WO2002010367A1 - Chimeric receptors and their uses - Google Patents

Abstract

The present invention provides chimeric plant receptors useful for modulating plant responses to pathogens. The receptors comprise a heterologous extracellular domain and a kinase domain from an RRK protein, such as Xa21.

Description

CHIMERIC RECEPTORS AND THEIR USES

FIELD OF THE INVENTION

The present invention relates generally to plant molecular biology. In particular, it relates to nucleic acids and methods for conferring disease resistance in plants.

BACKGROUND OF THE INVENTION Plant pathogens cause hundreds of millions of dollars in damage to crops in the United States ^'annually and cause significantly more damage worldwide.

Traditional plant breeding techniques have developed some plants that resist specific pathogens, but these techniques are limited to genetic transfer within^' breeding species and can be plagued with the difficulty of introducing non-agronomic traits that are linked to pathogen resistance. Furthermore, traditional breeding has focused on resistance to specific pathogens rather than general, or systemic, resistance to a wide spectrum of pathogens.

Loci conferring disease resistance have been identified in many plant species. Generic analysis of many plant-pathogen interactions has demonstrated that plants contain loci that confer resistance against specific races of a pathogen containing a complementary avirulefice gene. Molecular characterization of these genes should provide means for conferring disease .resistance to a wide variety of crop plants. Bacterial blight disease caused by Xanthc-monas spp. infects virtually all crop plants and leads to extensive crop losses worldwide. One source of resistance (Xa21) had been identified and cloned f om the wild species Orγza longistaminata (Song et al. Science 270: 1804-1806 (1995)). The Xa 21 gene is a member of a class of disease resistance genes referred to as^' "RRK genes". These genes encode RJRK polypeptides which typically comprise an extracellular LJΛR domain, a transmembrane domain, and a cytoplasrnic protein kmase domain (see, WO96/2237S and WO99/0 151 for a description of RRK disease resitance genes), Considerable effort has been directed toward cloning and engineering of plant genes conferring resistance to a variety of bacterial, fungal and viral diseases. Means of modifying plant responses to pathogens and other environmental signals is an active area of research and development. The present invention addresses these and other needs..

Receptor kinases mediate extracellular signals for diverse processes in plants and ariimals (see, e.g., Richter TE and Ronald PC. Plant Mol Biol.200042(1): 195-204; Hocking et al., Matrix Biol 1998 Apr;17(l):l-19; Gomez-Gomez L, and Boiler T. Mol Cell.2000 Jun;5(6): 1003-11; Itoh A, Brain Res Mol Brain Res. 1998 Nov 20;62(2):175- 86; Buchanan SG, Gene. 1998 May 12;211(2):235-44; Taguchi A, et al., Brain Res Mol Brain Res. 1996 Jan;35(l-2):31-40). Detailed mechanistic studies of both receptor tyrosine kinases and

kinases have been well documented in animal cells, where it has been shown that ligand binding to the extracellular domains of receptors induces receptor dimerization and stimulates receptor phosphotylation, resulting in the activation of intracellular signaling cascades (P. van der Geer et al, Annu. Rev. Cell Biol. 10:251 (1994); J. Massague, Cell 85:947 (1996); X, Feng and R. Deryneck, EMBO J. 16:3912 (1997)), In contrast, the study of plant receptor-like kinases (RLKs), all of which are serme/1hreκ>ninc kinases, is still in its infancy (K. Lease et al Curr. Opin. Plant Biol. 1 :388 (1998); P. Becraft, Trends Plant Sci. 3:384 (1998)). Despite the large numbers of putative RLKs encoded in the genomes of plants, how these receptors carry out signal transductiort has yet to be determined.

Of the various RLKs, the largest group is the leucine-rich repeat receptor kinases (LRR- RLKs). This class consists of at least 120 genes in Arabidopsis. A few LRR-RLKs are involved in diverse biological processes based on their mutant phenotypes. These processes include the control of meristem development (S. Clark et al. Cell 89:575 (1997)), disease resistance (W. Song, etal., Science 270:1804 (1995)), hormone signaling (J. Li and J. Chory, Cell 90:92 (1997)), and organ elongation and abscission (K. Torit, et al, Plant Cell 8:735 (1 96) and T. L, Lin et al. Genes Dev. 14:108 (2000)). However_, in no case is there biochemical evidence for the identity of a ligand, although genetic studies have provided some clues. On the basis of the similarity of mutant phenotypes and their adjacent expression domains within the meristem, CLAVATA3, a putative extracellular protein of 96 amino acids, has been proposed as the ligand of the LRR-RLK CLAVATA1 (J. Fletcher et al. Science 283:1911 (1999)). Likewise, genetic studies suggest that the steroid hormone brassinolide (BL), the most biologically active brassinosteroid, is the ligand for the BRli-encoded LRR-RLK (Li and Chory Cell (1997)). Thus, the LRR-RLKs might use either small molecules or proteins as ligands.

SUMMARY OF THE INVENTION The present invention provides nucleic acid molecules comprising a polynucleotide encoding a chi eric RRK receptor, the receptor comprising a heterologous extracellular domain and a kinase domain from a Xa2l polypeptide. The nucleic acid of the invention typically comprise a plant promoter operably linked to the polynucleotide encoding the chimeric RRK receptor. The promoter can be inducible or constitutive. Typically, the heterologous extracellular domain comprises an LRR domain, for example from Bril. An exemplary extracellular LRR domain sequence is at least about 70% identical to a sequence from about residue 681 to about residue 3332 in SEQ ID NO: 3. An exemplary kinase domain sequence is at least about 70% identical to a sequence from about residue 2121 to about residue 3918 in SEQ ID NO: I.

