997 | A |
| 5633435 | Barry et al. | May 1997 | A |
| 5633448 | Lebrun et al. | May 1997 | A |
| 5639024 | Mueller et al. | Jun 1997 | A |
| 5646024 | Leemans et al. | Jul 1997 | A |
| 5648477 | Leemans et al. | Jul 1997 | A |
| 5663312 | Chaturvedula | Sep 1997 | A |
| 5677437 | Teng et al. | Oct 1997 | A |
| 5677439 | Weis et al. | Oct 1997 | A |
| 5714331 | Buchardt et al. | Feb 1998 | A |
| 5719046 | Guerineau et al. | Feb 1998 | A |
| 5719262 | Buchardt et al. | Feb 1998 | A |
| 5721138 | Lawn | Feb 1998 | A |
| 5731180 | Dietrich | Mar 1998 | A |
| 5739180 | Taylor-Smith | Apr 1998 | A |
| 5746180 | Jefferson et al. | May 1998 | A |
| 5767361 | Dietrich | Jun 1998 | A |
| 5767373 | Ward et al. | Jun 1998 | A |
| 5780708 | Lundquist et al. | Jul 1998 | A |
| 5804425 | Barry et al. | Sep 1998 | A |
| 5824877 | Hinchee et al. | Oct 1998 | A |
| 5837848 | Ely et al. | Nov 1998 | A |
| 5859347 | Brown et al. | Jan 1999 | A |
| 5866775 | Eichholtz et al. | Feb 1999 | A |
| 5874265 | Adams et al. | Feb 1999 | A |
| 5879903 | Strauch et al. | Mar 1999 | A |
| 5898031 | Crooke | Apr 1999 | A |
| 5914451 | Martinell et al. | Jun 1999 | A |
| 5919675 | Adams et al. | Jul 1999 | A |
| 5928937 | Kakefuda et al. | Jul 1999 | A |
| 5939602 | Volrath et al. | Aug 1999 | A |
| 5969213 | Adams et al. | Oct 1999 | A |
| 5981840 | Zhao et al. | Nov 1999 | A |
| 5985793 | Sandbrink et al. | Nov 1999 | A |
| RE36449 | Lebrun et al. | Dec 1999 | E |
| 6040497 | Spencer et al. | Mar 2000 | A |
| 6056938 | Unger et al. | May 2000 | A |
| 6069115 | Pallett et al. | May 2000 | A |
| 6084089 | Mine et al. | Jul 2000 | A |
| 6084155 | Volrath et al. | Jul 2000 | A |
| 6107094 | Crooke | Aug 2000 | A |
| 6118047 | Anderson et al. | Sep 2000 | A |
| 6121513 | Zhang et al. | Sep 2000 | A |
| 6130366 | Herrera-Estrella et al. | Oct 2000 | A |
| 6140078 | Sanders et al. | Oct 2000 | A |
| 6153812 | Fry et al. | Nov 2000 | A |
| 6160208 | Lundquist et al. | Dec 2000 | A |
| 6177616 | Bartsch et al. | Jan 2001 | B1 |
| 6194636 | McElroy et al. | Feb 2001 | B1 |
| 6225105 | Sathasivan et al. | May 2001 | B1 |
| 6225114 | Eichholtz et al. | May 2001 | B1 |
| 6232526 | McElroy et al. | May 2001 | B1 |
| 6232536 | McElroy et al. | May 2001 | B1 |
| 6245968 | Boudec et al. | Jun 2001 | B1 |
| 6248876 | Barry et al. | Jun 2001 | B1 |
| 6252138 | Karimi et al. | Jun 2001 | B1 |
| RE37287 | Lebrun et al. | Jul 2001 | E |
| 6268549 | Sailland et al. | Jul 2001 | B1 |
| 6271359 | Norris et al. | Aug 2001 | B1 |
| 6282837 | Ward et al. | Sep 2001 | B1 |
| 6288306 | Ward et al. | Sep 2001 | B1 |
| 6288312 | Christou et al. | Sep 2001 | B1 |
| 6294714 | Matsunaga et al. | Sep 2001 | B1 |
| 6303374 | Zhang et al. | Oct 2001 | B1 |
| 6326193 | Liu et al. | Dec 2001 | B1 |
| 6329571 | Hiei | Dec 2001 | B1 |
| 6348185 | Piwnica-Worms | Feb 2002 | B1 |
| 6365807 | Christou et al. | Apr 2002 | B1 |
| 6384301 | Martinell et al. | May 2002 | B1 |
| 6385902 | Schipper et al. | May 2002 | B1 |
| 6399861 | Anderson et al. | Jun 2002 | B1 |
| 6403865 | Koziel et al. | Jun 2002 | B1 |
| 6414222 | Gengenbach et al. | Jul 2002 | B1 |
| 6421956 | Boukens et al. | Jul 2002 | B1 |
| 6426446 | McElroy et al. | Jul 2002 | B1 |
| 6433252 | Kriz et al. | Aug 2002 | B1 |
| 6437217 | McElroy et al. | Aug 2002 | B1 |
| 6453609 | Soll et al. | Sep 2002 | B1 |
| 6479291 | Kumagai et al. | Nov 2002 | B2 |
| 6506559 | Fire et al. | Jan 2003 | B1 |
| 6642435 | Rafalski et al. | Nov 2003 | B1 |
| 6644341 | Chemo et al. | Nov 2003 | B1 |
| 6645914 | Woznica et al. | Nov 2003 | B1 |
| 6768044 | Boudec et al. | Jul 2004 | B1 |
| 6843985 | Erickson, Jr. et al. | Jan 2005 | B2 |
| 6992237 | Habben et al. | Jan 2006 | B1 |
| 7022896 | Weeks et al. | Apr 2006 | B1 |
| 7026528 | Cheng et al. | Apr 2006 | B2 |
| 7056704 | Tuschl et al. | Jun 2006 | B2 |
| 7078196 | Tuschl et al. | Jul 2006 | B2 |
| RE39247 | Barry et al. | Aug 2006 | E |
| 7105724 | Weeks et al. | Sep 2006 | B2 |
| 7297541 | Moshiri et al. | Nov 2007 | B2 |
| 7304209 | Zink et al. | Dec 2007 | B2 |
| 7312379 | Andrews et al. | Dec 2007 | B2 |
| 7323310 | Peters et al. | Jan 2008 | B2 |
| 7371927 | Yao et al. | May 2008 | B2 |
| 7392379 | Le Pennec et al. | Jun 2008 | B2 |
| 7405347 | Hammer et al. | Jul 2008 | B2 |
| 7406981 | Hemo et al. | Aug 2008 | B2 |
| 7462379 | Fukuda et al. | Dec 2008 | B2 |
| 7485777 | Nakajima et al. | Feb 2009 | B2 |
| 7525013 | Hildebrand et al. | Apr 2009 | B2 |
| 7550578 | Budworth et al. | Jun 2009 | B2 |
| 7622301 | Ren et al. | Nov 2009 | B2 |
| 7657299 | Huizenga et al. | Feb 2010 | B2 |
| 7671254 | Tranel et al. | Mar 2010 | B2 |
| 7714188 | Castle et al. | May 2010 | B2 |
| 7738626 | Weese et al. | Jun 2010 | B2 |
| 7807791 | Sekar et al. | Oct 2010 | B2 |
| 7838263 | Dam et al. | Nov 2010 | B2 |
| 7838733 | Wright et al. | Nov 2010 | B2 |
| 7842856 | Tranel et al. | Nov 2010 | B2 |
| 7855323 | Huang et al. | Dec 2010 | B2 |
| 7884262 | Clemente et al. | Feb 2011 | B2 |
| 7910805 | Duck et al. | Mar 2011 | B2 |
| 7935869 | Pallett et al. | May 2011 | B2 |
| 7943819 | Baum et al. | May 2011 | B2 |
| 7973218 | McCutchen et al. | Jul 2011 | B2 |
| 8090164 | Bullitt et al. | Jan 2012 | B2 |
| 8097712 | Paldi et al. | Jan 2012 | B2 |
| 8143480 | Axtell et al. | Mar 2012 | B2 |
| 8158414 | Rommens et al. | Apr 2012 | B2 |
| 8507457 | Paldi et al. | Aug 2013 | B2 |
| 8548778 | Hart et al. | Oct 2013 | B1 |
| 8554490 | Tang et al. | Oct 2013 | B2 |
| 8598332 | Waterhouse et al. | Dec 2013 | B1 |
| 9006414 | Huang et al. | Apr 2015 | B2 |
| 9121022 | Sammons et al. | Sep 2015 | B2 |
| 9422557 | Ader | Aug 2016 | B2 |
| 9445603 | Baum et al. | Sep 2016 | B2 |
| 20010006797 | Kumagai et al. | Jul 2001 | A1 |
| 20010042257 | Connor-Ward et al. | Nov 2001 | A1 |
| 20020069430 | Kiaska et al. | Jun 2002 | A1 |
| 20020086356 | Tuschl et al. | Jul 2002 | A1 |
| 20020114784 | Li et al. | Aug 2002 | A1 |
| 20020123476 | Emanuele et al. | Sep 2002 | A1 |
| 20030017068 | Larrain et al. | Jan 2003 | A1 |
| 20030044443 | Erickson, Jr. et al. | Mar 2003 | A1 |
| 20030092651 | Norris et al. | May 2003 | A1 |
| 20030096980 | Froehler et al. | May 2003 | A1 |
| 20030140371 | Stevens et al. | Jul 2003 | A1 |
| 20030150017 | Mesa et al. | Aug 2003 | A1 |
| 20030154508 | Stevens et al. | Aug 2003 | A1 |
| 20030167537 | Jiang | Sep 2003 | A1 |
| 20030180945 | Wang et al. | Sep 2003 | A1 |
| 20040029275 | Brown et al. | Feb 2004 | A1 |
| 20040053289 | Allen et al. | Mar 2004 | A1 |
| 20040055041 | Labate et al. | Mar 2004 | A1 |
| 20040072692 | Hoffman et al. | Apr 2004 | A1 |
| 20040082475 | Hoffman et al. | Apr 2004 | A1 |
| 20040123347 | Hinchey et al. | Jun 2004 | A1 |
| 20040126845 | Eenennaam et al. | Jul 2004 | A1 |
| 20040133944 | Hake et al. | Jul 2004 | A1 |
| 20040147475 | Li et al. | Jul 2004 | A1 |
| 20040177399 | Hammer et al. | Sep 2004 | A1 |
| 20040216189 | Houmard et al. | Oct 2004 | A1 |
| 20040244075 | Cai et al. | Dec 2004 | A1 |
| 20040250310 | Shukla et al. | Dec 2004 | A1 |
| 20040259247 | Tuschl et al. | Dec 2004 | A1 |
| 20050005319 | della-Cioppa et al. | Jan 2005 | A1 |
| 20050044591 | Yao et al. | Feb 2005 | A1 |
| 20050080032 | Gross et al. | Apr 2005 | A1 |
| 20050095199 | Whyard et al. | May 2005 | A1 |
| 20050215435 | Menges et al. | Sep 2005 | A1 |
| 20050250647 | Hills et al. | Nov 2005 | A1 |
| 20060009358 | Kibler et al. | Jan 2006 | A1 |
| 20060021087 | Baum et al. | Jan 2006 | A1 |
| 20060040826 | Eaton et al. | Feb 2006 | A1 |
| 20060111241 | Gerwick, III et al. | May 2006 | A1 |
| 20060130172 | Whaley et al. | Jun 2006 | A1 |
| 20060135758 | Wu | Jun 2006 | A1 |
| 20060200878 | Lutfiyya et al. | Sep 2006 | A1 |
| 20060223708 | Hoffman et al. | Oct 2006 | A1 |
| 20060223709 | Helmke et al. | Oct 2006 | A1 |
| 20060247197 | Van De Craen et al. | Nov 2006 | A1 |
| 20060272049 | Waterhouse et al. | Nov 2006 | A1 |
| 20060276339 | Windsor et al. | Dec 2006 | A1 |
| 20070011448 | Chhabra et al. | Jan 2007 | A1 |
| 20070011775 | Allen et al. | Jan 2007 | A1 |
| 20070021360 | Nyce et al. | Jan 2007 | A1 |
| 20070026765 | Renn | Feb 2007 | A1 |
| 20070050860 | Andersen et al. | Mar 2007 | A1 |
| 20070050863 | Tranel et al. | Mar 2007 | A1 |
| 20070124836 | Baum et al. | May 2007 | A1 |
| 20070199095 | Allen et al. | Aug 2007 | A1 |
| 20070219151 | Satishchandran et al. | Sep 2007 | A1 |
| 20070232188 | Probasco | Oct 2007 | A1 |
| 20070250947 | Boukharov et al. | Oct 2007 | A1 |
| 20070259785 | Heck et al. | Nov 2007 | A1 |
| 20070269815 | Rivory et al. | Nov 2007 | A1 |
| 20070281900 | Cui et al. | Dec 2007 | A1 |
| 20070300329 | Allen et al. | Dec 2007 | A1 |
| 20080022423 | Roberts et al. | Jan 2008 | A1 |
| 20080050342 | Fire et al. | Feb 2008 | A1 |
| 20080092256 | Kohn | Apr 2008 | A1 |
| 20080113351 | Naito et al. | May 2008 | A1 |
| 20080155716 | Sonnewald et al. | Jun 2008 | A1 |
| 20080194512 | John et al. | Aug 2008 | A1 |
| 20080214443 | Baum et al. | Sep 2008 | A1 |
| 20080261303 | Kreutzer et al. | Oct 2008 | A1 |
| 20090011934 | Zawierucha et al. | Jan 2009 | A1 |
| 20090018016 | Duck et al. | Jan 2009 | A1 |
| 20090036311 | Witschel et al. | Feb 2009 | A1 |
| 20090054240 | Witschel et al. | Feb 2009 | A1 |
| 20090075921 | Ikegawa et al. | Mar 2009 | A1 |
| 20090098614 | Zamore et al. | Apr 2009 | A1 |
| 20090118214 | Paldi et al. | May 2009 | A1 |
| 20090137395 | Chicoine et al. | May 2009 | A1 |
| 20090165153 | Wang et al. | Jun 2009 | A1 |
| 20090165166 | Feng et al. | Jun 2009 | A1 |
| 20090172838 | Axtell et al. | Jul 2009 | A1 |
| 20090188005 | Boukharov et al. | Jul 2009 | A1 |
| 20090205079 | Kumar et al. | Aug 2009 | A1 |
| 20090215628 | Witschel et al. | Aug 2009 | A1 |
| 20090285784 | Raemaekers et al. | Nov 2009 | A1 |
| 20090293148 | Ren et al. | Nov 2009 | A1 |
| 20090298787 | Raemaekers et al. | Dec 2009 | A1 |
| 20090306189 | Raemaekers et al. | Dec 2009 | A1 |
| 20090307803 | Baum | Dec 2009 | A1 |
| 20100005551 | Roberts et al. | Jan 2010 | A1 |
| 20100048670 | Biard et al. | Feb 2010 | A1 |
| 20100068172 | Van De Craen | Mar 2010 | A1 |
| 20100071088 | Sela et al. | Mar 2010 | A1 |
| 20100099561 | Selby et al. | Apr 2010 | A1 |
| 20100100988 | Tranel et al. | Apr 2010 | A1 |
| 20100152443 | Hirai et al. | Jun 2010 | A1 |
| 20100154083 | Ross et al. | Jun 2010 | A1 |
| 20100192237 | Ren et al. | Jul 2010 | A1 |
| 20100247578 | Salama | Sep 2010 | A1 |
| 20110015084 | Christian et al. | Jan 2011 | A1 |
| 20110015284 | Dees et al. | Jan 2011 | A1 |
| 20110028412 | Cappello et al. | Feb 2011 | A1 |
| 20110035836 | Eudes et al. | Feb 2011 | A1 |
| 20110041400 | Trias Vila et al. | Feb 2011 | A1 |
| 20110053226 | Rohayem | Mar 2011 | A1 |
| 20110098180 | Michel et al. | Apr 2011 | A1 |
| 20110105327 | Nelson | May 2011 | A1 |
| 20110105329 | Song et al. | May 2011 | A1 |
| 20110112570 | Mannava et al. | May 2011 | A1 |
| 20110126310 | Feng et al. | May 2011 | A1 |
| 20110126311 | Velcheva et al. | May 2011 | A1 |
| 20110152339 | Brown et al. | Jun 2011 | A1 |
| 20110152346 | Karleson et al. | Jun 2011 | A1 |
| 20110152353 | Koizumi et al. | Jun 2011 | A1 |
| 20110160082 | Woo et al. | Jun 2011 | A1 |
| 20110166022 | Israels et al. | Jul 2011 | A1 |
| 20110166023 | Nettleton-Hammond et al. | Jul 2011 | A1 |
| 20110171176 | Baas et al. | Jul 2011 | A1 |
| 20110171287 | Saarma et al. | Jul 2011 | A1 |
| 20110177949 | Krapp et al. | Jul 2011 | A1 |
| 20110185444 | Li et al. | Jul 2011 | A1 |
| 20110185445 | Bogner et al. | Jul 2011 | A1 |
| 20110191897 | Poree et al. | Aug 2011 | A1 |
| 20110201501 | Song et al. | Aug 2011 | A1 |
| 20110296555 | Ivashuta et al. | Dec 2011 | A1 |
| 20110296556 | Sammons et al. | Dec 2011 | A1 |
| 20120036594 | Cardoza et al. | Feb 2012 | A1 |
| 20120053231 | Paldi et al. | Mar 2012 | A1 |
| 20120107355 | Harris et al. | May 2012 | A1 |
| 20120108497 | Paldi et al. | May 2012 | A1 |
| 20120128218 | Amyot et al. | May 2012 | A1 |
| 20120137387 | Baum et al. | May 2012 | A1 |
| 20120150048 | Kang et al. | Jun 2012 | A1 |
| 20120156784 | Adams, Jr. et al. | Jun 2012 | A1 |
| 20120157512 | Ben-Chanoch et al. | Jun 2012 | A1 |
| 20120164205 | Baum et al. | Jun 2012 | A1 |
| 20120185967 | Sela et al. | Jul 2012 | A1 |
| 20120198586 | Narva et al. | Aug 2012 | A1 |
| 20120230565 | Steinberg et al. | Sep 2012 | A1 |
| 20120258646 | Sela et al. | Oct 2012 | A1 |
| 20120297501 | Beghyn | Nov 2012 | A1 |
| 20120316220 | Ward et al. | Dec 2012 | A1 |
| 20130003213 | Kabelac et al. | Jan 2013 | A1 |
| 20130041004 | Drager et al. | Feb 2013 | A1 |
| 20130047297 | Sammons et al. | Feb 2013 | A1 |
| 20130047298 | Tang | Feb 2013 | A1 |
| 20130058890 | Raemaekers et al. | Mar 2013 | A1 |
| 20130060133 | Kassab et al. | Mar 2013 | A1 |
| 20130067618 | Ader et al. | Mar 2013 | A1 |
| 20130084243 | Goetsch et al. | Apr 2013 | A1 |
| 20130096073 | Sidelman | Apr 2013 | A1 |
