Patent Application
20150313238
The sequence listing that is contained in the file named “MONS317WOsequencelisting.txt”, which is 251 kilobytes as measured in Microsoft Windows operating system and was created on Oct. 11, 2013, is filed electronically herewith and incorporated herein by reference.
The methods and compositions generally relate to the field of plant disease control. More specifically, the invention relates to methods and compositions for treating or preventing symptoms associated with plant Tospovirus or Geminivirus infection.
Plant viruses of the genus Tospovirus and Geminivirus are economically important, causing reduced vegetative output and death of infected plants. Growers seeking to protect their crops from tospoviruses have traditionally attempted to guard their crops from the insect vectors, either with insecticide application, or with reflective mulches or plastic covers. Because these strategies have had limited success, and are expensive and labor intensive, alternative strategies for controlling Tospovirus and Geminivirus infection are needed.
The embodiments described herein relate to methods and compositions for the prevention or treatment of viral infection in a plant comprising the topical administration to a plant of a polynucleotide comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a viral gene. The polynucleotide may be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA).
In one aspect, the invention provides a method of treatment or prevention of a Tospovirus infection in a plant comprising: topically applying to said plant a composition comprising an antisense single-stranded DNA polynucleotide and a transfer agent, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential Tospovirus gene sequence or an RNA transcript thereof, wherein the symptoms of viral infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In some embodiments, the antisense single-stranded DNA polynucleotide comprises at least 18 contiguous nucleotides that are essentially complementary to a sequence selected from the group consisting of SEQ ID NOs:13-46. In one embodiment, the transfer agent is an organosilicone surfactant composition or compound contained therein. In another embodiment, the composition comprises more than one antisense single-stranded DNA polynucleotide complementary to all or a portion of an essential Tospovirus gene sequence, an RNA transcript of said essential Tospovirus gene sequence, or a fragment thereof. In another embodiment, the antisense single-stranded DNA polynucleotide is selected from the group consisting of SEQ NOs:1-12 or a fragment thereof. In another embodiment, the Tospovirus is selected from the group consisting of bean necrotic mosaic virus, Capsicum chlorosis virus, groundnut bud necrosis virus, groundnut ringspot virus, groundnut yellow spot virus, impatiens necrotic spot virus, iris yellow spot viris, melon yellow spot virus, peanut bud necrosis virus, peanut yellow spot virus, soybean vein necrosis-associated virus, tomato chlorotic spot virus, tomato necrotic ringspot virus, tomato spotted wilt virus, tomato zonate spot virus, watermelon bud necrosis virus, watermelon silver mottle virus, and zucchini lethal chlorosis virus. In another embodiment, the essential Tospovirus gene is selected from the group consisting of nucleocapsid gene (N), coat protein gene (CP), virulence factors NSm and NSs, and RNA-dependent RNA polymerase L segment (RdRp/L segment). In another embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:13-46. In another embodiment, composition is topically applied by spraying, dusting, or is applied to the plant surface as matrix-encapsulated DNA.
In another aspect, the invention provides a composition comprising an antisense single-stranded DNA polynucleotide and a transfer agent, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential Tospovirus gene sequence or an RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Tospovirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In some embodiments, the essential gene sequence is selected from the group consisting of SEQ ID NOs:13-46, or the transfer agent is an organosilicone composition, or the antisense single-stranded DNA polynucleotide is selected from the group consisting of SEQ ID NOs:1-12.
In another aspect, the invention provides a method of reducing expression of an essential Tospovirus gene comprising contacting a Tospovirus particle with a composition comprising an antisense single-stranded DNA polynucleotide and a transfer agent, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential gene sequence in said Tospovirus or an RNA transcript thereof, wherein the symptoms of Tospovirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:13-46. In another embodiment, the transfer agent is an organosilicone compound. In another embodiment, the antisense single-stranded DNA polynucleotide is selected from the group consisting of SEQ ID NOs:1-12 or fragment thereof.
In another aspect, the invention provides a method of identifying antisense single-stranded DNA polynucleotides useful in modulating Tospovirus gene expression when topically treating a plant comprising: a) providing a plurality of antisense single-stranded DNA polynucleotides that comprise a region complementary to all or a part of an essential Tospovirus gene or RNA transcript thereof; b) topically treating said plant with one or more of said antisense single-stranded DNA polynucleotides and a transfer agent; c) analyzing said plant or extract for modulation of symptoms of Tospovirus infection; and d) selecting an antisense single-stranded DNA polynucleotide capable of modulating the symptoms or occurrence of Tospovirus infection. In an embodiment, the transfer agent is an organosilicone compound.
In another aspect, the invention provides an agricultural chemical composition comprising an admixture of an antisense single-stranded DNA polynucleotide and a pesticide, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential Tospovirus gene sequence or RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Tospovirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In an embodiment, the pesticide is selected from the group consisting of anti-viral compounds, insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides.
In another aspect, the invention provides a method of treatment or prevention of a Tospovirus infection in a plant comprising: topically applying to said plant a composition comprising a double-stranded RNA polynucleotide and a transfer agent, wherein said double-stranded RNA comprises a polynucleotide that is essentially complementary to all or a portion of an essential Tospovirus gene sequence or an RNA transcript thereof, wherein the symptoms of viral infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In some embodiments, the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:13-46. In one embodiment, transfer agent is an organosilicone surfactant composition or compound contained therein. In another embodiment, the composition comprises more than one double-stranded RNA comprising a polynucleotide that is complementary to all or a portion of an essential Tospovirus gene sequence, an RNA transcript of said essential Tospovirus gene sequence, or a fragment thereof. In another embodiment, the double-stranded RNA polynucleotide comprises a polynucleotide that is essentially identical or essentially complementary to a nucleotide sequence as set forth in SEQ NOs:47-103, 448-483, or a fragment thereof. In some embodiments, the antisense polynucleotide of the dsRNA comprises a two (2) nucleotide overhang on the 3′ end that is complementary to the target gene. In another embodiment, the Tospovirus is selected from the group consisting of bean necrotic mosaic virus, Capsicum chlorosis virus, groundnut bud necrosis virus, groundnut ringspot virus, groundnut yellow spot virus, impatiens necrotic spot virus, iris yellow spot viris, melon yellow spot virus, peanut bud necrosis virus, peanut yellow spot virus, soybean vein necrosis-associated virus, tomato chlorotic spot virus, tomato necrotic ringspot virus, tomato spotted wilt virus, tomato zonate spot virus, watermelon bud necrosis virus, watermelon silver mottle virus, and zucchini lethal chlorosis virus. In another embodiment, the essential Tospovirus gene is selected from the group consisting of nucleocapsid gene (N), coat protein gene (CP), virulence factors NSm and NSs, and RNA-dependent RNA polymerase L segment (RdRp/L segment). In another embodiment, the essential Tospovirus gene is selected from the group consisting of SEQ ID NOs:13-46. In another embodiment, the composition is topically applied by spraying, dusting, or is applied to the plant surface as matrix-encapsulated RNA.
In another aspect, the invention provides a composition comprising a double-stranded RNA polynucleotide and a transfer agent, wherein said double-stranded RNA polynucleotide is complementary to all or a portion of an essential Tospovirus gene sequence or an RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Tospovirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:13-46. In another embodiment, the transfer agent is an organosilicone composition. In another embodiment, the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to a nucleotide sequence selected from the group consisting of SEQ NOs:47-103 and 448-483. In some embodiments, the antisense polynucleotide of the dsRNA comprises a two (2) nucleotide overhang on the 3′ end that is complementary to the target gene.
In another aspect, the invention provides a method of reducing expression of an essential Tospovirus gene comprising contacting a Tospovirus particle with a composition comprising a double-stranded RNA polynucleotide and a transfer agent, wherein said double-stranded RNA comprises a polynucleotide that is complementary to all or a portion of an essential gene sequence in said Tospovirus or an RNA transcript thereof, wherein the symptoms of Tospovirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:13-46. In another embodiment, the transfer agent is an organosilicone compound. In another embodiment, the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOs:47-103, 448-483, or fragment thereof. In some embodiments, the antisense polynucleotide of the dsRNA comprises a two (2) nucleotide overhang on the 3′ end that is complementary to the target gene.
In another aspect, the invention provides a method of identifying a double-stranded RNA polynucleotide useful in modulating Tospovirus gene expression when topically treating a plant comprising: a) providing a plurality of double-stranded RNA polynucleotides that comprise a region complementary to all or a part of an essential Tospovirus gene or RNA transcript thereof; b) topically treating said plant with one or more of said double-stranded RNA polynucleotides and a transfer agent; c) analyzing said plant or extract for modulation of symptoms of Tospovirus infection; and d) selecting a double-stranded RNA polynucleotide capable of modulating the symptoms or occurrence of Tospovirus infection. In one embodiment, the transfer agent is an organosilicone compound. In some embodiments, the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:13-46.
In another aspect, the invention provides an agricultural chemical composition comprising an admixture of a double-stranded RNA polynucleotide and a pesticide, wherein said double-stranded RNA comprises a polynucleotide that is essentially complementary to all or a portion of an essential Tospovirus gene sequence or RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Tospovirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the pesticide is selected from the group consisting of anti-viral compounds, insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides.