In other embodiments, the heterologous extracellular domain is a hevein domain from RCH10. An exemplary extracellular hevein domain sequence is at least about 70% identical to a sequence from about residue 1928 to about residue 2289 in SEQ , ID NO: 5. The kinase domain sequence is usually at least about 70% identical to a sequence from^'about residue 1870 to about residue 3918 in SEQ ID NO:l.

The present invention also provides transgenic plants comprising a recombinant expression cassette comprising a plant promoter operably linked to a polynucleotide sequence encoding a chimeric RRK receptor of the invention.

In an alternate embodiment of the present invention,the LRR domain can be from a plant source or animal source, including insects, mammals, amphibians, reptiles, fish and the like. LRRs from sources are preferable when inducible systems for plants are desired. Those of skill in the art will readily recognize sources other those listed herein for LRR domains. In addition, those of skill in the art will readily recognize ligands for such LRR domains.

In another embodiment of the invention, the chimeric receptor kinase may be engineered to include an "effector" or "action" domain that is capable of triggering cell growth, proliferation, differentiation, apoptosis, gene transcription, hypersensative response, etc. A variety of illustrative effector domains that may be used in practicing this invention are known by those skilled in the art, , In one embodiment, there are provided assays for identifying ligands for kinase receptors. In a prefered embodiment, there are provided assays which are cell-based assays in which a cell which expresses a membrane-bound form of one of the invention chimeric receptor kinases (e.g., NRG1, NRG2, and/or NRG3) on the cell surface is contacted with a test compound and the ability of the test compound to bind to a chimeric receptor kinase determined. The cell, for example, can be of plant origin, such as Arabidopsis or rice. In yet another embodiment, the cell can be heterologous to the construct, or at least to the LRR domain. Identification of the ligand can be determined by numerous ways, mcluding binding of the ligand to the receptor or activation of the kinase domain. Those of skill in the art will readily recognize alternative ways for identifying ligands employing the invention construct, for example, deteπrrining the ability of the test compound to bind to the chimeric receptor kinase can be accomplished by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the chimeric receptor kinase or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ^I2S1, ^S, ^,4C, or ³H, either directly or mdirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. Test compound binding may also determined by serological methods, such as Western blot, or enzyme linked immunoassay. In a preferred embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NRG1, NRG2 and or NRO3, or a biologically active is portion thereof, on the cell surface with a compound which binds NRG1, NRG2, and/or NRG3 to determine compounds that recognize the chimeric receptor kinase.

In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of one of the chimeric receptor kinases, including NRG1, NRG2, and/or NRG3, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the chimeric receptor kinase. Deterrruning the ability of the test compound to modulate the activity of the chimeric receptor kinase or a biologically active portion thereof can be accomplished, for example, by determining the ability of the chirneric receptor kinase to bindf to or i teract with a ©rgetmolec«te,e& a mofeeMtewrth w ϋab one ormone of chimeric receptor chiπase binds or interacts with in nature or otherwise, for example, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.

Determining the ability of the chimeric receptor kinase to bind to or interact with a chimeric receptor kinase target molecule can be accomplished by one of the methods described above for determining direct binding. Methods of measuring the activity of a kinase are well known by those skilled in the art. In a preferred embodiment, determining the ability of the chimeric receptor kinase to bind to or interact with a chimeric receptor kinase target molecule can be accomplished by determining the activity of the target molecule.

In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a chimeric receptor kittase, such as NRGl, NRG2, and/or NRG3, with a test compound and determining the ability of the test compound to bind to the chimeric receptor kinase. Binding of the test compound to the chimeric receptor kinase can be deteraύned either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the chimeric τeceptor kinase with a known compound which binds one or more of chimeric receptor kinases to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a chimeric receptor kinase, wherein deteπrtining the ability of the test compound to interact with a chimeric receptor kinase comprises determining the ability of the test compound to preferentially bind to the chimeric receptor kinase or biologically active portion thereof as compared to the known compound, e.g., to identify antagonists.

In another embodiment, an assay is a cell-free assay comprising contacting chimeric receptor kinase or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or uihibit) the activity of the chimeric receptor kinase or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of chimeric receptor kinase can be accomplished, for example, by determining the ability of the chimeric receptor kinase to bind to a chimeric receptor kinase target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of chimeric receptor kinase can be accomplished by determining the ability of the chimeric receptor kinase to further modulate a chimeric receptor kinase target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting the chimeric receptor kinase or biologically active portion thereof with a known compound which binds chimeric receptor kinase to form an assay rriixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a chimeric receptor kinase protein, wherein deteirπining the ability of the test compound to interact with a chimeric receptor kinase protein comprises determining the ability of the chimeric receptor kinase protein to preferentially bind to or modulate the activity of a chimeric receptor kinase target molecule.

The cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form of chimeric receptor kinase. In the case of cell-free assays comprising the membrane-bound form of the chimeric receptor kinase, it may be desirable to utilize a solubiliziπg agent such that the membrane-bound form of the chimeric receptor kinase is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucosidc, n-dodecylglucoside, n- dodecylmaltoside, octanoyl-N-methylglucarnide, decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit, Isotridecypoly(ethylene glycol ether)n, 3-[(3- cholamidopropyl)(hmethylammirιio]-l-propane sulfonate (CHAPS), 3-[(3- cholarmdoρroρyl)dimet ylaιrιminio]-2-hydroxy-l-proρaue sulfonate (CHAPSO), or N- dodecyl.dbd.N,N-dirnellιyl-3-arΛmonio-l-propane sulfonate.

