Maize chitinases and their use in enhancing disease...

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

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C800S286000, C800S298000, C800S320100, C800S312000, C800S322000, C800S306000, C800S320200, C800S320300, C800S320000, C800S314000, C435S418000, C435S419000, C435S412000, C435S415000, C435S416000, C435S200000, C536S023600, C536S023200

Reexamination Certificate

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06563020

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 INVENTION
Disease in plants is caused by biotic and abiotic causes. Biotic causes include fungi, viruses, insects, bacteria, and nematodes. Of these, fungi are the most frequent causative agents of disease in plants. Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution.
As noted, among the causative agents of infectious disease of crop plants, the phytopathogenic fungi play the dominant role. Plytopathogenic fungi cause devastating epidemics, as well as causing significant annual crop yield losses. Pathogenic fungi attack all of the approximately 300,000 species of flowering plants.
Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. Generally, the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose. Typically, this involved elaborate breeding to incorporate natural resistance mechanisms into elite breeding material. The sources of this natural resistance were often otherwise undesirable plant materials, and so extensive backcrossing and introgression was needed to recreate the desired background with the disease resistance. Sometimes even this was not obtained, as the resistance mechanism(s) were polygenic. In short, improving disease resistance by conventional breeding is expensive in both time and money.
Increasingly various genetic engineering strategies are being put forth to create enhanced disease resistance using recombinant DNA technology and transgenic plants. Sometimes this involves isolation of a resistance gene and then discreetly inserting it into a susceptible plant by transformation. Other strategies involve engineering elevated expression of antimicrobial compounds, reactive oxygen species, which are known to be antimicrobial and/or stimulators of plant defense systems.
The potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. What is needed in the art are compositions and methods for overcoming the conventional breeding method and existing genetic engineering strategies by providing discrete novel genes encoding antimicrobial/antifungal proteins. Chitinases are one such class of genes. These genes encode enzymes which hydrolyze beta-1,4-linkages in chitin, a polymer of N-acetyl-D-glucosamine. Chitin, the substrate of chitinase enzymes, is present in fungal cell walls and in the exoskeletons of insects, nematodes, and some other organisms. Consequently, chitinases have antibiotic action against such organisms, a variety of which are pathogenic on plants.
Chitinases are divided into two main groups: those of the glucosyl hydrolase family 19, which is specific to plants, and which exhibits only chitinase activity; and the glucosyl hydrolase family 18, which are chitinases, but which sometimes also have lysosyme activity. Lysozymes degrade mixed linked polymers of N-acetyl-glucosamine and N-acetyl-muramic acid. These polymers are found in bacterial cell walls. As such, those chitinases of the glucosyl hydrolase family
18
will also find utility in combating bacterial plant pathogens.
The glucosyl hydrolase family
19
is further divided into classes I, II, and IV. Class I chitinases have a signal peptide, a cysteine-rich chitin binding domain, an enzyme catalytic region, and a C-terminal extension directing the protein to the vacuole. Class II chitinases have a signal peptide and an enzyme catalytic region. Class IV chitinases have a signal peptide, an abbreviated cysteine-rich chitin binding domain, and an enzyme catalytic region. In addition, there are chitinases with minor modifications of these features.
The chitinases of glucosyl hydrolase family
18
are also known as class III chitinases. They have a signal peptide and a catalytic domain. They are structurally unrelated to the chitinases of glucosyl hydrolase family
19
.
The present invention describes novel maize chitinase genes represented by cDNAs. The chitinase genes of the present invention are useful in the control of pathogens. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
In the present invention, seven chitinases of the glucosyl hydrolase family
19
are presented as partial or full-length cDNAs/proteins named: ZmCht2 (SEQ ID NO: 1/SEQ ID NO:2). ZmCht7 (SEQ ID NO:5/SEQ ID NO:6), ZmCht11 (SEQ ID NO: 11/SEQ ID NO:12), ZmCht14 (SEQ ID NO:17/SEQ ID NO:18), ZmCht15 (SEQ ID NO:19/SEQ ID NO:20), ZmCht16 (SEQ ID NO:21/SEQ ID NO:22), and ZmCht17 (SEQ ID NO:23/SEQ ID NO:24). Five chitinases of glucosyl hydrolase family
18
are presented herein as partial or full-length cDNAs/proteins and are named: ZmCht6 (SEQ ID NO:3/SEQ ID NO:4), ZmCht9 (SEQ ID NO:7/SEQ ID NO:8), ZmCht10 (SEQ ID NO:9/SEQ ID NO:10), ZmCht12 (SEQ ID NO:13/SEQ ID NO: 14), and ZmCht13 (SEQ ID NO:15/SEQ ID NO:16).
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to maize chitinases. It is an object of the present invention to provide: 1) antigenic fragments of the proteins of the present invention; 2) transgenic plants comprising the nucleic acids of the present invention; 3) 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 (a) a polynucleotide having a specified sequence identity to a polynucleotide 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). 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. The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide of the present invention and to a polypeptide of the present invention having a specified number of contiguous amino acids. Also, the present invention relates to a polypeptide having a specific sequence identity to the polypeptide of the present invention. In addition, the present invention relates to a polypeptide encoded by a polynucleotide of the present invention.
In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant 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.
Finally, the present invention relates to methods of modulating the level of chitinase in a plant by a) introducing an expression cassette containing a polynucleotide of the present invention, b) culturing the plant cell under plant cell growing conditions, and c) inducing expression of the polynucleotide for a time sufficient to modulate the level of chitinase in the plant.
Definitions
Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. N

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