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
2001-09-17
2004-12-28
Kruse, David H (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
C800S279000, C800S288000
Reexamination Certificate
active
06835868
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to plants that are genetically engineered to express one or more peptides belonging to the temporin and/or dermaseptin families.
BACKGROUND OF THEE INVENTION
Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, and nematodes. These pathogens are responsible for significant crop losses worldwide, resulting from both infection of growing plants and destruction of harvested crops. The most widely practiced methods of reducing the damage caused by such pathogens involve the use of various chemical agents. Unfortunately, many pathogens develop resistance to such chemicals, and some pathogens (especially viruses) are not susceptible to control by chemical means. In addition, many of the chemical agents used are broad-spectrum toxins, and may cause serious environmental damage, as well as toxicity in humans.
Plant breeding and, more recently, genetic engineering techniques have also been employed to combat plant pathogens. In certain instances, breeders and molecular biologists have successfully engineered resistance to certain pathogens. In the last few years, a number of plant R (resistance) genes have been isolated from plants. When introduced into otherwise susceptible crops, these R genes produce enhanced resistance to certain pathogens. For example, U.S. Pat. No. 5,571,706 describes the isolation of the tobacco N gene, which confers enhanced resistance to Tobacco Mosaic Virus. However, while conventional breeding and genetic engineering approaches reported to date can successfully enhance pathogen resistance, they typically address problems caused by just one pathogen, or a small number of closely related pathogens. As a result, while crops produced using these approaches may have enhanced protection against one pathogen, conventional chemical agents must still be used to control others.
It would be of great agricultural benefit to be able to produce plants having enhanced resistance to a broad spectrum of pathogens, including bacterial and fungal pathogens. It is to such plants that the present invention is directed.
SUMMARY OF THE INVENTION
The present inventors have discovered that the expression of certain peptides in transgenic plants confers broad spectrum pathogen resistance, including enhanced resistance to both fungal and bacterial pathogens. The peptides in question are small, positively charged (cationic) peptides belonging to the temporin and dermaseptin families, which occur naturally in the skin of certain species of frog. Transgenic plants provided by the invention may be used in conventional agricultural applications, such as food crops. Alternatively, the plants may be harvested and processed to extract the expressed temporin and/or dermaseptin peptides, which may then be purified for use in medical and other applications.
The invention thus encompasses transgenic plants that express at least one dermaseptin or temporin peptide, and methods of making such plants. Parts of such plants, including seeds, fruit, stems, leaves and roots, may be utilized in conventional ways as food sources, or as a source of the dermaseptin or temporin peptides. Because all plant types are susceptible to one or more plant pathogens, the present invention may be usefully applied to produce broad-spectrum resistance in any plant type. Thus, the invention may be applied to both monocotyledonous, dicotyledenous and gymnosperm plants, including, but not limited to maize, wheat, rice, barley, soybean, beans in general, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut, clover, vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brussels sprouts, peppers; tree fruits such as citrus, apples, pears, peaches, apricots, walnuts; and flowers such as orchids, carnations and roses.
In its most basic form, the invention provides transgenic plants that express one or more dermaseptin and/or temporin peptides. Members of the dermaseptin and temporin peptide families are well known in the art. Examples of dermaseptins that may be used in the invention include, but are not limited to, the dermaseptins described by Mor. et al.,
Biochemistry,
30:8824-8830, 1991, Strahilevitz,
Biochemistry,
33:10951-10960, 1994 and Wechselberger,
Biochim. Biophys. Acta
1388: 279-283, 1998. Examples of temporins that may be used include, but are not limited to, the temporins described by Simmaco et al.,
Eur. J. Biochem.,
242:788-92, 1996. In their natural state (i.e., expressed in frog cells), both dermaseptin and temporin peptides are produced as precursor forms that are subsequently processed by proteolytic cleavage to form mature proteins. The mature forms of dermaseptins are typically about 27-34 amino acids in length, while the mature forms of temporins are typically about 10-13 amino acids in length. The invention contemplates the use of both the naturally occurring full-length (unprocessed) forms of these peptides, as well as the mature (processed) forms of the peptides and intermediate forms. In addition, synthetic forms of the peptides may also be employed. Synthetic forms of the peptides include any form that is not naturally occurring, and encompasses peptides that differ in amino acid sequence from the naturally occurring peptides, but which still retain dermaseptin or temporin biological activity. Such sequence variants will typically retain at least 40% amino acid sequence identity with at least one naturally occurring dermaseptin or temporin peptide.
Other synthetic forms of dermaseptins and temporins that may be employed include forms having N-terminal peptide extensions. Such peptide extensions may comprise portions of the precursor forms of dermaseptins or temporins that are usually removed during protein processing, or may be synthetic sequences. These N-terminal peptide extensions may serve to provide enhanced resistance to proteolytic cleavage, and may also enhance the antimicrobial activity of the peptides. Typically, these N-terminal extensions are of between 2 and 25 amino acids in length, although longer extensions may also be employed. Examples of N-terminal extension sequences that are utilized in certain embodiments include the peptide sequences MAMWK (amino acids 1-5 of SEQ ID NO: 28) and MASRH (amino acids 1-5 of SEQ ID NO. The AMWK sequence (amino acids 1-5 of SEQ ID NO: 28) is a naturally-occurring peptide extension; it is part of the full-length dermaseptin-b peptide sequence that is normally cleaved during processing. The ASRH (amino acids 1-5 of SEQ ID NO: 34) is a synthetic extension sequence. In each case, the N-terminal methionine is added to the extension peptide to ensure proper expression of the peptide.
While the fundamental aspect of the invention is based on the expression of temporin and dermaseptin peptides in transgenic plants, other amino acid sequences may be joined to the peptides in order to produce fusion peptides. Expression of such fusion peptides in transgenic plants may provide even more effective broad-spectrum pathogen resistance than expression of temporin or dermaseptin peptides alone, or may enhance stability of the expressed dermaseptin/temporin molecule to provide higher expression levels, and thereby facilitate purification of the peptide from plant tissues. Thus, in other embodiments, the invention provides transgenic plants that express a fusion peptide comprising:
(1) a first peptide sequence that is a dermaseptin or a temporin; and
(2) a second peptide sequence operably linked to the first peptide sequence.
The second peptide sequence is typically, but not necessarily, linked to the amino (N—) terminus of the first peptide sequence.
In certain embodiments, the second peptide sequence comprises an anionic (negatively charged) “pro-region” peptide sequence. Such pro-region peptides serve to X neutralize the cationic nature of the dermaseptin or temporin and may thus provide enhanced stability in the cellular environment. Thus, these
Kay William W.
Misra Santosh
Kruse David H
University of Victoria Innovation and Development Corporation
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