In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either chimeric receptor kinase or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to the chimeric receptor kinase, or interaction of the chimeric receptor kinase with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a to domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase chimeric receptor kinase fusion proteins or glutathione-S-transferase target fusion proteins can be adsorbed onto glutathione sopharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or the chimeric receptor kinase protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of chimeric receptor kinase binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the chimeric receptor kinase or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated chimeric receptor kinase or target molecules can be prepared from biotin- NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, III.), and immobilized in the wells of streptavidin-coated ,96 well plates (Pierce Chemical). Alternatively, antibodies reactive with chimeric receptor kinase or target molecules but which do not interfere with binding of the chimeric receptor kinase protein to its target molecule can be derivatized to the wells of the plate, and unbound target or chimeric receptor kinase trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-iιruτιobilized complexes, include immunodetection of complexes using antibodies reactive with the chimeric receptor kinase or target- molecule, as well s enzyme-linked assays which rely on detecting an enzymatic activity associated with the chimeric receptor kinase or target molecule. In yet another embodiment of the present invention, invention constructs can be employed to induce signaling for controlling plant development and/or disease resistance. By employing ligands, preferably exogenous ligands, invention constructs can be induced to control plant development^' and/or disease resistance. Preferred constructs will employ endogenous LRR domains which are induced, preferably by ligands not native to the plant.

Definitions

An "RRK gene" is member of a class of plant disease resistance genes which encode RRK polypeptides which typically comprise an extracellular LRR domain, a transmembrane domain, and a cytoplasmic protein kinase domain (as shown in e.g., WO 96/22375).

An "Xa21 polynucleotide sequence" is a subsequence or full length polynucleotide sequence of a ήceXa2I gene, which, when present in a transgenic plant confers resistance to Xanthomonas spp, (e.g., X. oryzae) on the plant Exemplary polynucleotides of the invention include the coding region of the sequences provided below. An X 2ϊ polynucleotide is typically at least about 3100 nucleotides to about 6500 nucleotides in length, usually from about 4000 to about 4500 nucleotides.

A "kinase domain from a Xa2l protein" is a sequence at least substantially identical to a sequence from about residue 708 to about residue 1004 in SEQ ID NO: 2 (see, also, GenBank Accession No. U37133).

A "heterologous extracellular domain" in a chimeric receptor of the invention is one that originates from a protein different from the protein from which the kinase domain of the receptor originates. Typically, the extracellular domain will comprise an LRR domain or a hevein domain.

An "LRR domain" is an extracellular domain which comprises a^" block of about 23 tandem Ieucine-rich repeats (LRR) with an average length of 24 amino acids. The LRR motif has been implicated in protein-protein interactions and ligand binding in a variety of proteins (see, WO 96/22375).

A "Bril polynucleotide sequence" is a subsequence or full length polynucleotide sequence from a Bril gene. A Bril gene encodes brassinosteroid receptor. Brassinosteroids are widely distributed natural products that promote growth and posses plant hormone activity. Brassinosteroids have a number of effects on plants including modulation of response to stress, reproductive processes. There is intensive research being performed to examine the binding proteins, mode of action in regulation of gene expression, biosynthesis, and secondary messengers of these compounds. Analysts of 18 Arabidopsis dwarf mutants unable to respond to exogenously added brassinosteroid led to the- cloning of a brassinosteroid receptor with kinase activity, referred to here as Bri 1 (see, Li et al. Celt 90:825-7 (1 97) and WO 98/59039). The encoded protein comprises an extracellular LRR domain containing 25 tandem leucine-rich repeats found in plant disease resistance genes such' as RRK genes.

A "RCH10 chitinase polynucleotide sequence" refers to a subsequence of full length polynucleotide sequence from a rice RCH10 chitinase gene (Zhu and Lamb Mol. Gen. Genet, 226:289-296 (1991). The extracellular domain of this protein comprises a lectin domain homologous to hevein from Hevea brasiliensis.

One of skill will recognize that, in the expression of the various domains from prior art proteins i the chimeric receptors of the invention need not be identical and may be "substantially identical" to a sequence of the protein from which it was derived. • As explained below, these variants are specifically covered by the above terms.

The phrase "nucleic acid sequence" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.

The term "promoter" refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA poly erase and other proteins to initiate transcription, A "plant promoter" is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.

The term "plant" includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiospcrms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.

A polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).

A polynucleotide "exogenous to" an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-media.ted transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a Ti (e.g. in Arabidopsis by vacuum infiltration) or Ro (for plants regenerated from transformed cells in vitro) generation transgenic plant. Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant "Recombinant" refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation e.g., by methods described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1 89) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)) of an. isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above. Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophbbicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero a d I. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).

The phrase "substantially identical," in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 70%, more preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.

One of skill in the art will recognize that two polypeptides can also be "substantially identical" if the two polypeptides are irnmunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 7. Acad Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WD, or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1 95 Supplement) (Ausub^'el)).

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1990) J. Mol. Biol 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res.25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches' to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be mcreased. Cumulative scores are calculated using, for nucleotide sequences,^" the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of One or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M~5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001,

A further mdication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is i munologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

^' "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codoπ, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide, Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG. which is ordinarily the only codon for methioniπe) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Gluta ic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylaianme(F), Tyrosiπe fY),Tryptophan(W). (see, e.g., Creighton, Proteins (1984)).

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.

The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or Ubrary DNA or RNA). The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10^aC lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Lower stringency conditions are generally selected to be about 15-30 °C below the T_m. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e,g., 10 to 50 nucleotides) and at least about 60"C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabihzing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

In the present invention, geπomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and at least one wash in 0.2X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 4.5*C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of chimeric receptors of the invention engineered using the LRR domain of Bril and the kinase domain of Xa21. In the Figure, TM = transmembrane region, JM « juxtamembrane region.

