Claims
- 1. A geminivirus silencing vector comprising a geminivirus B component comprising one or more heterologous DNA sequences, each of the heterologous DNA sequences having substantial sequence similarity to an endogenous plant gene.
- 2. The silencing vector of claim 1, wherein the silencing vector further comprises a geminivirus AL1 or AL3 gene.
- 3. The silencing vector of claim 1, wherein the silencing vector further comprises a geminivirus AL2 gene.
- 4. The silencing vector of claim 1, wherein the one or more heterologous DNA sequences have at least 85% sequence identity to an endogenous plant gene.
- 5. The silencing vector of claim 1, wherein each of the heterologous DNA sequences is at least about 20 base pairs in length, and further wherein the combined length of the heterologous DNA sequences does not exceed about 200 base pairs in length.
- 6. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is inserted into or replaces a segment following the stop codon of the BR1 gene.
- 7. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is inserted into or replaces a segment following the stop codon of the BL1 gene.
- 8. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is inserted into or replaces a segment of the intergenic region.
- 9. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is inserted into or replaces a segment of the common region.
- 10. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is inserted into or replaces a segment of the coding region of the BR1 gene.
- 11. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is inserted into or replaces a segment of the coding region of the BL1 gene.
- 12. The silencing vector of claim 1, wherein expression of the one or more heterologous DNA sequences modifies one or more observable plant phenotypic traits.
- 13. The silencing vector of claim 1, further comprising a heterologous DNA sequence that has substantial sequence similarity to a gene encoding a reporter protein.
- 14. The silencing vector of claim 1, wherein the silencing vector comprises two or more heterologous DNA sequences having substantial sequence similarity to an endogenous plant gene.
- 15. The silencing vector of claim 14, wherein the two or more heterologous DNA sequences have substantial sequence similarity to two or more non-homologous endogenous plant genes.
- 16. The silencing vector of claim 14, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to a endogenous plant gene encoding a non-translated RNA molecule or a fragment thereof.
- 17. The silencing vector of claim 14, wherein the two or more heterologous DNA sequences have substantial sequence similarity with two or more endogenous plant genes within a biochemical pathway.
- 18. The silencing vector of claim 1, wherein each of the heterologous DNA sequences is operably associated with a promoter.
- 19. The silencing vector of claim 1, wherein the silencing vector is phloem limited.
- 20. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is in the sense orientation.
- 21. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences is in an antisense orientation.
- 22. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to a fragment of an endogenous plant gene.
- 23. The silencing vector of claim 22, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to the coding region of an endogenous plant gene.
- 24. The silencing vector of claim 1, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to an endogenous plant promoter sequence.
- 25. The silencing vector of claim 1, wherein the silencing vector is a shuttle vector that replicates in a non-plant cell.
- 26. The shuttle vector of claim 25, wherein the shuttle vector replicates in a bacterial cell .
- 27. The shuttle vector of claim 26, wherein the shuttle vector is a plasmid.
- 28. A silencing vector comprising a geminivirus origin of replication, a geminivirus BR1 gene, and one or more heterologous DNA sequences, each of the heterologous DNA sequences having substantial sequence similarity to an endogenous plant gene.
- 29. The silencing vector of claim 28, wherein the silencing vector further comprises a geminivirus BR1 gene.
- 30. The silencing vector of claim 28, wherein the silencing vector further comprises an intergenic region from a geminivirus B component
- 31. The silencing vector of claim 28, wherein the silencing vector further comprises a geminivirus common region.
- 32. The silencing vector of claim 28, wherein the silencing vector further comprises DNA sequences encoding proteins sufficient to support the replication of said vector in a plant cell.
- 33. The silencing vector of claim 28, wherein the silencing vector further comprises a geminivirus AL3 gene.
- 34. The silencing vector of claim 28, wherein the silencing vector further comprises a geminivirus AL1 gene.
- 35. The silencing vector of claim 28, wherein the silencing vector further comprises a geminivirus AL2 gene.
- 36. The silencing vector of claim 28, wherein the silencing vector is phloem-limited.
- 37. The silencing vector of claim 28, wherein the one or more heterologous DNA sequences are inserted into or replace a segment of:
(a) the region downstream from the stop codon of the BR1 gene; or (b) the coding region of the BR1 gene.
- 38. The silencing vector of claim 28, wherein at least one of the heterologous DNA sequences is inserted into the silencing vector outside of the geminivirus sequences.
- 39. The silencing vector of claim 28, wherein expression of the one or more heterologous DNA sequences modifies one or more observable plant phenotypic traits.
- 40. The silencing vector of claim 28, wherein the one or more heterologous DNA sequences has substantial sequence similarity to two or more endogenous plant genes.
- 41. The silencing vector of claim 40, wherein the one or more heterologous DNA sequences has substantial sequence similarity to two or more non-homologous endogenous plant genes.
- 42. The silencing vector of claim 28, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to a fragment of an endogenous plant gene.