In still another aspect, the invention provides a method of treatment or prevention of a Geminivirus infection in a plant comprising: topically applying to said plant a composition comprising a double-stranded RNA polynucleotide and a transfer agent, wherein said double-stranded RNA comprises a polynucleotide that is complementary to all or a portion of an essential Geminivirus gene sequence, or an RNA transcript thereof, wherein the symptoms of viral infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the transfer agent is an organosilicone surfactant composition or compound contained therein. In another embodiment, the composition comprises more than one double-stranded RNA comprising a polynucleotide that is essentially complementary to all or a portion of an essential Geminivirus gene sequence, an RNA transcript of said essential Geminivirus gene sequence, or a fragment thereof. In another embodiment, the double-stranded RNA comprises a polynucleotide that is essentially identical or esstentially complementary to at least 18 nucleotides of a sequence selected from the group consisting of SEQ NOs:104-268 or a fragment thereof. In another embodiment, the Geminivirus is selected from the group consisting of Barley yellow dwarf virus, Cucumber mosaic virus, Pepino mosaic virus, Cotton curl leaf virus, Tomato yellow leaf curl virus, Tomato golden mosaic virus, Potato yellow mosaic virus, Pepper leaf curl virus, Bean golden mosaic virus, Bean golden mosaic virus, Tomato mottle virus. In still another aspect, the essential Geminivirus gene is selected from the group consisting of nucleocapsid gene (N), a coat protein gene (CP), virulence factors NSm and NSs, and RNA-dependent RNA polymerase L segment (RdRp/L segment), a silencing suppressor gene, movement protein (MP), Nia, CP-N, a triple gene block, CP-P3, MP-P4, C2, and AC2. In another embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:269-447. In another embodiment, the composition is topically applied by spraying, dusting, or is applied to the plant surface as matrix-encapsulated RNA.
In another aspect, the invention provides a composition comprising a double-stranded RNA polynucleotide and a transfer agent, wherein said double-stranded RNA comprises a polynucleotide that is essentially complementary to all or a portion of an essential Geminivirus gene sequence, such as one set forth as SEQ ID NOs:104-268, 269-447, or an RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Geminivirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:269-447. In another embodiment, the transfer agent is an organosilicone composition. In another embodiment, the double-stranded RNA polynucleotide is selected from the group consisting of SEQ NOs:104-268.
In another aspect, a method of reducing expression of an essential Geminivirus gene comprising contacting a Geminivirus particle with a composition comprising a double-stranded RNA polynucleotide and a transfer agent, wherein said double-stranded RNA comprises a polynucleotide that is essentially complementary to all or a portion of an essential gene sequence in said Geminivirus or an RNA transcript thereof, wherein the symptoms of Geminivirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:269-447. In another embodiment, the transfer agent is an organosilicone compound. In another embodiment, the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 nucleotides of a sequence selected from the group consisting of SEQ NOs:104-268 or fragment thereof.
In still another aspect, the invention provides a method of identifying a double-stranded RNA polynucleotide useful in modulating Geminivirus gene expression when topically treating a plant comprising: a) providing a plurality of double-stranded RNA polynucleotides that comprise a region complementary to all or a part of an essential Geminivirus gene or RNA transcript thereof; b) topically treating said plant with one or more of said double-stranded RNA polynucleotides and a transfer agent; c) analyzing said plant or extract for modulation of symptoms of Geminivirus infection; and d) selecting a double-stranded RNA polynucleotide capable of modulating the symptoms or occurrence of Geminivirus infection. In one embodiment, the transfer agent is an organosilicone compound. In some embodiments, the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:269-447. In some embodiments, the Geminivirus is Cucumber Mosiac Virus and the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the to group consisting of SEQ ID NOs:269-316. In some embodiments, the Geminivirus is Pepino Mosaic Virus and the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:317-349. In some embodiments, the Geminivirus is Tomato Yellow Curl Leaf Virus and the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:386-421. In some embodiments, the Gemini virus is Cotton Leaf Curl Virus and the double-stranded RNA comprises a polynucleotide that is essentially identical or essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:422-441.
In another aspect, the invention provides an agricultural chemical composition comprising an admixture of a double-stranded RNA polynucleotide and a pesticide, wherein said double-stranded RNA polynucleotide is complementary to all or a portion of an essential Geminivirus gene sequence or RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Geminivirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the pesticide is selected from the group consisting of anti-viral compounds, insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides.
In one aspect, the invention provides a method of treatment or prevention of a Geminivirus infection in a plant comprising: topically applying to said plant a composition comprising an antisense single-stranded DNA polynucleotide and a transfer agent, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential Geminivirus gene sequence or an RNA transcript thereof, wherein the symptoms of viral infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In some embodiments, the antisense single-stranded DNA polynucleotide comprises at least 18 contiguous nucleotides that are essentially complementary a sequence selected from the group consisting of SEQ ID NOs:104-268. In some embodiments, the antisense single-stranded DNA polynucleotide comprises at least 18 contiguous nucleotides that are essentially complementary a sequence selected from the group consisting of SEQ ID NOs:269-447. In one embodiment, the transfer agent is an organosilicone surfactant composition or compound contained therein. In another embodiment, the composition comprises more than one antisense single-stranded DNA polynucleotide complementary to all or a portion of an essential Geminivirus gene sequence, an RNA transcript of said essential Geminivirus gene sequence, or a fragment thereof. In another embodiment, the Geminivirus is selected from the group consisting of Barley yellow dwarf virus, Cucumber mosaic virus, Pepino mosaic virus, Cotton curl leaf virus, Tomato yellow leaf curl virus, Tomato golden mosaic virus, Potato yellow mosaic virus, Pepper leaf curl virus, Bean golden mosaic virus, Bean golden mosaic virus, and Tomato mottle virus. In still another aspect, the essential Geminivirus gene is selected from the group consisting of nucleocapsid gene (N), a coat protein gene (CP), virulence factors NSm and NSs, and RNA-dependent RNA polymerase L segment (RdRp/L segment), a silencing suppressor gene, movement protein (MP), Nia, CP-N, a triple gene block, CP-P3, MP-P4, C2, and AC2. In another embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:269-447. In another embodiment, the composition is topically applied by spraying, dusting, or is applied to the plant surface as matrix-encapsulated RNA.
In another aspect, the invention provides a composition comprising an antisense single-stranded DNA polynucleotide and a transfer agent, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential Geminivirus gene sequence or an RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Geminivirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In some embodiments, the essential gene sequence is selected from the group consisting of SEQ ID NOs:104-447, or the transfer agent is an organosilicone composition.
In another aspect, the invention provides a method of reducing expression of an essential Geminivirus gene comprising contacting a Geminivirus particle with a composition comprising an antisense single-stranded DNA polynucleotide and a transfer agent, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential gene sequence in said Geminivirus or an RNA transcript thereof, wherein the symptoms of Geminivirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In one embodiment, the essential gene sequence is selected from the group consisting of SEQ ID NOs:104-447. In another embodiment, the transfer agent is an organosilicone compound.
In another aspect, the invention provides a method of identifying antisense single-stranded DNA polynucleotides useful in modulating Geminivirus gene expression when topically treating a plant comprising: a) providing a plurality of antisense single-stranded DNA polynucleotides that comprise a region complementary to all or a part of an essential Geminivirus gene or RNA transcript thereof; b) topically treating said plant with one or more of said antisense single-stranded DNA polynucleotides and a transfer agent; c) analyzing said plant or extract for modulation of symptoms of Geminivirus infection; and d) selecting an antisense single-stranded DNA polynucleotide capable of modulating the symptoms or occurrence of Geminivirus infection. In an embodiment, the transfer agent is an organosilicone compound. In some embodiments, the antisense single-stranded DNA is essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:269-447. In some embodiments, the Geminivirus is Cucumber mosaic virus and the antisense single-stranded DNA is essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:269-316. In some embodiments, the Geminivirus is Pepino mosaic virus and the antisense single-stranded DNA is essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:317-349. In some embodiments, the Geminivirus is Tomato yellow leaf curl virus and the antisense single-stranded DNA is essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:386-421. In some embodiments, the Geminivirus is Cotton leaf curl virus and the antisense single-stranded DNA is essentially complementary to at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:422-441.
In another aspect, the invention provides an agricultural chemical composition comprising an admixture of an antisense single-stranded DNA polynucleotide and a pesticide, wherein said antisense single-stranded DNA polynucleotide is complementary to all or a portion of an essential Geminivirus gene sequence or RNA transcript thereof, wherein said composition is topically applied to a plant and wherein the symptoms of Geminivirus infection or development of symptoms are reduced or eliminated in said plant relative to a plant not treated with said composition when grown under the same conditions. In an embodiment, the pesticide is selected from the group consisting of anti-viral compounds, insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the function of the compositions and methods. The function may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The function can be more fully understood from the following description of the figures:
Provided are compositions and methods useful for treating or preventing viral infection in plants. Aspects of the methods and compositions disclosed herein can be applied to treat or prevent viral infection in plants in agronomic and other cultivated environments.
