Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-01-21
2002-09-10
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S279000, C800S287000, C800S290000, C800S298000, C800S301000, C800S306000, C536S023100, C536S024100, C435S069100, C435S410000, C435S418000, C435S419000, C435S243000, C435S252300
Reexamination Certificate
active
06448471
ABSTRACT:
The invention relates to a gene which is specifically expressed in nematode feeding structures, the promoter which regulates said expression and methods for making plants that are resistant, or at least less susceptible to plant parasitic nematodes, or their effects, as well as to cells, plants and parts thereof.
STATE OF THE ART
In International patent application WO92/17054, a method is disclosed for the identification and subsequent isolation of nematode responsive regulatory DNA sequences from
Arabidopsis thaliana.
In WO 92/21757 several nematode-inducible regulatory DNA sequences have been isolated from
Lycopersicon esculentum
, which are responsive to the root-knot nematode
Meloidogyne incognita
. Some of these regulatory sequences (LEMMI's, for
Lycopersicon eaculentum—Meloidogyne incognita
) are stimulated, whereas others appear to be repressed by the nematode. of one of the responsive sequences LEMMI-9, the gene which is expressed by said regulatory sequence was elucidated, which showed to be homologous to LEA, a seed storage protein.
Another regulatory sequence that is inducible by the root-knot nematode
Meloidogyne incognita
is disclosed in WO 93/06710. Also here the gene normally expressed by this TobRb7 regulatory sequence was elucidated and it appears to be coding for a water-pore which facilitates water transport from the xylem to the cell. Recently, it has been reported that antisense expression of this gene in tobacco (the plant in which it naturally occurs) gives a decrease in infectibility by nematodes (Opperman, C. H. and Conkling, M. H., Third Int. Nematol. Congr. Guadeloupe, 1996, S-62). A disadvantage of this regulatory sequence TobRb7 is that it is not activated by a number of cyst nematodes, among which the Heterodera and Globodera species. This makes the TobRB7 sequence unsuitable for use in chimerical constructs aiming at, for example, cyst nematode resistance in potato.
It is an object of the invention to provide a gene with its regulatory DNA sequences which is at least inducible by both cyst nematodes and which can be used to suppress the presence and/or the function of the naturally occurring protein inside the feeding structure of the nematode, thereby conferring resistance to nematodes.
SUMMARY OF THE INVENTION
The invention provides a polynucleotide obtainable from
Arabidopsis thaliana
that is encoding a protein and its regulatory sequence which is specifically expressed in nematode feeding structures. Preferably the protein is having an amino acid sequence according to SEQ ID NO:2, SEQ ID NO:4 or a mutein thereof. Preferably the nucleotide sequence according to the invention is the sequence represented in SEQ ID NO:1 or SEQ ID NO:3. Also part of the invention is the regulatory sequence capable of promoting root knot and cyst nematode-inducible transcription of an associated DNA sequence when re-introduced into a plant. Preferably this regulatory sequence is the sequence depicted from nucleotide 1 to 4037 in SEQ ID NO:1 or pieces thereof which still can direct specific expression. A still further preferred aspect of the invention comprises a regulatory DNA fragment that is substantially nematode feeding site-specific.
Further embodiments of the invention comprise chimerical DNA sequences comprising a DNA sequence coding for (part of) the nematode feeding site specific protein of the invention, but expressed in antisense order, in order to block endogenous mRNA coding for the endogenous protein and thus giving resistance to nematodes. The regulatory element can further be used in chimerical DNA sequences comprising in the direction of transcription a regulatory DNA fragment according to the invention and a DNA sequence to be expressed under the transcriptional control thereof and which is not naturally under transcriptional control of said DNA fragment. Preferred among the chimerical DNA sequences according to the invention are those wherein the DNA sequence to be expressed causes the production of a plant cell-disruptive substance, such as barnase. In a different embodiment the cell-disruptive substance comprises RNA complementary to RNA essential to cell viability. Yet in another embodiment the DNA sequence to be expressed causes the production of a substance toxic to the inducing nematode.
The invention finds further use in a replicon comprising a DNA fragment or chimerical DNA sequence according to the invention, a microorganism containing such a replicon, as well as plant cells having incorporated into their genome a chimerical DNA sequence according to the invention. Further useful embodiments are a root system of a plant essentially consisting of cells according to the invention, as well as full grown plants essentially consisting of cells according to the invention, preferably a dicotyledonous plant, more preferably a potato plant. Also envisaged are plants grafted on a root system according to the invention, as well as plant parts selected from seeds, flowers, tubers, roots, leaves, fruits, pollen and wood and crops comprising such plants.
The invention also encompasses the method of antisense expression of the protein of the invention, thereby blocking expression of the endogenous protein and thereby inhibiting the formation and/or further development of a nematode feeding structure which will give resistance to nematode infection.
The invention further provides the use of a fragment, portion or variant of a regulatory DNA according to the invention for making hybrid regulatory DNA sequences.
REFERENCES:
patent: WO 93/0251 (1993-05-01), None
patent: WO9746692 (1997-12-01), None
Barthels et al. The Plant Cell. 1997. Dec. issue. vol. 9: 2119-2134.*
Kim et al. Plant Molecular Biology. 1994. vol. 24: 105-117.*
Chen et al. Plant J. 1996. Dec. issue. 10:6 955-966.
Grundler Florian M. W.
Puzio Piotr S.
Bui Phuong T.
Ladas & Parry
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