CA2265441A1 - Protein kinases and uses thereof - Google Patents

Abstract

Nucleic acid molecules are disclosed that are induced upon pathogen invasion or elicitor treatment. Such molecules are functional in plants, plant tissue and in plant cells for inducible gene expression and altering the disease resistance phenotype of plants. Such molecules are, or are related to, sequences of calcium dependent protein kinase genes. Also disclosed are methods for obtaining transgenic plants containing such nucleic acid molecules and methods for using such molecules. Polypeptides encoded by such nucleic acids are also disclosed herein.

Description

?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109 PROTEIN KINASES AND USES THEREOFStatement as to Federallv Sponsored ResearchThe research reported herein was performed in partwith funding from the National Science Foundation of theUnited States Government. The United States Governmentmay have certain rights in this invention.Field of the InventionThis invention relates to nucleic acids encodingcalcium dependent protein kinases, polypeptides producedfrom such nucleic acids and transgenic plants expressingsuch nucleic acids.Background of the InventionIn plants, disease resistance to fungal,bacterial, and viral pathogens is associated with a plantresponse termed the hypersensitivity response (HR). Inthe HR, the site in the plant where the potentialphytopathogen invades undergoes localized cell death, andit is postulated that this localized plant cell deathcontains the invading microorganism or virus, therebyOther plantdefense responses include the production of phytoalexins,protecting the remainder of the plant.the production of lytic enzymes capable of avertingpathogen ingress and modifications to cell walls thatstrengthen it against physical and/or enzymatic attack.The HR of plants can include phytoalexinproduction as part of the response to invadingmicroorganisms. For example, tobacco (Nicotiana tabacum)produces sesquiterpenes in response to microbialinvaders, e.g., Pseudomonas lachrymans.A variety of compositions can serve as elicitorsof plant phytoalexin synthesis. These include one or?WO 99/02655101520253035CA 02265441 1999-03-05PCT/U S98/ 14109-2-more toxic ions, e.g., mercuric ions,defined compositions, metabolic inhibitors,other chemicallycell wallglycans, certain glycoproteins, certain enzymes, fungalchitosans, and certainspores, certain fatty acids,oligosaccharides derived from plant cell walls.e.g., (1983)and references cited therein.See,Sequeira, L. Annu. Rev. Microbiol. ;1:5l—79Cell wall fragments ofcertain Phytophthora species and cellulase fromTrichoderma viride but not Aspergillus japonicumpectolyase can also elicit the HR. Attack by other plantpathogens or an avirulent related strain can also inducethe HR.Elicitins are proteins produced by plant pathogensand potential plant pathogens. Elicitins can induce theHR in plants. Generally, localizedbut not necessarily,cell death is the result of the elicitin-induced responsein the infected These(or challenged) plant tissue.responses mediate full or partial resistance todestructive infection by the invading, potentially plantpathogenic microorganism. Amino acid and nucleotidecoding sequences for an elicitin of Phytophthoraparasitica have been published. (1993)Mol.Kamoun et al.Plant-Microbe Interactions 6:573-581.Plant pathogenic viruses including, but notlimited to, (TMV),Bacteria that infect plants also canTobacco Mosaic Virus induce the HR ininfected plants.induce HR and thereby disease resistance; representativee.g.,Plant pathogenic fungi generallybacteria eliciting HR include, Xanthomonas spp. andPseudomonas syringae.do not induce the HR response after attack on a hostplant, e.g., Phytophthora parasitica and Peronosporatabaciattackon tobacco hosts, but can induce the HR afteron a non—host plant.The signal transduction mechanisms involved inexpression of disease resistance are under investigation?