Several embodiments relate to methods and compositions for the prevention or treatment of Tospovirus infection in a plant comprising the topical administration of a polynucleotide comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Tospoviral gene. In some embodiments, the Tospoviral gene is selected from the group consisting of a nucleocapsid (N) gene, a suppressor (NSs) gene, a movement (NSm) gene, and a RNA dependent RNA polymerase (RdRp) gene. In some embodiments, methods and compositions for the prevention or treatment of Tospovirus infection in a plant comprising the topical administration of single-stranded (ss) DNA in antisense (as) orientation as set forth in SEQ ID NOs:1-12 (Tables 1-3) are provided. Also provided are methods and compositions for the prevention or treatment of Tospovirus infection in a plant comprising the topical administration of double-stranded (ds) RNA comprising a polynucleotide that is essentially identical or essentially complementary to a nucleotide sequence as set forth in SEQ ID NOs:47-103 (Table 5) or SEQ ID NOs:448-483 (Table 12). In some embodiments, the antisense polynucleotide of the dsRNA comprises a two (2) nucleotide overhang on the 3′ end that is complementary to the target gene. In certain embodiments, the methods and compositions of the invention provide regulation, repression, or delay and/or modulation of symptoms or disease caused by Tospovirus.
Several embodiments relate to methods and compositions for the prevention or treatment of Geminivirus infection in a plant comprising the topical administration of a polynucleotide comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a Geminiviral gene. In some embodiments, the Geminiviral gene is selected from the group consisting of a coat protien (CP) gene, a silencing suppressor gene, and a movement gene. Also provided are methods and compositions for the prevention or treatment of Geminivirus infection in a plant comprising the topical administration of dsRNA comprising a polynucleotide that is essentially identical or essentially complementary to a nucleotide sequence as set forth in SEQ ID NOs:104-268 (Table 6). Aspects of the methods and compositions can be applied to manage plant viral diseases in agronomic and other cultivated environments.
Compositions of the present invention may include ssDNA, dsDNA, ssRNA, or dsRNA polynucleotides and/or ssDNA, dsDNA, ssRNA, or dsRNA oligonucleotides designed to target single or multiple viral genes, or multiple segments of one or more viral genes, such as genes from a Tospovirus or other plant disease, including, but not limited to the viral gene sequences set forth in SEQ ID NOs:1-46 (Tables 1-4). In another embodiment, such polynucleotides and oligonucleotides may be designed to target single or multiple viral genes, or multiple segments of one or more viral genes, such as genes from a Geminivirus, including, but not limited to the viral gene sequences set forth in SEQ ID NOs:269-447 (Tables 7-11). In an embodiment, any viral gene from any plant virus may be targeted by compositions of the present invention. The target gene may include multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more Tospovirus species. In some embodiments, the polynucleotides or oligonucleotides are essentially identical or essentially complementary to a consensus nucleotide sequence.
Polynucleotides of the invention may be complementary to all or a portion of a viral gene sequence, including a promoter, intron, coding sequence, exon, 5′ untranslated region, and 3′ untranslated region. Compositions of the present invention further comprise a transfer agent that facilitates delivery of the polynucleotide of the invention to a plant, and may include solvents, diluents, a pesticide that complements the action of the polynucleotide, a herbicide or additional pesticides or that provides an additional mode of action different from the polynucleotide, various salts or stabilizing agents that enhance the utility of the composition as an admixture of the components of the composition.
In certain aspects, methods of the invention may include one or more applications of a polynucleotide composition and one or more applications of a transfer agent for conditioning of a plant or plant virus to permeation by polynucleotides or activity or stability of the polynucleotides. When the agent for conditioning to permeation is an organosilicone composition or compound contained therein, the polynucleotide molecules may be ssDNA, dsDNA, ssRNA, or dsRNA oligonucleotides; or ssDNA, dsDNA, ssRNA, or dsRNA polynucleotides, chemically modified DNA oligonucleotides or polynucleotides, or mixtures thereof.
In one embodiment, the present invention provides a method for controlling Tospovirus or Geminivirus infection of a plant including treatment of the plant with at least a first antisense ssDNA complementary to all or a portion of a target viral gene, wherein the polynucleotide molecules are capable of modulation of the target gene and controlling Tospovirus or Geminivirus infection. In another embodiment, the present invention provides a method for controlling Tospovirus or Geminivirus infection of a plant including treatment of the plant with at least a first antisense dsDNA complementary to all or a portion of a target viral gene, wherein the polynucleotide molecules are capable of modulation of the target gene and controlling Tospovirus or Geminivirus infection. In another embodiment, the invention provides a method for controlling Tospovirus or Geminivirus infection of a plant including treatment of the plant with at least a first dsRNA complementary to all or a portion of a target viral gene, wherein the polynucleotide molecules are capable of modulation of the target gene and controlling Tospovirus or Geminivirus infection.
In certain embodiments, a conditioning step to increase permeability of a plant to the polynucleotide may be included. The conditioning and polynucleotide application can be performed separately or in a single step. When the conditioning and polynucleotide application are performed in separate steps, the conditioning can precede or can follow the polynucleotide application within minutes, hours, or days. In some embodiments, more than one conditioning step or more than one polynucleotide molecule application can be performed on the same plant.
In specific embodiments of the method, a polynucleotide of the invention can be cloned or identified from (a) coding (protein-encoding), (b) non-coding (promoter and other gene related molecules), or (c) both coding and non-coding parts of the target viral gene. Non-coding parts may include DNA, such as promoter regions or an RNA transcribed by the DNA that provides RNA regulatory molecules, including but not limited to: introns, cis-acting regulatory RNA elements, 5′ or 3′ untranslated regions, and microRNAs (miRNA), trans-acting siRNAs, natural antisense siRNAs, and other small RNAs with regulatory function or RNAs having structural or enzymatic function including but not limited to: ribozymes, ribosomal RNAs, t-RNAs, aptamers, and riboswitches.
As used herein, “Tospovirus” refers to a virus from the genus Tospovirus, which may include bean necrotic mosaic virus, Capsicum chlorosis virus, groundnut bud necrosis virus, groundnut ringspot virus, groundnut yellow spot virus, impatiens necrotic spot virus, iris yellow spot viris, melon yellow spot virus, peanut bud necrosis virus, peanut yellow spot virus, soybean vein necrosis-associated virus, tomato chlorotic spot virus, tomato necrotic ringspot virus, tomato spotted wilt virus, tomato zonate spot virus, watermelon bud necrosis virus, watermelon silver mottle virus, or zucchini lethal chlorosis virus.
As used herein, a “Geminivirus” refers to a virus from the Geminiviridae Family of plant viruses. A Geminivirus may include, but is not limited to, Barley yellow dwarf virus (BYDW), Cucumber mosaic virus (CMV), Pepino mosaic virus (PepMV), Cotton curl leaf virus (CuCLV), Tomato yellow leaf curl virus (TYLCV), Tomato golden mosaic virus, Potato yellow mosaic virus, Pepper leaf curl virus (PepLCV), Bean golden mosaic virus (BGMV-PR), Bean golden mosaic virus (BGMV-DR), Tomato mottle virus (TMV), and the like.
The DNA or RNA polynucleotide compositions of the present invention are useful in compositions, such as liquids that comprise DNA or RNA polynucleotide molecules, alone or in combination with other components either in the same liquid or in separately applied liquids that provide a transfer agent. As used herein, a transfer agent is an agent that, when combined with a polynucleotide in a composition that is topically applied to a target plant surface facilitates the use of the polynucleotide in controlling a Tospovirus or Geminivirus. In one embodiment, the transfer agent enhances the ability of the polynucleotide to enter a plant cell. In certain embodiments, a transfer agent is therefore an agent that conditions the surface of plant tissue, e. g., leaves, stems, roots, flowers, or fruits, to permeation by the polynucleotide molecules into plant cells. The transfer of polynucleotides into plant cells can be facilitated by the prior or contemporaneous application of a polynucleotide-transferring agent to the plant tissue. In some embodiments the transferring agent is applied subsequent to the application of the polynucleotide composition. The polynucleotide transfer agent enables a pathway for polynucleotides through cuticle wax barriers, stomata and/or cell wall or membrane barriers into plant cells. Suitable transfer agents to facilitate transfer of the to polynucleotide into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents to facilitate transfer of the composition into a plant cell 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. Embodiments of the method can optionally include 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.
Embodiments of agents or treatments for conditioning of a plant to permeation by polynucleotides include emulsions, reverse emulsions, liposomes, and other micellar-like compositions. Embodiments of agents or treatments for conditioning of a plant to permeation by polynucleotides 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. Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, e.g., plant-sourced oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available on the worldwide web (internet) at herbicide.adjuvants.com can be used, e.g., paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine. Transfer agents include, but are not limited to, organosilicone preparations.
In certain embodiments, an organosilicone preparation that comprises an organosilicone compound comprising a trisiloxane head group is used in the methods and compositions provided herein. In certain embodiments, an organosilicone preparation that comprises an organosilicone compound comprising a heptamethyltrisiloxane head group is used in the methods and compositions provided herein. In certain embodiments of the methods and compositions provided herein, a to composition that comprises a polynucleotide molecule and one or more effective organosilicone compound 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) is used or provided.