| 20130097726 | Ader et al. | Apr 2013 | A1 |
| 20130212739 | Giritch et al. | Aug 2013 | A1 |
| 20130226003 | Edic et al. | Aug 2013 | A1 |
| 20130232646 | Baum et al. | Sep 2013 | A1 |
| 20130247247 | Ader et al. | Sep 2013 | A1 |
| 20130254940 | Ader et al. | Sep 2013 | A1 |
| 20130254941 | Ader et al. | Sep 2013 | A1 |
| 20130288895 | Ader et al. | Oct 2013 | A1 |
| 20130289097 | Paldi et al. | Oct 2013 | A1 |
| 20130318657 | Avniel et al. | Nov 2013 | A1 |
| 20130318658 | Ader et al. | Nov 2013 | A1 |
| 20130324842 | Mittal et al. | Dec 2013 | A1 |
| 20130326731 | Ader et al. | Dec 2013 | A1 |
| 20140018241 | Sammons et al. | Jan 2014 | A1 |
| 20140057789 | Sammons et al. | Feb 2014 | A1 |
| 20140109258 | Van De Craen et al. | Apr 2014 | A1 |
| 20140230090 | Avniel et al. | Aug 2014 | A1 |
| 20140274712 | Finnessy et al. | Sep 2014 | A1 |
| 20140275208 | Hu et al. | Sep 2014 | A1 |
| 20140296503 | Avniel et al. | Oct 2014 | A1 |
| 20140371298 | Paldi et al. | Dec 2014 | A1 |
| 20150096079 | Avniel et al. | Apr 2015 | A1 |
| 20150143580 | Beattie et al. | May 2015 | A1 |
| 20150159156 | Inberg et al. | Jun 2015 | A1 |
| 20150203867 | Beattie et al. | Jul 2015 | A1 |
| 20150240258 | Beattie et al. | Aug 2015 | A1 |
| 20160015035 | Tao | Jan 2016 | A1 |
| 20160029644 | Tao | Feb 2016 | A1 |
| 20170037407 | Gleit-Kielmanowicz et al. | Feb 2017 | A1 |
| 20170088838 | Inberg et al. | Mar 2017 | A1 |
| 20170183683 | Zheng et al. | Jun 2017 | A1 |
| 20170260522 | Zheng et al. | Sep 2017 | A1 |
| Number | Date | Country |
|---|---|---|
| 2008325989 | May 2009 | AU |
| AU 2008258254 | Jul 2014 | AU |
| 2806295 | Feb 2011 | CA |
| 1505504 | Jun 2004 | CN |
| 101139607 | Mar 2008 | CN |
| 101279950 | Oct 2008 | CN |
| 101279951 | Oct 2008 | CN |
| 101914540 | Dec 2010 | CN |
| 102822350 | Dec 2012 | CN |
| 288618 | Apr 1991 | DE |
| 10000600 | Jul 2001 | DE |
| 10116399 | Oct 2002 | DE |
| 10256353 | Jun 2003 | DE |
| 10256354 | Jun 2003 | DE |
| 10256367 | Jun 2003 | DE |
| 10204951 | Aug 2003 | DE |
| 10234875 | Feb 2004 | DE |
| 10234876 | Feb 2004 | DE |
| 102004054666 | May 2006 | DE |
| 102005014638 | Oct 2006 | DE |
| 102005014906 | Oct 2006 | DE |
| 102007012168 | Sep 2008 | DE |
| 102010042866 | May 2011 | DE |
| 0 375 408 | Jun 1990 | EP |
| 0 804 600 | Nov 1997 | EP |
| 1 157 991 | Nov 2001 | EP |
| 1 238 586 | Sep 2002 | EP |
| 1 416 049 | May 2004 | EP |
| 2 147 919 | Jan 2010 | EP |
| 2 160 098 | Nov 2010 | EP |
| 2 530 159 | Mar 2011 | EP |
| 2 305 813 | Apr 2011 | EP |
| 2 545 182 | Jan 2013 | EP |
| 2 703 489 | Mar 2014 | EP |
| 2 703 490 | Mar 2014 | EP |
| 2 706 114 | Mar 2014 | EP |
| 3 066 200 | Sep 2016 | EP |
| 2001253874 | Sep 2001 | JP |
| 2002080454 | Mar 2002 | JP |
| 2002138075 | May 2002 | JP |
| 2002145707 | May 2002 | JP |
| 2002220389 | Aug 2002 | JP |
| 2003064059 | Mar 2003 | JP |
| 2003096059 | Apr 2003 | JP |
| 2004051628 | Feb 2004 | JP |
| 2004107228 | Apr 2004 | JP |
| 2005008583 | Jan 2005 | JP |
| 2005239675 | Sep 2005 | JP |
| 2005314407 | Nov 2005 | JP |
| 2006232824 | Sep 2006 | JP |
| 2006282552 | Oct 2006 | JP |
| 2007153847 | Jun 2007 | JP |
| 2007161701 | Jun 2007 | JP |
| 2007182404 | Jul 2007 | JP |
| 2008074840 | Apr 2008 | JP |
| 2008074841 | Apr 2008 | JP |
| 2008133207 | Jun 2008 | JP |
| 2008133218 | Jun 2008 | JP |
| 2008169121 | Jul 2008 | JP |
| 2009-508481 | Mar 2009 | JP |
| 2009067739 | Apr 2009 | JP |
| 2009114128 | May 2009 | JP |
| 2009126792 | Jun 2009 | JP |
| 2009137851 | Jun 2009 | JP |
| WO 8911789 | Dec 1989 | WO |
| WO 9534659 | Dec 1995 | WO |
| WO 9534668 | Dec 1995 | WO |
| WO 96005721 | Feb 1996 | WO |
| WO 96033270 | Oct 1996 | WO |
| WO 96038567 | Dec 1996 | WO |
| WO 96040964 | Dec 1996 | WO |
| WO 9747193 | Dec 1997 | WO |
| WO 9749816 | Dec 1997 | WO |
| WO 99024585 | May 1999 | WO |
| WO 9926467 | Jun 1999 | WO |
| WO 9927116 | Jun 1999 | WO |
| WO 9932619 | Jul 1999 | WO |
| WO 9961631 | Dec 1999 | WO |
| WO 9967367 | Dec 1999 | WO |
| WO 0004176 | Jan 2000 | WO |
| WO 0032757 | Jun 2000 | WO |
| WO 00034035 | Jun 2000 | WO |
| WO 00044914 | Aug 2000 | WO |
| WO 0107601 | Feb 2001 | WO |
| WO 0134815 | May 2001 | WO |
| WO 2001085970 | Nov 2001 | WO |
| WO 0214472 | Feb 2002 | WO |
| WO 02066660 | Aug 2002 | WO |
| WO 03000679 | Jan 2003 | WO |
| WO 03004649 | Jan 2003 | WO |
| WO 03006422 | Jan 2003 | WO |
| WO 03012052 | Feb 2003 | WO |
| WO 03013247 | Feb 2003 | WO |
| WO 03016308 | Feb 2003 | WO |
| WO 2003014357 | Feb 2003 | WO |
| WO 03020704 | Mar 2003 | WO |
| WO 03022051 | Mar 2003 | WO |
| WO 03022831 | Mar 2003 | WO |
| WO 03022843 | Mar 2003 | WO |
| WO 03029243 | Apr 2003 | WO |
| WO 03037085 | May 2003 | WO |
| WO 03037878 | May 2003 | WO |
| WO 03045878 | Jun 2003 | WO |
| WO 03050087 | Jun 2003 | WO |
| WO 03051823 | Jun 2003 | WO |
| WO 03051824 | Jun 2003 | WO |
| WO 03051846 | Jun 2003 | WO |
| WO 03064625 | Aug 2003 | WO |
| WO 03076409 | Sep 2003 | WO |
| WO 03077648 | Sep 2003 | WO |
| WO 03087067 | Oct 2003 | WO |
| WO 03090539 | Nov 2003 | WO |
| WO 03091217 | Nov 2003 | WO |
| WO 03093269 | Nov 2003 | WO |
| WO 03104206 | Dec 2003 | WO |
| WO 2004002947 | Jan 2004 | WO |
| WO 2004002981 | Jan 2004 | WO |
| WO 2004005485 | Jan 2004 | WO |
| WO 2004009761 | Jan 2004 | WO |
| WO 2004011429 | Feb 2004 | WO |
| WO 2004022771 | Mar 2004 | WO |
| WO 2004029060 | Apr 2004 | WO |
| WO 2004035545 | Apr 2004 | WO |
| WO 2004035563 | Apr 2004 | WO |
| WO 2004035564 | Apr 2004 | WO |
| WO 2004037787 | May 2004 | WO |
| WO 2004049806 | Jun 2004 | WO |
| WO 2004062351 | Jul 2004 | WO |
| WO 2004067518 | Aug 2004 | WO |
| WO 2004067527 | Aug 2004 | WO |
| WO 2004074443 | Sep 2004 | WO |
| WO 2004077950 | Sep 2004 | WO |
| WO 2005000824 | Jan 2005 | WO |
| WO 2005003362 | Jan 2005 | WO |
| WO 2005007627 | Jan 2005 | WO |
| WO 2005007860 | Jan 2005 | WO |
| WO 2005040152 | May 2005 | WO |
| WO 2005047233 | May 2005 | WO |
| WO 2005047281 | May 2005 | WO |
| WO 2005061443 | Jul 2005 | WO |
| WO 2005061464 | Jul 2005 | WO |
| WO 2005068434 | Jul 2005 | WO |
| WO 2005070889 | Aug 2005 | WO |
| WO 2005089551 | Sep 2005 | WO |
| WO 2005095335 | Oct 2005 | WO |
| 2005110068 | Nov 2005 | WO |
| WO 2005107437 | Nov 2005 | WO |
| WO 2006006569 | Jan 2006 | WO |
| WO 2006024820 | Mar 2006 | WO |
| WO 2006029828 | Mar 2006 | WO |
| WO 2006029829 | Mar 2006 | WO |
| WO 2006037945 | Apr 2006 | WO |
| WO 2006050803 | May 2006 | WO |
| WO 2006074400 | Jul 2006 | WO |
| WO 2006090792 | Aug 2006 | WO |
| WO 2006123088 | Nov 2006 | WO |
| WO 2006125687 | Nov 2006 | WO |
| WO 2006125688 | Nov 2006 | WO |
| WO 2006138638 | Dec 2006 | WO |
| WO 2007003294 | Jan 2007 | WO |
| WO 2007007316 | Jan 2007 | WO |
| WO 2007024783 | Mar 2007 | WO |
| WO 2007026834 | Mar 2007 | WO |
| WO 2007035650 | Mar 2007 | WO |
| WO 2007038788 | Apr 2007 | WO |
| WO 2007039454 | Apr 2007 | WO |
| WO 2007050715 | May 2007 | WO |
| WO 2007051462 | May 2007 | WO |
| WO 2007070389 | Jun 2007 | WO |
| WO 2007071900 | Jun 2007 | WO |
| WO 2007074405 | Jul 2007 | WO |
| WO 2007074976 | Jul 2007 | WO |
| WO 2007077201 | Jul 2007 | WO |
| WO 2007077247 | Jul 2007 | WO |
| WO 2007080126 | Jul 2007 | WO |
| WO 2007080127 | Jul 2007 | WO |
| WO 2007083193 | Jul 2007 | WO |
| WO 2007096576 | Aug 2007 | WO |
| WO 2007051462 | Oct 2007 | WO |
| WO 2007119434 | Oct 2007 | WO |
| WO 2007134984 | Nov 2007 | WO |
| WO 2008007100 | Jan 2008 | WO |
| WO 2008009908 | Jan 2008 | WO |
| WO 2008029084 | Mar 2008 | WO |
| WO 2008042231 | Apr 2008 | WO |
| WO 2008059948 | May 2008 | WO |
| WO 2008063203 | May 2008 | WO |
| WO 2008071918 | Jun 2008 | WO |
| WO 2008074991 | Jun 2008 | WO |
| WO 2008084073 | Jul 2008 | WO |
| WO 2008100426 | Aug 2008 | WO |
| WO 2008102908 | Aug 2008 | WO |
| WO 2008148223 | Dec 2008 | WO |
| WO 2008152072 | Dec 2008 | WO |
| WO 2008152073 | Dec 2008 | WO |
| WO 2009000757 | Dec 2008 | WO |
| WO 2009005297 | Jan 2009 | WO |
| WO 2009029690 | Mar 2009 | WO |
| WO 2009035150 | Mar 2009 | WO |
| WO 2009037329 | Mar 2009 | WO |
| WO 2009046384 | Apr 2009 | WO |
| WO 2009060429 | May 2009 | WO |
| WO 2009063180 | May 2009 | WO |
| WO 2009068170 | Jun 2009 | WO |
| WO 2009068171 | Jun 2009 | WO |
| WO 2009086041 | Jul 2009 | WO |
| WO 2009090402 | Jul 2009 | WO |
| WO 2009091862 | Jul 2009 | WO |
| WO 2009115788 | Sep 2009 | WO |
| WO 2009116558 | Sep 2009 | WO |
| WO 2009125401 | Oct 2009 | WO |
| WO 2009152995 | Dec 2009 | WO |
| WO 2009158258 | Dec 2009 | WO |
| WO 2010012649 | Feb 2010 | WO |
| WO 2010026989 | Mar 2010 | WO |
| WO 2010034153 | Apr 2010 | WO |
| WO 2010049270 | May 2010 | WO |
| WO 2010049369 | May 2010 | WO |
| WO 2010049405 | May 2010 | WO |
| WO 2010049414 | May 2010 | WO |
| WO 2010056519 | May 2010 | WO |
| WO 2010063422 | Jun 2010 | WO |
| WO 2010069802 | Jun 2010 | WO |
| WO 2010078906 | Jul 2010 | WO |
| WO 2010078912 | Jul 2010 | WO |
| WO 2010093788 | Aug 2010 | WO |
| WO 2010104217 | Sep 2010 | WO |
| WO 2010108611 | Sep 2010 | WO |
| WO 2010112826 | Oct 2010 | WO |
| WO 2010116122 | Oct 2010 | WO |
| WO 2010119906 | Oct 2010 | WO |
| WO 2010128465 | Nov 2010 | WO |
| WO 2010130970 | Nov 2010 | WO |
| WO 2011001434 | Jan 2011 | WO |
| WO 2011003776 | Jan 2011 | WO |
| WO 2011021171 | Feb 2011 | WO |
| WO 2011035874 | Mar 2011 | WO |
| WO 2011045796 | Apr 2011 | WO |
| WO 2011065451 | Jun 2011 | WO |
| WO 2011067745 | Jun 2011 | WO |
| WO 2011075188 | Jun 2011 | WO |
| WO 2011080674 | Jul 2011 | WO |
| WO 2011112570 | Sep 2011 | WO |
| WO 2011132127 | Oct 2011 | WO |
| WO 2012001626 | Jan 2012 | WO |
| WO 2012056401 | May 2012 | WO |
| WO 2012092580 | Jul 2012 | WO |
| WO 2012164100 | Dec 2012 | WO |
| WO 2012177639 | Dec 2012 | WO |
| WO 2013010690 | Jan 2013 | WO |
| WO 2013010691 | Jan 2013 | WO |
| WO 2013025670 | Feb 2013 | WO |
| WO 2013039990 | Mar 2013 | WO |
| WO 2013040005 | Mar 2013 | WO |
| WO 2013040021 | Mar 2013 | WO |
| WO 2013040033 | Mar 2013 | WO |
| WO 2013040049 | Mar 2013 | WO |
| WO 2013040057 | Mar 2013 | WO |
| WO 2013040116 | Mar 2013 | WO |
| WO 2013040117 | Mar 2013 | WO |
| WO 2013153553 | Oct 2013 | WO |
| WO 2013175480 | Nov 2013 | WO |
| WO 2014022739 | Feb 2014 | WO |
| WO 2014106837 | Jul 2014 | WO |
| WO 2014106838 | Jul 2014 | WO |
| WO 2014151255 | Sep 2014 | WO |
| WO 2014164761 | Oct 2014 | WO |
| WO 2014164797 | Oct 2014 | WO |
| WO 2015001336 | Jan 2015 | WO |
| WO 2015010026 | Jan 2015 | WO |
| WO 2016018887 | Feb 2016 | WO |
| Entry |
|---|
| Thomas et al. 2001, The Plant Journal 25(4):417-425. |
| Yibrah et al. 1993, Hereditas 118:273-2890. |
| Arpaia et al., “Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado Potato Beetle (Leptinotarsa decemlineata Say),” Theor. Appl. Genet., 95:329-334 (1997). |
| Banerjee et al., “Efficient production of transgenic potato (S. tuberosum L. ssp. andigena) plants via Agrobacteriutn tumefaciens-mediated transformation,” Plant Sci., 170(4):732-738 (2006). |
| Baum et al., “Control of coleopteran insect pests through RNA interference,” Nature Biotechnol., 25(11):1322-1326 (2007). |
| Becker et al., “Fertile transgenic wheat from microprojectile bombardment of scutellar tissue,” Plant J. , 5(2), pp. 299-307 (1994). |
| Bytebier et al., “T-DNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis,” Proc. Natl. Acad. Sci. USA., 84:5345-5349 (1997). |
| CERA. (2012). GM Crop Database. Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C., available electronically at www.cera-gmc.org/?action=gm_crop_database. |
| Chakravarty et al., “Genetic Transformation in Potato: Approaches and Strategies,” Amer. J. Potato Res., 84(4):301-311 (2007). |
| Cheng et al., “Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens,” Plant Cell Rep., 15(9): 653-657 (1996). |
| Davidson and Ellington, “Engineering regulatory RNAs,” Trends Biotechnol., 23(3):109-112 (2005). |
| De Framond et al., “Mini-Ti: A New Vector Strategy for Plant Genetic Engineering,” Biotechnology, 1:262-269 (1983). |
| Du et al., “A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites,” Nucleic Acids Res., 33(5):1671-1677 (2005). |
| Gallic et al., “Identification of the motifs within the tobacco mosaic virus 5′-leader responsible for enhancing translation,” Nucleic Acids Res., 20(17):4631-4638 (1992). |
| Gong et al., “Silencing of Rieske iron-sulfur protein using chemically synthesized siRNA as a potential biopesticide against Plutella xylostella,” Pest Manag. Sci., 67:514-520 (2011). |
| Hoekema et al., “A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid,” Nature, 303:179-180 (1983). |
| International Search Report dated Oct. 1, 2015 in corresponding International Application No. PCT/US2015/022985. |
| Invitrogen' s “Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques,” (Life Technologies, Carlsbad, CA)(2002). |