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-3-and some of the genetic and biochemical features havee.g., et al.,However, many aspects of signalbeen outlined. See,268:66l—667 (1995) .transduction pathways and the role of many specificStaskawicz, B. Sciencecomponents are not well understood.There is a long felt need in the art for methodsof protecting plants, particularly crop plants, frominfection by plant pathogens. Especially important fromthe standpoint of economic and environmental concerns arebiological or "natural" methods rather than those whichdepend on the application of chemicals to crop plants.There is also a need in the art for plant polynucleotidesequences for enhancing and/or improving diseaseresistance in plants.Summary of the InventionNucleic acids of the present invention are based(CDPK)Induction ofon novel calcium dependent protein kinase genesand their corresponding proteins.expression of these novel CDPK genes is surprisinglyrapid, i.e., mRNA transcription of such genes can beobserved as soon as 30 minutes after elicitor-mediatedThus,genes disclosed herein are among those genes that areinduction of plant defense responses. the novelmost rapidly induced in response to signals indicating aninvading plant pathogen.An isolated polynucleotide is disclosed herein,that comprises the nucleotide sequence of SEQ ID NO:1 andits complement, and an RNA analog of SEQ ID NO:l or itscomplement. Such a polynucleotide can also be a nucleicacid fragment of the above that is at least 20nucleotides in length and that hybridizes under stringentconditions to genomic DNA encoding the polypeptide ofFigure 3. The polynucleotide can comprise, for example,?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-4-nucleotides 1 to 170, nucleotides 160 to 560, ornucleotides 550 to 920 of Figure 2.A nucleic acid construct as disclosed hereincomprises a polynucleotide of the invention. In such aconstruct, a polynucleotide of the invention can beoperably linked to one or more elements that regulatetranscription of the polynucleotide, for example, aregulatory element induced in response to a plantpathogen such as a fungus (e.g., Phytophthora), abacterium (e.g., Pseudomonas),or a virus (e.g., TobaccoMosaic Virus) as described herein.In other embodiments,such induction is mediated by an elicitor by(e-9..fungal or bacterial elicitors).Further aspects of the present invention aretransgenic plant cells, plant tissues, and plants thathave been genetically engineered to contain and express apolynucleotide of the invention, for example, a codingsequence,or an antisense sequence. The construct canfurther comprises a regulatory element operably linked toe.g.,The plant can be a dicotyledonous plant, e.g.,the polynucleotide, an inducible regulatoryelement.a member of the Solanaceae family such as Nicotianatabacum. The plant can also be a monocotyledonous plant,a gymnosperm,A transgenic plant is disclosed herein thator a conifer.contains a polynucleotide expressing a polypeptide havingfrom about 250 to about 550 amino acids. The polypeptidecomprises an amino acid sequence substantially identicalto the amino acid sequence of Figure 3.A method of using a polynucleotide is disclosedherein. The method comprises the step of hybridizing thepolynucleotide discussed above to DNA or RNA from aplant. The method can further comprise the steps ofidentifying a segment of the plant DNA or RNA that hasabout 70% or greater sequence identity to the?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/ 14109_.5_polynucleotide, and the step of cloning at least aportion of the DNA or RNA segment. The cloned portionmay further comprise DNA flanking the segment having 70%or greater sequence identity.In another aspect, the invention features a methodThe methodcomprises the steps of introducing a polynucleotide ofof altering disease resistance in a plant.the invention into a plant cell; and producing a plantcontaining the polynucleotide from the plant cell.Expression of the polynucleotide alters diseaseresistance in the plant. For example, the nucleic acidconstruct may further comprise an inducible regulatoryelement operably linked to the polynucleotide andexpression may be induced by the regulatory element uponexposure of the plant to an elicitor or plant pathogen.In another aspect, the invention features anisolated polypeptide, having from about 250 to about 550amino acids and comprising an amino acid sequencesubstantially identical to Figure 3.An inducible regulatory element is a DNA sequenceeffective for regulating the expression of apolynucleotide that is operably linked to that regulatoryelement. For example, a CDPK gene product associatedwith a plant defense response (e.g., a hypersensitiveresponse) can be operably linked to a developmentally-regulated regulatory element. Also included in this termare regulatory elements that are sufficient to rendergene expression inducible in response to disease-associated external signals or agents (e.g., pathogen- orelicitor-induced signals or agents as described herein).Also included in this term are those regulatory elementsflanking a novel CDPK gene and involved in rapidinduction of transcription of such a novel gene. Ingeneral, defense response regulatory elements are located?