Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-04-09
2003-11-25
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C424S404000, C530S324000, C530S370000
Reexamination Certificate
active
06653280
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an antifungal polypeptide, AlyAFP, obtainable from flowers of plants in the genus Alyssum, and methods for controlling pathogenic fungi employing this antifungal polypeptide. The antifungal polypeptide can be applied directly to a plant, applied to a plant in the form of microorganisms that produce the polypeptide, or plants themselves can be genetically modified to produce the polypeptide. The present invention also relates to DNA sequences, microorganisms, plants, and compositions useful in these methods.
2. Description of Related Art
A number of plant polypeptides and proteins exhibiting antifungal activity against a variety of plant pathogenic fungi have been isolated (Bowles (1990)
Annu. Rev. Biochem.
59:873-907; Brears et al. (1994)
Agro
-
Food
-
Industry Hi
-
Tech.
10-13). These antifungal polypeptides and proteins, encompassing several classes including chitinases, cysteine-rich chitin-binding proteins, &bgr;-1,3-glucanases, permatins (including zeamatins), thionins, ribosome-inactivating proteins, and non-specific lipid transfer proteins, are believed to play important roles in plant defense against fungal infection.
Recently, another group of plant proteins has been found to function as defensins in combatting infections by plant pathogens (PCT International Publication WO 93/05153). Two small cysteine-rich proteins isolated from radish seed, Rs-AFP1 and Rs-AFP2, were found to inhibit the growth of many pathogenic fungi when the pure protein was added to an in vitro antifungal assay medium. Transgenic tobacco plants containing the gene encoding Rs-AFP2 protein were found to be more resistant to attack by fungi than non-transformed plants.
Proteins similar to radish seed Rs-AFP2 have been isolated from seeds of many other plants (PCT International Publication WO 93/05153; Broekaert et al. (1995)
Plant Physiol.
108:1353-1358). All the proteins in this group share similarity in their amino acid sequence, but differ in their antifungal activities against various fungi, especially in the presence of different mono- and divalent salts in the assay medium, which more closely resembles the physiological condition in plant cells: the antifungal activity of some antifungal proteins is dramatically reduced in the presence of 1 mM CaCl
2
and 50 mM KCl (Terras et al. (1992)
J. Biol. Chem.
267:15301-15309). The usefulness of an antifungal protein for genetically engineering plant disease resistance can be greatly influenced by the sensitivity of the antifungal activity to salt concentration, since metal ions such K
+
, Na
+
, Ca
2+
, and Mg
2+
are required for normal physiological functions and are therefore abundantly present in plant cells.
The use of natural protein products to control plant pathogens has been demonstrated, for example, in European Patent Application 0 392 225.
SUMMARY OF THE INVENTION
The present inventors have discovered a new polypeptide, AlyAFP, exhibiting broad spectrum antifungal activity against plant pathogenic and other fungi. In one aspect, the present invention provides an isolated antifungal polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, and biologically functional equivalents thereof.
AlyAFP, or biologically functional equivalents thereof, can be isolated from plants, or produced or synthesized by any suitable method known in the art, including direct chemical synthesis, synthesis in heterologous biological systems such as microbial, plant, and animal systems, tissue cultures, cell cultures, or in vitro translation systems.
The present invention also provides isolated DNA sequences encoding the antifungal polypeptides of the present invention, and genetic constructs and methods for inserting such DNA sequences into host cells for the production of the polypeptides encoded thereby.
The present invention also provides microorganisms and plants transformed with DNA nucleotide sequences encoding the antifungal polypeptides according to the present invention.
The present invention provides transformed plants that express antifungal polypeptides according to the invention, as well as plants that co-express these antifungal polypeptides along with other antifungal, antibacterial, or antiviral pathogenesis-related peptides, polypeptides, or proteins; insecticidal proteins, e.g.,
Bacillus thuringiensis
(B.t.) proteins; and proteins involved in improving the quality of plant products or agronomic performance of plants. Simultaneous co-expression of multiple antifungal proteins in plants is advantageous in that it exploits more than one mode of action to control fungal damage. This can minimize the possibility of developing resistant fungal strains, broaden the scope of resistance, and potentially result in a syngergistic antifungal effect, thereby enhancing the level of resistance. Note WO 92/17591, for example, in this regard.