| Isaacs et al., “Engineered riboregulators enable post-transcriptional control gene expression,” Nat. Biotechnol., 22(7):841-847 (Jul. 2004). |
| Kertbundit et al., “In vivo random β-glucuronidase gene fusions in Arabidopsis thaliana,” Proc. Natl. Acad. Sci. USA., 88:5212-5216 (1991). |
| Lee et al., “Aptamer database,” Nucleic Acids Res., 32(Database issue):D95-100 (2004). |
| Li et al., “Establishment of a highly efficient transformation system for pepper (Capsicum annuum L.),” Plant Cell Reports, 21:785-788 (2003). |
| Lu et al, “RNA silencing in plants by the expression of siRNA duplexes,” Nucleic Acids Research, 32(21):e171 (2004). |
| Lu et al., “OligoWalk: an online siRNA design tool utilizing hybridization thermodynamics,” Nucleic Acids Res., 36(Web Server issue):W104-108 (2008). |
| Mandal et al., “Adenine riboswitches and gene activation by disruption of a transcription terminator,” Nature Struct. Mol. Biol., 11:29-35 (2004). |
| Mandal et al., “Gene regulation by riboswitches,” Nature Rev. Mol. Cell Biol., 5:451-463 (2004). |
| Mao et al., “Silencing a botton bollworm P450 monoosygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol,” Nature Biotechnol., 25(11):1307-1313 (2007). |
| Masoud et al., “Constitutive expression of an inducible β-1,3-glucanase in alfalfa reduces disease severity caused by the oomycete pathogen Phytophthora megasperma f. spmedicaginis, but does not reduce disease severity of chitin-containing fungi,” Transgen. Res., 5:313-323 (1996). |
| Pitino et al., “Silencing of Aphid Genes by dsRNA Feeding from Plants,” PLoS One,6(10):e25709 (2011). |
| Pridgeon et al., “Topically Applied AaelAP1 Double-Stranded RNA Kills Feman Adults of Aedes aegypti,” J. Med. Entomol., 45(3):414-420 (2008). |
| Reynolds et al., Rational siRNA design for RNA Interference,: Nature Biotechnol., 22:326-330 (2004). |
| Sindhu et al., “Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success,” J. Exp. Botany, 60(1):315-324 (2009). |
| Steeves et al., “Transgenic soybeans expressing siRNAs specific to a major sperm protein gene suppress Heterodera glycines reproduction,” Funci. Plant Biol., 33(11):991-999 (2006). |
| Sun et al., “A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics,” Plant Cell Physiol., 47(3):426-431 (2006). |
| Zhang et al., “Transgenic rice plants produced by electroporation-mediated plasmid uptake into photoplasts,” Plant Cell Rep., 7(6):379-384 (1988). |
| Advisory Action dated Feb. 22, 2013, in U.S. Appl. No. 13/332,430. |
| Agricultural Chemical Usage 2006 Vegetables Summary, Agricultural Statistics Board, NASS, USDA, pp. 1-372 (2007). |
| Agrios, Plant Pathology (Second Edition), 2:466-470 (1978). |
| Amarzguioui et al., “An algorithm for selection of functional siRNA sequences,” Biochemical and Biophysical Research Communications, 316:1050-1058 (2004). |
| Amdam et al., Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA, BMC Biotechnology, 3(1):1-8 (2003). |
| An et al., “Transient RNAi Induction against Endogenous Genes in Arabidopsis Protoplasts Using in Vitro-Prepared Double-Stranded RNA,” Biosci Biotechnol Biochem, 69(2):415-418 (2005). |
| Andersen et al., “Delivery of siRNA from lyophilized polymeric surfaces,” Biomaterials, 29:506-512 (2008). |
| Anonymous, “Agronomy Facts 37: Adjuvants for enhancing herbicide performance,” n.p., 1-8, (Jan. 26, 2000), Web, (Jan. 21, 2014). |
| Aoki et al., “In Vivo Transfer Efficiency of Antisense Oligonucleotides into the Myocardium Using HVJ—Liposome Method,” Biochem Biophys Res Commun, 231:540-545 (1997). |
| Arpaia et al., “Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado Potato Beetle (Leptinotarsa decemlineata Say),” (1997) Theor. Appl. Genet., 95:329-334 (1997). |
| Artymovich, “Using RNA interference to increase crop yield and decrease pest damage,” MMG 445 Basic Biotech., 5(1):7-12 (2009). |
| Ascencio-Ibanez et al., “DNA abrasion onto plants is an effective method for geminivirus infection and virus-induced gene silencing,” Journal of Virological Methods, 142:198-203 (2007). |
| Axtell et al., “A Two-Hit Trigger for siRNA Biogenesis in Plants,” Cell, 127:565-577 (2006). |
| Bachman et al., “Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte),” Transgenic Res., pp. 1-16 (2013). |
| Bart et al., “A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts,” Plant Methods, 2(13):1-9 (2006). |
| Baulcombe, “RNA silencing and heritable epigenetic effects in tomato and Arabidopsis,” Abstract 13th Annual Fall Symposium, Plant Genomes to Phenomes, Donald Danforth Plant Science Center, 28-30 (2011). |
| Baum et al., “Control of coleopteran insect pests through RNA interference,” Nature Biotechnology, 25(11):1322-1326 (2007). |
| Baum et al., “Progress Towards RNAi-Mediated Insect Pest Management” Advances in Insect Physiology, 47:249-295 (2014). |
| Bayer et al., “Programmable ligand-controlled riboregulators of eukaryotic gene expression,” Nature Biotechnol., 23(3):337-343 (2005). |
| Beal, et al., “Second Structural Motif for Recognition of DNA by Oligonucleotide-Directed Triple-Helix Formation,” Science, 251:1360-1363 (1992). |
| Becker et al., “Fertile transgenic wheat from microprojectile bombardment of scutellar tissue,” The Plant Journal, 5(2):299-307 (1994). |
| Bhargava et al., “Long double-stranded RNA-mediated RNA interference as a tool to achieve site-specific silencing of hypothalamic neuropeptides,” Brain Research Protocols, 13:115-125 (2004). |
| Bhatia et al., “Aphid resistance in Brassica crops: Challenges, biotechnological progress and emerging possibilities,” Biotechnology Advances 29:879-955 (2011). |
| Boletta et al., “High Efficient Non-Viral Gene Delivery to the Rat Kidney by Novel Polycationic Vectors,” J. Am Soc. Nephrol., 7:1728 (1996). |
| Bolognesi et al., “Characterizing the Mechanism of Action of Double-Stranded RNA Activity against Western Corn Rootworm(Diabrotica virgifera virgifera LeConte),” PLoS ONE 7(10):e47534 (2012). |
| Breaker et al., “A DNA enzyme with Mg2+-dependent RNA phosphoesterase activity,” Chemistry and Biology, 2:655-660 (1995). |
| Brodersen et al., “The diversity of RNA silencing pathways in plants,” Trends in Genetics, 22(5):268-280 (2006). |
| Busch et al., “RNAi for discovery of novel crop protection products,” Pflanzenschutz-Nachrichten Bayer, 58(1):34-50 (2005). |
| Campbell et al., “Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase,” Parasites & Vectors, 3(1):73, pp. 1-10 (2010). |
| Carthew, “Gene silencing by double-stranded RNA,” Curr Opin Cell Biol., 13(2):244-248 (2001). |
| Chabannes et al., “In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels,” The Plant Journal, 28(3):271-282 (2001). |
| Chabbouh et al., “Cucumber mosaic virus in artichoke,” FAO Plant Protection Bulletin, 38:52-53 (1990). |
| Chang et al., “Dual-target gene silencing by using long, sythetic siRNA duplexes without triggering antiviral responses,” Molecules and Cells, 27(6)″689-695 (2009). |
| Chang et al., “Cellular Internalization of Fluorescent Proteins via Arginine-rich Intracellular Delivery Peptide in Plant Cells,” Plant Cell Physiol., 46(3):482-488 (2005). |
| Chen et al., “Exploring MicroRNA-Like Small RNAs in the Filamentous Fungus Fusarium oxysporum,” PLOS One, 9(8):e104956:1-10 (2014). |
| Christiaens et al., “The challenge of RNAi-mediated control of hemipterans,” Current Opinion in Insect Science, 6:15-21 (2014). |
| CN101914540 Patent Diclosure, “Introduction of RNA into plant by interference,” (2010). |
| Colorado Potato Beetle, Leptinotarsa decemlineata,Transcriptome, PLoS One, 9(1):e86012 (2014). |
| Communication pursuant to Article 94(3) EPC dated Jan. 14, 2016, in European Patent Application No. 12 832 415.9. |
| Communication pursuant to Article 94(3) EPC dated Jun. 26, 2015, in European Patent Application No. 11 753 916.3. |
| Communication pursuant to Article 94(3) EPC dated Mar. 18, 2016, in European Patent Application No. 12 832 160.1. |
| Communication pursuant to Article 94(3) EPC dated Mar. 24, 2016, in European Patent Application No. 12 831 684.1. |
| Communication pursuant to Article 94(3) EPC dated Mar. 4, 2016, in European Patent Application No. 12 830 932.5. |
| Communication pursuant to Article 94(3) EPC dated Mar. 9, 2016, in European Patent Application No. 12 831 166.9. |
| Communication pursuant to Article 94(3) EPC dated Oct. 23, 2015, in European Patent Application No. 12 831 945.6. |
| Concise Descriptions of Relevance filed by a third party dated Nov. 29, 2012, in U.S. Appl. No. 13/042,856. |
| Constan et al., “An outer envelope membrane component of the plastid protein import apparatus plays an essential role in Arabidopsis,” The Plant Journal, 38:93-106 (2004). |
| Cooney et al., “Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in Vitro,” Science ,241:456-459 (1988). |
| COST Action FA0806 progress report “Plant virus control employing RNA-based vaccines: A novel non-transgenic strategy” (2010). |
| Cox-Foster et al., “A Metagenomic Survey of Microbes in Honey Bee Colony Collapse Disorder,” Science, 318(5848):283-287 (2007). |
| Dalakoums et al., “Induction of Silencing in Plants by High-Pressure Spraying of In vitro—Synthesized Small RNAs,” Frontiers in Plant Science, 7(1327):1-5 (2016). |
| Dalmay et al., “An RNA-Depenedent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus,” Cell, 101:543-553 (2000). |
| Davidson et al., “Engineering regulatory RNAs,” TRENDS in Biotechnology, 23(3):109-112 (2005). |
| Dawson et al., “cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts,” Proc. Natl. Acad. Sci. USA, 83:1832-1836 (1986). |
| De Framond, “MINI-Ti: A New Vector Strategy for Plant Genetic Engineering,” Nature Biotechnology, 1:262-269 (1983). |
| Desai et al., “Reduction in deformed wing virus infection in larval and adult honey bees (Apis mellifera L.) by double-stranded RNA ingestion,” Insect Molecular Biology, 21(4):446-455 (2012). |
| Di Prisco et al. “Varroa Destructor Is an Effective Vector of Israeli Acute Paralysis Virus in the Honeybee, Apis mellifera”, Journal of General Virology, 92: 151-155 (2011). |
| Di Stilio et al., “Virus-Induced Gene Silencing as a Tool for Comparative Functional Studies in Thalictrum,” PLoS One, 5(8):e12064 (2010). |
| Diallo et al., “Long Endogenous dsRNAs Can Induce Complete Gene Silencing in Mammalian Cells and Primary Cultures,” Oligonucleotides, 13:381-392 (2003). |
| Dietemann et al, “Varroa destructor: research avenues towards sustainable control,” Journal of Apicultural Research, 51(1):125-132 (2012). |
| Dietzl et al., “A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila,” Nature, 448:151-157 (2007). |
| Du et al., “A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites,” Nucleic Acids Research, 33(5):1671-1677 (2005). |
| Dunoyer et al., “Small RNA Duplexes Function as Mobile Silencing Signals Between Plant Cells,” Science, 328:912-916 (2010). |
| Eamens et al., “RNA Silencing in Plants: Yesterday, Today, and Tomorrow,” Plant Physiology, 147(2):456-468 (2008). |
| Ellington et al., “In vitro selection of RNA molecules that bind specific ligands,” Nature, 346:818-822 (1990). |
| Eudes et al., “Cell-penetrating peptides,” Plant Signaling & Behavior, 3(8):549-5550 (2008). |
| European Cooperation in the field of Scientific and Technical Research—Memorandum of Understanding for COST Action FA0806 (2008). |
| European Search Report and the European Search Opinion dated Feb. 3, 2014, in European Patent Application No. 13156180.5. |
| European Search Report dated Feb. 3, 2014, in European Patent Application No. 13156180.4. |
| European Search Report dated Feb. 6, 2014, in European Patent Application No. 13156183.9. |
| European Search Report dated Jun. 29, 2018, in European Patent Application No. 18157745.3. |
| European Search Report dated Sep. 7, 2017, in European Patent Application No. 17152830.0. |
| Examination Report dated Mar. 1, 2018, in Australian Patent Application No. 2013264742. |
| Extended European Search Report dated Feb. 2, 2015, in European Patent Application No. 12 830 932.5. |
| Extended European Search Report dated Feb. 27, 2015, in European Patent Application No. 12 832 160.1. |
| Extended European Search Report dated Feb. 3, 2015, in European Patent Application No. 12 831 945.6. |
| Extended European Search Report dated Feb. 6, 2014, in European Patent Application No. 13156183.9. |
| Extended European Search Report dated Jan. 20, 2016, in European Patent Application No. 13 794 339.5. |
| Extended European Search Report dated Jan. 21, 2015, in European Patent Application No. 12 832 415.9. |
| Extended European Search Report dated Jan. 29, 2015, in European Patent Application No. 12 831 567.8. |
| Extended European Search Report dated Jun. 29, 2015, in European Patent Application No. 12 831 494.5. |
| Extended European Search Report dated Mar. 17, 2015, in European Patent Application No. 12 831 684.1. |
