Claims for Patent: 6,008,436

Summary for Patent: 6,008,436

Title:	Nematode-resistant transgenic plants
Abstract:	Nematode-resistant transgenic plants are disclosed. The plants comprise plant cells containing a DNA construct comprising a transcription cassette, which construct comprises, in the 5\' to 3\' direction, a promoter operable in the plant cells, and a DNA comprising at least a portion of a DNA sequence encoding a nematode-inducible transmembrane pore protein in either the sense or antisense orientation. Intermediates for producing the same along with methods of making and using the same are also disclosed. In an alternate embodiment of the invention, the sense or antisense DNA is replaced with a DNA encoding an enzymatic RNA molecule directed against the mRNA transcript of a DNA sequence encoding a nematode-inducible transmembrane pore protein.
Inventor(s):	Conkling; Mark A. (Fuquay-Varina, NC), Opperman; Charles H. (Raleigh, NC), Acedo; Gregoria N. (Durham, NC), Song; Wen (Raleigh, NC)
Assignee:	North Carolina State University (Raleigh, NC)
Application Number:	08/654,025
Patent Claims:	1. A DNA construct comprising a transcription cassette, which construct comprises, in the 5' to 3' direction, a promoter operable in a plant cell, and a DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein, said DNA segment in the opposite orientation for expression, where said DNA sequence encoding a nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNA of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 2. A DNA construct according to claim 1, which DNA sequence encoding a nematode-inducible transmembrane pore protein encodes a TobRB7 nemtode-inducible transmembrane pore protein. 3. A DNA construct according to claim 1, which DNA segment in the opposite orientation for expression includes an intron-exon junction. 4. A DNA construct according to claim 1, which DNA segment in the opposite orientation for expression has SEQ ID NO:3. 5. A DNA construct according to claim 1, which promoter is constitutively active in plant cells. 6. A DNA construct according to claim 1, which promoter is selectively active in plant root tissue cells. 7. A DNA construct according to claim 1, which promoter is a Cauliflower Mosaic Virus 35S promoter. 8. A DNA construct according to claim 1, which promoter is activated by a plant-parasitic nematode. 9. A DNA construct according to claim 1, which promoter is a nematode-responsive element selected from the group consisting of: (i) isolated DNA having the sequence given herein as SEQ ID NO:5; and (ii) isolated DNA which hybridizes to isolated DNA of (i) above and which encodes a nematode responsive element. 10. A DNA construct according to claim 1, which promoter is an RB7 nematode-responsive element. 11. A DNA construct according to claim 1 carried by a plant transformation vector. 12. A DNA construct according to claim 1 carried by a plant transformation vector, which plant transformation vector is an Agrobacterium tumefaciens vector. 13. A nematode-resistant transgenic plant comprising plant cells containing a DNA construct comprising a transcription cassette, which construct comprises, in the 5' to 3' direction, a promoter operable in said plant cells, and a DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein, said DNA segment in the opposite orientation for expression, where said DNA sequence encoding a nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 14. A plant according to claim 13, which plant is a dicot. 15. A plant according to claim 13, which plant is a dicot selected from the group consisting of tobacco, potato, soybean, peanuts, pineapple, and cotton. 16. A plant according to claim 13, which plant is a member of the family Solanacae. 17. A plant according to claim 13, which DNA sequence encoding a nematode-inducible transmembrane pore protein encodes a TobRB7 nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 18. A plant according to claim 13, which promoter is constitutively active in plant cells. 19. A plant according to claim 13, which promoter is selectively active in plant root tissue cells. 20. A plant according to claim 13, which promoter is activated by a plant-parasitic nematode. 21. A plant according to claim 13, which promoter is a nematode-responsive element selected from the group consisting of: (i) isolated DNA having the sequence given herein as SEQ ID NO:5; and (ii) isolated DNA which hybridizes to isolated DNA of (i) above and which encodes a nematode responsive element. 22. A crop comprising a plurality of plants according to claim 13 planted together in an agricultural field. 23. A method of combatting plant parasitic nematodes in an agricultural field, comprising planting the field with a crop of plants according to claim 13. 24. A method of making a recombinant pathogen-resistant plant, said method comprising: providing a plant cell capable of regeneration; transforming said plant cell with a DNA construct comprising a transcription cassette, which construct comprises, in the 5' to 3' direction, a promoter operable in said plant cell, and a DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein, said DNA segment in either (a) the opposite orientation for expression or (b) the proper orientation for expression, and where said DNA sequence encoding a nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein; and then regenerating a recombinant nematode-resistant plant from said transformed plant cell. 25. A method according to claim 24, wherein said plant cell resides in a plant tissue capable of regeneration. 26. A method according to claim 24, wherein said transforming step is carried out by bombarding said plant cell with microparticles carrying said transcription cassette. 27. A method according to claim 24, wherein said transforming step is carried out by infecting said cells with an Agrobacterium tumefaciens containing a Ti plasmid carrying said transcription cassette. 