Figure 2 is a bar graph showing that brassinosteroid treatment induces cell death in plant cells expressing the chimeric receptors of the invention as compared to controls expressing mutant chimeric receptors and in untransformed cells (TP309). ^' Figure 3 is a schematic representation of chimeric receptors of the invention engineered using the hevein domain of RCH10 and the kinase domain of Xa21 In the Figure, TM = transmembrane region, JM = juxtamembrane region..

Figure 4 is a bar graph showing that chitin treatment induces cell death in plant cells expressing the chimeric receptors of the invention as compared to controls expressing mutant chimeric receptors and in untransformed cells (TP309).

Figure 5A is a graph iUustrating cell death, oxidative burst, and defense pathways are initiated in the XA21 cell lines inoculated with oo. XA21 and Taipei 309 cell lines were inoculated with 107 cells/ml of the incompatible Phillipine Race 6 strain. (P6) (gray bars) and the compatible Korean Race 1 strain (Kl) (open bars) 5 days after transfer to fresh medium and grown foran additional 24 hours.

Figure 5B is a graph illustrating cells were inoculated for 1 hour with P6 and Kl (107 cells/ml). H2O2 levels in media were assayed (23) with at least three repeats.

Figure S& graphs mRNA levels estimated with a Phospholmager System (Molecular Dynamics, Sunnyvale, California). Levels at time 0 were set as onefold for RCH10 and PAL and 10-fold for OsCatB. Curves indicate incompatible (■) and compatible (p) interactions.

Figure 6 is a schematic diagram of crώneric receptor kinases NRGl, NRG2, and NRG3 and mutant controls NRGlmL and NRGl K. The XA21 and BRll protein structures are labeled in white and gray, respectively, with signal peptides indicated in dark gray. These chimeras were constructed by in vitro mutagenesis (A BRll DNA fragment encoding the presumed extracellular, transmembrane (TM), and juxtamembrane (JM) domains [amino acids 1 to 879 (J. Li and J. Chory, Cell)] was fused with the Xa21 fragment encoding the predicted kinase domain (amino acids 708 to 1025 (7)] to make NRGl. NRG2 consisted of amino acids 1 to 769 of BRll and the XA21 TM, JM, and kinase domains with amino acids 625 to 1025. NRG3 consisted of amino acids 1 to 834 of BRll and amino acids 684 to 1025 of XA21. NRGlmL contains a mutation (Gly6l 1 Glu) corresponding to the allele bril-113 (J. Li and J. Chory, Cell). NRGlmK is a mutation of XA21 (Lys737 Glu) obtained by in vitro mutagenesis with the primer (5'- GTTGCAGTGGAGGTACTAA-3') corresponding to the Xa21 sequence 2197 to 2215 (W. Song, et al., Science).) and driven by the cauliflower mosaic virus 35S promoter in rice cells (K. ToriL et l,, Plant Cell).

Fig. 7 A and B are graphs illustrating BL induction of cell death, oxidative burst, and defense pathway activation in NRGl cell lines. Figure 7A shows cell suspensions (14): NRGl -30, NRGl -34, NRGlmL, NRGlrnK, and wild-type Taipei 309 treated with 2 μM BL for 24 hours. Cell death was assayed as described in Fig.5 A. Figure 7B shows NRGl and control cell lines treated for 30 min with 2 μM BL for H2O2 production assay with gray bars for treatment and open bars for nontreatment.

Figure 7C graphs RNA levels estimated as in Fig.5C. Cell lines are NRGl -30 <■), NRG1-34 ( ), NRGlmL (Q), NRGlmK (O), and Taipei 309 (Δ).

Figure 8 is a graph depicting BL dose response for RCH10 induction in NRGl cell lines. Cells were treated with 0 to 4 μM BL. RNA was extracted 6 hours after treatment, and transcript levels were deteπrαned (24), Cell lines are NRGl -30 (■), NRG1-34 (•), NRGlmL <^Q), NRGlmK (O), and Taipei 309 (Δ).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS This invention relates to chimeric RRK receptors and genes encoding them. In exemplary receptors of the invention, a kinase domain from an Xa21 gene is linked to extracellular domains from heterologous proteins to allow induction of plant disease resistance in response to desired environmental signals. The chimeric receptors of the invention can be used for the discovery of new ligands of the extracellular domains, new functions of the receptor kinases and for engineering new pathogen resistance in plants.

Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used.for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA lϊgase, DNA polymerase, restriction endonucleases and the like are performed accordmg to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et at., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989). The isolation of genes useful in production of chimeric receptors of the invention may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here and in WO 96/22375 and WO 99/09151 can be used to isolate RRK resistance genes. Bril genes can be prepared using the sequences disclosed in WO 98/59039. Similarly, chitinase genes can be prepared using sequence in Zhu and smbMol Gen. Genet. 226:289-296 (1991). Typically, the sequences described here and in the prior art are used to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library; mRNA is isolated from the desired organ, such as leaf and a cDNA library which contains the desired gene transcript is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which desired genes orhomologs are expressed. Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the Λ&fiT nd related genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries, PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.

Appropriate primers and probes for identifying desired sequences from plant tissues are generated from comparisons of the sequences provided herein. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D„ Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).

Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al, Cold Spring Harbor Syntp. Quant. Biol 47:411-418 (1982), and Adams et al, J. Am. Ghent. Soc. 105:661

(1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. Isolated sequences prepared as described herein can then be used to provide chimeric receptor gene expression and therefore, for example, enhanced pathogen resistance in desired plants. One of skill will recognize that the nucleic acid encoding a functional protein need not have a sequence identical to the exemplified gene or domains disclosed here. Thus, the sequences encoding domains described here need not be full length or identical to those described here,, so long as the desired functional domain of the protein is expressed. Modified protein chains can be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.