| Extended European Search Report dated Mar. 3, 2015, in European Patent Application No. 12 831 166.9. |
| Extended European Search Report dated May 23, 2018, in European Patent Application No. 15826865.6. |
| Extended European Search Report dated Nov. 7, 2017, in European Patent Application No. 15811092.4. |
| Extended European Search Report dated Nov. 8, 2017, in European Patent Application No. 15737282.2. |
| Extended European Search Report dated Oct. 8, 2013, in European Patent Application No. 11753916.3. |
| Extended European Search Report dated Sep. 29, 2016, in European Patent Application No. 14778840.0. |
| Extended European Search Report dated Apr. 13, 2018, in European Patent Application No. 15812530.0. |
| Extended European Search Report dated Mar. 15, 2018, in European Patent Application No. 17181861.0. |
| Fairbairn et al. “Host-Delivered RNAi: An Effective Strategy to Silence Genes in Plant Parasitic Nematodes”, Planta, 226(6):1525-1533 (2007) Abstract. |
| Fernandez et al., “Uptake of Hydrophilic Solutes Through Plant Leaves: Current State of Knowledge and Perspectives of Foliar Fertilization,” Critical Reviews in Plant Sciences, 28:36-38 (2009). |
| Fiala et al., “Reversible Downregulation of Protein Kinase A during Olfactory Learning Using Antisense Technique Impairs Long-Term Memory Formation in the Honeybee, Apis mellifera,” J. Neuroscience, 19:10125-10134 (1999). |
| Final Office Action dated Apr. 7, 2016, in U.S. Appl. No. 13/619,980. |
| Final Office Action dated Aug. 1, 2013, in U.S. Appl. No. 13/318,636. |
| Final Office Action dated Dec. 17, 2015, in U.S. Appl. No. 14/335,135. |
| Final Office Action dated Feb. 17, 2016, in U.S. Appl. No. 13/612,929. |
| Final Office Action dated Feb. 4, 2016, in U.S. Appl. No. 13/612,936. |
| Final Office Action dated Jun. 30, 2016, in U.S. Appl. No. 13/901,326. |
| Final Office Action dated Mar. 18, 2011, in U.S. Appl. No. 12/222,949. |
| Final Office Action dated Mar. 2, 2016, in U.S. Appl. No. 13/612,995. |
| Final Office Action dated Mar. 21, 2016, in U.S. Appl. No. 13/612,925. |
| Final Office Action dated May 26, 2016, in U.S. Appl. No. 14/532,596. |
| Final Office Action dated Nov. 10, 2015, in U.S. Appl. No. 13/612,985. |
| Final Office Action dated Nov. 10, 2016, in U.S. Appl. No. 13/583,302. |
| Final Office Action dated Nov. 19, 2015, in U.S. Appl. No. 13/612,941. |
| Final Office Action dated Nov. 30, 2015, in U.S. Appl. No. 13/612,948. |
| Final Office Action dated Nov. 7, 2013, in U.S. Appl. No. 13/042,856. |
| Final Office Action dated Oct. 15, 2012, in U.S. Appl. No. 13/332,430. |
| Final Office Action dated Oct. 20, 2016, in U.S. Appl. No. 14/480,199. |
| Final Office Action dated Oct. 22, 2015, in U.S. Appl. No. 14/608,951. |
| Final Office Action dated Sep. 9, 2016, in U.S. Appl. No. 13/612,954. |
| Final Office Action dated Sep. 9, 2016, in U.S. Appl. No. 14/603,347. |
| Final Office Action dated Sep. 9, 2016, in U.S. Appl. No. 14/608,951. |
| Fire et al., “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans,” Nature, 391:806-811 (1998). |
| First Examination Report dated Apr. 23, 2013, in New Zealand Patent Application No. 601784. |
| First Examination Report dated Jul. 28, 2014, in New Zealand Patent Application No. 627060. |
| First Office Action dated Aug. 31, 2015, in Chinese Patent Application No. 201280053985.3. |
| First Office Action dated Feb. 2, 2016, in Chinese Patent Application No. 201380039346.6. |
| First Office Action dated Jul. 7, 2015, in Chinese Patent Application No. 201280054820.8. |
| First Office Action dated Mar. 12, 2015, in Chinese Patent Application No. 201280053984.9. |
| First Office Action dated Mar. 2, 2015, in Chinese Patent Application No. 201280054819.5. |
| First Office Action dated May 27, 2015, in Chinese Patent Application No. 201280054179.8. |
| First Office Action dated Sep. 9, 2015, in Chinese Patent Application No. 201280055409.2. |
| Foley et al., “The distribution of Aspergillus spp. Opportunistic parasites in hives and their pathogenicity to honey bees,” Veterinary Microbiology, 169:203-210 (2014). |
| Fraley et al., “Liposome-mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts: A sensitive assay for monitoring liposome-protoplast interactions,” Proc Natl Acad Sci U S A., 79(6):1859-1863 (1982). |
| Friedberg, “Automated protein function prediction—the genomic challenge,” Briefings in Bioinformatics, 7(3):225-242 (2006). |
| Fukuhara et al., “Enigmatic Double-Stranded RNA in Japonica Rice,” Plant Molecular Biology, 21:1121-1130 (1993). |
| Fukuhara et al., “The Unusual Structure of a Novel RNA Replicon in Rice,” The Journal of Biological Chemistry, 270(30):18147-18149 (1995). |
| Fukuhara et al., “The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties,” Archives of Virology, 151:995-1002 (2006). |
| Fukunaga et al., “dsRNA with 5′ overhangs v contributes to endogenous and antiviral RNA silencing pathways in plants,” The EMBO Journal, 28(5):545-555 (2009). |
| Further Examination Report dated May 16, 2014, in New Zealand Patent Application No. 601784. |
| Gallie et al., “Identification of the motifs within the tobacco mosaic virus 5′—leader responsible for enhancing translation,” Nucleic Acids Res., 20(17):4631-4638 (1992). |
| Gan et al., “Bacterially expressed dsRNA protects maize against SCMV infection,” Plant Cell Rep, 29(11):1261-1268 (2010). |
| Gao et al., “Down-regulation of acetolactate synthase compromises 01-1-mediated resistance to powdery mildew in tomato,” BMC Plant Biology, 14 (2014). |
| Gao et al., “Nonviral Methods for siRNA Delivery,” Molecular Pharmaceutics, 6(3):651-658 (2008). |
| Garbian et al., “Bidirectional Transfer of RNAi between Honey Bee and Varroa destructor: Varroa Gene Silencing Reduces Varroa Population,” 8(12):1- 9:e1003035 (2012). |
| Gaskin et al., “Novel organosillicone adjuvants to reduce agrochemical spray volumes on row crops,” New Zealand Plant Protection, 53:350-354 (2000). |
| Gill et al. “Stripped-Down DNA Repair in a High Reduced Parasite”, BMC Molecular Biology, 8(24): 1-14 (2007). |
| Gomez-Zurita et al., “Recalibrated Tree of Leaf Beetles (Chrysomelidae) Indicates Independent Diversification of Angiosperms and Their Insect Herbivores,” PLoS One, 4(e360):1-8 (2007). |
| Gong et al., “Silencing of Rieske iron—sulfur protein using chemically synthesised siRNA as a potential biopesticide against Plutella xylostella,” Pest Manag Sci, 67:514-520 (2011). |
| Gossamer Threads, Compendium of Herbicide Adjuvants: Organo-Silicone Surfactant, p. 1-4 (1998). |
| Hagio, “Chapter 25: Direct Gene Transfer into Plant Mature Seeds via Electroporation After Vacuum Treatment,” Electroporation and Sonoporation in Developmental Biology, p. 285-293 (2009). |
| Hamilton et al., “Two classes of short interfering RNA in RNA silencing,” EMBO J., 21(17):4671-4679 (2002). |
| Han et al., “Molecular Basis for the Recognition of Primary microRNAs by the Drosha-DGCR8 Complex,” Cell, 125(5):887-901 (2006). |
| Hannon, “RNA interference,” Nature, 481:244-251 (2002). |
| Henderson et al., “U.S. National Bee Colony Loss Survey, www.beesurvey.com, Preliminary Findings With Respect to Colony Collapse Disorder (CCD),” Bee Alert Technology, Inc. (2007). |
| Heneberg et al., “Assemblage of filamentous fungi associated with aculeate hymenopteran brood in reed galls,” Journal of Invertebrate Pathology, 133:95-106 (2016). |
| Herman et al., “A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression,” J. Biol. Chem., 280: 24759-24767 (2005). |
| Hess, “Surfactants and Additives,” 1999 Proceedings of the California Weed Science Society, 51:156-172 (1999). |
| Hewezi et al., “Local infiltration of high- and low-molecular-weight RNA from silenced sunflower (Helianthus annuus L.) plants triggers post-transcriptional gene silencing in non-silenced plants,” Plant Biotechnology Journal, 3:81-89 (2005). |
| Himber et al., “Transitivity-dependant and -independent cell-to-cell movement of RNA silencing,” The EMBO Journal, 22(17):4523-4533 (2003). |
| Hsieh et al., “A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens,” Nucleic Acids Res., 32(3):893-901 (2004). |
| Hu et al., “High efficiency transport of quantum dots into plant roots with the aid of silwet L-77,” Plant Physiology and Biochemistry, 48:703-709 (2010). |
| Huang et al., “Engineering broad root-know resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene,” Proc. Natl. Acad. Sci. USA, 103(39):14302-14306 (2006). |
| Huesken et al., “Design of a genome-wide siRNA library using an artificial neural network,” Nature Biotechnology, 23(8): 995-1001 (2005). |
| Hunter et al., “Large-Scale Field Application of RNAi Technology Reducing Israeli Acute Paralysis Virus Disease in Honey Bees (Apis mellifera, Hymenoptera: Apidae),” PLoS Pathogens, 6(12):e1001160-1-el 001160-10 (2010). |
| Hunter et al., “RNA Interference Strategy to suppress Psyllids & Leafhoppers,” International Plant and Animal Genome XIX, 15-19 (2011). |
| Huvenne et al., “Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review,” Journal of Insect Physiology, 56:227-235 (2010). |
| Ichihara et al., “Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities,” Nucleic Acids Res., 35(18):e123 (2007). |
| International Preliminary Report on Patentability (Chapter II) dated Jul. 24, 2015, in International Application No. PCT/US2014/047204. |
| International Preliminary Report on Patentability dated Apr. 26, 2012, in International Application No. PCT/IL2010/000844. |
| International Preliminary Report on Patentability dated Feb. 1, 2010, in International Application No. PCT/IL2008/001440. |
| International Preliminary Report on Patentability dated Feb. 21, 2012, in International Application No. PCT/IB2010/053776. |
| International Preliminary Report on Patentability dated Nov. 17, 2011, in International Application No. PCT/IB2010/051980. |
| International Preliminary Report on Patentability dated Oct. 23, 2014, in International Application No. PCT/IL2013/050321. |
| International Preliminary Report on Patentability dated Sep. 11, 2012, in International Application No. PCT/US2011/027528. |
| International Preliminary Report on Patentability dated Sep. 11, 2014, in International Application No. PCT/IL2013/050447. |
| International Rice Genome Sequencing Project, The map-based sequence of the rice genome, Nature, 436(11):793-800 (2005). |
| International Search Report and the Written Opinion dated Feb. 25, 2013, in International Application No. PCT/US2012/054883. |
| International Search Report and the Written Opinion dated Feb. 27, 2013, in International Application No. PCT/US2012/054814. |
| International Search Report and the Written Opinion dated Feb. 27, 2013, in International Application No. PCT/US2012/054842. |
| International Search Report and the Written Opinion dated Feb. 27, 2013, in International Application No. PCT/US2012/054894. |
| International Search Report and the Written Opinion dated Feb. 27, 2013, in International Application No. PCT/US2012/054974. |
| International Search Report and the Written Opinion dated Feb. 27, 2013, in International Application No. PCT/US2012/054980. |
| International Search Report and the Written Opinion dated Jul. 15, 2014, in International Application No. PCT/US2014/025305. |
| International Search Report and the Written Opinion dated Jul. 22, 2014, in International Application No. PCT/IL2013/051083. |
| International Search Report and the Written Opinion dated Jul. 22, 2014, in International Application No. PCT/IL2013/051085. |
| International Search Report and the Written Opinion dated Jul. 24, 2014, in International Application No. PCT/US2014/026036. |
| International Search Report and the Written Opinion dated May 10, 2011, in International Application No. PCT/US2011/027528. |
| International Search Report and the Written Opinion dated Oct. 1, 2013, in International Application No. PCT/IL2013/050447. |
| International Search Report and Written Opinion dated Aug. 25, 2014, in International Application No. PCT/US2014/023503. |
| International Search Report and Written Opinion dated Aug. 27, 2014, in International Application No. PCT/US2014/023409. |
| International Search Report and Written Opinion dated Dec. 31, 2015, in International Application No. PCT/US2015/042415. |
| International Search Report and Written Opinion dated Feb. 23, 2015, in International Application No. PCT/US2014/063832. |
| International Search Report and Written Opinion dated Feb. 24, 2011, in International Application No. PCT/IL2010/000844. |
| International Search Report and Written Opinion dated Jul. 19, 2010, in International Application No. PCT/IB2010/051980. |
| International Search Report and Written Opinion dated Jul. 8, 2015, in International Application No. PCT/US2015/011408. |
| International Search Report and Written Opinion dated Mar. 26, 2015, in International Application No. PCT/US2014/069535. |
