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
1999-01-14
2001-04-03
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
C435S320100, C435S419000, C536S023600, C800S279000
Reexamination Certificate
active
06211440
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INETION
Disease resistance genes are defined as Mendelian factors that cosegregate with the resistance trait. The genes Hm1 and Hm2 control resistance to
Cochliobolus carbonum
Nelson race 1 and are among the first disease resistance genes to be described. The disease caused by
C. carbonum
race 1 can be devastating, resulting in yield losses of 80% or more due to plant death and grain mold. In 1938, this disease was first reported in Indiana on an inbred line, Pr (Ullstrup, A. J. (1941)
Phytopathology
31, 508-521, which was bred in Iowa from an open-pollinated cultivar, Proudfit Reid (Gerdes, J. T., Behr, C. F., Coors, J. O. & Tracy, W. F. (1993)
Compilation of North American Maize Breeding Germplasm
(Am. Soc. Agron., Madison, Wis.)). Disease symptoms included grayish tan necrotic spots with concentric rings on the foliage, and infection often resulted in severe molding of the ears and premature killing of the plant. Hm1 and Hm2 are the only disease resistance genes that are known to be fixed at a high frequency in maize germplasm.
Genetic studies revealed that pathogenicity of
C. carbonum
race 1 is determined by a single locus, Tox2, which also confers the ability to produce HC toxin (Scheffer, R. P. & Ullstrup, A. J. (1965)
Phytopathology
55, 1037-1038; Scheffer, R. P., et al., (1967)
Phytopathology
57, 1288-1289). HC toxin is a cyclic tetrapeptide of the structure cyclo(D-prolyl-L-alanyl-D-alanyl-L-Aeo), where the unusual amino acid Aeo stands for 2-amino-9,10-epoxy-8-oxodecanoic acid (Gross, M. L, et al., (1982)
Tetrahedron Lett.
23, 5381-5384, Walton, J. D., et al., (1982)
Biochem. Biophys. Res. Commun.
107, 785-794), and appears to be the sole determinant of pathogenicity. Genetic variants that do not produce HC toxin are unable to colonize much beyond the site of penetration and, therefore, cause only chlorotic or necrotic flecks on leaves (Conistock, J. C. & Scheffer, R. P. (1973)
Phytopathology
63, 24-29, Panaccione, D. C., et al., (1992)
Proc. NatL Acad. Sci. USA
39, 6590-6594). The Tox2 locus has been cloned and found to encode the enzymes required for the biosynthesis of HC toxin (Panaccione, et al. supra; Scott-Craig, J. S., et al., (1992)
J. Biol Chem.
267, 26044-26049). It is not clear how HC toxin allows colonization of susceptible maize. However, by virtue of its inhibitory action on histone deacetylases, HC toxin may interfere with the induction of defense genes in maize, thereby leaving the plant vulnerable to colonization by the pathogen (Brosch, et al.,
Plant Cell
7:1941-1950 (1995)).
C. carbonum
race 1 is one of the most aggressive pathogens of maize. Fortunately, most maize germplasm is resistant. The dominant gene, Hm1 confers complete protection (Ullstrup, A. J. (1941)
J. Agric. Res.
63, 331-334; Meeley, R. B., et al.,
Advances in Molecular Genetics of Plant
-
Microbe Interactions,
463-467, Nester and Verma (eds.) Kluwer Academic Publishers, Netherlands), and it maps to the long arm of chromosome 1 (1L). Another gene, Hm2, provides partial, adult plant resistance, and it maps to 9L (Nelson, O. E. & Ulistrup, A. J. (1964)
J. Hered.
55, 195-199). The cloning of the Hm1 gene has revealed that, by encoding HC toxin reductase (HCTR), Hm1 inactivates HC toxin, and this result is sufficient to prevent infection (Johal, G. S. & Briggs, S. P. (1992)
Science
258, 985-987; Meeley, R. B., et al., (1992)
Plant Cell
4, 71-77; Briggs, et al., U.S. Pat. No. 5,589,611. The foregoing references are herein incorporated by reference.).
In the present invention the Hm2 polynucleotide sequence has been cloned and sequenced. The Hm2 polynucleotide functions to inactivate HC toxin and other cyclic tetrapeptides such as cyl-2, chlamydocin, apicidin and WF-3161 (Walton, J. D.,
Biochemistry of Peptide Antibiotics,
179-203, Kleinkauf and Dohren (eds.), Walter de Gruyter Berlin, New York (1990); Darkan-Rattray et al,
Proc. Natl. Acad. Sci. USA
93:13143-13147 (1996). The foregoing references are herein incorporated by reference.).
Therefore, the Hm2 polynucleotide can be used to prevent fungal infection in plants or as a selectable marker gene in plant transformation.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to Hm2. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention and methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a); and, (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter or expression cassettes containing the promoter of the present invention operably linked to a heterologous gene.
In another aspect, the present invention is directed to a host cell into which has been introduced the expression cassette.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having a specified number of contiguous amino acids encoded by an isolated nucleic acid of the present invention.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of specified length, which selectively hybridizes under stringent conditions to a polynucleotide of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In another aspect, the present invention relates to an expression cassette comprising a nucleic acid amplified from a library as referred to supra, wherein the nucleic acid is operably linked to a promoter. In some embodiments, the present invention relates to a host cell transfected with this expression cassette. In some embodiments, the present invention relates to a protein of the present invention which is produced from this host cell.
In yet another aspect, the present invention relates to a transgenic plant comprising an expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. The present invention also provides transgenic seed from the transgenic plant.
Additionally, the Hm2 polynucleotides can be inserted using conventional transformation methods into the genomes of plants, which lack the gene and are susceptible to disease caused by fungal pathogens utilizing HC toxin or other cyclic tetrapeptide toxins. Resulting transformants are resistant to the disease.
Further, the Hm2 polynucleotide can be used in conjunction with the HC-toxin or other cyclic tetrapeptide toxins in a selectable marker system for use in plant transformation. When the cloned Hm2 polynucleotide is linked to appropriate regulatory sequences for expression in plant cells and cotransformed into plant cells along with another quantitative or qualitative trait which is not selectable, it confers upon transfonnants a resistance to HC toxin or other cyclic tetrapeptide toxins by virtue of the production of the Hm2 polypeptide. The cells can continue to grow on medium containing the isolated toxin. Nontransformed cells do not express the Hm2 polypeptide and are rapidly killed by the toxin the pathogen produces. The net effect is a tissue culture containing only transformed cells, which can then be regenerated by known methods to form transformed shoots and even who
Briggs Steven P.
Johal Gurmukh
Multani Dilbag Singh
Nelson Amy J.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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