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-5-5’ to the coding region of a gene, but are not solimited.By "tissue—specific" is meant capable ofpreferentially increasing expression of a gene product(e.g., an mRNA molecule or polypeptide) in one tissue(e.g., xylem tissue) as compared to another tissue (e.g.,phloem). By "cell—specific" is meant capable ofpreferentially increasing expression of a gene product(e.g.,(e.g., a parenchyma cell) as compared to another cellan mRNA molecule or polypeptide) in one cell(e.g., an epidermal cell).A "pathogen" is an organism whose infection of, orassociation with, cells of viable plant tissue can resultin a disease. An "elicitor" is any molecule that iscapable of initiating a plant defense response. Examplesof elicitors include,e-9-Icompositions,without limitation, one or moretoxic ions, mercuric ions, other chemically definedmetabolic inhibitors, cell wall glycans,certain glycoproteins, certain enzymes, fungal spores,chitosans, certain fatty acids, and certainoligosaccharides derived from plant cell walls, and(e.g.,By "operably linked" is meant that twoelicitins harpin, cryptogein, and parasiticein).polynucleotides are connected in such a way as to permitthe two polynucleotides to achieve a desired functionalactivity, for example, linking of an inducible regulatorysequence and a coding sequence to achieve gene expressionwhen the appropriate inducer molecules are present.Unless otherwise defined, all technical andscientific terms used herein have the same meaning asCommonly understood by one of ordinary skill in the artAlthough methods andmaterials similar or equivalent to those described hereinto which this invention belongs.can be used in the practice or testing of the presentinvention, suitable methods and materials are described?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-7-below. All publications, patent applications, patents,and other references mentioned herein are incorporated byreference in their entirety. theIn case of conflict,present specification, including definitions, willcontrol. In addition, the materials, methods, andexamples are illustrative only and not intended to belimiting.Other features and advantages of the inventionwill be apparent from the following description of thepreferred embodiments thereof, and from the claims.Brief Description of the DrawingsFigure I is a representation of the nucleotidesequences of the primers FokinB and RecalIV.Figure 2 is a representation of the DNA sequence(SEQ ID NO:l)cell suspension culture derived from a tobacco cultivarKY14 explant,of a partial CDNA clone isolated from aafter growth in the presence of theelicitin parasiticein.Figure 3 is a representation of the deduced aminoacid sequence of the DNA sequence of Figure 2, using thestandard one letter amino acid code.Figure 4 is a schematic comparison of the aminoacid sequence of Figure 3 to that of a soybean CDPK.Detailed Description of the InventionThe present invention relates to isolatedpolynucleotides (nucleic acids) that are induced in plantcells in response to invasion by a potential plantpathogen and/or treatment with an elicitor or elicitor-mimicking chemical signals. Such nucleic acids typically(CDPK)Induction ofencode a calcium dependent protein kinasepolypeptide or CDPK—related polypeptide.the novel polynucleotides disclosed herein corresponds intime to that of plant defense response genes, whereas?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-8-other CDPK genes appear to be induced less rapidly.Induction of gene expression for such novel genes is morerapid than that of genes involved in developmentallye.g.,processes such as floral development.regulated processes in plants, developmentallyregulatedInduction of the novel CDPK genes disclosed herein isalso more rapid than that of many genes involved inresponses to abiotic stress, such as salt or waterstress.A polynucleotide of the present invention can bein the form of RNA or in the form of DNA, including cDNA,synthetic DNA or genomic DNA. The DNA can be double-stranded or single—stranded and, if single—stranded, canbe either a coding strand or non—coding strand. An RNAanalog of SEQ ID NO:1 may be, for example, mRNA or acombination of ribo— and deoxyribonucleotides.A polynucleotide of the invention can encode apolypeptide including an amino acid sequencesubstantially similar or identical to that of Figure 3.In some embodiments, a polynucleotide may be a variant ofthe nucleic acid shown in SEQ ID NO:1, e.g.,due to the degeneracycan have adifferent nucleotide sequence that,of the genetic code, encodes the same amino acid sequenceas the polypeptide of Figure 3.A polynucleotide of the invention can furtherinclude additional nucleic acid sequences. ‘For example,a nucleic acid fragment encoding a secretory or leaderamino acid sequence can be fused in—frame to the aminoterminal end of a polypeptide comprising the amino acidsequence of Figure 3. Other nucleic acid fragments areknown in the art that encode amino acid sequences usefulfor fusing in—frame to the CDPK polypeptides disclosede.g., U.S. 5,629,193.further include one or more regulatory elements operablyherein. See, A polynucleotide canlinked to a CDPK polynucleotide disclosed herein.?