Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
2001-06-29
2004-06-15
Nelson, Amy J. (Department: 1638)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C536S023200, C536S023600, C435S468000, C435S320100, C800S278000
Reexamination Certificate
active
06750057
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the fields of molecular biology, biochemistry, plant pathology, and agriculture. More particularly, the invention relates to proteins and polynucleotides associated with resistance to microbial plant pathogens.
BACKGROUND
Disease resistance in plants is often controlled by host recognition of specific pathogen determinants. Resistance (R) gene products in a host plant are believed to function as receptors that recognize effector proteins produced directly or indirectly by a pathogen's avirulence genes. Over the past decade, a number of dominant R genes have been characterized from diverse plants. The proteins encoded by these genes can be grouped into several classes based on structure: serine/threonine kinases, proteins with a nucleotide binding site and leucine-rich repeats (NBS-LRR), presumed extracellular LRR-containing proteins with or without a transmembrane domain, and serine/threonine receptor-like kinases. Baker et al., Science 276, 726 (1997); Staskawicz et al., Science 268, 661 (1995); and Wang et al., Plant Cell 10, 765 (1998). Based on their structures, such proteins are thought to mediate their function by modulating intracellular signaling pathways.
The action of R genes is usually associated with a number of defense responses. One such response common in many different plant types is the hypersensitive response (HR). HR is characterized by rapid cell death at the site of the infection that can often be visualized as a dry brown lesion. While the intracellular signaling events that mediate resistance and HR are not completely characterized, it has been hypothesized that recognition of a pathogen-produced effector protein induces a negative regulatory pathway that can degrade the R protein. Such a pathway would regulate the intensity and duration of cell death and other elicited intracellular signals that contribute to the resistance response. Boyes et al., Proc. Natl. Acad. Sci. U.S.A. 95, 15849 (1998); S. G. Moller and N.-H. Chua, J. Mol. Biol. 293, 219 (1999).
One possible molecular mechanism for the negative regulatory pathway is ubiquitin-mediated protein degradation, which plays an important role in controlling the abundance of numerous short-lived proteins. A. Hershko and A. Ciechanover, Annu. Rev. Biochem. 67, 425 (1998); A. Ciechanover, EMBO J. 17, 7151 (1998). In this process, ubiquitin is activated by the ubiquitin-activating enzyme E1, transferred to the ubiquitin-conjugating enzyme E2, and finally linked to a target substrate by the ubiquitin ligase E3. Id. Upon attachment by ubiquitin, the target protein is subjected to degradation by the 26S proteasome. The specificity of the degradation pathway is determined by E3, which binds to the targeted substrate. Characterization of a number of E3s in animal systems indicates that a zinc-binding domain, RING (for Really Interesting New Gene) finger, is essential for many ubiquitin-mediated protein degradation processes. P. S. Freemont, Curr. Biol. 10, R84 (2000); R. J. Deshaies, Annu. Rev. Cell Dev. Biol. 15, 435 (1999).
A model for studying R-mediated plant pathogen resistance is that of rice (
Oryza sativa
) and the bacterial pathogen
Xanthomonas oryzae
, pv.
oryzae
(Xoo). In this system, the rice R gene Xa21 confers resistance to bacterial blight disease caused by Xoo. Transgenic cell lines expressing the protein encoded by Xa21, i.e. XA21, respond with cell death after inoculation with an avirulent strain of Xoo such as Philippine race 6 (strain pXO99 AZ), but not with virulent strain of Xoo such as Korean race 1 (strain DY890931). He et al., Science 288:2360 (2000). Although XA21 is known to be a receptor-like kinase with serine/threonine specificity [U.S. Pat. No. 5,952,485; and Song et al., Science 270, 661 (1995)], the mechanisms by which it mediates resistance and cell death are not completely understood. Identification of molecules that interact with and/or modify XA21 should help clarify these mechanisms, and provide new tools for engineering broad-spectrum, durable disease resistance in rice and other crop plants.
SUMMARY
The invention relates to the discovery of Xb3, a polynucleotide encoding XB3 (for XA21 Binding Protein 3), a protein that interacts with the XA21 kinase. Xb3 was identified in a yeast two-hybrid assay in which a rice cDNA library was screened using the XA21 kinase as bait. The cloned Xb3 was subsequently sequenced. Based on the nucleotide sequence, it was determined that Xb3 encodes a 450 amino acid protein (i.e., XB3) that has a myristoylation site, 8 imperfect copies of ankyrin repeats, and a RING finger motif. Functional studies indicated that XB3 is a substrate for the XA21 serine/threonine kinase, and binds XA21 via its ankyrin repeat domain. Other studies indicated that XB3's RING finger domain ubiquitinates itself, and is required for ubiquitination of XA21. In vivo protein assays indicated that XA21 is rapidly degraded in response to infection with an avirulent strain of Xoo, but not with a virulent strain. Taken together, these results suggest that XB3 serve as an E3 that negatively regulates pathogen resistance and cell death through ubiquitin-mediated protein degradation of XA21.
Accordingly, the invention features a purified nucleic acid that includes a nucleotide sequence which encodes a naturally occurring protein that: (a) shares at least 80% sequence identity with SEQ ID NO:2 and (b) has at least one functional activity of native XB3. For example, nucleic acids having a nucleotide sequence whose complement hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO:1; those encoding a protein having the amino acid sequence of SEQ ID NO:2; and those encoding a protein that specifically binds to XA21 are featured in the invention.
In another aspect, the invention features a vector including a nucleic acid of the invention. The vector can have a nucleic acid operably linked to one or more expression control sequences. Also within the invention is a cell containing a nucleic acid of the invention.
Proteins that include an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO:2 and have at least one functional activity of native XB3 (e.g., purified protein whose amino acid sequence is SEQ ID NO:2) are included in the invention. Also featured are purified proteins containing one or more of amino acid residues 1-10, 11-305, and/or 319-385 of SEQ ID NO:2; as well as fusion proteins containing one of the foregoing proteins fused to a heterologous polypeptide.
Within the invention is a purified antibody that specifically binds to a protein of the invention. Such antibody can include a detectable label.
In still another aspect, the invention features several methods, including a screening method for identifying a substance that modulates binding of an XB3 protein to XA21. This method includes the steps of: providing a sample containing the XB3 protein; adding to the sample a candidate substance; adding to the sample XA21; and detecting an increase or decrease in binding of the XB3 protein to XA21 in the presence of the candidate substance, compared to the binding of the XB3 protein to XA21 in the absence of the candidate substance, as an indication that the candidate substance modulates binding of XB3 protein to XA21.
Also within the invention is a method of producing an XB3 protein. This method includes the steps of: providing a cell transformed with an isolated nucleic acid comprising a nucleotide sequence that encodes an XB3 protein; culturing the cell under conditions that allow expression of the XB3 protein; and collecting the XB3 protein from the cultured cell.
Another method within the invention is a screening method for identifying a substance that modulates expression of a gene encoding XB3. This method includes the steps of: providing a test cell; contacting the test cell with a candidate substance; and detecting an increase or decrease in the expression level of the gene encoding XB3 in the presence of the candidate substance, compare
Pi Li-Ya
Song Wen-Yuan
Akerman & Senterfitt
Kruse David H.
Nelson Amy J.
University of Florida
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