To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared, Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, for example, Weising et al Ann. Rev. Genet. 22:421-477 (1988),

A DNA sequence coding for the desired chimeric receptor polypeptide will be used to construct a recombinant expression cassette which can be introduced into the desired plant. An expression cassette will typically comprise a polynucleotide encoding the chimeric receptor polynucleotide operably linked to transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the polynucleotide in the intended tissues of the transformed plant

For example, a plant promoter fragment may be employed which will direct expression of the chimeric receptor in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill. Constitutive promoters and regulatory elements can also be isolated from genes that are expressed constitutively or at least expressed in most if not all tissues of a plant. Such genes include, for example, ACT11 from Arabidopsis (Huang et al. Plant Mot. Biol 33:125-139 (1996)), Caβ orn Arabidopsis (GenBankNo. U43147, 2hong et al, Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al Plant Physiol 104:1167-1176 (1994)), GPcl from maize (GenBank No. X15596, Martinez et al J. Mol. Biol 208:551-565 (1989)), and Gpc2 from maize (GenBank No, U45855, Manjunath et al, Plant Mol Biol 33:97 '-112 (1997)). Alternatively, the plant promoter may direct expression of a nucleic acid of the invention in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise envfronmental or developmental control (i.e. inducible promoters). Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, elevated temperature, the presence of light, or application of chemicals hormones. Tissue-specific promoters can be inducible. Similarly, tissue-specific promoters may only promote transcription within a certain time frame of developmental stage within that tissue. Other tissue specific promoters may be active throughout the life cycle of a particular tissue. One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue- ' specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.

Examples of promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves, roots, fruit, seeds, or flowers. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive vo. certain locations.

The endogenous promoters from RRK genes or other genes used to construct the desired chimeric receptors can be used to direct expression. Methods of identifying promoters are known. Typically, the 5' portions of the genomic clones are analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (e.g, TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (orT) N G, J,Messing et al., in Genetic Engineering in Plants, pp,221-227 (Kosage, Meredith and Hollaender, eds. 1983).

If proper polypeptide expression is desired, a polyadenylation region at the 3'-end of the chimeric receptor coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.

The vector comprising the desired sequences will typically comprise a marker gene which confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G4I8, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.

DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporarion and micTOinjecfion of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.

Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al EMBO. J. 3:2717-2722 (1984). Electroporation techniques are described in Frornm et al. Proc. Nad. Acάd. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et at. Nature 327:70-73 (1987). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell £NA when the cell is infected by the bacteria. Agrobacterium tumefaciens-xasdisXed transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al Science 233:496-498 (1984), and Fraley et al Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to Plants; Potrykus, ed, (Spr ger-Verlag, Berlin 1995).

Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased seed mass. Such ^" regeneration techniques rely on manipulation of certain phytohorrnones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMiililan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp.21-73, CRC Press, Boca Raton, 1985, Regeneration can also be obtained from plant callus, explants, organs, or parts thereof Such regeneration techniques are described generally in Klee et al, Ann. Rev. of Plant Phys. 38:467-486 (1987).

The nucleic acids of the invention can be used to confer desired traits on essentially any plant Thus, the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrutlus, Capsicum. Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hord um, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Mains, Manihot, Mjorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachio, Pisum, Pyrus, Prun s, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis. Vigna, and Zea,

One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Using known procedures one of skill can screen for plants of the invention by detecting the mRNA or chimeric receptor proteins of the invention in transgenic plants. Means for detecting and quantitatiπg mRNAs or proteins are well known in the art.

Plants with enhanced resistance to desired pathogens or other traits can be selected in many ways. One of ordinary skill in the art will recognize that the following methods are but a few of the possibilities. One method of selecting plants with enhanced resistance is to determine resistance of a plant to a specific plant pathogen. Possible pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) (1988)). One of skill in the art will recognize that resistance responses of plants vary depending on many factors, including what pathogen or plant is used, Generally, enhanced resistance is measured by the reduction or elimination of disease symptoms when compared to a control plant. In some cases, however, enhanced resistance can also be measured by the production of the hypersensitive response (HR) of the plant (see, e.g., Staskawicz et a Science 268(5211): 661-7 (1995)), Plants with enhanced resistance can produce an enhanced hypersensitive response relative to control plants,

Enhanced resistance can also be determined by measuring the increased expression of a gene operably linked a defense related promoter. Measurement of such expression can be measured by quantitating the accumulation of RNA or subsequent . protein product (e.g., using northern or western blot techniques, respectively (see, e.g. , Sambrook et al and Ausubel et al). A possible alternate strategy for measuring defense gene promoter expression involves operably linking a reporter gene to the promoter. Reporter gene constructs allow for ease of measurement of expression from the promoter of interest Examples of reporter genes include: β-gal, GUS (see, e.g., Jefferson, R. A„ et al, EMBO J 6". ^'390Ϊ-3907 (1987), green fluorescent protein, luciferase, and others. One of ordinary skill in the art will recognize that the following methods are but a few of the possibilities. One method of selecting plants with enhanced resistance is to determine resistance of a plant to a specific plant stress condition such as heat, cold, or nutritional deprivation. Other possible stress conditions include, but are not limited to, chemicals, metabolic changes (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) (1988)). One of skill in the art will recognize that resistance responses of plants vary depending on many factors, including what particular stress condition or plant is used. Generally, enhanced resistance is measured by the reduction or elimination of stress symptoms when compared to a control plant

The following Examples are offered by way of illustration, not limitation.