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-9-The present invention also includespolynucleotides that selectively hybridize to a CDPKpolynucleotide sequence disclosed herein. Hybridizationmay involve Southern analysis (Southern blotting), amethod by which the presence of DNA sequences in a targetnucleic acid mixture are identified by hybridization to alabeled oligonucleotide or DNA fragment probe. Southernanalysis typically involves electrophoretic separation ofDNA digests on agarose gels, denaturation of the DNAafter electrophoretic separation, and transfer of the DNAto nitrocellulose, nylon, or another suitable membranesupport for analysis with a radiolabeled, biotinylated,or enzyme-labeled probe as described in sections 9.37-9.52 of Sambrook et al., secondedition, Cold Spring Harbor Laboratory, Plainview, NY.(1989) Molecular Cloning,A polynucleotide can hybridize under moderatestringency conditions or under high stringency conditionsto a polynucleotide disclosed herein. High stringencyconditions are used to identify nucleic acids that have ahigh degree of homology or sequence identity to theprobe. High stringency conditions can include the use ofa denaturing agent such as formamide duringe.g.,albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mMsodium phosphate buffer at pH 6.5 with 750 mM NaCl, and75 mM sodium citrate at 42°C. Another example is the use5 X SSC (0.75 M NaCl, 0.075 M sodiumhybridization, 50% formamide with 0.1% bovine serumof 50% formamide,citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5 x Denhardt’s solution, sonicated salmonsperm DNA (50 pg/ml), 0.1% SDS,at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.and 10% dextran sulfateAlternatively, low ionic strength and high temperaturecan be employed for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate (0.lX SSC); 0.1%lauryl.sulfate (SDS) at 65°C.sodium?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-10-Moderate stringency conditions are hybridizationconditions used to identify nucleic acids that have lesshomology or identity to the probe than do nucleic acidsidentified under high stringency conditions. Moderatestringency conditions can include the use of higher ionicstrength and/or lower temperatures for washing of thehybridization membrane, compared to the ionic strengthand temperatures used for high stringency hybridization.For example, a wash solution comprising 0.060 MNaCl/0.0060 M sodium citrate (4X SSC) and 0.1% sodiumlauryl sulfate (SDS) can be used at 50°C, with a lastwash in 1X SSC, at 65°C. Alternatively, a hybridizationwash in 1X SSC at 37°C can be used.Hybridization can also be done by Northernanalysis (Northern blotting), a method used to identifyRNAs that hybridize to a probe. The probe is labeledwith a radioisotope such as ”P, by biotinylation or withThe RNA to be analyzed can beelectrophoretically separated on an agarose oran enzyme .polyacrylamide gel, transferred to nitrocellulose,and hybridized with thenylon,or other suitable membrane,probe, using standard techniques well known in the artsuch as those described in sections 7.39-7.52 of Sambrooket al., supra.It is generally preferred that a probe of at leastabout 20 nucleotides in length be used, preferably atleast about 50 nucleotides, more preferably at leastabout 100 nucleotides. If a relatively short probe is tobe used, the nucleotide sequence of the probe preferablyavoids regions conserved among plant CDPK genes (proteinkinase domains and calcium—binding domains), to morereadily distinguish the rapidly induced CDPK genesdisclosed herein from more slowly induced CDPK genes,constitutive CDPK genes or low—level constitutive CDPKgenes. Nevertheless, probes containing such conserved?WO 99/02655l01520253035CA 02265441 1999-03-05PCT/US98/14109-11..regions can be used, provided that there are sufficientnon-conserved regions present in the probe that are morespecific for the novel polynucleotides disclosed herein.A polynucleotide of the invention has at leastabout 70% sequence identity, preferably at least about80% sequence