Disarmed Agrobacterium Strains, Ri-Plasmids, and Methods of Transformation Based Thereon

• US 20070292953A1
• Filed: 08/31/2005
• Published: 12/20/2007
• Est. Priority Date: 09/02/2004
• Status: Active Grant

• Current Assignee: BASF Plant Science GmbH (BASF SE)
• Original Assignee: BASF Plant Science GmbH (BASF SE)
• Inventors: Mankin, Luke, Hill, Dwight-Steven, Olhoft, Paula, Toren, Effie, Xing, Liqun, Fu, Huihua, Ireland, Lesley, Jia, Hongmei, Song, Hee-Sook, Nea, Larry, Wenck, Allan, Brown, Jeffrey
• Granted Patent: US 7,989,678 B2
• Time in Patent Office: Days
• US Class Current: 435/469
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation

Abstract

The present invention relates “disarmed” strain variants of Agrobacterium strain K599 (NCPPB 2659), “disarmed” plasmid variants of the Ri-plasmid pRi2659, and derivatives thereof, and methods employing these strains and plasmids in plant transformation.

Claims

1. A method for producing a transgenic plant cell comprising the steps of:

a) providing bacteria of a transgenic, non-pathogenic strain variant of Agrobacterium strain K599 (NCPPB

     2659) or of a derivative of said strain, wherein said strain variant is capable to infect plant cells but is lacking hairy root phenotype inducing properties and wherein said strain variant is further comprising a transgenic T-DNA, and

b) co-cultivating a plant cell with said bacteria, and

c) isolating or selecting plant cells comprising stably integrated into their genome said transgenic T-DNA.

3. The method of claim 1, wherein said non-pathogenic strain variant is capable to infect plant cells, to mediate T-DNA transfer into plant cells, and to mediate T-DNA insertion into the plant genome, but is lacking hairy root phenotype inducing properties.

4. The method of claim 1, wherein the derivative of Agrobacterium strain K599 (NCPPB2659) is a soil borne, plant pathogenic bacterium, characterized by a 16S-23S rRNA intergenic sequence comprising at least one sequence motive selected from the group consisting of sequence motifs described by SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

5. The method of claim 1, wherein said non-pathogenic strain variant comprises a non-pathogenic plasmid variant of the Ri-plasmid pRi2659 or of a derivative of said plasmid.

6. The method of claim 5, wherein the non-pathogenic plasmid variant is comprising at least one sequence selected from the group of sequences described by

a) a sequence comprising a sequence described by SEQ ID NO:

24, or a sequence of at least 100 consecutive nucleotides of the sequence described by SEQ ID NO;

24, and

b) a sequences having a sequence identity of at least 90% to a sequence as described by SEQ ID NO;

24 or a sequence of at least 1000 consecutive nucleotides of the sequence described by SEQ ID NO;

24, and,

c) a sequence hybridizing under conditions equivalent to binding or hybridization at 68°

C. in a solution consisting of 5×

SSPE, 1% SDS, 5×

Denhardt'"'"'s reagent and 100 μ

g/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×

SSPE, and 0.1% SDS at 68°

C. to a probe consisting of at least 100 consecutive nucleotides of a sequence as described by SEQ ID NO;

24 or the complementary sequence thereto.

9. The method of claim 1, wherein said transgenic T-DNA comprises at least one plant expressible selectable marker gene.

2. A method for producing a transgenic plant comprising the steps of:

a) providing bacteria of a transgenic, non-pathogenic strain variant of Agrobacterium strain K599 (NCPPB

     2659) or of a derivative of said strain, wherein said strain variant is capable to infect plant cells but is lacking hairy root phenotype inducing properties and wherein said strain variant is further comprising a transgenic T-DNA, and

b) co-cultivating a plant, plant cell or plant tissue with said bacteria, and

c) isolating or selecting and—

optionally—

regenerating plants comprising stably integrated into their genome said transgenic T-DNA.

7. The method of claim 2, wherein said plant cell, plant tissue, or plant is derived from a plant selected from the group of monocotyledonous plants, dicotyledonous plants, and gymnosperm plants.