| International Search Report and Written Opinion dated May 23, 2017, in International Application No. PCT/US2017/015061. |
| International Search Report and Written Opinion dated May 26, 2016, in International Application No. PCT/US2016/014344. |
| International Search Report and Written Opinion dated Nov. 24, 2015, in International Application No. PCT/US2015/037522. |
| International Search Report and Written Opinion dated Nov. 27, 2015, in International Application No. PCT/US2015/037015. |
| International Search Report and Written Opinion dated Nov. 30, 2010, in International Application No. PCT/IB2010/053776. |
| International Search Report and Written Opinion dated Oct. 1, 2015 in International Application No. PCT/US2015/022985. |
| International Search Report and Written Opinion dated Oct. 17, 2016, in International Application No. PCT/US2016/030579. |
| International Search Report and Written Opinion dated Oct. 28, 2013, in International Application No. PCT/IL2013/050321. |
| International Search Report dated Aug. 13, 2009, in International Application No. PCT/IL2008/001440. |
| International Search Report dated Mar. 12, 2013, in International Application No. PCT/US2012/054789. |
| Invitation to Pay Additional Fees dated May 6, 2014, in International Application No. PCT/IL2013/051083. |
| Invitation to Pay Additional Fees dated May 6, 2014, in International Application No. PCT/IL2013/051085. |
| Invitation to Pay Additional Fees dated Nov. 25, 2014, in International Application No. PCT/US2014/047204. |
| Invitation to Pay Additional Fees dated Sep. 8, 2015, in International Application No. PCT/US2015/037015. |
| Invitation to Pay Additional Fees dated Sep. 9, 2015, in International Application No. PCT/US2015/037522. |
| Isaacs et al., “Engineered riboregulators enable post-transcriptional control of gene expression,” Nature Biotechnology, 22(7):841-847 (2004). |
| Jarvis et al, “An Arabidopsis mutant defective in the plastid general protein import apparatus,” Science, 282:100-103 (1998). |
| Ji et al., “Regulation of small RNA stability: methylation and beyond,” Cell Research, 22:624-636 (2012). |
| Jin et al., “Posttranslational Elevation of Cell Wall Invertase Activity by Silencing its Inhibitor in Tomato Delays Leaf Senescence and Increases Seed Weight and Fruit Hexose Level,” The Plant Cell, 21:2072-2089 (2009). |
| Jones-Rhoades et al., “MicroRNAs and Their Regulatory Roles in Plants,” Annu. Rev. Plant Biol., 57:19-53 (2006). |
| Kam et al., “Nanotube Molecular Transporters: Internalization of Carbon Nanotube—Protein Conjugates into Mammalian Cells,” J. Am. Chem. Soc., 126(22):6850-6851 (2004). |
| Katoh et al., “Specific residues at every third position of siRNA shape its efficient RNAi activity,” Nucleic Acids Res., 35(4): e27 (2007). |
| Khodakovskaya et al., “Carbon Nanotubes Are Able to Penetrate Plant Seed Coat and Dramatically Affect Seed Germination and Plant Growth,” ACS Nano, 3(10):3221-3227 (2009). |
| Khovorova et al., “Rational siRNA design for RNA interference,” Nature Biotechnol., 22 :326-330 (2004). |
| Kikkert et al., “Stable Transformation of Plant Cells by Particle Bombardment/Biolistics,” Methods in Molecular Biology, 286:61-78 (2005). |
| Kim et al., “Optimization of Conditions for Transient Agrobacterium-Mediated Gene Expression Assays in Arabidopsis,” Plant Cell Reports, 28:1159-1167 (2009). |
| Kim et al., “Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy,” Nature Biotechnology, 23(2):222-226 (2005). |
| Klahre et al., “High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants,” Proc. Natl. Acad. Sci. USA, PNAS, 99(18):11981-11986 (2002). |
| Kondylis et al., “The Golgi apparatus: Lessons from Drosophila,” FEBS Letters 583:3827-3838 (2009). |
| Kronenwett et al., “Oligodeoxyribonucleotide Uptake in Primary Human Hematopoietic Cells Is Enhanced by Cationic Lipids and Depends on the Hematopoietic Cell Subset,” Blood, 91(3):852-862 (1998). |
| Kumar et al., “Sequencing, De Novo Assembly and Annotation of the Colorado Potato Beetle, Leptinotarsa decemlineata, Transcriptome,” PLOS One, 9(1):e86012:1-17 (2014). |
| Kusaba, “RNA interference in crop plants,” Curr Opin Biotechnol, 15(2):139-143 (2004). |
| Lee et al., “A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.” Nature Genetics, 33:40-48 (2003). |
| Lee et al., “Aptamer Database,” Nucleic Acids Research, 32:D95-D100 (2004). |
| Li et al, “RNA interference in Nilaparvata lugens (Homoptera:Delphacidae) based on dsRNA ingestion,” Pest Manag. Sci., 67:852-859 (2011). |
| Li et at., “Long dsRNA but not siRNA initiates RNAi in western corn rootworm larvae and adults,” Journal of Applied Entomology, 139(6):432-445 (2015). |
| Liu et al., “Prevention of Chinese Sacbrood Virus Infection in Apis Ceram. Using RNA Interference,” Current Microbiology, 61(5):422-428 (2010). |
| Liu et al., “Carbon Nanotubes as Molecular Transporters for Walled Plant Cells,” Nano Letters, 9(3):1007-1010 (2009). |
| Liu et al., “Comparative study on the interaction of DNA with three different kinds of surfactants and the formation of multilayer films,” Bioelectrochemistiy, 70:301-307 (2007). |
| Liu et al., “DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli,” BMC Biotechnology, 10:85 (2010). |
| Liu et al., “RNA interference in Nilaparvata lugens (Homoptera : Delphacidae) based on dsRNA ingestion,” Pest Manag. Sci., 67:852-859 (2011). |
| Liu, “Influence of Sugars on the Foliar Uptake of Bentazone and Glyphosate,” New Zealand Plant Protection, 55:159-162 (2002). |
| Liu, “The Transformation of Nucleic Acid Degradants in Plants,” China Organic Fertilizers, Agriculture Press, ISBN: 7-1091634 (with English translation). |
| Llave et al., “Endogenous and Silencing-Associated Small RNAs in Plants,” The Plant Cell, 14:1605-1619 (2002). |
| Lu et al., “Oligo Walk: an online siRNA design tool utilizing hybridization thermodynamics,” Nucleic Acids Research, 36:W104-W108 (2008). |
| Lu et al., “RNA silencing in plants by the expression of siRNA duplexes,” Nucleic Acids Res., 32(21):e171 (2004). |
| Luft, “Making sense out of antisense oligodeoxynucleotide delivery: getting there is half the fun,” J Mol Med, 76:75-76 (1998). |
| Luque et al., “Water Permeability of Isolated Cuticular Membranes: A Structural Analysis,” Archives of Biochemistry and Biophysics, 317(2):417-422 (1995). |
| Maas et al., “Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts,” Plant Cell Reports, 8:148-149 (1989). |
| MacKenzie et al., “Transgenic Nicotiana debneyii expressing viral coat protein are resistant to potato virus S infection,” Journal of General Virology, 71:2167-2170 (1990). |
| Maggi et al., “Resistance Phenomena to Amitraz From Population of the Ectoparasitic Mite Varroa Destructor of Argentina,” Parasitology Research, 107(5):1189-1192 (2010). |
| Maher III et al., “Inhibition of DNA binding proteins by oligonucleotide-directed triple helix formation,” Science, 245(4919):725-730 (1989). |
| Makkouk et al., “Virus Diseases of Peas, Beans, and Faba Bean in the Mediterranean region,” Adv Virus Res, 84:367-402 (2012). |
| Manoharan, “Oligonucleotide Conjugates as Potential Antisense Drugs with Improved Uptake, Biodistribution, Targeted Delivery, and Mechanism of Action,” Antisense & Nucleic Acid Drug Development, 12:103-128 (2002). |
| Mao et al., “Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol.,” Nat Biotechnol., 25(11):1307-13 (2007). |
| Maori et al., “Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra- and inter-species recombination,” Journal of General Virology, 88:3428-3438 (2007). |
| Maori et al., “Israel Acute Paralysis Virus of Bees, Complete Genome,” GenBank EMBL, EBI Dbfetch, XP002533679, Accession No. EF219380, Nov. 21, 2007. |
| Maori et al., “Reciprocal sequence exchange between non-retro viruses and hosts leading to the appearance of new host phenotypes,” Virology, 362(2):342-349 (2007). |
| Maori et al., “IAPV, a bee-affecting virus associated with Colony Collapse Disorder can be silenced by dsRNA ingestion,” Insect Molecular Biology, 18(1):55-60 (2009). |
| Masoud et al., “Constitutive expression of an inducible β-1,3-glucanase in alfalfa reduces disease severity caused by the oomycete pathogen Phytophthora megasperma f. sp medicaginis, but does not reduce disease severity of chitincontaining fungi,” Transge. |
| Matveeva et al., “Prediction of antisense oligonucleotide efficacy by in vitro methods,” Nature Biotechnology, 16:1374-1375 (1998). |
| McGinnis, “RNAi for functional genomics in plants,” Brief Funct Genomics, 9(2):111-7 (2010). |
| Meins et al., “RNA Silencing Systems and Their Relevance to Plant Development,” Annu. Rev. Cell Dev. Biol., 21:297-318 (2005). |
| Melnyk et al., “Intercellular and systemic movement of RNA silencing signals,” The EMBO Journal, 30:3553-3563 (2011). |
| Migge et al., “Greenhouse-grown conditionally lethal tobacco plants obtained by expression of plastidic glutamine synthetase antisense RNA may contribute to biological safety,” Plant Science 153:107-112 (2000). |
| Molnar et al., “Plant Virus-Derived Small Interfering RNAs Originate redominantly from Highly Structured Single-Stranded Viral RNAs,” Journal of Virology, 79(12):7812-7818 (2005). |
| Molnar et al., “Small Silencing RNAs in Plants Are Mobile and Direct Epigenetic Modification in Recipient Cells,” Science, 328:872-875 (2010). |
| Moriyama et al., “Double-stranded RNA in rice: a novel RNA replicon in plants,” Molecular & General Genetics, 248(3):364-369 (1995). |
| Moriyama et al., “Stringently and developmentally regulated levels of a cytoplasmic double-stranded RNA and its high-efficiency transmission via egg and pollen in rice,” Plant Molecular Biology, 31:713-719 (1996). |
| Morrissey et al., “Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs,” Nat Biotechnol. 23(8):1002-1007 (2005). |
| Moser et al., “Sequence—Specific Cleavage of Double Helical DNA by Triple Helix Formation,” Science, 238:645-646 (1987). |
| Mount et al., “Gene and Metabolite Regulatory Network Analysis of Early Developing Fruit Tissues Highlights New Candidate Genes for the Control of Tomato Fruit Composition and Development,” Plant Physiology, 149:1505- 1528 (2009). |
| Nakayashiki et al. “Evolution and Diversification of RNA Silencing Proteins in Fungi”, Journal of Molecular Evolution, 63(1):127-135 (2006). |
| Non-Final Office Action dated Apr. 11, 2013, in U.S. Appl. No. 13/042,856. |
| Non-Final Office Action dated Apr. 15, 2014, in U.S. Appl. No. 13/446,557. |
| Non-Final Office Action dated Apr. 29, 2016, in U.S. Appl. No. 13/583,302. |
| Non-Final Office Action dated Aug. 10, 2016, in U.S. Appl. No. 13/612,995. |
| Non-Final Office Action dated Aug. 12, 2015, in U.S. Appl. No. 13/612,936. |
| Non-Final Office Action dated Aug. 13, 2015, in U.S. Appl. No. 13/612,929. |
| Non-Final Office Action dated Aug. 19, 2016, in U.S. Appl. No. 13/612,925. |
| Non-Final Office Action dated Aug. 19, 2016, in U.S. Appl. No. 13/612,929. |
| Non-Final Office Action dated Aug. 3, 2016, in U.S. Appl. No. 14/015,715. |
| Non-Final Office Action dated Aug. 5, 2016, in U.S. Appl. No. 14/015,785. |
| Non-Final Office Action dated Aug. 8, 2016, in U.S. Appl. No. 13/612,936. |
| Non-Final Office Action dated Dec. 17, 2015, in U.S. Appl. No. 14/532,596. |
| Non-Final Office Action dated Feb. 10, 2016, in U.S. Appl. No. 13/901,326. |
| Non-Final Office Action dated Feb. 23, 2016, in U.S. Appl. No. 14/603,347. |
| Non-Final Office Action dated Feb. 23, 2016, in U.S. Appl. No. 14/608,951. |
| Non-Final Office Action dated Feb. 5, 2014, in U.S. Appl. No. 13/446,557. |
| Non-Final Office Action dated Jan. 7, 2013, in U.S. Appl. No. 13/318,636. |
| Non-Final Office Action dated Jul. 23, 2015, in U.S. Appl. No. 14/335,135. |
| Non-Final Office Action dated Jul. 30, 2014, in U.S. Appl. No. 13/042,856. |
| Non-Final Office Action dated Jun. 5, 2015, in U.S. Appl. No. 13/612,948. |
| Non-Final Office Action dated Jun. 8, 2015, in U.S. Appl. No. 13/612,941. |
| Non-Final Office Action dated Mar. 1, 2016, in U.S. Appl. No. 13/612,954. |
| Non-Final Office Action dated Mar. 21, 2018, in U.S. Appl. No. 13/619,980. |
| Non-Final Office Action dated Mar. 30, 2015, in U.S. Appl. No. 13/583,302. |
| Non-Final Office Action dated May 15, 2015, in U.S. Appl. No. 14/608,951. |
| Non-Final Office Action dated May 22, 2015, in U.S. Appl. No. 13/612,985. |
| Non-Final Office Action dated May 30, 2012, in U.S. Appl. No. 13/332,430. |
| Non-Final Office Action dated May 4, 2015, in U.S. Appl. No. 13/932,051. |
| Non-Final Office Action dated Nov. 9, 2016, in U.S. Appl. No. 14/901,003. |
| Non-Final Office Action dated Oct. 3, 2016, in U.S. Appl. No. 14/403,491. |
| Non-Final Office Action dated Sep. 1, 2015, in U.S. Appl. No. 13/612,954. |