1. Construction of Chimeras For the Bril;;Xa21 chimeric receptor construction, the Bril sequence from

681 to 3332 (see, Li et al. Cell 90:929-938 (1997)), and the Xa21 sequence from 2121 to 3918 (see, Song et al Science 270:1804-1806 (1995)) were fused (see. Figure 1). Two sites, Nhel nd Kpnl, were introduced into Bril residues 681 to 688 and 3327 to 3332 by in vitro mutagenesis. The primer used to introduce the Nhel site is SRG-1 , 5'-TCTCTCTCACAGCTAGCTGAGAAATGAAG-3\ and the primer for the Kpnl site is SRG-2, 5'-TTCTTCAGGGTACCAATGG-3\ Kpnl site was also introduced into Xa2l residues 2119 to 2124, using primer SRG-3, 5'-ACAGATGGAACCGCGCCGAC-3\ The Nhel/Kpnl fragment of Bril and the Kpnl Pstl fragment of Xa2l were ligated, and the resultant construct was called NRGl . Using NRGl, a Bril LRR mutant construct called NRGlmL was made by using the Bril mutant binl-3 that has a single amino acid change (G to E) at the residue 2524 (see, Li et al Cell 90: 929-938 (1997)). Kinase domain mutant construct, NRGlm was made by in vitro mutagenesis of t &Xa21 kinase domain, changing the amino acid residue K to E in the site 2206 with primer SRG- 13, 5'-GTTGCAGTGGAGGTACTAA-3'. The mutant constructs NRGlmL and NRGlmK were used as controls.

For the Hevein: :Xa21 chimeric receptor construction, the Hevein domain from rice chitinase RCH10 sequence 1928 to 2289 (see Zhu and Lamb. Mot Gen Gent 226:289-286 (1991)) and the ήcQXa21 sequence from 1870 to 3918 were fused to get the chimeric receptor NRG6 (see, Figure 3). The Hevein domain of RCH10 was altered using in vitro mutagenesis with primer SRG-9, 5'-

ATCTTATATTTAGCTAGCACACCATGAGA-3' that introduced aNhel site. A Xmal site was introduced into Xa21 residues 1870-1875 using primer SRG-6, 5'- GGATCTCAATCCCCGGGAATGCCAAACTC-3'. The 0.36 kb Nhel/Xmal fragment containing the Hevein domain afRCHlO was combined with the Xmal/Pstl fragment of Xa21, resulting in the chimeric receptor NRG6. A mutant construct NRG9m that consists of the Hevein domain and Xa21 mutant kinase domain with the K to E substitution was made as control.

Rice Transgene and Cell Suspension Lines

All the constructs were inserted into the sites of Xbal and Sail of the vector ρBΪ321 under the control of the CaMV35S promoter and a NOS terminator. Rice transformation was carried out by microprojectile bombardment of embryogenic callus (see Chen et al. Plant Cell Reports 18: 25-31 (1998)). Transgenic calli carrying hygromycϊn resistance were screened by PCR and Northern blot analysis. Cell suspension lines were made from transgenic calli.

Cell Death and Defense Gene Activation For the brassinolide inducing cell death assay, Bril::Xa21 cell lines were treated with 2uM brassinolide for 24 hr. Cell cultures were staining with 0.05% Evans blue for 15 min and rinsed completely to remove excess unbound dye. Dye bound to cells was solubilized in 50% methanol with 1% SDS for 30 min at 50°C and quantified by absorbance at 600nm. Cell death was presented as dye binding increase against non- treated controls (see, Figures 2 and 4).

For the chitin inducing cell death assay, Hevein::Xa2l cell lines were treated with lOOng per ml dissoluble chitin (≥5mers) for 24hr. Dye binding increase was measured as above.

Rice defense gene (RCHIO and PAL) activation was assayed by Northern blot analysis. RNA samples were prepared from different time points after brassinolide or chitin treatment. A 1.2 kb Nsil/HindlU fragment of RCHIO (see Zhu and Lamb. Mol Gen Gent226:289-286 (1991)) and a 0.4 kb Sphl/Xhol fragment of rice PAL gene ZB8 (see Zhu et al Plant Mol Biol 29: 535-550 (1 95)) were used as probes.

To determine if BRll plays a direct role in BL perception, we developed a cell- based assay using the XA2Ϊ LRR-RLK from rice. XA2I confers resistance to Xanthomonas oryzae pv. oryzae (Xoo) (W. Song, et al., Science (1995)). Most incompatible plantpathogen interactions lead to a hypersensitive response (HR) that includes an oxidative burst, defense gene activation, and cell death (G. Martin, Curr. Opin. Plant Biol.2:273 (1999); A. Levine et al. Cell 79:583 (1994)). Thus, XA21 signaling outputs may provide a facile assay for determining the mechanism of LRR- RLK signaling. Figure 1 shows that stably transformed O. sativa ssp. Japonioa var. Taipei 309 cells expressing full-length XA21 from its native promoter exhibit race-specific defense responses (L. Chen et al., Plant Cell Rep. 18:25 (1998)). XA21 expression initiated cell death in lines that were inoculated with the incompatible Xoo Phillipine Race 6 (P6) strain PXO99A, but not when inoculated with the compatible Korean Race 1 (Kl) strain DY89031 (Fig. 5A). XA21 and Taipei 309 cell lines were inoculated with 107 cells/ml of the incompatible Phillipine Race 6 strain (P6) and the compatible Korean Race 1 strain (Kl) 5 days after transfer to fresh medium and grown for an additional 24 hours. Cells were stained with Evans blue, and dye binding increases over uninoculated .controls were quantified as an indicator of cell death. The experiment was repeated five times.

