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
1999-08-26
2002-06-04
Achutamurthy, Ponnathapu (Department: 1652)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C435S195000, C536S023200
Reexamination Certificate
active
06399858
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of agricultural microbiology and plant disease control. In particular, this invention provides a novel gene from
Stenotrophomonas maltophilia,
which encodes a chitinolytic enzyme useful for biocontrol of plant pathogenic fungi.
BACKGROUND OF THE INVENTION
Various scientific articles are referred to in brackets throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
The use of chemical pesticides remains one of the most effective methods for control of plant diseases. However, concerns for health and environmental safety have increased pressure to reduce the use of chemicals in agriculture. Biological control using beneficial microbes is one alternative to chemicals that have yet to be fully investigated for plant disease control. However, in order for biocontrol to achieve full acceptance as disease control method, inconsistent performance of biocontrol agents in field conditions must be overcome. A major factor that contributes to inconsistent performance is the lack of efficient expression of biocontrol traits at the appropriate times and sites in the biosphere.
Bacteria that function in fungal pathogen antagonism by proposed lytic activity are described in the literature, dating as early as the 1960s. Investigative studies on the mechanisms by which these and similar strains function focus largely on the characterization of degradative enzymes. However, there are only limited studies that focus on the role of chitinases in biocontrol activity of lytic bacteria.
Stenotrophomonas maltophilia
represents a rhizosphere bacterium of potential agronomic importance.
Stenotrophomonas maltophilia
strain 34S1 (Sm34S1) is capable of controlling summer patch disease of Kentucky bluegrass (
Poa pratensis
) caused by the root infecting fungus,
Magnaporthe poae
(Kobayashi et al., 1995, Soil Biol. Biochem. 27:1479-1487). The bacterium expresses several traits likely to contribute to biocontrol activity, including the production of several extracellular enzymes capable of degrading fungal cell wall components, including chitinase, protease and lipase (Kobayashi et al, 1995, supra; O'Brien and Davis, 1982, J. Clin. Microbiol. 16:417-421; Nord, et al., 1975, Med. Microbiol. Immunol. 161:79-87).
Chitinases digest chitin, the predominant polysaccharide in fungal cell walls. Chitin is an unbranched polymer of &bgr;-1,4-linked N-acetyl-D-glucosamine. Chitin is enzymatically degraded to N-acetylglucosamine by two distinct enzymes: an endochitinase [poly (1,4-N-acetyl-b-D-glucosaminidase) glycanohydrolase; EC 3.2.1.14], which liberates mostly dimers and a chitobiase (N-acetyl-b-glucosaminidase; EC 3.2.1.30), which-reduces dimers to monomers for metabolic utilization. Characterized bacterial chitinases are endochitinases, and have been shown to rupture hyphal tips as well as inhibit spore germination (Inbar and Chet, 1991, Soil Biol. Biochem. 23:973-978; Lim, et al., 1991, Environ. Microbiol. 57:510-516).
Bacterial chitinases, as well as the genes that encode them and the bacterial strains that harbor them (either native strains or genetically engineered strains) are useful tools for biological control of fungal plant pathogens. Such tools could be used to minimize a fungal plant disease without the use, or with reduced use, of ecologically detrimental fungicides. Accordingly, it is of agronomic, economic and ecological importance to provide isolated genes encoding such chitinases, as well as the chitinases produced from such genes and bacterial strains that express the genes.
SUMMARY OF THE INVENTION
According to one aspect of the present invention, an isolated nucleic acid from Stenotrophomonas is provided. This isolated nucleic acid confers to bacteria in which it exists naturally, or into which it is transformed, the ability to delay the onset of symptoms of fungal infection in plants. The nucleic acid molecule is preferably isolated from
Stenotrophomonas maltophilia.
The isolated nucleic acid molecule described above encodes a chitinase. In a preferred embodiment, the isolated nucleic acid comprises the chiA gene from
S. maltophilia
strain 34S1, and in a most preferred embodiment, the isolated nucleic acid comprises part or all of SEQ ID NO:1.
According to another aspect of the invention, a biologically pure culture of Stenotrophomonas is provided that has the chiA gene in its genome. In a preferred embodiment, the culture is
S. maltophilia,
and in a most preferred embodiment, is strain 34S1.
According to another aspect of the invention, an isolated polypeptide is provided that is produced by expression of at least 30 consecutive nucleic acids of the aforementioned Stenotrophomonas isolated nucleic acid molecules. In a preferred embodiment, the polypeptide has an amino acid sequence comprising part or all of SEQ ID NO:2. Also included in this aspect are antibodies immunologically specific to this isolated polypeptide.
According to another aspect of the invention, an isolated nucleic acid molecule is provided that is at least 70% identical to SEQ ID NO:1 or ORFI of SEQ ID NO:1, a sequence encoding an amino acid sequence at least 60% similar to SEQ ID NO:2, and a nucleic acid molecule that hybridizes to SEQ ID NO:1 at moderate stringency.
According to another aspect of the invention, various recombinant DNA molecules are provided comprising variants of SEQ ID NO:1 and encoding variants of SEQ ID NO:2. These recombinant molecules include an expression cassette, a cloning vector and an Agrobacterium binary vector.
According to another aspect of the invention is a cell that has been transformed with a nucleic acid that is a variant of SEQ ID NO:1 or encodes a variant of SEQ ID NO:2. In a preferred embodiment, the cell transformed is a bacterial, yeast or plant cell. A more preferred embodiment is a biologically pure culture of this cell. Particularly preferred embodiments are a transgenic plant regenerated from the transformed plant cell, a seed from the plant and a plant from the seed.
According to another aspect of the invention, a method for protecting plants from fungal infection is provided. This method comprises introducing the chiA polypeptide into the environment of the plant. In a preferred embodiment, the chiA polypeptide is added as an isolated polypeptide. In another preferred embodiment, the chiA gene is expressed in an organism in the environment of the plant. In a more preferred embodiment, the organism is a transgenic micro-organism, a transgenic plant, a transgenic fungus or a strain of
Stenotrophomonas maltophilia.
In another preferred embodiments, the organism is introduced into the soil around the plant or topically applied to the leaves of the plant.
According to another aspect of the invention, the transcription regulatory region of the
Stenotrophomonas maltophilia
chiA gene is provided. In a preferred embodiment, this regulatory region is disposed between nucleotides 1 and 306 of SEQ ID NO:1.
Other features and advantages of the present invention will be better understood by reference to the drawings, detailed description and examples that follow.
REFERENCES:
Fuji T., Multiple domain structure in a chitinase gene (chiC) of Streptomyces Iivdans, J. Gen. Microbiol. 1993, 139, 677-686.*
Kobayashi, D.Y. 1994. Abstract 139, Phytopathology 84:1081.
Kobayashi, D. and Holtman, M.A. 1997. Abstract, In Proceedings of the Sixth Annual Rutgers Turfgrass Symposium. Center for Turfgrass Science. J. Derr, ed. p. 18.
Kobayashi et al., 1995, Soil Biol. Biochem. 27:1479-1487.
Kobayashi and El-Barrad, 1996, Cur. Microbiol. 32:106-110.
Holtman M.A. and Kobayashi D.Y. 1997. Abstract, Phytopathology 87 (Supplement):S43.
Licata & Tyrrell P.C.
Rutgers, the State University
Walicka Malgorzata A
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