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
2001-10-23
2004-04-13
Nelson, Amy J. (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, C435S320100, C435S419000, C536S024100
Reexamination Certificate
active
06720480
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of the genetic manipulation of plants, particularly the modulation of gene activity and development in plants and increased disease resistance.
BACKGROUND OF THE INVENTION
Disease in plants is caused by biotic and abiotic causes. Biotic causes include fungi, viruses, bacteria, and nematodes. An example of the importance of plant disease is illustrated by phytopathogenic fungi, which cause significant annual crop yield losses as well as devastating epidemics. Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. Pathogenic fungi attack all of the approximately 300,000 species of flowering plants, however, a single plant species can be host to only a few fungal species, and similarly, most fungi usually have a limited host range. Generally, the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose. However, the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. Molecular methods of crop protection have the potential to implement novel mechanisms for disease resistance and can also be implemented more quickly than traditional breeding methods. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack.
A host of cellular processes enable plants to defend themselves against disease caused by pathogenic agents. These defense mechanisms are activated by initial pathogen infection in a process known as elicitation. In elicitation, the host plant recognizes a pathogen-derived compound known as an elicitor; the plant then activates disease gene expression to limit further spread of the invading microorganism. It is generally believed that to overcome these plant defense mechanisms, plant pathogens must find a way to suppress elicitation as well as to overcome more physically-based barriers to infection, such as reinforcement and/or rearrangement of the actin filament networks near the cell's plasma membrane.
Thus, the present invention solves needs for enhancement of the plant's defensive elicitation response via a molecularly based mechanism which can be quickly incorporated into commercial crops.
SUMMARY OF THE INVENTION
Nucleic acids and proteins relating to plant disease resistance are provided. In particular, proteinase inhibitor-like nucleic acid and protein sequences and a nucleotide sequence comprising an inducible promoter are provided. The nucleic acid sequences can be used to alter the level, tissue, or timing of expression of the plant genes to achieve enhanced disease resistance. Transgenic plants comprising the nucleic acids of the present invention are also provided. Methods for modulating the expression of the nucleic acids in a transgenic plant are additionally disclosed.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising (a) a polynucleotide encoding proteinase inhibitor-like polypeptides of the present invention; (b) a polynucleotide comprising at least 20 contiguous bases of the polynucleotides of the present invention; (c) a polynucleotide having at least 60% sequence identity to the polynucleotides of the present invention; and (d) a polynucleotide complementary to a polynucleotide of (a) through (c). The isolated nucleic acid can be DNA. The isolated nucleic acid can also be RNA.
In another aspect, the present invention relates to vectors comprising the polynucleotides of the present invention. Also, the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter. In addition, the present invention relates to recombinant expression cassettes.
In another aspect, the present invention is directed to a host cell having stably incorporated the the recombinant expression cassette.
In yet another aspect, the present invention relates to a transgenic plant or plant cell comprising a recombinant expression cassette with a promoter operably linked to any of the isolated nucleic acids of the present invention. Preferred plants containing the recombinant expression cassette of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice barley, and millet. The present invention also provides transgenic seed from the transgenic plant.
In another aspect, the present invention relates to an isolated protein comprising (a) a proteinase inhibitor-like polypeptide; (b) a polypeptide comprising at least 20 contiguous amino acids of SEQ ID NO: 2; (c) a polypeptide comprising at least 60% sequence identity to SEQ ID NO: 2; (d) a polypeptide encoded by a nucleic acid of the present invention; (e) a polypeptide characterized by SEQ ID NO: 2; and (f) a conservatively modified variant of SEQ ID NO: 2.
In a further aspect, the present invention relates to a method of modulating the level of protein in a plant by introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; culturing the plant cell under plant growing conditions to produce a regenerated plant; and inducing expression of the polynucleotide for a time sufficient to modulate the protein of the present invention in the plant. Preferred plants of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet. The level of protein in the plant can either be increased or decreased.
In another aspect, the present invention further relates to recombinant expression cassettes, comprising an inducible promoter of the present invention operably linked to a nucleotide sequence of interest. In addition, the present invention relates to the recombinant expression cassettes comprising such DNA constructs.
In further aspect, the present invention relates to a method of modulating the level of a nucleotide sequence of interest in a plant by introducing into a plant cell a recombinant expression cassette comprising a nucleotide sequence of interest operably linked to the proteinase inhibitor-like promoter of the invention; and culturing the plant cell under plant growing conditions to produce a regenerated plant. Exposure of the transformed plant to a stimulus regulates transcription of the nucleotide sequence of interest. Preferred plants of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.
DETAILED DESCRIPTION OF THE INVENTION
Compositions of the invention comprise a proteinase inhibitor-like sequence. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO: 2. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example those set forth in SEQ ID NO:1 and fragments and variants thereof.
The present invention provides a novel proteinase inhibitor-like sequence. Proteinase inhibitor-like sequences have antifungal and antimicrobial activity. Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. Other plant tissues also express proteinase inhibitors. Members of both the Bowmann-Birk and the double-headed proteinase inhibitor family have been shown to be induced in response to wounding, and hence have been implicated in the plant defense response. Thus, increased expression of the genes that encode the proteinase inhibitor-like proteins of the present invention find use in enhancing disease resistance of plants. It is further recognized that the decreased expression of the sequences of the invention may lead to nutrient enhancement.
By “disease resistance” is intended that the plants avoid the disease s
Crane Virginia C.
Simmons Carl R.
Kubelik Anne
Nelson Amy J.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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