Organosilicone preparations used in the methods and compositions provided herein can comprise one or more effective organosilicone compounds. As used herein, the phrase “effective organosilicone compound” is used to describe any organosilicone compound that is found in an organosilicone preparation that enables a polynucleotide to enter a plant cell. In certain embodiments, an effective organosilicone compound can enable a polynucleotide to enter a plant cell in a manner permitting a polynucleotide mediated suppression of a target gene expression in the plant cell. In general, effective organosilicone compounds include, but are not limited to, compounds that can comprise: i) a trisiloxane head group that is covalently linked to, ii) an alkyl linker including, but not limited to, an n-propyl linker, that is covalently linked to, iii) a poly glycol chain, that is covalently linked to, iv) a terminal group. Trisiloxane head groups of such effective organosilicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an n-propyl linker. Poly glycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Poly glycol 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. Effective organosilicone compounds are believed to include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane.
In certain embodiments, an organosilicone preparation that is commercially available as Silwet® L-77 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. can be used to prepare a polynucleotide composition. In certain embodiments where a Silwet L-77 organosilicone preparation is used as a pre-spray treatment 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 polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation comprising 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) is used or provided.
In certain embodiments, any of the commercially available organosilicone preparations provided such as the following Breakthru S 321, Breakthru S 200 Cat#67674-67-3, Breakthru OE 441 Cat#68937-55-3, Breakthru S 278 Cat #27306-78-1, Breakthru S 243, Breakthru S 233 Cat#134180-76-0, available from manufacturer Evonik Goldschmidt (Germany), Silwet® HS 429, Silwet® HS 312, Silwet® HS 508, Silwet® HS 604 (Momentive Performance Materials, Albany, N.Y.) can be used as transfer agents in a polynucleotide composition. In certain embodiments where an organosilicone preparation is used as a pre-spray treatment of plant leaves or other 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 polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation 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) is used or provided.
Delivery of a polynucleotide according to the invention can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a ssDNA, dsDNA, ssRNA, or dsRNA molecule provided herein and (2) complexing a ssDNA, dsDNA, ssRNA, or dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged, nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidyl glycerol, dioleoyl phosphatidylethanolamine or liposomes comprising dihydrosphingomyelin (DHSM). Numerous lipophilic agents are commercially available, including Lipofectin® (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene™ (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some eases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15:647-652, 1997) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am Soc. Nephrol. 7:1728, 1996). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey et al. (Nat Biotechnol. 23(8):1002-7, 2005).
The following definitions and methods are provided to guide those of ordinary skill in the art. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Where a term is provided in the singular, the inventors also contemplate aspects described by the plural of that term.
By “non-transcribable” polynucleotides is meant that the polynucleotides do not comprise a complete polymerase II transcription unit.
As used herein “solution” refers to homogeneous mixtures and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions.
A “trigger” or “trigger polynucleotide” is a DNA polynucleotide molecule that is homologous or complementary to a target gene polynucleotide. The trigger polynucleotide molecules modulate expression of the target gene when topically applied to a plant surface with a transfer agent, whereby a virus-infected plant that is treated with said composition is able to sustain its growth or development or reproductive ability, or said plant is less sensitive to a virus as a result of said polynucleotide-containing composition relative to a plant not treated with a composition containing the trigger molecule. A plant treated with such a composition may be resistant to viral expression as a result of said polynucleotide-containing composition relative to a plant not treated with a composition containing the trigger molecule. Trigger polynucleotides disclosed herein may be generally described in relation to the target gene sequence in an antisense (complementary) or sense orientation as ssDNA, dsDNA, ssRNA, or dsRNA molecules or nucleotide variants and modified nucleotides thereof depending on the various regions of a gene being targeted.
It is contemplated that the composition may contain multiple DNA or RNA polynucleotides and/or pesticides that include, but are not limited to, anti-viral compounds, insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides. Essential genes are genes in a plant that provide key enzymes or other proteins, for example, a biosynthetic enzyme, metabolizing enzyme, receptor, signal transduction protein, structural gene product, transcription factor, or transport protein; or regulating RNAs, such as, microRNAs, that are essential to the growth or survival of the organism or cell or involved in the normal growth and development of the plant (Meinke et al., Trends Plant Sci. 2008:13(9):483-91). Essential genes in a virus may include genes responsible for capsid production, virus assembly, infectivity, budding, and the like. The suppression of an essential gene in a virus affects the function of a gene product that enables viral infection in a plant. The compositions may include various trigger DNA or RNA polynucleotides that modulate the expression of an essential gene in a Tospovirus.
As used herein, the term “DNA,” “DNA molecule,” or “DNA polynucleotide molecule” refers to a ssDNA or dsDNA molecule of genomic or synthetic origin, such as a polymer of deoxyribonucleotide bases or a DNA polynucleotide molecule. As used herein, the term “DNA sequence,” “DNA nucleotide sequence,” or “DNA polynucleotide sequence” refers to the nucleotide sequence of a DNA molecule. Unless otherwise stated, nucleotide sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations §1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
As used herein, the term “RNA,” “RNA molecule,” or “RNA polynucleotide molecule” refers to a ssRNA or dsRNA molecule of genomic or synthetic origin, such as a polymer of ribonucleotide bases or an RNA polynucleotide molecule. As used herein, the term “RNA sequence,” “RNA nucleotide sequence,” or “RNA polynucleotide sequence” refers to the nucleotide sequence of an RNA molecule. Unless otherwise stated, nucleotide sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations §1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
As used herein, “polynucleotide” refers to a DNA or RNA molecule containing multiple nucleotides and generally also refers to “oligonucleotides” (a polynucleotide molecule of typically 50 or fewer nucleotides in length). Embodiments 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), for example, oligonucleotides as set forth by SEQ ID NOs:1-12, 47-268, and 448-483 or fragments thereof. A target gene comprises any polynucleotide molecule in a plant cell or fragment thereof for which the modulation of the expression of the target gene is provided by the methods and compositions. A gene has noncoding genetic elements (components) that provide for the function of the gene, these elements are polynucleotides that provide gene expression regulation, such as, a promoter, an enhancer, a 5′ untranslated region, intron regions, and a 3′ untranslated region. Oligonucleotides and polynucleotides can be made to any of the genetic elements of a gene and to polynucleotides spanning the junction region of a genetic element, such as, an intron and exon, the junction region of a promoter and a to transcribed region, the junction region of a 5′ leader and a coding sequence, the junction of a 3′ untranslated region and a coding sequence.
Polynucleotide compositions used in the various embodiments include compositions including oligonucleotides or polynucleotides, or a mixture of both, of DNA or RNA, or chemically modified oligonucleotides or polynucleotides or a mixture thereof. In some embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, US Patent Publication 20110171287, US Patent Publication 20110171176, and US Patent Publication 20110152353, US Patent Publication, 20110152346, US Patent Publication 20110160082, herein incorporated in its entirety by reference hereto. For example, including, but not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide 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 (for example, fluorescein or rhodamine) or other label (for example, biotin).
The term “gene” refers to components that comprise chromosomal DNA, RNA, plasmid DNA, cDNA, intron and exon DNA, artificial DNA polynucleotide, or other DNA that encodes a peptide, polypeptide, protein, or RNA transcript molecule, and the genetic elements flanking the coding sequence that are involved in the regulation of expression, such as, promoter regions, 5′ leader regions, 3′ untranslated region that may exist as native genes or transgenes in a plant genome. The gene or a fragment thereof is isolated and subjected to polynucleotide sequencing methods that determines the order of the nucleotides that comprise the gene. Any of the components of the gene are potential targets for a trigger oligonucleotide and polynucleotides.
The trigger polynucleotide molecules are designed to modulate expression by inducing regulation or suppression of a viral gene and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence of a viral gene or to the sequence of RNA transcribed from a viral gene of a plant, the sequence thereof determined by isolating the gene or a to fragment of the gene from the plant, which can be coding sequence or non-coding sequence. Effective molecules that modulate expression are referred to as “a trigger molecule, or trigger polynucleotide”. By “essentially identical” or “essentially complementary” is meant that the trigger polynucleotides (or at least a portion of a polynucleotide) are designed to hybridize to the endogenous gene noncoding sequence or to RNA transcribed (known as messenger RNA or an RNA transcript) from the endogenous gene to effect regulation or suppression of expression of the endogenous gene. Trigger molecules are identified by “tiling” the gene targets with partially overlapping probes or non-overlapping probes of antisense polynucleotides that are essentially identical or essentially complementary to the nucleotide sequence of an endogenous gene. Multiple target sequences can be aligned and sequence regions with homology in common, according to the methods, are identified as potential trigger molecules for the multiple targets. Multiple trigger molecules of various lengths, for example 18-25 nucleotides, 26-50 nucleotides, 51-100 nucleotides, 101-200 nucleotides, 201-300 nucleotides or more can be pooled into a few treatments in order to investigate polynucleotide molecules that cover a portion of a gene sequence (for example, a portion of a coding versus a portion of a noncoding region, or a 5′ versus a 3′ portion of a gene) or an entire gene sequence including coding and noncoding regions of a target gene. Polynucleotide molecules of the pooled trigger molecules can be divided into smaller pools or single molecules in order to identify trigger molecules that provide the desired effect.
