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
2003-05-12
2004-04-06
Fox, David T. (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
C435S418000, C435S419000, C536S023700, C800S320100, C800S320200, C800S317300, C800S288000
Reexamination Certificate
active
06717035
ABSTRACT:
BACKGROUND OF THE INVENTION
As reported in WO98/08932, 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 the native toxin genes.
Of the separate toxins isolated from
Photorhabdus luminescens
(W-14), those designated Toxin A and Toxin B are especially potent 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.
Toxin B is similarly comprised of two different subunits. The native gene tcbA (SEQ ID NO:2) encodes protoxin TcbA (see SEQ ID NO:2). As determined by mass spectrometry, TcbA is processed by one or more proteases to provide Toxin B. More specifically, TcbA is an approximately 280.6 kDA (2504 aa) protein that is processed to provide TcbAii, an approximately 207.7 kDA (1844 aa) protein encoded by nucleotides 262-5793 of SEQ ID NO:2 and TcbAiii, an approximately 62.9 kDA (573 aa) protein encoded by nucleotides 5794-7512 of SEQ ID NO:2.
The native tcdA and tcbA genes are not well suited for high level expression in plants. They encode multiple destabilization sequences, mRNA splice sites, polyA addition sites and other possibly detrimental sequence motifs. In addition, the codon compositions are not like those of plant genes. WO98/08932 gives general guidance on how the toxin genes could be reengineered to more efficiently expressed in the cytoplasm of plants, and describes how plants can be transformed to incorporate the Photorhabdus toxin genes into their genomes.
SUMMARY OF THE INVENTION
In a preferred embodiment, the invention provides novel polynucleotide sequences that encode TcdA and TcbA. The novel sequences have base compositions that differ substantially from the native genes, making them more similar to plant genes. The new sequences are suitable for use for high expression in both monocots and dicots, and this feature is designated by referring to the sequences as the “hemicot” criteria, which is set forth in detail hereinafter. Other important features of the sequences are that potentially deleterious sequences have been eliminated, and unique restriction sites have been built in to enable adding or changing expression elements, organellar targeting signals, engineered protease sites and the like, if desired.
In a particularly preferred embodiment, the invention provides polynucleotide sequences that satisfy hemicot criteria and that comprise a sequence encoding an endoplasmic reticulum signal or similar targeting sequence for a cellular organelle in combination with a sequence encoding TcdA or TdbA.
More broadly, the invention provides engineered nucleic acids encoding functional Photorhabdus toxins wherein the sequences satisfy hemicot criteria.
The invention also provides transgenic plants with genomes comprising a novel sequence of the invention that imparts functional activity against insects.
BRIEF DESCRIPTION OF SEQUENCES
SEQ ID NO:1 is the native tcdA DNA sequence together with the corresponding encoded amino acid sequence for TcdA.
SEQ ID NO:2 is the native tcbA DNA sequence together with the corresponding encoded amino acid sequence for TcbA.
SEQ ID NO:3 is an artificial sequence encoding TcdA that is suitable for expression in monocot and dicot plants.
SEQ ID NO:4 is an artificial sequence encoding TcbA that is suitable for expression in monocot and dicot plants.
SEQ ID NO:5 is an artificial hemicot sequence that encodes the 21 amino acid ER signal peptide of 15 kDa zein from Black Mexican Sweet maize.
SEQ ID NO:6 is an artificial hemicot sequence that encodes for the full-length native TcdA protein (amino acids 22-2537) fused to the modified 15 kDa zein endoplasmic reticulum signal peptide (amino acids 1-21).
DETAILED DESCRIPTION
The native Photorhabdus toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus. Of particular interest are the proteins produced by the species
Photorhabdus luminescens
. The protein complexes have a molecular size of approximately 1,000 kDa and can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities. The toxins exhibit significant toxicity upon ingestion by a number of insects.
A unique feature of Photorhabdus is its bioluminescence. Photorhabdus may be isolated from a variety of sources. One such source is nematodes, more particularly nematodes of the genus Heterorhabditis. Another such source is from human clinical samples from wounds, see Farmer et al. 1989 J. Clin. Microbiol. 27 pp. 1594-1600. These saprohytic strains are deposited in the American Type Culture Collection (Rockville, Md.) ATCC #s 43948, 43949, 43950, 43951, and 43952, and are incorporated herein by reference. It is possible that other sources could harbor Photorhabdus bacteria that produce insecticidal toxins. Such sources in the environment could be either terrestrial or aquatic based.
The genus Photorhabdus is taxonomically defined as a member of the Family Enterobacteriaceae, although it has certain traits a typical of this family. For example, strains of this genus are nitrate reduction negative, yellow and red pigment producing and bioluminescent. This latter trait is otherwise unknown within the Enterobacteriaceae. Photorhabdus has only recently been described as a genus separate from the Xenorhabdus (Boemare et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This differentiation is based on DNA—DNA hybridization studies, phenotypic differences (e.g., presence (Photorhabdus) or absence (Xenorhabdus) of catalase and bioluminescence) and the Family of the nematode host (Xenorhabdus; Steinernematidae, Photorhabdus; Heterorhabditidae). Comparative, cellular fatty-acid analyses (Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401) support the separation of Photorhabdus from Xenorhabdus.
Currently, the bacterial genus Photorhabdus is comprised of a single defined species,
Photorhabdus luminescens
(ATCC Type strain #29999, Poinar et al., 1977, Nematologica 23, 97-102). A variety of related strains have been described in the literature (e.g., Akhurst et al. 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol. 43 pp. 249-255; Putz et al. 1990, Appl. Environ. Microbiol., 56, 181-186).
The following toxin producing Photorhabdus strains have been deposited:
strain
accession number
date of deposit
W-14
ATCC 55397
Mar. 5, 1993
WX1
NRRL B-21710
Apr. 29, 1997
WX2
NRRL B-21711
Apr. 29, 1997
WX3
NRRL B-21712
Apr. 29, 1997
WX4
NRRL B-21713
Apr. 29, 1997
WX5
NRRL B-21714
Apr. 29, 1997
WX6
NRRL B-21715
Apr. 29, 1997
WX7
NRRL B-21716
Apr. 29, 1997
WX8
NRRL B-21717
Apr. 29, 1997
WX9
NRRL B-21718
Apr. 29, 1997
WX10
NRRL B-21719
Apr. 29, 1997
WX11
NRRL B-21720
Apr. 29, 1997
WX12
NRRL B-21721
Apr. 29, 1997
WX14
NRRL B-21722
Apr. 29, 1997
WX15
NRRL B-21723
Apr. 29, 1997
H9
NRRL B-21727
Apr. 29, 1997
Hb
NRRL B-21726
Apr. 29, 1997
Hm
NRRL B-21725
Apr. 29, 1997
HP88
NRRL B-21724
Apr. 29, 1997
NC-1
NRRL B-21728
Apr. 29, 1997
W30
NRRL B-21729
Apr. 29, 1997
WIR
NRRL B-21730
Apr. 29, 1997
B2
NRRL B-21731
Apr. 29, 1997
ATCC 43948
ATCC 55878
Nov. 5, 1996
ATCC 43949
ATCC 55879
Nov. 5, 1996
ATCC 43950
ATCC 55880
Nov. 5, 1996
ATCC 53951
ATCC 558
Guo Lining
Herman Rod A.
Merlo Ann Owens
Merlo Donald J.
Petell James K.
Dow AgroSciences LLC
Fox David T.
Kruse David H
Stuart Donald R.
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