| Non-Final Office Action dated Sep. 11, 2015, in U.S. Appl. No. 13/612,925. |
| Non-Final Office Action dated Sep. 23, 2010, in U.S. Appl. No. 12/222,949. |
| Non-Final Office Action dated Sep. 4, 2015, in U.S. Appl. No. 13/612,995. |
| Non-Final Office Action dated Sep. 6, 2016, in U.S. Appl. No. 14/335,135. |
| Nookaraju et al., “Molecular approaches for enhancing sweetness in fruits and vegetables,” Scientia Horticulture, 127:1-15 (2010). |
| Nord-Larsen et al., “Cloning, characterization and expression analysis of tonoplast intrinsic proteins and glutamine synthetase in ryegrass (Lolium perenne L.),” Plant Cell Reports, 28(10):1549-1562 (2009). |
| Nowak et al., “A new and efficient method for inhibition of RNA viruses by DNA interference,” The FEBS Journal, 276:4372-4380 (2009). |
| Nunes et al., “A non-invasive method for silencing gene transcriptoin in honeybees maintained under natural conditions,” Insect Biochemistry and Molecular Biology, 39:157-160 (2009). |
| Office Action dated 2015, in Chinese Patent Application No. 201080056585.9. |
| Office Action dated Apr. 13, 2016, in Chinese Patent Application No. 201280053985.3. |
| Office Action dated Apr. 15, 2014, in U.S. Appl. No. 13/446,557. |
| Office Action dated Aug. 1, 2013, in U.S. Appl. No. 13/318,636. |
| Office Action dated Aug. 1, 2017, in European Patent Application No. 12 830 932.5. |
| Office Action dated Aug. 14, 2017, in Israeli Patent Application No. 235878. |
| Office Action dated Aug. 22, 2017, in Korean Patent Application No. 10-2012-7023415. |
| Office Action dated Aug. 25, 2015, in New Zealand Patent Application No. 700791. |
| Office Action dated Aug. 25, 2016, in Eurasian Patent Application No. 201201264. |
| Office Action dated Aug. 28, 2013, in Chinese Patent Application No. 201180012795.2. |
| Office Action dated Aug. 3, 2015, in Chinese Patent Application No. 201080056585.9 (English translation attached). |
| Office Action dated Aug. 3, 2017, in Chinese Patent Application No. 201480014392.5 (with English translation). |
| Office Action dated Aug. 3, 2017, in European Patent Application No. 12 831 684.1. |
| Office Action dated Aug. 8, 2017, in Chilean Patent Application No. 201501874. |
| Office Action dated Dec. 13, 2016, in Ukrainian Patent Application No. a 2014 03843. |
| Office Action dated Dec. 14, 2016, in Ukrainian Patent Application No. a 2014 03850. |
| Office Action dated Dec. 15, 2016, in Ukrainian Patent Application No. a 2014 03845. |
| Office Action dated Dec. 15, 2016, in Ukrainian Patent Application No. a 2014 03849. |
| Office Action dated Dec. 15, 2016, in Ukrainian Patent Application No. a 2014 03852. |
| Office Action dated Dec. 18, 2014, in Israeli Patent Application No. 216154 (with English translation). |
| Office Action dated Dec. 21, 2015, in Israeli Patent Application No. 240416 (with English translation). |
| Office Action dated Dec. 27, 2016, in Ukrainian Patent Application No. a 2012 11548. |
| Office Action dated Dec. 31, 2014, in Israeli Patent Application No. 205594 (with English translation). |
| Office Action dated Dec. 5, 2017, in Japanese Patent Application No. 2016-502033. |
| Office Action dated Feb. 17, 2011, in European Patent Application No. 08847971.2. |
| Office Action dated Feb. 17, 2014, in European Patent Application No. 08847971.2. |
| Office Action dated Feb. 17, 2014, in Mexican Patent Application No. MX/a/2012/010479. |
| Office Action dated Feb. 21, 2018, in Mexican Patent Application No. MX/a/2015/012632 (with English translation). |
| Office Action dated Feb. 24, 2014, in Eurasian Patent Application No. 201201264. |
| Office Action dated Feb. 5, 2015, in European Patent Application No. 13156183.9. |
| Office Action dated Feb. 6, 2015, in European Patent Application No. 10719620.6. |
| Office Action dated Jan. 19, 2014, in Israeli Patent Application No. 205594 (with English translation). |
| Office Action dated Jan. 26, 2015, in Israeli Patent Application No. 205594 (with English translation). |
| Office Action dated Jan. 26, 2015, in Israeli Patent Application No. 219193 (with English translation). |
| Office Action dated Jan. 30, 2015, in Mexican Patent Application No. Mx/a/2012/004378 (with English translation). |
| Office Action dated Jan. 7, 2013, in U.S. Appl. No. 13/318,636. |
| Office Action dated Jul. 11, 2017, in Mexican Patent Application No. MX/a/2015/013118 (with English translation). |
| Office Action dated Jul. 12, 2013, in European Patent Application No. 08847971.2. |
| Office Action dated Jul. 18, 2016, in Indonesian Patent Application No. W00201203610. |
| Office Action dated Jul. 2, 2013, in Chinese Patent Application No. 201080056585.9. |
| Office Action dated Jul. 23, 2015, in Ukrainian Patent Application No. 201211548. |
| Office Action dated Jul. 3, 2017, in Mexican Patent Application No. MX/a/2015/012632 (with English translation). |
| Office Action dated Jul. 6, 2017, in Mexican Patent Application No. MX/a/2015/013103 (with English translation). |
| Office Action dated Jun. 29, 2012, in European Patent Application No. 08847971.2. |
| Office Action dated Jun. 20, 2016, in Chinese Patent Application No. 201280054819.5. |
| Office Action dated Jun. 24, 2016, in Chinese Patent Application No. 201280053984.9. |
| Office Action dated Jun. 28, 2010, in U.S. Appl. No. 12/222,949. |
| Office Action dated Jun. 8, 2015, in European Patent Application No. 13156185.4. |
| Office Action dated Mar. 10, 2014, in European Patent Application No. 13156180.4. |
| Office Action dated Mar. 10, 2014, in European Patent Application No. 13156180.5. |
| Office Action dated Mar. 16, 2017, in Chinese Patent Application No. 201280054819.5. |
| Office Action dated Mar. 17, 2014, in European Patent Application No. 13156183.9. |
| Office Action dated Mar. 18, 2011, in U.S. Appl. No. 12/222,949. |
| Office Action dated Mar. 19, 2012, in Israeli Patent Application No. 205594 (English Translation attached). |
| Office Action dated Mar. 19, 2015, in Canadian Patent Application No. 2,704,858. |
| Office Action dated Mar. 20, 2015, in Indian Patent Application No. 1150/MUMNP/2010. |
| Office Action dated Mar. 8, 2018 (with English translation), in Chilean Patent Application No. 201403192. |
| Office Action dated May 12, 2014, in Chinese Patent Application No. 201080056585.9. |
| Office Action dated May 12, 2014, in Mexican Patent Application No. MX/a/2012/004378 (English Translation attached). |
| Office Action dated May 29, 2015, in European Patent Application No. 13156180.4. |
| Office Action dated May 29, 2015, in European Patent Application No. 13156180.5. |
| Office Action dated May 3, 2016, in Chilean Patent Application No. 201601057. |
| Office Action dated May 30, 2012, in U.S. Appl. No. 13/332,430. |
| Office Action dated Nov. 10, 2014, in European Patent Application No. 10779855.5. |
| Office Action dated Nov. 15, 2016, in Mexican Patent Application No. MX/a/2014/003068 (with English translation). |
| Office Action dated Nov. 21, 2012, in U.S. Appl. No. 13/318,636. |
| Office Action dated Nov. 6, 2014, in Australian Patent Application No. 2010244122. |
| Office Action dated Oct. 15, 2012, in U.S. Appl. No. 13/332,430. |
| Office Action dated Oct. 21, 2015, in European Patent Application No. 13723252.6. |
| Office Action dated Oct. 23, 2013, in Australian Patent Application No. 2008325989. |
| Office Action dated Oct. 31, 2013, , in Mexican Patent Application No. MX/a/2012/004378 (English Translation attached). |
| Office Action dated Oct. 31, 2013, in Mexican Patent Application No. MX/a/2012/004378. |
| Office Action dated Oct. 8, 2013, in European Patent Application No. 10719620.6. |
| Office Action dated Sep. 29, 2014, European Patent Application No. 13156180.4. |
| Office Action dated Sep. 29, 2014, in European Patent Application No. 13156180.5. |
| Office Action dated Sep. 1, 2015, in European Patent Application No. 10779855.5. |
| Office Action dated Sep. 23, 2010, in U.S. Appl. No. 12/222,949. |
| Office Action dated Sep. 28, 2015, in U.S. Appl. No. 13/932,051. |
| Office Action dated Sep. 29, 2014, in European Patent Application No. 13156180.4. |
| Office Action dated Sep. 4, 2015, in European Patent Application No. 13156183.9. |
| Office Action dated Sep. 5, 2016, in Ukrainian Patent Application No. a 2014 03846. |
| Office Action dated Sep. 6, 2017, in Chinese Patent Application No. 2014800154012 (with English translation). |
| Office Action dated Nov. 3, 2014, in Chinese Patent Application No. 201180012795.2. |
| Office Action dated Jan. 6, 2015, in Japanese Patent Application No. 2012-557165. |
| Office Action dated Nov. 19, 2014, in Eurasian Patent Application No. 201201264/28. |
| Office Action dated Oct. 5, 2015, in Eurasian Patent Application No. 201201264/28. |
| Ongvarrasopone et al., “A Simple and Cost Effective Method to Generate dsRNA for RNAi Studies in Invertebrates,” Science Asia, 33:35-39 (2007). |
| Orbović et al., “Foliar-Applied Surfactants and Urea Temporarily Reduce Carbon Assimilation of Grapefruit Leaves,” J. Amer. Soc. Hort. Sci., 126(4):486-490 (2001). |
| Ouellet et al., “Members of the Acetohydroxyacid Synthase Muligene Family of Brassica napus Have Divergent Patterns of Expression,” The Plant Journal, Blackwell Scientific Publications, Oxford, GB, 2(3):321-330 (1992). |
| Paddison et al., “Stable suppression of gene expression by RNAi in mammalian cells,” Proc. Natl Acad. Sci. USA, 99(3):1443-1448 (2002). |
| Palacios et al., “Genetic Analysis of Israel Acute Paralysis Virus: Distinct Clusters Are Circulating in the United States,” Journal of Virology, 82(13):6209-6217 (2008). |
| Palauqui et al., “Activation of systemic acquired silencing by localised introduction of DNA,” Current Biology, 9:59-66 (1999). |
| Paldi et al. “Effective Gene Silencing in a Microsporidian Parasite Associated With Honeybee (Apis mellifera) Colony Declines”, Applied and Environmental Microbiology, 760(17):5960-5964 (2010). |
| Parera et al., “Dehydration Rate after Solid Matrix Priming Alters Seed Performance of Shrunken-2 Corn,” J. Amer. Soc. Hort. Sci., 119(3):629-635 (1994). |
| Partial European Search Report dated Dec. 6, 2017, in European Patent Application No. 17181861.0. |
| Partial International Search Report dated Jul. 24, 2013, International Application No. PCT/2013/050321. |
| Partial International Search Report dated May 13, 2009, in International Application No. PCT/IL2008/001440. |
| Partial Supplementary European Search Report dated Mar. 2, 2015, in European Patent Application No. 12 831 494.5. |
| Partial Supplementary European Search Report dated Jan. 11, 2018, in European Patent Application No. 15812530.0. |
| Patent Examination Report dated Nov. 6, 2014, in Australian Patent Application No. 2010244122. |
| Patent Examination Report dated Oct. 23, 2013, in Australian Patent Application No. 2008325989. |
| Patent Examination Report No. 1 dated Feb. 8, 2016, in Australian Patent Application No. 2014262189. |
| Patent Examination Report No. 1 dated Jun. 17, 2016, in Australian Patent Application No. 2012308659. |
| Patent Examination Report No. 1 dated Jun. 17, 2016, in Australian Patent Application No. 2012308660. |
| Patent Examination Report No. 1 dated Jun. 8, 2017, in Australian Patent Application No. 2012308686. |
| Patent Examination Report No. 1 dated Nov. 11, 2013, in Australian Patent Application No. 2011224570. |
| Paungfoo-Lonhienne et al., “DNA is Taken up by Root Hairs and Pollen, and Stimulates Root and Pollen Tube Growth,” Plant Physiology, 153:799-805 (2010). |
| Paungfoo-Lonhienne et al., “DNA uptake by Arabidopsis induces changes in the expression of CLE peptides which control root morphology,” Plant Signaling & Behavior, 5(9):1112-1114 (2010). |
| Pei et al., “On the art of identifying effective and specific siRNAs,” Nature Methods, 3(9):670-676 (2006). |
| Peretz et al., “A Universal Expression/Silencing Vector in Plants,” Plant Physiology, 145:1251-1263 (2007). |
| Pitino et al., “Silencing of Aphid Genes by dsRNA Feeding from Plants,” PLos One, 6:e25709 (2011). |
| Pornprom et al., “Glutamine synthetase mutation conferring target-site-based resistance to glufosinate in soybean cell selections,” Pest Manag Sci, 2009; 65(2):216-222 (2009). |
| Powles et al., “Evolution in Action: Plants Resistant to Herbicides,” Annual Review of Plant Biology, 61(1):317-347 (2010). |
| Pratt et al., “Amaranthus rudis and A. tuberculatus, One Species or Two?,” Journal of the Torrey Botanical Society, 128(3):282-296 (2001). |
| Preston et al., “Multiple effects of a naturally occurring proline to threonine substitution within acetolactate synthase in two herbicide-resistant populations of Lactuca serriola,” Pesticide Biochem. Physiol., 84(3):227-235 (2006). |
| Price et al. “RNAi-Mediated Crop Protection Against Insects”, Trends in Biotechnology, XP022757296, 26(7):393-400 (2008). |