The target gene ssDNA polynucleotide molecules, including SEQ ID NOs:1-12, or dsRNA molecules, including SEQ ID NOs:47-268 and 448-483 may be sequenced by any number of available methods and equipment known in the art. Some of the sequencing technologies are available commercially, such as the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.) and the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) and Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.). In addition to the single molecule sequencing performed using sequencing-by-synthesis of Helicos Biosciences, other single molecule sequencing technologies are encompassed and include the SMRT™ technology of Pacific Biosciences, the Ion Torrent™ technology, and nanopore sequencing being developed for example, by to Oxford Nanopore Technologies. A viral target gene comprising DNA or RNA can be isolated using primers or probes essentially complementary or essentially homologous to the target gene or a fragment thereof. A polymerase chain reaction (PCR) gene fragment can be produced using primers essentially complementary or essentially homologous to a viral gene or a fragment thereof that is useful to isolate a viral gene from a plant genome. Various sequence capture technologies can be used to isolate additional target gene sequences, for example, including but not limited to Roche NimbleGen® (Madison, Wis.) and Streptavdin-coupled Dynabeads® (Life Technologies, Grand Island, N.Y.) and US20110015084, herein incorporated by reference in its entirety.
Embodiments of functional single-stranded or double-stranded polynucleotides have sequence complementarity that need not be 100 percent, but is at least sufficient to permit hybridization to RNA transcribed from the target gene or DNA of the target gene to form a duplex to permit a gene silencing mechanism. Thus, in embodiments, a polynucleotide fragment is designed to be complementary to all or a portion of an essential target Tospovirus or Geminivirus gene sequence. For instance, the fragment may be essentially identical or essentially complementary to a sequence of 18 or more contiguous nucleotides in either the target viral gene sequence or messenger RNA transcribed from the target gene. By “essentially identical” is meant having 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 target gene or RNA transcribed from the target gene; by “essentially complementary” is meant having 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. In some embodiments, polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to one allele or one family member of a given target gene (coding or non-coding sequence of a gene); in other embodiments the polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to multiple alleles or family members of a given target gene.
“Identity” refers to the degree of similarity between two polynucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there are 100 matched amino acids between a 200 and a 400 amino acid protein, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
Trigger molecules for specific viral gene family members can be identified from coding and/or non-coding sequences of gene families of a plant virus or multiple plant viruses, by aligning and selecting 200-300 polynucleotide fragments from the least homologous regions among the aligned sequences and evaluated using topically applied polynucleotides (antisense ssDNA or dsRNA) to determine their relative effectiveness in providing the anti-viral phenotype. In some embodiments, the viral gene family is Tospovirus and the sequences are selected from SEQ ID NOs:13-46. In some embodiments, the viral gene family is Cucumber mosaic virus and the sequences are selected from SEQ ID NOs:269-316. In some embodiments, the viral gene family is Pepino mosaic virus and the sequences are selected from SEQ ID NOs:317-349. In some embodiments, the viral gene family is Barley yellow dwarf virus and the sequences are selected from SEQ ID NOs:350-385. In some embodiments, the viral gene family is Tomato yellow leaf curl virus and the sequences are selected from SEQ ID NOs:386-421. In some embodiments, the viral gene family is Cotton leaf curl virus and the sequences are selected from SEQ ID NOs:422-441. The effective segments are further subdivided into 50-60 polynucleotide fragments, prioritized by least homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by least homology, and again evaluated for induction of the anti-viral phenotype. Once relative effectiveness is determined, the fragments are utilized singly, or again evaluated in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the anti-viral phenotype.
Trigger molecules for broad anti-viral activity can be identified from coding and/or non-coding sequences of gene families of a plant virus or multiple plants viruses, by aligning and selecting 200-300 polynucleotide fragments from the most homologous regions amongst the aligned sequences and evaluated using topically applied polynucleotides (antisense ssDNA or dsRNA) to determine their relative effectiveness in inducing the anti-viral phenotype. In some embodiments, the viral gene family is Tospovirus and the sequences are selected from SEQ ID NOs:13-46. In some embodiments, the viral gene family is Cucumber mosaic virus and the sequences are selected from SEQ ID NOs:269-316. In some embodiments, the viral gene family is Pepino mosaic virus and the sequences are selected from SEQ ID NOs:317-349. In some embodiments, the viral gene family is Barley yellow dwarf virus and the sequences are selected from SEQ ID NOs:350-385. In some embodiments, the viral gene family is Tomato yellow leaf curl virus and the sequences are selected from SEQ ID NOs:386-421. In some embodiments, the viral gene family is Cotton leaf curl virus and the sequences are selected from SEQ ID NOs:422-441. The effective segments are subdivided into 50-60 polynucleotide fragments, prioritized by most homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by most homology, and again evaluated for induction of the anti-viral phenotype. Once relative effectiveness is determined, the fragments may be utilized singly, or in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the anti-viral phenotype.
Methods of making polynucleotides are well known in the art. Chemical synthesis, in vivo synthesis and in vitro synthesis methods and compositions are known in the art and include various viral elements, microbial cells, modified polymerases, and modified nucleotides. Commercial preparation of oligonucleotides often provides two deoxyribonucleotides on the 3′ end of the sense strand. Long polynucleotide molecules can be synthesized from commercially available kits. Long polynucleotide molecules can also be assembled from multiple DNA fragments. In to some embodiments design parameters such as Reynolds score (Reynolds et al. Nature Biotechnology 22, 326-330 (2004), Tuschl rules (Pei and Tuschl, Nature Methods 3(9):670-676, 2006), i-score (Nucleic Acids Res 35:e123, 2007), i-Score Designer tool and associated algorithms (Nucleic Acids Res 32:936-948, 2004. Biochem Biophys Res Commun 316:1050-1058, 2004, Nucleic Acids Res 32:893-901, 2004, Cell Cycle 3:790-5, 2004, Nat Biotechnol 23:995-1001, 2005, Nucleic Acids Res 35:e27, 2007, BMC Bioinformatics 7:520, 2006, Nucleic Acids Res 35:e123, 2007, Nat Biotechnol 22:326-330, 2004) are known in the art and may be used in selecting polynucleotide sequences effective in gene silencing. In some embodiments the sequence of a polynucleotide is screened against the genomic DNA of the intended plant to minimize unintentional silencing of other genes.
Ligands can be tethered to a ssDNA or dsRNA polynucleotide. Ligands in general can include modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids (e.g., cholesterol, a bile acid, or a fatty acid (e.g., lithocholic-oleyl, lauroyl, docosnyl, stearoyl, palmitoyl, myristoyl oleoyl, linoleoyl), steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., polyethylene glycol (PEG), PEG-40K, PEG-20K and PEG-5K. Other examples of ligands include lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters and ethers thereof, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl; e.g., lauroyl, docosnyl, stearoyl, oleoyl, linoleoyl 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dodecanoyl, lithocholyl, 5β-cholanyl, N,N-distearyl-lithocholamide, 1,2-di-O-stearoylglyceride, dimethoxytrityl, or phenoxazine) and PEG (e.g., PEG-5K, PEG-20K, PEG-40K). Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues.
The method of the invention may be applied to plants that are or are not transgenic. Non-limiting examples of transgenic plants include those that comprise one or more transgene conferring a trait selected from the group consisting of insect resistance, pesticide resistance, enhanced shelf life, fruit coloring, fruit ripening, fruit sweetness, nutritional value, and the like.