Examples of plants transformed to express B.t. genes are disclosed in European Patent Publication 0 385 962, which corresponds to U.S. Ser. No. 07/476,661, filed Feb. 12, 1990, by Fischhoff et al.
Non-limiting examples of DNAs that can be co-expressed along with DNAs encoding the polypeptides of the present invention include 1) DNAs encoding enzymes such as: glucose oxidase (which converts glucose to gluconic acid, concomitantly producing hydrogen peroxide which confers broad spectrum resistance to plant pathogens); pyruvate oxidase; oxylate oxidase; cholesterol oxidase; amino acid oxidases; and other oxidases that use molecular oxygen as a primary or secondary substrate to produce peroxides, including hydrogen peroxide; 2) pathogenesis related proteins such as SAR8.2a and SARB.2b proteins; the acidic and basic forms of tobacco PR-1a, PR-1b, PR-1c, PR-1′, PR-2, PR-3, PR-4, PR-5, PR-N, PR-O, PR-O′, PR-P, PR-Q, PR-S, and PR-R proteins; chitinases such as tobacco basic chitinase and cucumber chitinase/lysozyme; peroxidases such as cucumber basic peroxidase; glucanases such as tobacco basic glucanase; osmotin-like proteins; 3) viral capsid proteins and replicases of plant viruses; 4) plant R-genes (resistance genes), such as Arabidopsis RPS2 (Bent et al. (1994)
Science
265:1856-1860), Arabidopsis RPM1 (Grant et al. (1995)
Science
269:843-846), tobacco N-gene and N′-gene (Whitham et al. (1994)
Cell
78:1101-1115), tomato Cf-9 (Jones et al. (1994)
Science
266:789-793), flax L
6
(Ellis et al. (1995)
Proc. Natl. Acad. Sci. U.S.A.
92: 4185), and rice Xa21 (Song et al. (1995)
Science
270: 1804-1806). These genes can be expressed using constitutive promoters, tissue-specific promoters, or promoters inducible by fungal pathogens or other biological or chemical inducers; 5) pathogen Avr genes, such as
Cladosporium fulvum
Avr9 (Van Den Ackerveken et al. (1992)
Plant J.
2:359), that can be expressed using pathogen- or chemical-inducible promoters; and 6) genes that are involved in the biosynthesis of salicylic acid, such as benzoic acid 2-hydroxylase (Leon et al. (1995)
Proc. Natl. Acad. Sci. USA
92:10413-10417).
A number of publications have discussed the use of plant and bacterial glucanases, chitinases, and lysozymes to produce transgenic plants exhibiting increased resistance to various microorganisms such as fungi. These include EP 0 292 435, EP 0 290 123, WO 88/00976, U.S. Pat. No. 4,940,840, WO 90/07001, EP 0 392 225, EP 0 307 841, EP 0 332 104, EP 0 440 304, EP 0 418 695, EP 0 448 511, and WO 91/06312. The use of osmotin-like proteins is discussed in WO 91/18984.
In accomplishing the foregoing, there is provided in accordance with various aspects of the present invention:
An isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
An isolated DNA molecule encoding this isolated polypeptide. The isolated DNA molecule can be a cDNA molecule comprising the nucleotide sequence shown in SEQ ID NO:12. Alternatively, this cDNA molecule can comprise nucleotides 116 to 269 of the nucleotide sequence shown in SEQ ID NO:12.
A recombinant, double-stranded DN
Liang Jihong
Rosenberger Cindy Annette
Shah Dilip Maganlal
Wu Yonnie Shun
Ball Timothy K.
Hoerner, Jr. Dennis R.
Low Christopher S. F.
Mayes Laurie
Monsanto Technology LLC
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