8. The method of claim 7, wherein said plant is from a genus selected from the group consisting of Medicago, Lycopersicon, Brassica, Cucumis, Solanum, Juglans, Gossypium, Malus, Vitis, Antirrhinum, Populus, Fragaria, Arabidopsis, Picea, Capsicum, Chenopodium, Dendranthema, Pharbitis, Pinus, Pisum, Oryza, Zea, Triticum, Triticale, Secale, Lolium, Hordeum, Glycine, Pseudotsuga, Kalanchoe, Beta, Helianthus and Nicotiana.

10. An isolated nucleotide sequence selected from the group of sequences described by

a) a sequence comprising a sequence described by SEQ ID NO:

24, or a sequence of at least 100 consecutive nucleotides of the sequence described by SEQ ID NO;

24,

b) a sequence having a sequence identity of at least 90% to a sequence as described by SEQ ID NO;

24 or a sequence of at least 1000 consecutive nucleotides of the sequence described by SEQ ID NO;

24, and,

c) a sequence hybridizing under conditions equivalent to binding or hybridization at 68°

C. in a solution consisting of 5×

SSPE, 1% SDS, 5×

Denhardt'"'"'s reagent and 100 μ

g/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×

SSPE, and 0.1% SDS at 68°

C. to a probe consisting of at least 100 consecutive nucleotides of a sequence as described by SEQ ID NO;

24 or the complementary sequence thereto.

23. A cell or non-human organism comprising a nucleotide sequence of claim 10.

24. The cell or non-human organism of claim 23, wherein said cell or organism is selected from the group consisting of bacteria, yeasts, plants, mammals, and insects.

25. The cell or non-human organism of claim 23, wherein said cell or organism is a soil born bacterium of the genus Rhizobiaceae.

11. A non-pathogenic plasmid variant of pRi2659 or of a derivative of said Ri-plasmid, said plasmid variant providing the functions required for plant cell infection and transformation, but lacking sequences causing the hairy root phenotype.

12. The non-pathogenic plasmid variant of claim 11, described by an isolated nucleotide sequence selected from the group of sequences described by

a) a sequence comprising a sequence described by SEQ ID NO:

24, or a sequence of at least 100 consecutive nucleotides of the sequence described by SEQ ID NO;

24;

b) a sequence having a sequence identity of at least 90% to a sequence as described by SEQ ID NO;

24 or a sequence of at least 1000 consecutive nucleotides of the sequence described by SEQ ID NO;

24; and

c) a sequence hybridizing under conditions equivalent to binding or hybridization at 68°

C. in a solution consisting of 5×

SSPE, 1% SDS, 5×

Denhardt'"'"'s reagent and 100 μ

g/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×

SSPE, and 0.1% SDS at 68°

C. to a probe consisting of at least 100 consecutive nucleotides of a sequence as described by SEQ ID NO;

24 or the complementary sequence thereto.

13. The non-pathogenic plasmid variant of claim 11, wherein the derivative is encoding a virD2 protein having a amino acid sequence identify of at least 85% with the sequence described by SEQ ID NO:

112.

14. The non-pathogenic plasmid variant of claim 11, wherein said variant is comprising the sequences required for plant cell infection and transformation of the native, pathogenic pRi2659 or its derivative but is lacking sequences of the T-DNA mediating the hairy root phenotype.

15. The non-pathogenic plasmid variant of claim 11, wherein the entire T-DNA including the borders is deleted from the native plasmid.

16. The non-pathogenic plasmid variant of claim 11, wherein the deleted T-DNA corresponds to the sequence described by the sequence from about base 538 to about base 15,519 of SEQ ID NO:

4 or from about base 3644 to about 18577 base of SEQ ID NO;

26.