In specific embodiments of the invention, a plant disease control composition as provided herein may further be provided in a composition formulated for application to a plant that comprises at least one other active ingredient. Examples of such active ingredients may include, but are not limited to, an insecticidal protein such as a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphearicus insecticidal protein. In another non-limiting example, such an active ingredient is a herbicide, such as one or more of acetochlor, acifluorfen, acifluorfen-sodium, aclonifen, acrolein, alachlor, alloxydim, allyl alcohol, ametryn, amicarbazone, amidosulfuron, aminopyralid, amitrole, ammonium sulfamate, anilofos, asulam, atraton, atrazine, azimsulfuron, BCPC, beflubutamid, benazolin, benfluralin, benfuresate, bensulfuron, bensulfuron-methyl, bensulide, bentazone, benzfendizone, benzobicyclon, benzofenap, bifenox, bilanafos, bispyribac, bispyribac-sodium, borax, bromacil, bromobutide, bromoxynil, butachlor, butafenacil, butamifos, butralin, butroxydim, butylate, cacodylic acid, calcium chlorate, cafenstrole, carbetamide, carfentrazone, carfentrazone-ethyl, CDEA, CEPC, chlorflurenol, chlorflurenol-methyl, chloridazon, chlorimuron, chlorimuron-ethyl, chloroacetic acid, chlorotoluron, chlorpropham, chlorsulfuron, chlorthal, chlorthal-dimethyl, cinidon-ethyl, cinmethylin, cinosulfuron, cisanilide, clethodim, clodinafop, clodinafop-propargyl, clomazone, clomeprop, clopyralid, cloransulam, cloransulam-methyl, CMA, 4-CPB, CPMF, 4-CPP, CPPC, cresol, cumyluron, cyanamide, cyanazine, cycloate, cyclosulfamuron, cycloxydim, cyhalofop, cyhalofop-butyl, 2,4-D, 3,4-DA, daimuron, dalapon, dazomet, 2,4-DB, 3,4-DB, 2,4-DEB, desmedipham, dicamba, dichlobenil, ortho-dichlorobenzene, para-dichlorobenzene, dichlorprop, dichlorprop-P, diclofop, diclofop-methyl, diclosulam, difenzoquat, difenzoquat metilsulfate, diflufenican, diflufenzopyr, dimefuron, dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P, dimethipin, dimethylarsinic acid, to dinitramine, dinoterb, diphenamid, diquat, diquat dibromide, dithiopyr, diuron, DNOC, 3,4-DP, DSMA, EBEP, endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron, ethametsulfuron-methyl, ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-P, fenoxaprop-P-ethyl, fentrazamide, ferrous sulfate, flamprop-M, flazasulfuron, florasulam, fluazifop, fluazifop-butyl, fluazifop-P, fluazifop-P-butyl, flucarbazone, flucarbazone-sodium, flucetosulfuron, fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam, flumiclorac, flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen, fluoroglycofen-ethyl, flupropanate, flupyrsulfuron, flupyrsulfuron-methyl-sodium, flurenol, fluridone, fluorochloridone, fluoroxypyr, flurtamone, fluthiacet, fluthiacet-methyl, fomesafen, foramsulfuron, fosamine, glufosinate, glufosinate-ammonium, glyphosate, halosulfuron, halosulfuron-methyl, haloxyfop, haloxyfop-P, HC-252, hexazinone, imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, imazosulfuron, indanofan, iodomethane, iodosulfuron, iodosulfuron-methyl-sodium, ioxynil, isoproturon, isouron, isoxaben, isoxachlortole, isoxaflutole, karbutilate, lactofen, lenacil, linuron, MAA, MAMA, MCPA, MCPA-thioethyl, MCPB, mecoprop, mecoprop-P, mefenacet, mefluidide, mesosulfuron, mesosulfuron-methyl, mesotrione, metam, metamifop, metamitron, metazachlor, methabenzthiazuron, methylarsonic acid, methyldymron, methyl isothiocyanate, metobenzuron, metolachlor, S-metolachlor, metosulam, metoxuron, metribuzin, metsulfuron, metsulfuron-methyl, MK-66, molinate, monolinuron, MSMA, naproanilide, napropamide, naptalam, neburon, nicosulfuron, nonanoic acid, norflurazon, oleic acid (fatty acids), orbencarb, orthosulfamuron, oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone, oxyfluorfen, paraquat, paraquat dichloride, pebulate, pendimethalin, penoxsulam, pentachlorophenol, pentanochlor, pentoxazone, pethoxamid, petrolium oils, phenmedipham, phenmedipham-ethyl, picloram, picolinafen, pinoxaden, piperophos, potassium arsenite, potassium azide, pretilachlor, primisulfuron, primisulfuron-methyl, prodiamine, profluazol, profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop, propazine, propham, propisochlor, propoxycarbazone, propoxycarbazone-sodium, propyzamide, prosulfocarb, prosulfuron, pyraclonil, pyraflufen, pyraflufen-ethyl, pyrazolynate, pyrazosulfuron, pyrazosulfuron-ethyl, pyrazoxyfen, pyribenzoxim, pyributicarb, pyridafol, pyridate, pyriftalid, pyriminobac, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyrithiobac-sodium, quinclorac, quinmerac, quinoclamine, quizalofop, quizalofop-P, rimsulfuron, sethoxydim, siduron, simazine, simetryn, SMA, sodium arsenite, sodium azide, sodium chlorate, sulcotrione, sulfentrazone, sulfometuron, sulfometuron-methyl, sulfosate, sulfosulfuron, sulfuric acid, tar oils, 2,3,6-TBA, TCA, TCA-sodium, tebuthiuron, tepraloxydim, terbacil, terbumeton, terbuthylazine, terbutryn, thenylchlor, thiazopyr, thifensulfuron, thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone, tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron, tribenuron-methyl, tricamba, triclopyr, trietazine, trifloxysulfuron, trifloxysulfuron-sodium, trifluralin, triflusulfuron, triflusulfuron-methyl, trihydroxytriazine, tritosulfuron, [3-[2-chloro-4-fluoro-5-(-methyl-6-trifluoromethyl-2,4-dioxo-,2,3,4-t-etrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetic acid ethyl ester (CAS RN 353292-3-6), 4-[(4,5-dihydro-3-methoxy-4-methyl-5-oxo)-H-,2,4-triazolylcarbonyl-sulfamoyl]-5-methyl-thiophene-3-carboxylic acid (BAY636), BAY747 (CAS RN 33504-84-2), topramezone (CAS RN 2063-68-8), 4-hydroxy-3-[[2-[(2-methoxyethoxy)methyl]-6-(trifluoro-methyl)-3-pyridi-nyl]carbonyl]-bicyclo[3.2.]oct-3-en-2-one (CAS RN 35200-68-5), and 4-hydroxy-3-[[2-(3-methoxypropyl)-6-(difluoromethyl)-3-pyridinyl]carbon-yl]-bicyclo[3.2]oct-3-en-2-one.
The trigger DNA or RNA polynucleotide and/or oligonucleotide molecule compositions are useful in compositions, such as liquids that comprise the polynucleotide molecules at low concentrations, alone or in combination with other components, for example one or more herbicide molecules, either in the same solution or in separately applied liquids that also provide a transfer agent. While there is no upper limit on the concentrations and dosages of polynucleotide molecules that can useful in the methods, lower effective concentrations and dosages will generally be sought for efficiency. 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, or seed. In one embodiment, a useful treatment for herbaceous plants using 25-mer oligonucleotide molecules is about 1 nanomole (nmol) of oligonucleotide molecules per plant, for example, from about 0.05 to 1 nmol 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. Very large plants, trees, or to vines may require correspondingly larger amounts of polynucleotides. To illustrate embodiments, the factor 1×, when applied to oligonucleotide molecules is arbitrarily used to denote a treatment of 0.8 nmol of polynucleotide molecule per plant; 10×, 8 nmol of polynucleotide molecule per plant; and 100×, 80 nmol of polynucleotide molecule per plant.
An agronomic field in need of virus control may be treated by application of an agricultural chemical composition directly to the surface of the growing plants, such as by a spray. For example, the method is applied to control virus infection in a field of crop plants by spraying the field with the composition. The composition can be provided as a tank mix with one or more pesticidal or herbicidal chemicals to control pests and diseases of the crop plants in need of pest and disease control, a sequential treatment of components (generally the polynucleotide containing composition followed by the pesticide), or a simultaneous treatment or mixing of one or more of the components of the composition from separate containers. Treatment of the field can occur as often as needed to provide virus control and the components of the composition can be adjusted to target specific Tospoviruses or Geminiviruses through utilization of specific polynucleotides or polynucleotide compositions capable of selectively targeting the specific virus to be controlled. The composition can be applied at effective use rates according to the time of application to the field, for example, preplant, at planting, post planting, or post harvest. The polynucleotides of the composition can be applied at rates of 1 to 30 grams per acre depending on the number of trigger molecules needed for the scope of virus infection in the field.
Crop plants in which virus control may be needed include but are not limited to corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, barley, and wheat; vegetable plants including, but not limited to, tomato, sweet pepper, hot pepper, melon, watermelon, cucumber, zucchini, eggplant, cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish, potato, Brussels sprouts, tomatillo, peanut, garden beans, dry beans, or okra; culinary plants including, but not limited to, basil, parsley, coffee, or tea; or fruit plants including but not limited to apple, pear, cherry, peach, plum, apricot, banana, plantain, table grape, wine grape, citrus, avocado, mango, or berry; a tree grown for ornamental or commercial use, including, but not limited to, a fruit or nut tree; ornamental plant (e.g., an ornamental flowering plant or shrub or turf to grass), such as iris and impatiens. The methods and compositions provided herein can also be applied to plants produced by a cutting, cloning, or grafting process (i.e., a plant not grown from a seed) including fruit trees and plants that include, but are not limited to, avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes, as well as various ornamental plants.
