Rac-like genes and methods of use

Details Rac-like genes and methods of use Rac-like genes and methods of use

1999-09-08

2003-04-29

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S278000, C800S298000, C800S295000, C800S286000, C800S320000, C800S320100, C435S069100, C435S320100, C435S419000, C435S468000, C435S252300, C435S254200, C435S325000, C536S023200, C536S023600, C536S024100, C536S024500

Reexamination Certificate

active

06555732

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 to enhance disease resistance.
BACKGROUND OF THE INVENTION
Rho, rac, and cdc42 are members of a family of small GTP (guanosine triphosphate) binding proteins which function as molecular switches in regulating a variety of cellular processes in both plants and animals. One such process is the regulation of NADPH oxidase and the oxidative burst response which are involved in the defense response of both plants and animals to pathogens (Kwong, et al.,
Journal of Biol Chem
, 270, No. 34: 19868-19872 (1995); Dusi, et al.,
Biochem J
, 314: 409-412 (1996); Diekmann, et al.,
Science
265: 531-533 (1994); Purgin, et al.,
The Plant Cell
9: 2077-2091 (1997); Kleinberg, et al.,
Biochemistry
, 33: 2490-2495 (1994); Prigmore, et al.,
Journal of Biol Chem
27, No. 18: 10717-10722 (1995); Irani, et al.,
Science
275: 1649-1652 (1997); Low, et al.,
Advances in Molecular Genetics of Plant
-
Microbe Interactions
3: 361-369 (1994) eds. M. J. Daniels, Kluwer Acadmic Publishers, Netherlands; Mehdy, et al.,
Plant Physiol
. 105: 467472 (1994); Sundaresan, et al.,
Biochem J
318: 379-382 (1996)). The GTP binding proteins also function in altering the cytoskeleton and in cell transformation (for a review see Symon, M.,
TIBS
21: 178-181 (1996)). In plants, a Rho-like GTPase has been found to control pollen tube growth (Lin et al,
The Plant Cell
9:1647-1659 (1997). Additionally, the GTP-binding proteins have been found to be regulators of transciptional activation (Hill, et al.,
Cell
81: 1159-1170 (1995); Chandra, et at.,
Proc. Natl. Acad. Sci. USA
93: 13393-13397 (1996)). Recently, it has been shown in mice that Rac proteins are involved in the growth and death of mammalian T cells (Lores, et al.,
Oncogene
15: 601-605 (1997)). Clearly, this family of GTP binding proteins control multiple functions in a plant or animal cell and are integral in the cellular defense against pathogens.
In plants, the Rho family is restricted to one large family of Rac-like proteins (Winge, et al.,
Plant Molec Biology
, 35: 483-495 (1997)). Recently, it has been proposed that these proteins be given their own Rho subfamily designation, Rop (Lin, et al., supra). The plant Rac proteins are small, approximately 200 amino acid, soluble and show sequence homology. Plant Racs are activated by the binding of GTP and also have GTPase activity that allows them to cycle off to the inactive state. Various effector proteins can either increase or decrease the level of activation of Rac by promoting or inhibiting GTPase activity. In addition, single amino acid changes in Rac itself can alter the ability of Rac to cycle between active and inactive states. A change of glycine to valine at residue 12 in the highly conserved mammalian Racs results in total loss of GTPase activity, so that when the mutant Rac binds GTP it stays activated permanently, in other words a “dominant positive Rac is formed”. Conversely, changing residue 18 from threonine to alanine causes loss of ability to bind GTP and hence causes permanent inactivation of Rac, in other words a “dominant negative Rac is formed”. (See for example, Xuemi, et al.,
Biochemistry
, 36: 626-632 (1997).)
The Rac proteins from plants show sequence homology with other Rac family members. In
Arabidopsis thaliana
, five Rac cDNAs have been cloned and sequenced. The Rac proteins in
A. thaliana
are all highly conserved, and the N-terminal portion, including the effector domain, share considerable homology to the animal Rac proteins (Winge, et al., supra). In plants the Rac proteins seem to be involved in the oxidative burst observed when plants are infected by a pathogen or an avirulent strain of a pathogen, inducing the disease response pathway, sometimes including the hypersensitivity response (HR). In the hypersensitivity response, cells contacted by the pathogen, and often neighboring cells, rapidly collapse and dry in a necrotic fleck. Other HR responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various antibiotic small molecules and proteins. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting the spread of infection.
Disease in plants is caused by biotic and abiotic causes. Biotic causes include fungi, viruses, bacteria, and nematodes. Of these, fungi are the most frequent causative agent of disease on plants. Abiotic causes of disease in plants include extremes in temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution. A host of cellular processes enables plants to defend themselves from disease caused by pathogenic agents. These processes apparently form an integrated set of resistance mechanisms that is activated by initial infection and then limits further spread of the invading pathogenic microorganism.
As noted, among the causative agents of infectious disease of crop plants, the phytopathogenic fungi play the dominant role. Phytopathogenic fungi cause devastating epidemics, as well as causing significant annual crop yield losses. All of the approximately 300,000 species of flowering plant species can be host to only a few fungal species, and similarly, most fungi usually have a limited-host range.
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. However, the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack. Therefore molecular regulation of the plant Rac proteins is important in improving disease resistance in plants. The present invention provides five newly identified plant Rac genes and methods for modulating the expression of the plant Rac genes.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to Rac proteins that function as molecular switches. It is an object of the present invention to provide antigenic fragments of the proteins of the present invention. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention. Additionally, it is an object of the present invention to provide 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 encoding a polypeptide of the present invention; (b) a polynucleotide amplified from a
Zea mays
nucleic acid library using the primers of the present invention; (c) a polynucleotide comprising at least 25 contiguous bases of the polynucleotides of the present invention; (d) a polynucleotide having at least 64% sequence identity to the polynucleotides of the present invention; (e) a polynucleotide which hybridizes under stringent hybridization conditions to the polynucleotides of the present invention; (f) a polynucleotide selected from SEQ ID NOS: 1, 3, 5, 7, 9, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 (g) a polynucleotide encoding a maize Rac polypeptide and (h) a polynucleotide complementary to a polynucleotide of (a) through (g). The isolated nucleic acid can be DNA. The isolated nucleic acid can also be RNA.
In another aspect, the present invention relates to vectors comprising the polynucleotides of the present invention. Also the present invention relates to recombinant expression cassettes, comprising a nuclei

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