- 43. The silencing vector of claim 28, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to the entire coding region of an endogenous plant gene.
- 44. The silencing vector of claim 28, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to an endogenous plant promoter sequence or a fragment thereof.
- 45. The silencing vector of claim 28, wherein the silencing vector is a shuttle vector that replicates in a non-plant cell.
- 46. A silencing vector comprising a geminivirus origin of replication, a geminivirus BL1 gene, and one or more heterologous DNA sequences, each of the heterologous DNA sequences having substantial sequence similarity to an endogenous plant gene.
- 47. The silencing vector of claim 46, wherein the one or more heterologous DNA sequences are inserted into or replace a segment of:
(a) the region downstream from the stop codon of the BL1 gene; or (b) the coding region of the BL1 gene.
- 48. A plant cell comprising the silencing vector of claim 1.
- 49. A plant comprising the plant cell of claim 48.
- 50. A silencing vector comprising a geminivirus genomic component comprising one or more heterologous DNA sequences, each of the DNA sequences having substantial sequence similarity to an endogenous plant gene, wherein at least one of the heterologous DNA sequences is inserted into or replaces the 3′ noncoding region of a viral gene.
- 51. The silencing vector of claim 50, wherein the silencing vector comprises a geminivirus A component.
- 52. The silencing vector of claim 50, wherein the silencing vector comprises a geminivirus B component.
- 53. A method of silencing the expression of one or more endogenous plant genes, comprising inoculating a plant cell with the silencing vector of claim 1.
- 54. The method of claim 53, wherein the plant cell is selected from the group consisting of a mesophyll cell, epidermis cell, cortical cell, parenchymal cell, guard cell, xylem cell, floral cell, fruit cell, seed coat cell, meristematic cell, apical cell, sclerenchyma cell, and colenchyma cell.
- 55. The method of claim 53, wherein the silencing vector is phloem limited.
- 56. The method of claim 55, wherein the silencing vector comprises a phloem limiting mutation.
- 57. The method of claim 53, wherein the silencing vector is not phloem limited.
- 58. The method of claim 53, further comprising inoculating the plant cell with an additional vector comprising DNA sequences encoding proteins sufficient to support the replication of the silencing vector in a plant cell.
- 59. The method of claim 58, wherein the additional vector comprises a geminivirus A component.
- 60. The method of claim 59, wherein the additional vector comprising the geminivirus A component is a silencing vector.
- 61. The method of claim 53, wherein the plant cell is stably transformed with and expresses the geminivirus AL1, AL2, or AL3 gene.
- 62. A method of silencing the expression of one or more plant genes, comprising inoculating a plant cell with the silencing vector of claim 28.
- 63. The method of claim 62, wherein the silencing vector comprises geminivirus BR1 and BL1 genes.
- 64. A method of silencing the expression of one or more plant genes, comprising inoculating a plant cell with the silencing vector of claim 46.
- 65. A method of silencing expression of one or more endogenous plant genes, comprising inoculating a plant with the silencing vector of claim 1.
- 66. The method of claim 65, wherein expression of the one or more plant genes is systemically silenced in the plant.
- 67. A method of silencing expression of one or more endogenous plant genes, comprising inoculating a plant with the silencing vector of claim 28.
- 68. The method of claim 67, wherein expression of the one or more plant genes is systemically silenced in the plant.
- 69. A method of silencing expression of one or more endogenous plant genes, comprising inoculating a plant with the silencing vector of claim 46.
- 70. The method of claim 69, wherein expression of the one or more plant genes is systemically silenced in the plant.
- 71. A method of screening an isolated plant DNA sequence for function, comprising:
inoculating a plant with a silencing vector according to claim 1, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to the isolated plant DNA sequence; and comparing the inoculated plant to control plant tissue; wherein differences between the inoculated and control plant tissues indicate the function of the isolated plant DNA sequence.
- 72. The method of claim 71, wherein the inoculated plant comprises the control plant tissue.
- 73. A method of screening for the function of one or more endogenous plant genes, comprising:
inoculating a plant with a silencing vector according to claim 1, wherein at least one of the heterologous DNA sequences has substantial sequence similarity to an endogenous plant gene; and comparing the inoculated plant with control plant tissue; wherein differences between the inoculated and control plant tissues indicate the function of the one or more plant genes.
- 74. The method of claim 73, wherein the inoculated plant comprises the control plant tissue.
RELATED APPLICATION INFORMATION
[0001] This application claims the benefit of U.S. Provisional Application No. 60/210,105, filed Jun. 7, 2000, which is incorporated by reference herein in its entirety.
STATEMENT OF FEDERAL SUPPORT
[0002] This invention was made with government support under United States Department of Agriculture grant number NRICGP 97-35303-4538 and Tri-Agency Training Grant number NSF BIR9420689 from the National Science Foundation, United States Department of Energy, and the United States Department of Agriculture. The United States government has certain rights to this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60210105 |
Jun 2000 |
US |