The trigger polynucleotide compositions may also be used as mixtures with various agricultural chemicals and/or insecticides, miticides and fungicides, pesticidal and biopesticidal agents. Examples include, but are not limited to, azinphos-methyl, acephate, isoxathion, isofenphos, ethion, etrimfos, oxydemeton-methyl, oxydeprofos, quinalphos, chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, cyanophos, dioxabenzofos, dichlorvos, disulfoton, dimethylvinphos, dimethoate, sulprofos, diazinon, thiometon, tetrachlorvinphos, temephos, tebupirimfos, terbufos, naled, vamidothion, pyraclofos, pyridafenthion, pirimiphos-methyl, fenitrothion, fenthion, phenthoate, flupyrazophos, prothiofos, propaphos, profenofos, phoxime, phosalone, phosmet, formothion, phorate, malathion, mecarbam, mesulfenfos, methamidophos, methidathion, parathion, methyl parathion, monocrotophos, trichlorphon, EPN, isazophos, isamidofos, cadusafos, diamidaphos, dichlofenthion, thionazin, fenamiphos, fosthiazate, fosthietan, phosphocarb, DSP, ethoprophos, alanycarb, aldicarb, isoprocarb, ethiofencarb, carbaryl, carbosulfan, xylylcarb, thiodicarb, pirimicarb, fenobucarb, furathiocarb, propoxur, bendiocarb, benfuracarb, methomyl, metolcarb, XMC, carbofuran, aldoxycarb, oxamyl, acrinathrin, allethrin, esfenvalerate, empenthrin, cycloprothrin, cyhalothrin, gamma-cyhalothrin, lambda-cyhalothrin, cyfluthrin, beta-cyfluthrin, cypermethrin, alpha-cypermethrin, zeta-cypermethrin, silafluofen, tetramethrin, tefluthrin, deltamethrin, tralomethrin, bifenthrin, phenothrin, fenvalerate, fenpropathrin, furamethrin, prallethrin, flucythrinate, fluvalinate, flubrocythrinate, permethrin, resmethrin, ethofenprox, cartap, thiocyclam, bensultap, acetamiprid, imidacloprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, nitenpyram, chlorfluazuron, diflubenzuron, teflubenzuron, triflumuron, novaluron, noviflumuron, bistrifluoron, fluazuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, chromafenozide, tebufenozide, halofenozide, methoxyfenozide, diofenolan, cyromazine, pyriproxyfen, buprofezin, methoprene, hydroprene, kinoprene, triazamate, endosulfan, chlorfenson, chlorobenzilate, dicofol, bromopropylate, acetoprole, fipronil, ethiprole, pyrethrin, rotenone, nicotine sulphate, BT (Bacillus Thuringiensis) agent, spinosad, abamectin, acequinocyl, amidoflumet, amitraz, etoxazole, chinomethionat, clofentezine, fenbutatin oxide, dienochlor, cyhexatin, spirodiclofen, spiromesifen, tetradifon, tebufenpyrad, binapacryl, bifenazate, pyridaben, pyrimidifen, fenazaquin, fenothiocarb, fenpyroximate, fluacrypyrim, fluazinam, flufenzin, hexythiazox, propargite, benzomate, polynactin complex, milbemectin, lufenuron, mecarbam, methiocarb, mevinphos, halfenprox, azadirachtin, diafenthiuron, indoxacarb, emamectin benzoate, potassium oleate, sodium oleate, chlorfenapyr, tolfenpyrad, pymetrozine, fenoxycarb, hydramethylnon, hydroxy propyl starch, pyridalyl, flufenerim, flubendiamide, flonicamid, metaflumizole, lepimectin, TPIC, albendazole, oxibendazole, oxfendazole, trichlamide, fensulfothion, fenbendazole, levamisole hydrochloride, morantel tartrate, dazomet, metam-sodium, triadimefon, hexaconazole, propiconazole, ipconazole, prochloraz, triflumizole, tebuconazole, epoxiconazole, difenoconazole, flusilazole, triadimenol, cyproconazole, metconazole, fluquinconazole, bitertanol, tetraconazole, triticonazole, flutriafol, penconazole, diniconazole, fenbuconazole, bromuconazole, imibenconazole, simeconazole, myclobutanil, hymexazole, imazalil, furametpyr, thifluzamide, etridiazole, oxpoconazole, oxpoconazole fumarate, pefurazoate, prothioconazole, pyrifenox, fenarimol, nuarimol, bupirimate, mepanipyrim, cyprodinil, pyrimethanil, metalaxyl, mefenoxam, oxadixyl, benalaxyl, thiophanate, thiophanate-methyl, benomyl, carbendazim, fuberidazole, thiabendazole, manzeb, propineb, zineb, metiram, maneb, ziram, thiuram, chlorothalonil, ethaboxam, oxycarboxin, carboxin, flutolanil, silthiofam, mepronil, dimethomorph, fenpropidin, fenpropimorph, spiroxamine, tridemorph, dodemorph, flumorph, azoxystrobin, kresoxim-methyl, metominostrobin, orysastrobin, fluoxastrobin, trifloxystrobin, dimoxystrobin, pyraclostrobin, picoxystrobin, iprodione, procymidone, vinclozolin, chlozolinate, flusulfamide, dazomet, methyl isothiocyanate, chloropicrin, methasulfocarb, hydroxyisoxazole, potassium hydroxyisoxazole, echlomezol, D-D, carbam, basic copper chloride, basic copper sulfate, copper nonylphenolsulfonate, oxine copper, DBEDC, anhydrous copper sulfate, copper sulfate pentahydrate, cupric hydroxide, inorganic sulfur, wettable sulfur, lime sulfur, zinc sulfate, fentin, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hypochlorite, silver, edifenphos, tolclofos-methyl, fosetyl, iprobenfos, dinocap, pyrazophos, carpropamid, fthalide, tricyclazole, pyroquilon, diclocymet, fenoxanil, kasugamycin, validamycin, polyoxins, blasticiden S, oxytetracycline, mildiomycin, streptomycin, rape seed oil, to machine oil, benthiavalicarbisopropyl, iprovalicarb, propamocarb, diethofencarb, fluoroimide, fludioxanil, fenpiclonil, quinoxyfen, oxolinic acid, chlorothalonil, captan, folpet, probenazole, acibenzolar-S-methyl, tiadinil, cyflufenamid, fenhexamid, diflumetorim, metrafenone, picobenzamide, proquinazid, famoxadone, cyazofamid, fenamidone, zoxamide, boscalid, cymoxanil, dithianon, fluazinam, dichlofluanide, triforine, isoprothiolane, ferimzone, diclomezine, tecloftalam, pencycuron, chinomethionat, iminoctadine acetate, iminoctadine albesilate, ambam, polycarbamate, thiadiazine, chloroneb, nickel dimethyldithiocarbamate, guazatine, dodecylguanidine-acetate, quintozene, tolylfluanid, anilazine, nitrothalisopropyl, fenitropan, dimethirimol, benthiazole, harpin protein, flumetover, mandipropamide and penthiopyrad.
All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The following Examples are presented for the purposes of illustration and should not be construed as limitations. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed herein and still obtain a like or similar result without departing from the spirit and scope.
Single-stranded DNA (ssDNA) fragments in antisense (as) orientation were identified and mixed with a transfer agent and other components. This composition was topically applied to lettuce plants to effect repression of the target INSV nucleocapsid (N) gene to reduce or eliminate symptoms of viral infection in the plants. The procedure was as follows.
Growing lettuce plants (Lactuca sativa, c.v. SVR3606-L4) were topically treated with a composition for inducing suppression of a target gene in a plant. The composition included: (a) an agent to enable permeation of the polynucleotides into the plant, and (b) at least one polynucleotide strand including at least one segment of 17-25 contiguous nucleotides of the target gene in antisense orientation. Lettuce plants were topically treated with an adjuvant solution comprising antisense ssDNA, essentially homologous or essentially complementary to the INSV N protein coding sequence. Plants were grown and treated in growth chambers [22° C., 8 hour light (˜50 μmol), 16 hour dark cycles].
Lettuce plants were germinated for approximately 16-21 days prior to assay. Single leaves of lettuce plants (40 plants total) were infected with approximately 200 nanograms (100 ng/μL in phosphate buffer) of INSV virus. Approximately 3 hours after virus infection, 20 plants were sprayed with a mixture of oligonucleotides in solution (SEQ ID NO:1 and SEQ ID NO:2, mixed together) using an airbrush at 20 psi. The sequences of the antisense ssDNA oligonucleotides are listed in Table 1. The remaining 20 plants were not treated with oligonucleotides and served as the control.
The final concentration of each oligonucleotide or polynucleotide was 20 nMoles for ssDNA (in 0.1% Silwet L-77, 2% ammonium sulfate, 5 mM sodium phosphate buffer, pH 6.8) unless otherwise stated. The spray solution was applied to the plant to provide a total of 200-300 μL volume. The fresh weight of aerial tissue was measured (see
Leaf punctures harvested from untreated or treated plants lettuce plants (
As shown in
In this example, lettuce plants that were untreated (null) or that had been infected with INSV virus and treated with ss antisense oligonucleotides were measured using a portable chlorophyll fluorometer (PAM-2500). This measurement gives an effective yield of photosystem II (PSII) function, a measure of overall yield. A group of six randomly picked non-treated and six randomly picked treated plants were measured at leaf number 2, 4, 6 and 8. The leaf number is indicative of the age of the lettuce head with the youngest leaf (leaf 2) being inside the forming lettuce head and the oldest leaf (leaf 8) located on the outside of the forming lettuce head. Plants treated with ss antisense DNA oligos exhibited the most protection on the outer leaves compared to untreated (null) plants.
Single-stranded or double-stranded DNA or RNA fragments in sense or antisense orientation, or both, were identified and mixed with a transfer agent and other components. This composition was topically applied to tomato plants to effect expression of the target TSWV nucleocapsid or capsid genes to reduce or eliminate symptoms of viral infection in the plants. The procedure was as follows.
Tomato plants (Solanum lycopersicum HP375) and pepper plants (c.v. Yolo Wonder B) were grown in a cage outdoors. Pepper plants infected with TSWV, a negative-sense RNA virus, were transplanted from a breeder's infected pepper field in the center of the rows containing either tomato or pepper plants. Any subsequent infection was due to thrips transmitting TSWV from the infected center plants, thus mimicking a natural TSWV infection (see
Plants at the 2-5 fully expanded leaf stage were used in these assays. Seven or 8 plants were treated as control (virus infection only) and 7 or 8 plants were treated with polynucleotides. Two fully expanded leaves per plant were treated with the polynucleotide/Silwet L-77 solution. The final concentration for each oligonucleotide or polynucleotide was 10 nmoles for ssDNA (in 0.1% Silwet L-77, 2% ammonium sulfate, 5 mM sodium phosphate buffer, pH 6.8) unless otherwise stated. Twenty microliters of the solution was applied to the top surface of each of the two leaves to to provide a total of 40 μL for each plant.