| Pridgeon et al., “Topically Applied AaeIAP1 Double-Stranded RNA Kills Feman Adults of Aedes aegypti,” J. Med. Entomol., 45(3):414-420 (2008). |
| Rajur et al., “Covalent Protein—Oligonucleotide Conjugates for Efficient Delivery of Antisense Molecules,” Bioconjug Chem., 8:935-940 (1997). |
| Rakoczy-Trojanowska, “Alternative Methods of Plant Transformation—a short review,” Cellular & Molecular Biology Letters, 7:849-858 (2002). |
| Reddy et al., “Aminomethylphosphonic Acid Accumulation in Plant Species Treated with Glyphosate,” J. Agric. Food Chem., 56(6):2125-2130 (2008). |
| Reither et al., “Specificity of DNA triple helix formation analyzed by a FRET assay,” BMC Biochemistry, 3:27 (2002). |
| Requisition by the Examiner and Examination Search Report dated Mar. 19, 2015, in Canadian Patent Application No. 2,704,858. |
| Rey et al., “Diversity of Dicotyledenous-Infecting Geminiviruses and Their Associated DNA Molecules in Southern Africa, Including the South-West Indian Ocean Islands,” Viruses, 4:1753-1791 (2012). |
| Reynolds et al., “Rational siRNA design for RNA interference,” Nature Biotechnology, 22:326-330 (2004). |
| Richardson et al., “Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane,” Frontiers in Plant Science, 5:1-14 (2014). |
| Riggins et al., “Characterization of de novo transcriptome for waterhemp (Amaranthus tuberculatus) using GS-FLX 454 pyrosequencing and its application for studies of herbicide target-site genes,” Pest Manag. Sci., 66:1042-1052 (2010). |
| Robalino et al., “Double-Stranded RNA and Antiviral Immunity in Marine Shrimp: Inducible Host Mechanisms and Evidence for the Evolution of Viral Counter-Responses,” Developmental & Comparative Immunology, 31: 539-547 (2007). |
| Roberts, “Fast-track applications: The potential for direct delivery of proteins and nucleic acids to plant cells for the discovery of gene function,” Plant Methods, 1(12):1-3 (2005). |
| Rose et al., “Functional polarity is introduced by Dicer processing of short substrate RNAs,” Nucleic Acids Research, 33(13):4140-4156 (2005). |
| Rothnie et al., Pararetroviruses and Retroviruses: A Comparative Review of Viral Structure and Gene Expression Strategies, Advances in Virus Research, 44:1-67 (1994). |
| Ryabov et al., “Cell-to-Cell, but Not Long-Distance, Spread of RNA Silencing That Is Induced in Individual Epidermal Cells,” Journal of Virology, 78(6):3149-3154 (2004). |
| Salanenka et al., “Seedcoat Permeability: Uptake and Post-germination Transport of Applied Model Tracer Compounds,” HortScience, 46(4):622-626 (2011). |
| Santoro et al., “A general purpose RNA-cleaving DNA enzyme,” Proc. Natl. Acad. Sci. USA, 94:4262-4266 (1997). |
| Santosh et al., “RNA interference for the control of whiteflies (Bemisia tabaci) by oral route,” Journal of Biosciences, 36(1):153-161 (2011). |
| Sathasivan et al., “Nucleotide sequence of a mutant acetolactate synthase gene from an imidazolinone-resistant Arabidopsis thaliana var. Columbia,” Nucleic Acids Research, 18(8):2188-2193 (1990). |
| Schönherr, “Water Permeability of Isolated Cuticular Membranes: The Effect of pH and Cations on Diffusion, Hydrodynamic Permeability and Size of Polar Pores in the Cutin Matrix,” Planta, 128:113-126 (1976). |
| Schwab et al., “RNA silencing amplification in plants: Size matters,” PNAS, 107(34):14945-14946 (2010). |
| Schweizer et al., “Double-stranded RNA interferes with gene function at the single-cell level in cereals,” The Plant Journal, 24(6):895-903 (2000). |
| Scott et al., “Towards the elements of successful insect RNAi,” Journal of Insect Physiology, 59(12):1212-1221 (2013). |
| Scott et al., Botanical Insecticides for Controlling Agricultural Pests: Piperamides and the Colorado Potato Beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), Archives of Insect Biochemistry and Physiology, 54:212-225 (2003). |
| Search Report dated Jul. 24, 2017, in Chinese Patent Application No. 201480014392.5 (with English translation). |
| Search Report dated Oct. 20, 2017, in Chinese Patent Application No. 201380039346.6. |
| Second Chinese Office Action dated Jun. 10, 2014, in Chinese Patent Application No. 201180012795.2. |
| Second Office Action dated Feb. 25, 2016, in Chinese Patent Application No. 201280054179.8. |
| Second Office Action dated Mar. 4, 2016, in Chinese Patent Application No. 201280054820.8. |
| Second Office Action dated May 12, 2014, in Chinese Patent Application No. 201080056585.9. |
| Seidman et al., “The potential for gene repair via triple helix formation,” J Clin Invest., 112(4):487-494 (2003). |
| Selvarani et al., “Evaluation of seed priming methods to improve seed vigour of onion (Allium cepa cv. Aggregatum) and carrot (Daucus carota),” Journal of Agricultural Technology, 7(3):857-867 (2011). |
| Senthil-Kumar et al., “A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing,” New Phytologist, 176:782-791 (2007). |
| Sharma et al., “A simple and efficient Agrobacterium-mediated procedure for transformation of tomato,” J. Biosci., 34(3):423 433 (2009). |
| Shen et al., “The Role of Varroa Mites in Infections and Deformed Wing Virus (DWV) in Honey,” Available Online Aug. 18, 2005. |
| Shintani et al., “Antisense Expression and Overexpression of Biotin Carboxylase in Tobacco Leaves,” Plant Physiol., 114:881-886 (1997). |
| Showalter, “Structure and Function of Plant Cell Wall Proteins,” The Plant Cell, 5:9-23 (1993). |
| Sijen et al., “On the Role of RNA Amplification in dsRNA-Triggered Gene Silencing,” Cell, 107:465-476 (2001). |
| Silwet L-77 Spray Adjuvant for agricultural applications, product description from Momentive Performance Materials, Inc. (2003). |
| Sindhu et al., “Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success,” J. Exp. Botany, 60:315-324 (2008). |
| Siomi et al. “On the Road to Reading the RNA-Interference Code”, Nature, 457(7228): 396-404, Jan. 22, 2009. Abstract. |
| Slamovits et al. “Genome Compaction and Stability in Microsporidian Intracellular Parasites”, Current Biology, 14(10): 891-896, May 25, 2004. |
| Small, “RNAi for revealing and engineering plant gene functions,” Current Opinion in Biotechnology, 18:148-153 (2007). |
| Snead et al., “Molecular basis for improved gene silencing by Dicer substrate interfering RNA compared with other siRNA variants,” Nucleic Acids Research, 41(12):6209-6221 (2013). |
| Soares et al., “Capillary feeding of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks,” Insect Mol. Biol., 14(4):443-452 (2005). |
| Song et al., “Herbicide,” New Heterocyclic Pesticide, Chemical Industry Press, 354-356 (2011). |
| Standifer et al., “Supplemental Feeding of Honey Bee Colonies,” Agriculture Information Bullentin No. 413, USDA, pp. 1-8 (1977). |
| Statement of Grounds and Particulars dated Sep. 1, 2017, in Australian Patent No. 2014262189. |
| Steeves et al., “Transgenic soybeans expressing siRNAs specific to a major sperm protein gene suppress Heterodera glycines reproduction,” Funct. Plant Biol., 33:991-999 (2006). |
| Stevens et al., “New Formulation Technology—SILWET® Organosilicone Surfactants Have Physical and Physiological Properties Which Enhance the Performance of Sprays,” Proceedings of the 9th Australian Weeds Conference, pp. 327-331 (1990). |
| Stevens, “Formulation of Sprays to Improve the Efficacy of Foliar Fertilisers,” New Zealand Journal of Forestry Science, 24(1):27-34 (1994). |
| Stevens, “Organosilicone Surfactants as Adjuvants for Agrochemicals,” Journal of Pesticide Science, 38:103-122 (1993). |
| Stock et al., “Possible Mechanisms for Surfactant-Induced Foliar Uptake of Agrochemicals,” Pestic. Sci., 38:165-177 (1993). |
| Strat et al., “Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs,” Nucleic Acids Research, 34(13):3803-3810 (2006). |
| Street “Why is DNA (and not RNA) a stable storage form for genetic information?,” Biochemistry Revisited, pp. 1-4 (2008). |
| Sudarsan et al., “Metabolite-binding RNA domains are present in the genes of eukaryotes,” RNA, 9:644-647 (2003). |
| Summons to Attend Oral Proceedings Pursuant to Rule 115(1) EPC, dated Aug. 7, 2017, in European Patent Application No. 12832160.1. |
| Sun, “Characterization of Organosilicone Surfactants and Their Effects on Sulfonylurea Herbicide Activity,” Thesis Submitted to the Faculty of the Virginia Polytechnic Institute and State University dated Apr. 5, 1996. |
| Supplementary European Search Report dated Jan. 17, 2018, in European Patent Application No. 15773480.7. |
| Supplementary Partial European Search Report dated Jan. 11, 2018 in European Appln. 15 82 6865. |
| Supplementary Partial European Search Report dated Oct. 16, 2017, in European Patent Application No. 15773480.7. |
| Takasaki et al., “An Effective Method for Selecting siRNA Target Sequences in Mammalian Cells,” Cell Cycle, 3:790-795 (2004). |
| Tang et al., “Efficient delivery of small interfering RNA to plant cells by a nanosecond pulsed laser-induced stress wave for posttranscriptional gene silencing,” Plant Science, 171:375-381 (2006). |
| Tank Mixing Chemicals Applied to Peanut Crops: Are the Chemicals Compatible?, College of Agriculture & Life Sciences, NC State University, AGW-653, pp. 1-11 (2004). |
| Templeton et al., “Improved DNA: liposome complexes for increased systemic delivery and gene expression,” Nature Biotechnology, 15:647-652 (1997). |
| Tenllado et al., “Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infection,” BMC Biotechnology, 3(3):1-11 (2003). |
| Tenllado et al., “Double-Stranded RNA-Mediated Interference with Plant Virus Infection,” Journal of Virology, 75(24):12288-12297 (2001). |
| Tenllado et al., “RNA interference as a new biotechnological tool for the control of virus diseases in plants,” Virus Research, 102:85-96 (2004). |
| Tepfer, “Risk assessment of virus resistant transgenic plants,” Annual Review of Phytopathology, 40:467-491 (2002). |
| Terenius et al., RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design, Journal of Insect Physiology, 57(2):231-245 (2011). |
| Third Office Action dated Nov. 25, 2014, in Chinese Patent Application No. 201080056585.9. |
| Third Party Submission filed on Nov. 29, 2012 in U.S. Appl. No. 13/042,856. |
| Thomas et al., “Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector,” The Plant Journal, 25(4):417-425 (2001). |
| Tian et al., “Developmental Control of a Lepidopteran Pest Spodoptera exigua by Ingestion of Bacteria Expressing dsRNA of a Non-Midgut Gene,” PLoS ONE, 4:e6225, pp. 1-14 (2009). |
| Timmons et al., “Specific interference by ingested dsRNA,” Nature, 395:854 (1998). |
| Tomari et al., “Perspective: machines for RNAi,” Genes & Dev., 19:517-529 (2005). |
| Töpfer et al., “Uptake and Transient Expression of Chimeric Genes in Seed-Derived Embryos,” Plant Cell, 1:133-139 (1989). |
| Toriyama et al., “Transgenic Rice Plants After Direct Gene Transfer Into Protoplasts,” Bio/Technology, 6:1072-1074 (1988). |
| Tran et al., “Control of specific gene expression in mammalian cells by co-expression of long complementary RNAs,” FEBS Lett.;573(1-3):127-134 (2004). |
| Tranel et al., “Resistance of weeds to ALS-inhibiting herbicides: what have we learned?,” Weed Science, 50:700-712 (2002). |
| Translation dated Jan. 15, 2015 of Office Action dated Dec. 18, 2014 From the Israel Patent Office Re. Application No. 216154. |
| Translation dated Jan. 20, 2015 of Office Action dated Dec. 31, 2014 From the Israel Patent Office Re. Application No. 205594. |
| Translation of Office Action dated Jul. 2, 2013 From the State Intellectual Property Office of the People's Republic of China Re. Application No. 201080056585.9. |
| Tsugawa et al., “Efficient transformation of rice protoplasts mediated by a synthetic polycationic amino polymer,” Theor Appl Genet, 97:1019-1026 (1998). |
| Turina et al., “Tospoviruses in the Mediterranean Area,” Advances in Virus Research, 84:403-437 (2012). |
| Turner et al., “RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding,” Insect Mol. Biol., 15(3):383-391 (2006). |
| Tuschl, “Expanding small RNA interference,” Nature Biotechnol., 20: 446-448 (2002). |
| Tuschl, “RNA Interference and Small Interfering RNAs,” ChemBiochem. 2(4):239-245 (2001). |
| Ui-Tei et al., “Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference,” Nucleic Acids Res., 32(3): 936-948 (2004). |
| Ullu et al., “RNA Interference in Protozoan Parasites,” Cellular Microbiology, 6(6):509-519 (2004). |
| Ulrich et al., “Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target,” BMC genomics, 16(1):671 (2015). |
| Unnamalai et al., “Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells,” FEBS Letters, 566:307-310 (2004). |
| Unniraman et al., “Alternate Paradigm for Intrinsic Transcription Termination in Eubacteria,” The Journal of Biological Chemistry, 276(45)(9):41850-41855 (2001). |
