Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
1999-07-20
2001-06-26
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C800S278000, C800S287000, C800S285000, C800S288000, C800S306000, C800S317200, C536S023100, C536S024100, C536S023700, C536S023600, C435S069100, C435S418000, C435S419000, C435S320100, C435S468000, C435S480000, C435S199000
Reexamination Certificate
active
06252138
ABSTRACT:
BACKGROUND OF THE INVENTION
(i) Field of the Invention
This invention pertains to new pathogen-induced promoters, particularly nematode-induced promoters, characterized by their selective induction of expression in the pathogen infection sites, particularly nematode infection sites of a plant. Provided in this invention are these new promoters, as well as chimeric gene constructs comprising these promoters as regulatory elements. Also provided herein are transgenic plants transformed to be less susceptible to nematode infection by the selectively induced expression of either a nematocidal toxin or a plant cell cytotoxic molecule upon nematode infection.
(ii) Description of Related Art
Plant-parasitic nematodes are small worm-like animals which feed on roots, stem or leaf tissues of living plants. They are present whenever plants are cultivated. Parasitic nematodes can cause significant plant yield losses, the most striking effect being a general reduction in plant growth. Often nematodes act as vectors for plant viruses. Plant diseases caused by nematodes include root galling, root lesions, root rot, stubby roots, stunting and wilting. In 1984, monetary losses, when all crops are considered, exceed US $ 100 billion annually (Sasser and Freckman, 1987). Ectoparasitic nematodes, such as the dagger (Xiphinema spp.) nematodes, live outside the plant and pierce the plant cells with their stylet in order to feed. Migratory endoparasitic nematodes, such as the lesion (Pratylenchus spp.) nematodes, live and feed inside the plant, migrating through the plant tissues. Sedentary endoparasitic nematodes, such as the root-knot (Meloidogyne spp.) and cyst (Globodera spp. and Heterodera spp.) nematodes, live and feed either completely or partially inside the plant, inducing specialized fixed feeding sites called giant cells, syncytia or nurse cells in susceptible plants. Such fixed feeding sites serve as food transfer cells for the various developmental stages of the nematodes. Syncytia originate in the pericycle, endodermis or adjacent cortex (Jones. 1981).
Several methods were designed to obtain nematode resistant plants by expression of chimeric genes. One strategy involves the nematode induced expression of nematocidal products, particularly peptides or proteins, preferably at the site of nematode feeding. Other strategies were designed to eliminate the formation or reduce the metabolic activity of the nematode feeding structures, e.g. by selective destruction of the feeding structures through the expression of cytotoxic molecules in the plant under control of a nematode feeding site-selective promoter. In some applications, a molecule inactivating the cytotoxic molecule is expressed at low levels throughout the plant to prevent cell killing in other plant cells due to background expression (WO 92/21757, WO 93/10251).
To expand the applicability of the abovementioned strategies, new nematode-induced promoters with improved timing, selectivity, and strength, are continuously searched for (e.g. Goddijn et al., 1993).
WO 95/32288 describes nucleic acid sequences, isolated by subtractive cDNA library construction, which are preferentially expressed in feeding site cells.
WO 93/18170 describes in general terms the isolation of nematode-responsive promoters and the use thereof to provide root knot nematode resistance to plants.
WO 93/10251 describes a method for obtaining plants with reduced susceptibility to plant-parasitic nematodes by providing chimeric genes that disrupt or at least retard the formation of a nematode feeding structure.
WO 92/04453 describes a method of controlling nematodes, the method including the steps of identification of a gene induced within a successfully infected plant by nematode infection of said plant and modifying the gene to confer nematode resistance to the plant.
WO 92/15690 and U.S. Pat. No. 5,494813 are directed to methods of controlling plant parasitic nematodes using proteinase inhibitors.
WO 92/21757 provides nematode-responsive plant promoters, particularly useful in the production of transgenic plants which can produce fixed feeding site cells that become capable of killing, disabling or repelling nematodes or that are themselves killed or rendered unsuitable for nematodes to feed upon when nematodes infect the plants.
Gurr et al. (1991) describe a gene identified by a library screen with cDNA probes with an expression pattern that correlated with event in the immediate vicinity of the pathogen after syncytial establishment.
Goddijn et al. (1993) describe down regulation in the feeding structures of a number of viral and bacterial promoters which are highly active in non-infected roots, and also describe a number of transgenic plant lines obtained by interposon tagging, using a T-DNA with a promoterless uidA (gus) gene, with either downregulated or enhanced gus expression in nematode feeding sites.
Opperman et al. (1994) describe cis-acting sequences from the TobRB7 promoter mediating induction by the nematode, and report the exclusive expression in the developing feeding site of reporter genes driven by the nematode-responsive promoter sequences.
SUMMARY OF THE INVENTION
In accordance with the invention isolated DNA fragments are provided that comprise the nucleotide sequence of SEQ ID No. 5 from nucleotide position 1055 to 1417, or the nucleotide sequence of SEQ ID No. 4 from nucleotide position 46 to 408, particularly an isolated DNA fragment comprising the nucleotide sequence of SEQ ID No. 4 from nucleotide position 46 to 573, especially an isolated DNA fragment comprising the DNA sequence of SEQ ID No. 8, or variant isolated DNA fragments which comprise a nucleotide sequence which is essentially similar to one of the provided sequences, in particular which has 90% sequence similarity with the nucleotide sequence of SEQ ID No. 5 from nucleotide position 1055 to 1417 or which has 90% sequence similarity with the nucleotide sequence of SEQ ID No 4 from nucleotide position 46 to 408.
Also in accordance with the invention, isolated DNA fragments are provided, particularly an isolated DNA fragment comprising the DNA sequence of the about 528 bp Sspl-Pvull fragment of plasmid pARM1a (LMBP3638), an isolated DNA fragment comprising the DNA sequence of the about 3 kb Pstl-Styl fragment of plasmid pch/ARM1 D3500 (LMBP3635), an isolated DNA fragment comprising the about 1.3 kb Smal fragment of plasmid pARM1a1300 (LMBP3636), or an isolated DNA fragment comprising the about 3.7 kb Smal fragment of plasmid pARM1 a3500 (LMBP3637).
Further in accordance with the invention chimeric genes are provided, comprising the following operably linked DNA fragments:
a. a plant-expressible promoter comprising the isolated DNA fragments of this invention
b. a foreign DNA region
c. a 3′ end formation and polyadenylation signal functional in plant cells.
Preferred chimeric genes of this invention are those, wherein the foreign DNA region encodes a proteinase inhibitor, or barnase. Further in accordance with this invention plant cells, plants and seeds comprising such chimeric genes are provided.
This invention further provides a method for preventing nematode-attack of a plant, comprising planting a plant harbouring an introduced foreign DNA which comprises any of the chimeric DNAs of this invention in a field susceptible to infection by nematodes; a method for combatting plant pathogens, particularly a root pathogen, which comprises expressing a foreign DNA in a plant under the control of a promoter comprising the cited DNA fragments, and the use of a promoter comprising the isolated DNA fragments of this invention to express a gene, i.e. produce a protein in fixed feeding sites or specialized root cells of a nematode infected plant.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENT
Throughout the description and examples the following definitions apply:
“Nematode feeding cells” or “fixed feeding sites” should be understood as specialized feeding sites (such as giant cells, syncytia and nurse cells or hypertrophic cells and, if present, galls), the formation of which is indu
Barthels Nathalie
Gheysen Godelieve
Karimi Mansour
Fox David T.
Hunton and Williams
Ibrahim Medina A.
Plant Genetic Systems N.V.
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