Pathogen-induced cell death is often accompanied by an oxidative burst (A. Levine et al. Cell 79:583 (1994)). A small, but highly reproducible oxidative burst was observed in the XA21 cell line inoculated for 1 hour with the incompatible P6, compared with inoculation with the compatible Kl strain (Fig. SB). Cells were inoculated for 1 hour with P6 and Kl (107 cells/ml). One hundred-microliter samples were added to 1 ml of reaction buffer [0,25 mM FeSO4, 0.25 mM (NH4)2SO4, 2S mM H2SO4, 1.25 μM xylenol orange, and 1 mM sorbitol] at room temperature for 1 hour. Absorbance was measured at 560 nm, and H2O2 levels were calculated by reference to standards with at least three repeats. After treatment, media were filtered to remove cells with 0,2-μm syringe filters. (Z. Jiang, J. V, Hunt, S. P. Wolff, Anal, Biochem. 202, 383 (1 92)). This small increase of H2O2 levels, although not as large as those reported for other plants, is consistent with the levels of H2O2 that we have seen in rice.

Activation of XA21 signaling leads to rapid and strong induction of transcription of the rice defense genes chitinase RCHIO (Q, Zhu and C. Lamb, Mol. Gen. Genet. 226:289 (1991)) and phenylalanine ammonia-lyase (PAL) (Q. Zhu, et al„ Plant Mol. Biol. 29:535 (1995)) in the incompatible interaction with Xoo (Fig, SC), whereas the compatible interaction shows a weaker and slower accumulation of these transcripts. This race-specific difference correlates to whole plant assays. The expression of a rice catalasc B gene (OsCatB) was strongly down-regulated in the XA21 cell line inoculated with the incompatible strain (Fig. 5C), as seen in whole plants (S. Yi et al. Mol. Cell 9:320 (1999)). Northern blotting shows changes in the expression of defense genes RCH10, PAL, and OsCatB over a time course of 0 to 24 hours after Xoo inoculation. RNA was extracted from the XA21 cell lines at 0 to 24 hours after inoculation with P6 and Kl (107 cells per milliliter), with the TRIzol reagent (Life Technologies, Gaithersburg, MD). Thirty micrograms of total RNA was used in each lane. A l.2~kb fragment of rice chitinase RCHIO (A. Levine et al. Cell 79:583 (1994)), a 400-base pair f agment of PAL (Q. Zhu, et al., Plant Mol. Biol.29:535 (1995)), and a cDNA clone of OsCatB were used as probes. The filters were reprobed with 18S Arabidopsis rDNA for normalization. mRNA levels were estimated with a Phospholmager System (Molecular Dynamics, Sunnyvale, California). Levels at time 0 were set as onefold for RCHIO and PAL and 10- fold for OsCatB. Taken together, these results establish the rice cell culture system as an excellent reporter of the signaling output of LRR-RLKs.

To test the mechanism by which BRll signals, we constructed several chimeric receptors between BRll and XA21 (Fig.6A; NRGl, NRG2, and NRG3). Of the three receptors, only one, NRGl, consisting of BRIl's extracellular and transmembrane domains and 65 amino acids of the intracellular domain (juxtamembrane domain) fused to the kinase domain of XA21, was able to elicit the HR (Figs. 6 and 7, discussed below). As controls, we also constructed mutant versions of the NRGl chimeric receptor. Previous studies have implicated the importance of a 70-amino acid island embedded between the 21st and 22nd LRR of BRIl's extracellular domain for BRll function (J, Li and J. Chory, Cell), One naturally occurring allele of BRll, bril-113, is a mutation of glycine at position 61 l in this domain to glutamate (Gly6l 1 Glu). The mutant chimeric receptor, NRGlmL, incorporates this change into the NRGl construct. We also constructed a kinase domain mutant of XA21 (Lys73 Glu) in the chimeric receptor NRGlmK, which lacks kinase activity in vitro, Transgenic cell lines were established by transfoπning the rice line Taipei 309 (L. Chen et al. Plant Cell Rep. (1998)). Northern and Western blotting confirmed that two of the NRGl-expressing lines, NRGl -30 and NRG1-34, and the mutant receptor, NRGlmK, were expressed at comparable levels in the cell lines. Northern hybridization shows mRNA accumulation of each chimeric gene, with a 1.3-kb DNA fragment of the Xa21 kinase domain as aprobe. Western blot shows the expression of BRI1-XA21 chimeric proteins NRGlmL accumulated to higher levels. A BRll DNA fragment encoding the presumed extracellular, transmembrane (TM), and juxtamembrane (JM) domains [amino acids 1 to 879 (J, Li and J, Chory, Cell)) was fused with the Xa21 fragment encoding the predicted kinase domain [amino acids 708 to 1025 (W. Song, et al., Science)] to make NRGl , NRG2 consisted of amino acids 1 to 769 of BRll and the XA21 TM, JM, and kinase domains with amino acids 625 to 1025. NRG3 consisted of amino acids 1 to 834 of BRll and amino acids 684 to 1025 of XA2L NRGlmL contains a mutation (Gly611 Glu) corresponding to the allele bril-113 (J. Li and J. Chory, Cell). NRGlmK is a mutation of XA21 (Lys737 Glu) obtained by in vitro mutagenesis with the primer (S'-GTTGCAGTGGAGGTACTAA-3') corresponding to the Xa21 sequence 2197 to 2215 (W. Song, et al., Science). Regenerated NRGl transgenic plants were dwarfed and sterile and exhibited partial resistance to Xoo after BL treatment as compared with controls.