In this example, growing pepper plants (c.v. Yolo Wonder B) were inoculated with cucumber mosaic virus (CMV), a positive strand RNA virus, and the plants were separated into two groups. The experimental group was then topically treated with a mixture of at least one polynucleotide strand including at least one segment of 17-25 contiguous nucleotides of the target gene in either antisense or sense orientation. The trigger molecules in the topical adjuvant solution comprised dsRNA and ssDNA essentially homologous or essentially complementary to the CMV capsid coding sequence. The sequences of the trigger molecules used in each treatment are shown in Table 3.
Pepper plants at the 2-5 fully expanded leaf stage were used in the assays. Seven or 8 plants were used as the control (virus infection only) and 7 or 8 plants were treated with virus followed by a polynucleotide trigger solution. Two fully expanded leaves per plant were treated with the polynucleotide/Silwet L-77 solution. One set of plants was treated with a mixture of polynucleotides comprising SEQ ID NOs:5-8 and another set of plants was treated with a mixture of polynucleotides comprising SEQ ID NOs:9-12. The final concentration for each oligonucleotide or polynucleotide was 5 nmol for ssDNA (in 0.1% Silwet L-77, 2% ammonium sulfate, 5 mM sodium phosphate buffer, pH 6.8) unless otherwise stated. Twenty microliters of the solution was applied to the top surface of each of the two leaves to provide a total of 40 μL for each plant.
As shown in
In this example, growing onion plants were inoculated with iris yellow spot virus (IYSV), and the plants were separated into two groups (31 plants per group). The experimental group was then topically treated with a mixture of at least one polynucleotide strand including at least one segment of 17-25 contiguous nucleotides of the target gene in antisense orientation. The trigger molecules in the topical adjuvant solution comprised ssDNA essentially homologous or essentially complementary to an IYSV coding sequence. The results of treatment of onion plants with antisense ssDNA are shown in
In Table 4 of this example, the sequences of genes of Tospovirus isolates considered to be commercially relevant because of yield losses in tomato, pepper, potato, or soybean were identified and constitute SEQ ID NOs:13-46.
A computer alignment was used to identify highly conserved areas within the Nucleocapsid (N), Silencing Suppressor (NSs), Movement (NSm), and RNA-dependent RNA polymerase genes (SEQ ID NOs:47-103 in Table 5) to serve as candidates for antisense ssDNA or dsRNA polynucleotides homologous to the gene sequence for topical application treatment to control Tospovirus infection (Table 5). These polynucleotides can be tested on Tospovirus-infected tomato plants to control viral infection.
Solanum lycopersicum
Eustoma grandiflorum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Glycine max
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
In Table 6 of this example, a commonly used computer algorithm was used to identify highly conserved regions in the coat protein (CP), Movement Protein (MP), and Silencing Suppressor protein, of plant virus isolates that are commercially relevant in agriculture. These viruses may be of different families, such as Geminiviruses (i.e., Cotton leaf curl virus, Barley yellow dwarf virus), or Bromoviruses (i.e., CMV), or Potexviruses (i.e., PepMV). The triggers identified in to Table 6 constitue SEQ ID NOs:104-268 and can be topically applied with a transfer agent to tomato, or pepper plants to test the efficacy against infection by the respective viruses.
In this example, the sequences of the Coat Protein (CM) Movement Protein (MP) or Silencing Suppressor (S) for different Cucumber Mosaic Viruses were identified and can be seen in Table 7. Topical application of ss antisense DNA or dsRNA sequences derived from the listed sequences (SEQ ID NOs:269-316) will be performed in pepper plants infected by Cucumber Mosaic Virus (CMV) using a transfer reagent and the plants will be scored by ELISA analysis and visual to assessment for reduction of symptoms.
Spinacia oleracea
Capsicum sp
Capsicum sp
Capsicum sp
Capsicum sp
Raphanus sativus
Solanum lycopersicum
In this example the sequences of the Coat Protein (CM) and Movement Protein (MP) for different Pepino Mosaic Virus isolates were identified and can be seen in Table 8. Topical application of ss antisense DNA or dsRNA sequences derived from the listed sequences (SEQ ID NOs:317-349) will be performed in tomato plants infected by Pepino Mosaic Virus (PepMV) using a transfer reagent and the plants will be scored by ELISA analysis and visual assessment for reduction of symptoms.
Solanum lycopersicum
Solanum lycopersicum
Lycopersicon esculentum
Solanum lycopersicum
Lycopersicon esculentum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Lycopersicon esculentum
Lycopersicon esculentum
Lycopersicon esculentum
Lycopersicon esculentum
Lycopersicon esculentum
Lycopersicon esculentum
Lycopersicon esculentum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Lycopersicon esculentum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
In this example, the sequences of the Coat Protein (CM), Movement Protein (MP), and Silencing Suppressor (SS) for different Barley yellow dwarf virus isolates were identified and are set forth in Table 9. Topical application of antisense ssDNA or dsRNA sequences derived from the listed sequences (SEQ ID NOs:350-385) can be performed in barley plants infected by BYDV using a transfer reagent and the plants can be scored by ELISA analysis and visual assessment for reduction of symptoms.
In this example, the sequences of the Coat Protein (CM), Movement Protein (MP), and Complement (C2) protein for different Tomato yellow leaf curl virus isolates were identified and are set forth in Table 10. Topical application of antisense ssDNA or dsRNA sequences derived from the listed sequences (SEQ ID NOs:386-421) can be performed in tomato plants infected by TYLCV using a transfer reagent and the plants scored by ELISA analysis and visual assessment for reduction of to symptoms.
Stellaria aquatica
Lycopersicon esculentum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Solanum lycopersicum
Lycopersicon esculentum
Lycopersicon esculentum
Lycopersicon esculentum
Lycopersicon esculentum
Solanum lycopersicum
Lycopersicon esculentum
In this example the sequences of the Coat Protein (CM), Movement Protein (MP) and AC2 protein for different Cotton Leaf Curl Virus isolates were identified and can be seen in Table 11. Topical application of ss antisense DNA or dsRNA sequences derived from the listed sequences (SEQ ID NOs:422-447) will be performed in cotton plants infected by CLCuV using a transfer reagent and the plants will be scored by ELISA analysis and visual assessment for reduction of symptoms.
Gossypium hirsutum
Hibiscus rosa-sinensis
Hibiscus cannabinus
Hibiscus rosa-sinensis
Hibiscus rosa-sinensis
Abelmoschus esculentus (okra)
Hibiscus rosa-sinensis
Hibiscus rosa-sinensis (Rose Mallow)
Gossypium hirsutum subsp. Latifolium
Gossypium hirsutum
Gossypium hirsutum
In this example, growing pepper plants (c.v. Yolo Wonder B) were inoculated with tomato spotted wilt virus (TSWV), a negative strand ssRNA virus, and the plants were separated into different groups. The experimental group was topically treated with a liquid composition containing at least one dsRNA polynucleotide comprising an approximately 100 bp sequence that is homologous to a transcript of the nucleocapsid (N), suppressor (NSs) or movement (NSm) gene of TSWV and its complement. The sequences of the sense strand of the trigger molecules used in the experiments outlined in this Example are shown in Table 12.
Plants were sown in a growth chamber [22° C., 8 hour light (˜50 μmol), 16 hour dark cycles] and transferred to a green house a couple of days before treatment. Pepper plants at the 2-5 fully expanded leaf stage were used in this assay. The experimental setup consisted of between 20-24 plants per treatment. Treatments consisted of: (a) healthy controls (no viral infection), (b) virus only control (no polynucleotide solution), (c) formulation only (no polynucleotides), or (d) experimental application with polynucleotide/Silwet L-77 trigger solution comprising a trigger molecule selected from the list of SEQ ID NOs:448-483 following virus to infection. Virus infection was carried out using standard mechanical inoculation technique and using Tomato spotted wilt virus (TSWV) or Cucumber mosaic virus (CMV), a positive strand RNA virus unrelated to TSWV. The final concentration used for each dsRNA polynucleotide was between 14.2-15.15 pmol/plant (in 0.1% Silwet L-77, 2% ammonium sulfate, 5 mM sodium phosphate buffer, pH 6.8). One thousand micro-liters of the polynucleotide/Silwet L-77 solution was applied using an airbrush (Badger 200G) at 10 psi to each plant group. Plants were arranged in the greenhouse following a randomized complete block design and monitored visually for symptom development. Plant height and ELISA analysis were both carried out at 32 days post-infection (32 DPI). ELISA analysis was performed on supernatant extracts from control and systemic leaf tissue punctures using an antibody to TSWV nucleocapsid (N) protein. The experiment was repeated twice (see Tables 13-17).
Plants treated with polynucleotide trigger sequence T25748 corresponding to SEQ ID NO:448 in the TSWV Nucleocapsid (N) gene were significantly taller than plants treated with other polynucleotides. This is also shown in
In this experiment treatment with trigger sequence T25748 (SEQ ID NO:448) was the best performer of the “BC” group.
This application claims the benefit of U.S. Provisional Patent Application No. 61/714,733, filed Oct. 16, 2012, and U.S. Provisional Patent Application No. 61/786,032, filed Mar. 14, 2013, which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US13/65193 | 10/16/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61786032 | Mar 2013 | US | |
| 61714733 | Oct 2012 | US |