| Unniraman et al., “Conserved Economics of Transcription Termination in Eubacteria,” Nucleic Acids Research, 30(3):675-684 (2002). |
| Upadhyay et al., RNA interference for the control of whiteflies (Bemisia tabaci) by oral route, J. Biosci., 36(1):153-161 (2011). |
| Urayama et al., “Knock-down of OsDCL2 in Rice Negatively Affects Maintenance of the Endogenous dsRNA Virus, Oryza sativa Endornavirus,” Plant and Cell Physiology, 51(1):58-67 (2010). |
| van de Wetering et al., “Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector,” EMBO Rep., 4(6):609-615 (2003). |
| Vasil et al., “Herbicide Resistant Fertile Transgenic Wheat Plants Obtained by Microprojectile Bombardment of Regenerable Embryogenic Callus,” Bio/Technology,10:667-674 (1992). |
| Vaucheret, “Post-transcriptional small RNA pathways in plants: mechanisms and regulations,” Genes Dev., 20:759-771 (2006). |
| Vencill et al., “Resistance of Weeds to Herbicides,” Herbicides and Environment, 29:585-594 (2011). |
| Verma et al., “Modified oligonucleotides: synthesis and strategy for users,” Annu. Rev. Biochem., 67:99-134 (1998). |
| Vermeulen et al., “The contributions of dsRNA structure to Dicer specificity and efficiency,” RNA, 11(5):674-682 (2005). |
| Vert et al., “An accurate and interpretable model for siRNA efficacy prediction,” BMC Bioinformatics, 7:520 (2006). |
| Voinnet et al., “Systemic Spread of Sequence-Specific Transgene RNA Degradation in Plants Is Initiated by Localized Introduction of Ectopic Promoterless DNA,” Cell, 95:177-187 (1998). |
| Voinnet, “Origin, Biogenesis, and Activity of Plant MicroRNAs,” Cell, 136:669-687 (2009). |
| Wakelin et al., “A target-site mutation is present in a glyphosate-resistant Lolium rigidum population,” Weed Res. (Oxford), 46(5):432-440 (2006). |
| Walton et al., “Thermodynamic and Kinetic Characterization of Antisense Oligodeoxynucleotide Binding to a Structured mRNA,” Biophysical Journal, 82:366-377 (2002). |
| Walton et al., “Prediction of antisense oligonucleotide binding affinity to a structured RNA target,” Biotechnol Bioeng 65(1):1-9 (1999). |
| Wan et al., “Generation of Large Numbers of Independently Transformed Fertile Barley Plants,” Plant Physiol., 104:37-48 (1994). |
| Wang et al. “Molecular Characterization of an Arachnid Sodium Channel Gene From the Varroa Mite (Varroa Destructor)”, Insect Biochemistry and Molecular Biology, XP002621492, 33(7): 733-739, Jul. 2003. Abstract. |
| Wang et al. “Tracking Anonymous Peer-to-Peer VoIP Calls on the Internet”, ACM, CCS'05, Alexandria, VA, USA, Nov. 7-11, 2005, 11 P., 2005. |
| Wang et al., “Foliar uptake of pesticides-Present status and future challenge,” ScienceDirect, 87:1-8 (2007). |
| Wang et al., “Principle and technology of genetic engineering in plants,” in Plant genetic engineering principles and techniques, Beijing: Science Press, pp. 313-315 (1998). |
| Wang et al. ,“Silkworm Coatomers and Their Role in Tube Expansion of Posterior Silkgland,” PLoS ONE 5(10): E133252 (2010). |
| Wardell, “Floral Induction of Vegetative Plants Supplied a Purified Fraction of Deoxyribonucleic Acid from Stems of Flowering Plants,” Plant Physiol, 60:885-891 (1977). |
| Wardell,“Floral Activity in Solutions of Deoxyribonucleic Acid Extracted from Tobacco Stems,” Plant Physiol, 57:855-861 (1976). |
| Waterhouse et al., “Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA,” Proc Natl Acad Sci USA, 95 13959-13964 (1998). |
| Welch et al., “Expression of ribozymes in gene transfer systems to modulate target RNA levels,” Curr Opin Biotechnol. 9(5):486-496 (1998). |
| Whyard et al., “Ingested double-stranded RNAs can act as species-specific insecticides,” Insect Biochem. Mol. Biol., 39(11):824-832 (2009). |
| Widholm et al., “Glyphosate selection of gene amplification in suspension cultures of 3 plant species,” Phyisologia Plantarum, 112:540-545 (2001). |
| Wiesman et al., “Novel cationic vesicle platform derived from vernonia oil for efficient delivery of DNA through plant cuticle membranes,” Journal of Biotechnology, 130:85-94 (2007). |
| Wild Carrot Noxious Weed Control Board (NWCB) of Washington State (2010) <www.nwcb.wa.govidetail.asp?weed=46>. |
| Williams “Unique Physiology of Host-Parasite Interactions in Microsporidia Infections,” Cellular Microbiology, 11(11):1551-1560 (2009). |
| Williams et al., “Genome Sequence Surveys of Brachiola Algerae and Edhazardia Aedis Reveal Micriosporidia With Low Gene Densities,” BMC Genomics, 9(200):1-9 (2008). |
| Wilson, et al., “Transcription termination at intrinsic terminators: The role of the RNA hairpin,” Proc. Natl. Acad. Sci. USA, 92:8793-8797 (1995). |
| Winkler et al., “Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression,” Nature, 419:952-956 (2002). |
| Written Opinion dated Apr. 7, 2016, in Singapore Patent Application No. 201206152-9. |
| Written Opinion dated Aug. 13, 2009, in International Application No. PCT/IL2008/001440. |
| Written Opinion dated Mar. 6, 2017, in Singaporean Patent Application No. 2012061529. |
| Written Opinion dated May 8, 2014, in International Application No. PCT/IL2013/050447. |
| Written Opinion dated Sep. 1, 2014, in Singapore Patent Application No. 201206152-9. |
| Yadav et al., “Host-Generated Double Stranded RNA Induces RNAi in Plant-Parasitic Nematodes and Protects the Host From Infection,” Molecular & Biochemical Parasitology, 148:219-222 (2006). |
| Yao et al., “Development of RNAi Methods for Peregrinus maidis, the Corn Planthopper,” PLOS One, 8(8):1-11 (2013). |
| Yin et al., “Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system,” Appl. Microbiol. Biotechnol., 84(2):323-333 (2009). |
| YouTube video by General Electric Company “Silwet Surfactants,” screen shot taken on Jan. 11, 2012 of video of www.youtube.com/watch?v=WBw7nXMqHk8 (uploaded Jul. 13, 2009). |
| Zhang et al., “Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method,” Nature Protocols, 1(2):1-6 (2006). |
| Zhang et al., “Cationic lipids and polymers mediated vectors for delivery of siRNA,” Journal of Controlled Release, 123:1-10 (2007). |
| Zhang et al., “Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts,” The Plant Cell Rep., 7:379-384 (1988). |
| Zhang, “Artificial trans-acting small interfering RNA: a tool for plant biology study and crop improvements,” Planta, 239:1139-1146 (2014). |
| Zhang, Chapter 10: New Characteristics of Pesticide Research & Development, p. 209 (2010). |
| Zhao et al., “Phyllotreta striolata (Coleoptera: Chiysomelidae) Arginine kinase cloning and RNAi-based pest control,” European Journal of Entomology, 105(5):815-822 (2008). |
| Zhao et al., “PsOrl, a potential target for RNA interference-based pest management,” Insect Molecular Biology 20(1):97-104 (2011). |
| Zhu et al., “Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata,” Pest Manag Sci, 67:175-182 (2010). |
| Zotti et al., “RNAi technology for insect management and protection of beneficial insects from diseases: lessons, challenges and risk assessments,” Neotropical Entomology, 44(3):197-213 (2015). |
| Bachman et al., “Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte),” Transgenic Res., 22(6):1207-22 (2013). |
| Chu et al., “Potent RNAi by short RNA triggers,” RNA, 14:1714-1719 (2008). |
| Extended European Search Report dated Nov. 15, 2018, in European Patent Application No. 1818863 3.4. |
| Hawkins et al., “Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells,” Nucleic Acids Research, 37(9):2984-2995 (2009). |
| Qiang et al., “Cloning and bioinformatics analysis of an ubiquitin gene of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae),” African Journal of Biotechnology, 10(60):12852-12858 (2011). |
| Akiyoshi et al., “Genomic Survey of the Non-Cultivatable Opportunistic Human Pathogen, Enterocytozoon bieneusi,” PLoS Pathog, 5(l):el000261:l-14 (2009). |
| Alarcon-Reverte et al., “Resistance to ACCase-Inhibiting Herbicides in the Weed Lolium Multiflorum,” Comm. Appl. Biol. Sci, 73(4):899:902 (2008). |
| Al-Kaff et al., “Plants rendered herbicide-susceptible by cauliflower mosaic virus-elicited suppression of a 35S promoter-regulated transgene,” Nature Biotechnology, 18:995-999 (2000). |
| Ambrus et al., “The diverse roles of RNA helicases in RNAi,” Cell Cycle, 8(21):3500-3505 (2009). |
| Amdam et al., “The hive bee to forager transition in honeybee colonies: the double repressor hypothesis,” Journal of Theoretical Biology, 223:451-464 (2003). |
| Amdam et al., “Altered Physiology in Worker Honey Bees (Hymenoptera: Apidae) Infested with the Mite Varroa destructor (Acari: Varroidae): A Factor in Colony Loss During Overwintering?” J. Econ. Entomol., 97(3):741-747 (2004). |
| Andersson et al., “A novel selection system for potato transformation using a mutated AHAS gene,” Plant Cell Rep, 22:261-267 (2003). |
| Araujo et al., “RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection,” Insect Biochemistry and Molecular Biology, 36:683-693 (2006). |
| Aronstein et al., “SID-I is implicated in systemic gene silencing in the honey bee,” Journal of Apicultural Research and Bee World, 45(I):20-24 (2006) |
| Aronstein et al., “Characterization of a honey bee Toll related receptor gene Aml8w and its potential involvement in antimicrobial immune defense,” Apidologie, 36:3-14 (2005). |
| Database Accession No. BT006855, “Homo sapiens calmodulin 3 (phosphorylase kinase, delta) mRNA” pp. 1-2 (2003). |
| Database Accession No. EF219380, “SV 1; linear; mRNA; STD; VRL; 9499 BP.,” pp. 1-5 (2007). |
| Extended European Search Report dated Jan. 14, 2019, in European Patent Application No. 16789940.0. |
| Extended European Search Report dated Mar. 4, 2019, in European Patent Application No. 18 20 7017.7. |
| Final Office Action dated Sep. 28, 2015, in U.S. Appl. No. 13/932,051. |
| GenBank Bankit, <https://www.ncbi.nlm.nih.gov/WebSub/?tool=genbank[Sep. 27, 2016 6:27:51 AM]> pp. 1-3 (2016). |
| Heath et al., “RNA Interference Technology to Control Pest Sea Lampreys—A Proof-of-Concept,” PLOS One, 9(2):e88387:l-9 (2014). |
| International Search Report and Written Opinion dated Aug. 13, 2009, in International Application No. PCT/IL2008/001440. |
| International Search Report and Written Opinion dated Mar. 26, 2015, in International Application No. PCT/US2014/069353. |
| Invitation to Pay Additional Fees dated Jul. 24, 2013, in International Application No. PCT/IL/2013/050321. |
| Invitation to Pay Additional Fees dated May 13, 2009, in International Application No. PCT/IL2008/001440 |
| Non-Final Office Action dated Jun. 28, 2010, in U.S. Appl. No. 12/222,949. |
| Non-Final Office Action dated Nov. 21, 2012, in U.S. Appl. No. 13/318,636. |
| Patel et al., “The Making of a Queen: TOR Pathway Is a Key Player in Diphenic Caste Development,” Plos One, 2(6):e509:l-7 (2007). |
| Patent Examination Report No. 1 dated Nov. 6, 2014, in Australian Patent Application No. 2010244122. |
| Patent Examination Report No. 1 dated Oct. 23, 2013, in Australian Patent Application No. 2008325989. |
| Pridgeon et al., “Topically Applied AaelAPl Double-Stranded RNA Kills Female Adults of Aedes aegypti” J. Med. Entomol., 45(3):414-420 (2008). |
| Reddy et al.“Organosilicone Adjuvants Increased the Efficacy of Glyphosate for Control of Weeds in Citrus (Citrus spp.)” HortScience 27(9): 1003-1005 (1992). |
| Second Office Action dated May 12, 2014, in Chinese Patent Application No. 201080056585.9, with English translation of same. |
| Shen et al., “The role of varroa mites in infections of Kashmir bee virus (KBV) and deformed wing virus (DWV) in honey bees,” Virology, 342(1): 141-149 (2005). |
| Siomi et al., “On the Road to Reading the RNA-Interference Code”, Nature, 457(7228): 396-404 (2009). |
| Slamovits et al., “Genome Compaction and Stability in Microsporidian SiIntracellular Parasites”, Current Biology, 14(10): 891-896 (2004). |
| Supplementary Partial European Search Report date Jan. 11, 2018 in European Appln. 15826865. |
| Third Office Action dated November 25, 2014, in Chinese Patent Application No. 201080056585.9, with English translation of same. |
| Wang et al., “Tracking Anonymous Peer-to-Peer VoIP Calls on the Internet,” ACM, CCS'05, Alexandria, VA, USA, pp. 1-11 (2005). |
| Wang et al., “Molecular Characterization of An Arachnid Sodium Channel Gene From the Varroa Mite (Varroa Destructor)”, Insect Biochemistry and Molecular Biology, 33(7): 733-739 (2003). |
| Yibrah et al., “Antisense RNA inhibition of uidA gene expression in transgenic plants: Evidence for interaction between first and second transformation events,” Hereditas, 118:273-280 (1993). |
| Zrachya et al., “Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus,” Transgenic Res., 16:385-398 (2007). |
| Number | Date | Country | |
|---|---|---|---|
| 20170183683 A1 | Jun 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61973484 | Apr 2014 | US |