17. The non-pathogenic plasmid variant of claim 11, wherein said plasmid variant hybridizes under high-stringency conditions equivalent to binding or hybridization at 68°

C. in a solution consisting of 5×

SSPE, 1% SDS, 5×

Denhardt'"'"'s reagent and 100 μ

g/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×

SSPE, and 0.1% SDS at 68°

C. with the entire, native, pathogenic Ri-plasmid pRi2659 of the pathogenic Agrobacterium strain K599 (NCPPB

     2659), but does not hybridize under said high-stringency conditions with the sequence from about base 538 to about base 15,519 of the sequence characterized by SEQ ID NO;

4 or from about base 3644 to about 18577 base of the sequence characterized by SEQ ID NO;

26.

18. The non-pathogenic plasmid variant of claim 11, wherein the derivative of pRi2659 is a plasmid able to mediate T-DNA transfer from a soil borne bacterium into a plant cell further characterized by

a) having a sequence identity of at least 90% with the DNA encoding the native pRi2659 plasmid (as comprised in Agrobacterium strain K599 (NCPPB2659) or

b) hybridizing under high stringency conditions equivalent to binding or hybridization at 68°

C. in a solution consisting of 5×

SSPE, 1% SDS, 5×

Denhardt'"'"'s reagent and 100 μ

g/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×

SSPE, and 0.1% SDS at 68°

C. with the native pRi2659 plasmid.

19. A transgenic T-DNA flanked by at least one T-DNA border from the Agrobacterium rhizogenes pRi2659 plasmid, said transgenic T-DNA comprising no sequences causing a hairy root phenotype.

20. The transgenic T-DNA of claim 19, wherein said border sequence is described by SEQ ID NO:

18 or 19.

21. The transgenic T-DNA of claim 19, wherein said transgenic T-DNA comprises at least one expression cassette for conferring to said plant an agronomically valuable trait or at least one marker gene, which allows for selection and/or identification of transformed plants, plant cells or tissues.

22. A transgenic vector comprising a transgenic T-DNA of claim 19.

26. A non-pathogenic strain variant of Agrobacterium strain K599 (NCPPB 2659) or a derivative thereof, wherein said strain variant is capable to infect plant cells but is lacking hairy root phenotype inducing properties.

28. The non-pathogenic strain variant of claim 26, wherein said non-pathogenic strain variant is capable to infect plant cells, to mediate T-DNA transfer into plant cells, and to mediate T-DNA insertion into the plant genome, but is lacking hairy root phenotype inducing properties.

29. The non-pathogenic strain variant of claim 26, wherein the derivative of Agrobacterium strain K599 (NCPPB2659) is a soil borne, plant pathogenic bacterium, characterized by a 16S-23S rRNA intergenic sequence comprising at least one sequence motive selected from the group consisting of sequence motifs described by SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

30. The non-pathogenic strain variant of claim 26, wherein said strain variant comprises a non-pathogenic plasmid variant of the Ri-plasmid pRi2659 or of a derivative thereof.

32. The non-pathogenic strain variant of claim 26, further comprising one or more characteristics selected from the group consisting of presence of mutant or chimeric virA or virG genes or presence of super-virulent plasmids.

27. A transgenic, non-pathogenic strain variant of Agrobacterium strain K599 (NCPPB 2659) or of a derivative of said strain, wherein said strain variant is capable to infect plant cells but is lacking hairy root phenotype inducing properties and wherein said strain variant is further comprising a transgenic T-DNA.

31. (canceled)

33. A polypeptide comprising an amino acid sequence selected from the group consisting of:

a) the sequence as described by SEQ ID NO;

112 or sequences of at least 200 consecutive amino acids thereof, and

b) a sequence having a sequence identity of at least 85% with the sequence described by SEQ ID NO;

112.

35. A nucleotide sequence comprising a sequence encoding the polypeptide of claim 33.

34. A polypeptide comprising an amino acid sequence selected from the group consisting of:

a) the sequence as described by any of SEQ ID NO;

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 126, 128, 129, 130, 131, 132, 133, 134, 136, 137, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, or 187 or a sequence of at least 200 consecutive amino acids thereof, and

b) a sequence having a sequence identity of at least 85% with a sequence described by any of SEQ ID NO;

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 126, 128, 129, 130, 131, 132, 133, 134, 136, 137, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, or 187.

36. A nucleotide sequence comprising a sequence encoding the polypeptide of claim 34.