Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2001-03-26
2003-10-28
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S278000, C800S298000, C800S295000, C800S317100, C800S317200, C800S288000, C800S320000, C800S320100, C800S320200, C800S314000, C536S023700, C536S023100
Reexamination Certificate
active
06639129
ABSTRACT:
BACKGROUND OF THE INVENTION
As reported in W098/08932, protein toxins from the genus Photorhabdus have been shown to have oral toxicity against insects. The toxin complex produced by
Photorhabdus luminescens
(W-14), for example, has been shown to contain ten to fourteen proteins, and it is known that these are produced by expression of genes from four distinct genomic regions: tca, tcb, tcc, and tcd. WO98/08932 discloses nucleotide sequences for many of the native toxin genes.
Of the separate toxins isolated from
Photorhabdus luminescens
(W-14), those designated Toxin A and Toxin B have been the subject of focused investigation for their activity against target insect species of interest, for example corn rootworm. Toxin A is comprised of two different subunits. The native gene tcda (SEQ ID NO:1) encodes protoxin TcdA (see SEQ ID NO:1). As determined by mass spectrometry, TcdA is processed by one or more proteases to provide Toxin A. More specifically, TcdA is an approximately 282.9 kDA protein (2516 aa) that is processed to provide TcdAii, an approximately 208.2 kDA (1849 aa) protein encoded by nucleotides 265-5811 of SEQ ID NO:1, and TcdAiii, an approximately 63.5 kDA (579 aa) protein encoded by nucleotides 5812-7551 of SEQ ID NO:1.
WO 01/11029 discloses nucleotide sequences that encode TcdA and TcbA and have base compositions that have been altered from that of the native genes to make them more similar to plant genes. Also disclosed are transgenic plants that express Toxin A and Toxin B.
We have observed that heterologous expression of Toxin A does not afford the level of oral toxicity to insects that is observed for the native toxin. It would be very valuable if means could be found to enhance the level of toxicity of heterologously expressed Toxin A.
SUMMARY OF THE INVENTION
The present invention provides nucleotide sequences for two genes, tcdB and tccC2, from the tcd genomic region of
Photorhabdus luminescens
W-14. These sequences were not previously known. We have discovered that coexpression of tcdB and tccC2 with tcdA in heterologous hosts results in enhanced levels of oral insect toxicity compared to that obtained when tcdA is expressed alone in such heterologous hosts. Coexpression of tcdB and tccC2 with tcdA or tcbA, or with any other functionally equivalent toxin gene in the same family as tcdA and tcbA, to provide enhanced oral insect activity falls within the scope of the invention.
SUMMARY OF THE INVENTION
SEQ ID NO: 1 is the DNA sequence for tcdA from
Photorhabdus luminescens
W-14.
SEQ ID NO: 2 is the amino acid sequence for TcdA from
Photorhabdus luminescens
W-14.
SEQ ID NO: 3 is the DNA sequence for tcdB from
Photorhabdus luminescens
W-14.
SEQ ID NO: 4 is the amino acid sequence for TcdB from
Photorhabdus luminescens
W-14.
SEQ ID NO: 5 is the DNA sequence for tccC2 from
Photorhabdus luminescens
W-14.
SEQ ID NO: 6 is the amino acid sequence for TccC2 from from
Photorhabdus luminescens
W-14.
SEQ ID NO: 7 is the DNA sequence for tcbA from from
Photorhabdus luminescens
W-14.
SEQ ID NO: 8 is the amino acid sequence for TcbA from
Photorhabdus luminescens
W-14.
REFERENCES:
patent: WO98/08932 (1998-03-01), None
patent: WO01/11029 (2001-02-01), None
Morgan, J. Alun et al., “Cloning and expression of insecticidal toxin genes from Xenorhabdus species”, SIP 1999 Program & Asbstracts, XXXII Meeting, University of California at Irvine, Aug. 22-27, 1999.
Morgan, J Alun W., et al., Sequence Analysis of Insecticidal Genes fromXenorhabdus nematophilusPMF1296, Applied and Environmental Microbiology, 67: 2062-2069 (May 2001).
Bowen David J.
ffrench-Constant Richard H.
Rocheleau Thomas A.
Waterfield Nicholas R.
Ibrahim Medina A.
McElwain Elizabeth F.
Stuart Donald R.
Wisconsin Alumni Research Foundation
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