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
2000-05-22
2004-08-31
Kubelik, Anne R. (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
Reexamination Certificate
active
06784337
ABSTRACT:
the present invention disclouses a method of improving pathogen resistance or tolerance of a plant in that the plant is transformed with a transgene encoding a fusion protein of two or more proteins or protein domains which are capable of improving pathogen resistance or tolerance when expressed on their own. The invention is exemplified by codelivery of two distinct proteinase inhibitors as a fusion protein in
Arabidopsis thaliana
leading to improved resistance or tolerance of plant parasitic nematodes. For purposes of the present invention, it is recognized that transgenic plants obtained according to the present invention can be tolerant or resistant not only to nematodes but also to viruses, fungi, bacteria, insects, mites, and the like.
Nematodes are the principal animal parasites of plants causing global losses to agriculture estimated at >$100 billion each year. Improved plant resistance to parasitic nematodes is highly desirable to reduce the need for nematicides some of which belong to the most unacceptable pesticides used in agriculture. There are several possible approaches for developing transgenic plants with improved nematode resistance which include anti-invasion and migration strategies, feeding-cell attenuation and anti-nematode feeding strategies (Atkinson et al., Tibtech 13:369-374, 1995). This latter approach can utilize proteinase inhibitors (PIs) which are an important element of natural plant defence strategies (Ryan, Annu Rev Phytopathol 28:425-49, 1990). There are ten PI groups characterised from plants spanning all four classes of proteinases, namely cysteine-, serine-, metallo- and aspartyl-proteinases (Richardson, Methods in Plant Biochemistry 5: 259-305, 1991). EP-A-502 730 discloses that effective, PI-based transgenic defences can be achieved for nematodes. One of the preferred attributes of Pis in nematode control is their small size. The potential of Pis for transgenic crop protection is enhanced by a lack of harmful effects of many PIs when consumed by humans. cDNAs encoding cysteine and serine digestive proteinases of a cyst nematode have been cloned, their major proteolytic activity has been localised to the intestine, and the Pis CpTI and oryzacystatin (Oc-I) have been shown to be effective against these proteinases. Site-directed mutagenesis led to an improved K
i
of Oc-I following the deletion of one amino acid. This modified cystatin (Oc-I&Dgr;86) has enhanced efficacy as a transgene against potato cyst-nematode (Urwin et al., Plant J 8:121-131, 1995). When expressed in Arabidopsis it limits growth of both the cyst nematode Heterodera schachtii, and the root-knot nematode Meloidogyne incognita.
Progeny of a cross of transgenic tobacco expressing CpTI and pea lectin, respectively, showed additive efficacy against tobacco budworm (Boulter et al. Crop Protection, 9:351-354, 1990). Tandem promoter/gene constructs might achieve a similar result without the need for crossing plants. Nature suggests at least two further alternative ways of achieving expression of more than one inhibitor, namely bifunctional inhibitors (Wen et al, Plant Mol Biol 18:813-814, 1992) and multi-domain Pis (Waldron et al., Plant Mol Biol 23:801-812, 1993).
It is the object of the present invention to provide methods for the improvement of pathogen resistance or tolerance by delivering more than one resistance or tolerance effector protein. For the purpose of the present invention resistance describes the effect of an introduced transgene to restrict or prevent pathogen multiplication in or on the transgenic plant. Tolerance relates to the ability of the transgenic plant to withstand or recover from damaging effects of pathogen attack and to yield well. Both resistance to and tolerance of a pathogen result in reduction of damage to the crop caused by the pathogen.
The invention thus provides:
A method of improving pathogen resistance or tolerance in a plant and its descendant plants comprising integrating into the genome of said plant a gene encoding a fusion protein comprising
(a) a first protein or protein domain with anti-pathogenic activity:
(b) a linker peptide; and
(c) a second protein or protein domain with anti-pathogenic activity.
In particular, the invention provides methods, genes and proteins as mentioned before, wherein
further proteins or protein domains with anti-pathogenic activity are fused to the fusion protein by linker peptides
at least one of the proteins or protein domains with antipathogenic activity has proteinase inhibitor activity
at least one of the proteins or protein domains with anti-pathogenic activity is the proteinase inhibitor Oc-I&Dgr;D86
at least one of the proteins or protein domains with antipathogenic activity is the proteinase inhibitor CpTI
the gene is functionally linked to a promoter sequence driving expression preferentially in plant roots
the linker peptide comprises an amino acid sequence which is proteolytically cleaved by the plant
the linker peptide comprises an amino acid sequence which is proteolytically stable in the plant
the linker peptide is characterized by comprising the amino acid sequence QASSYTAPQPQ (SEQ ID NO:2)
the linker peptide is characterized by comprising the amino acid sequence VILGVGPAKIQFEG (SEQ ID NO:1)
the linker peptide is characterized by comprising the amino acid sequence QASIEGRYTAPQPQ (SEQ 1D NO: 11)
nematode resistance or tolerance is improved
The invention further provides transgenic plants that are obtainable by the method mentioned before. In particular, the invention provides:
a plant expressing a fusion protein encoded by a DNA molecule according to the invention
In addition, the invention allows use of the DNA molecules described to improve pathogen resistance or tolerance of a plant and its descendant plants.
To assist in the understanding of the present invention frequently used terms are explained in more detail below:
A plant refers to any plant, particularly to seed plants. The structural and physiological unit of plants are plant cells, comprising a protoplast and a cell wall. The term ‘plant cell’ refers to any cell which is either part of or derived from a plant. Examples of cells include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; the cells of undifferentiated tissue such as callus or tumors; differentiated cells of seeds, embryos, propagules or pollen. In particular, the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
A group of plant cells can be organized into a structural and functional unit called plant tissue. This term includes, but is not limited to, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units.
Plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
A plant or cell having stably incorporated recombinant DNA in its genome will be referred to as transgenic plant or cell.
Transformation refers to the introduction of a nucleic acid into a cell, particularly to the stable integration of a DNA molecule into the genome of an organism of interest.
Said recombinant DNA refers to one or more DNA molecules formed by joining DNA segments from different sources and obtained using recombinant DNA technology as described, for example, by Sambrook et al., in: “Molecular Cloning-A Laboratory Manual”, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA (1989). Recombinant DNA technology produces recombinant DNA in vitro and transfers it into cells where it can be expressed or propagated (See, Concise Dictionary of Biomedicine and Molecular Biology, Ed. Juo, CRC Press, Boca Raton (1996)), for example, transfer of DNA into a protoplast(s) or cell(s) in various forms, including, for example, (1) naked DNA in circular, linear or super
Atkinson Howard John
McPherson Michael John
Urwin Peter Edward
Kakefuda Mary
Kubelik Anne R.
Syngenta Participations (AG)
LandOfFree
Method of improving nematode resistance in plants via... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of improving nematode resistance in plants via..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of improving nematode resistance in plants via... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3313273