We found that NRGl could initiate the HR upon addition of BL using two different cell lines, NRG1-30 and NRG1-34 (Fig. 7). Cell death was observed after treatment for 24 hours with 2 μM BL (Fig. 7A), whereas very little cell death occurred in the Taipei 309 control, NRGlmL (Gly611 Glu), or NRGlmK (Lys737 Glu) cells. The magnitude of increase in cell death was comparable to that seen in the incompatible pathogen-XA21 interaction (Fig.5 ). Likewise, we observed a detectable oxidative burst in the NRGl cell lines within 30 min of EL treatment (Fig. 3B). Changes in expression of defense genes were monitored in the wild-type and mutant receptor lines (Fig.7C). We observed an accumulation of both RCHIO and PAL mRNAs in response to 2 μM BL in both NRGl lines, with peak levels (five- to eightfold) occurring 4 to 8 hours after BL treatment (Fig. 7C). In contrast, neither NRGl L, NRGlmK, nor the Taipei 309 control cells showed an induction of RCHIO or PAL mRNAs (Fig. 7C). Accumulation of OsCatB RNA was inhibited inNRGl-30 and NRG1-34 cell lines 2 to 12 hours after BL treatment (Fig. 7C). We did not detect cell death in transgenic cell lines carrying the XA21 wild-type protein or overexpressing the XA21 kinase domain after BL treatment.

A BL dose-response curve was constructed with RCHIO RNA accumulation as a reporter. We saw RCHIO induction using concentrations of BL as low as 10 nM; the response began to saturate at about 2 μM BL (Fig. 8). These BL concentrations are physiologically relevant, being consistent with those for rescue of the Arabidopsis BL biosynthetic mutant, det2 (J. Li et al. Science 272:398 (1996)). These three assays indicate that the BRI1-XA21 chimeric receptor can recognize BL to activate cell death, the oxidative burst, and defense gene induction. Moreover, both the extracellular/transmembrane/juxtamembrane domains of BRll an the XA21 kinase domain are required for these responses.

The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordmary skill in the art and are encompassed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED TS-

1. A heterologous nucleic acid molecule comprising: a LRR domain, a transmembrane domain, and a cytoplasmic protein kinase domain.

2. The heterologous nucleic acid of claim 1 , wherein the LRR domain is from receptor or receptor-like kinase.,

3. The heterologous nucleic acid of claim 2, wherein the polynucleotide sequence comprises an extracellular LRR domain sequence at least about 70% identical to a sequence from about residue 681 to about residue 3332 in SEQ ID NO: 3.

4. The heterologous nucleic acid of claim 3 wherein the extracellular LRR domain sequence consists of a sequence from about residue 681 to residue 3331 as shown in SEQ ID NO: 3.

5. The heterologous nucleic acid of claim 1, wherein the polynucleotide sequence comprises a kinase domain sequence at least about 70% identical to a sequence from about residue 2121 to about residue 3918 in SEQ ID NO: 1.

6. The heterologous nucleic acid of claim 1, wherein the heterologous extracellular domain is from RCHIO.

7. The heterologous nucleic acid of claim 6, wherein the polynucleotide sequence comprises an extrdcellular hevein domain sequence at least about 70% identical to a sequence from about residue 1928 to about residue 2289 in SEQ ID NO: 5.

8. The heterologous nucleic acid of claim 7, wherein the extracellular hevein domain consists of a sequence from about residue 1928 to about residue 2289 as shown in SEQ ID NO: 5.

9. The heterologous nucleic acid of claim 7, wherein the polynucleotide sequence comprises a kinase domain sequence at least about 70% identical to a sequence from about residue 1870 to about residue 3918 in SEQ ID NO: 1.

10. The heterologous nucleic acid of claim 1, further coprising a plant promoter operably linked to the polynucleotide encoding the chimeric RRK receptor.

11. The heterologous nucleic acid of claim 10, wherein the promoter is a constitutive promoter.

12. An expression vector comprising the heterologous nucleic acid molecule of claim 1.

13. A host cell transformed or transfectcd with the expression vector of claim 12.

14. A transgenic plant comprising the heterologous nucleic acid molecule of claim 1.

15. The transgenic plant of claim 14, wherein the heterologous extracellular domain comprises a LRR domain.

16. The transgenic plant of claim 14, wherein the heterologous extracellular LRR domain is from a Bril.

17. The transgenic plant of claim 16, wherein the polynucleotide sequence comprises an extracellular LRR domain sequence at least about 70% identical to a sequence from about residue 681 to about residue 3332 in SEQ 3D NO: 3.

18. The transgenic plant of claim 16, wherein the polynucleotide sequence comprises a kinase domain sequence at least about 70% identical to a sequence from about residue 2121 to about residue 3918 in SEQ ID NO: 1.

19. The transgeuic plant of claim 14, wherein the heterologous extracellular hevein domain from RCHIO.

20. The transgenic plant of claim 1 , wherein the polynucleotide sequence comprises an extracellular hevein domain sequence at least about 70% identical to a sequence from about residue 1928 to about residue 2289 in SEQ ID NO: 5.

21. The transgenic plant of claim 19, wherein the polynucleotide sequence comprises a kinase domain sequence at least about 70% identical to a sequence from about residue 1870 to about residue 3918 in SEQ ID NO: 1.

22. The transgenic plant of claim 19, further comprising a plant promoter operably linked to the polynucleotide encoding the RRK receptor.

23. The transgenic plant of claim 1 , wherein the promoter is a constitutive promoter.

24 . The seed of a plant transformed or transfected with the expression vector of claim 12.

25 . A method for identifying ligands for receptor or receptor-like kinase, comprising exposing the heterologous nucleic acid of claim 1 to a test compound and determining if said test compound has bound to the LRR domain of claim 1.

26. A method for controlling plant development and or disease resistance, said method comprising contacting the product of the heterologous nucleic acid of claim 1 to a ligand therefor.

27 . A chimeric receptor or receptor-like kinase, comprising the expression product of the heterologous nuceic acid of claim 1.