28. A nematode-resistant transgenic plant comprising plant cells containing a heterologous DNA construct comprising a transcription cassette, which heterologous construct comprises in the 5' to 3' direction, a promoter operable in said plant cells, and a DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein, said DNA segment in the proper orientation for expression, and where said DNA sequence encoding a nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 29. A plant according to claim 28, which plant is a dicot. 30. A plant according to claim 28, which plant is a dicot selected from the group consisting of tobacco, potato, soybean, peanuts, pineapple, and cotton. 31. A plant according to claim 28, which plant is a member of the family Solanacae. 32. A plant according to claim 28, which DNA sequence encoding a nematode-inducible transmembrane pore protein encodes a TobRB7 nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 33. A plant according to claim 28, which DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein is a sense DNA segment in the proper orientation for expression. 34. A plant according to claim 28, which DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein is an antisense DNA segment in the opposite orientation for expression. 35. A plant according to claim 28, which promoter is constitutively active in plant cells. 36. A plant according to claim 28, which promoter is selectively active in plant root tissue cells. 37. A plant according to claim 28, which promoter is activated by a plant-parasitic nematode. 38. A plant according to claim 28, which promoter is a nematode-responsive element selected from the group consisting of: (i) isolated DNA having the sequence given herein as SEQ ID NO:5; and (ii) isolated DNA which hybridizes to isolated DNA of (i) above and which encodes a nematode responsive element. 39. A crop comprising a plurality of nematode-resistant transgenic plants planted together in an agricultural field, said nematode-resistant transgenic plants comprising plant cells, said plant cells containing a DNA construct comprising a transcription cassette, which construct comprises, in the 5' to 3' direction, a promoter operable in said plant cells, and a DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein, said DNA segment in the proper orientation for expression, and where said DNA sequence encoding a nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 40. A method of combatting plant parasitic nematodes in an agricultural field, comprising planting the field with a crop of nematode-resistant transgenic plants comprising plant cells, said plant cells containing a DNA construct comprising a transcription cassette, which construct comprises, in the 5' to 3' direction, a promoter operable in said plant cells, and a DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein, said DNA segment in the proper orientation for expression, where said DNA sequence encoding a nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 41. A transgenic tobacco plant comprising tobacco plant cells containing a heterologous DNA construct comprising a transcription cassette, which construct comprises, in the 5' to 3' direction, a promoter operable in said tobacco plant cells, and a heterologous DNA sequence encoding a nematode-inducible transmembrane pore protein; said promoter selected from the group consisting of the Cauliflower Mosaic Virus 35S promoter and the promoter encoded by SEQ ID NO:5; said heterologous DNA sequence selected from the group consisting of DNA having the sequence given herein as SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:6; said tobacco plant resistant to a nematode selected from the group consisting of Meloidogyne incognita and Meloidogyne arenaria. 42. A DNA construct comprising a transcription cassette, which construct comprises, in the 5' to 3' direction, a promoter operable in a plant cell, and a DNA segment comprising at least 15 nucleotides of a DNA sequence encoding a nematode-inducible transmembrane pore protein, said DNA segment in the proper orientation for expression, where said DNA sequence encoding a nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 43. A DNA construct according to claim 42, which DNA sequence encoding a nematode-inducible transmembrane pore protein encodes a TobRB7 nematode-inducible transmembrane pore protein is selected from the group consisting of: (a) isolated DNA having the sequence given herein as SEQ ID NO:1 or SEQ ID NO:6; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a nematode inducible transmembrane pore protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in nucleotide sequence due to the degeneracy of the genetic code, and which encode a nematode-inducible transmembrane pore protein. 44. A DNA construct according to claim 42, which DNA segment includes an intron-exon junction. 45. A DNA construct according to claim 42, which DNA segment has SEQ ID NO:3. 46. A DNA construct according to claim 42, which promoter is constitutively active in plants. 47. A DNA construct according to claim 42, which promoter is selectively active in plant root tissue cells. 48. A DNA construct according to claim 42, which promoter is a Cauliflower Mosaic Virus 35S promoter. 49. A DNA construct according to claim 42, which promoter is activated by a plant-parasitic nematode. 50. A DNA construct according to claim 42, which promoter is a nematode-responsive element selected from the group consisting of: (i) isolated DNA having the sequence given herein as SEQ ID NO:5; (ii) isolated DNA which hybridizes to isolated DNA of (i) above and which encodes a nematode responsive element. 51. A DNA construct according to claim 42, which promoter is an RB7 nematode-responsive element. 52. A DNA construct according to claim 42 carried by a plant transformation vector. 53. A DNA construct according to claim 42 carried by a plant transformation vector, which plant transformation vector is an Agrobacterium tumefaciens vector.

Details for Patent 6,008,436

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2013-01-21
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2013-01-21
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2013-01-21