identity, more preferably at least about 90%sequence identity to SEQ ID NO:1. Sequence identity canbe determined, for example, by computer programs designedto perform single and multiple sequence alignments.Polynucleotides having at least about 70% nucleotidesequence identity to the polynucleotide of SEQ ID NO:1are included in the invention and can be identified byhybridization under conditions of moderate stringency.Polynucleotides having at least about 80% sequenceidentity, or atleast about 95%least about 90% sequence identity, or atsequence identity to the polynucleotideof SEQ ID NO:l can be identified by high stringencyhybridization.A polynucleotide of the invention can be obtainedby chemical synthesis, isolation and cloning from plantgenomic DNA, or other means known to the art, includingthe use of polymerase chain reaction (PCR) technologycarried out using oligonucleotides corresponding toportions of SEQ ID NO:l. PCR refers to a procedure ortechnique in which target nucleic acid is amplified in amanner similar to that described in U.S. Patent No.4,683,195, incorporated herein by reference, andsubsequent modifications of the procedure describedtherein. Generally, sequence information from the endsof the region of interest or beyond are employed todesign oligonucleotide primers that are identical orsimilar in sequence to opposite strands of the templateto be amplified. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA,and CDNA transcribed from total cellular RNA,?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-12-and the like.Alternatively, it is contemplated that a CDNA library (inbacteriophage or plasmid sequences,an expression vector) can be screened with CDPK—specificantibody prepared using peptide sequence(s) fromhydrophilic regions of the CDPK sequence of Figure 3 andtechnology known in the art.The novel polynucleotides of the invention can befound in substantially all plants, including members ofthe Leguminaceae (e.g., soybean), members of themembers of the(e.g., Arabidopsis thaliana) andSolanaceae (e.g., N. tabacum),Brassicaceae familymembers of the Graminaceae (e.g., Zea mays). Preferably,polynucleotides of the invention are selected from theSolanaceae family.In some embodiments, a polynucleotide of theinvention is identified and isolated from a plant basedon nucleotide sequence homology and on the rapidinduction of expression after elicitor or pathogentreatment. For example, DNA:DNA hybridization underconditions of moderate to high stringency with apolynucleotide probe disclosed herein allows theidentification of corresponding genes from other plant(e.g., CDNA)prepared from a tissue shortly after induction of defensespecies. Use of a target nucleic acidresponses facilitates the isolation of the novelpolynucleotides disclosed herein, because suchpolynucleotides typically are more rapidly induced thanother CDPK genes.A nucleic acid construct comprises apolynucleotide as disclosed herein, and typically islinked to another, different polynucleotide. Forexample, a full—length CDPK coding sequence can beoperably fused in—frame to a nucleic acid fragment thatencodes a leader sequence, secretory sequence or other?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-13-additional amino acid sequences that may be usefullylinked to a polypeptide or peptide fragment.In some embodiments, a nucleic acid constructincludes a polynucleotide of the invention operablylinked to at least one suitable regulatory sequence insense or antisense orientation. Regulatory sequencestypically do not themselves code for a gene product.Instead, regulatory sequences affect the expression levelof the coding sequence. Examples of regulatory sequencesare known in the art and include, without limitation,minimal promoters and promoters of genes induced inresponse to elicitors. Native regulatory sequences ofthe polynucleotides disclosed herein can be readilyisolated by those skilled in the art and used inconstructs of the invention. other examples of suitableregulatory sequences include enhancers or enhancer-likeelements, introns, 3’ non—coding regions such as poly Asequences and other regulatory sequences discussedherein. Molecular biology techniques for preparing suchchimeric genes are known in the art.Polypeptides of the invention have from about 250e-9-,acids to about 508 amino acids, or from about 308 aminoto about 550 amino acids, from about 300 aminoacids to about 500 amino acids. A polypeptide of theinvention typically contains protein kinase domains aswell as calcium-binding site domains. Such domains42 to 49, 191to 202, 227 to 238, 264 to 274, and 297 to 307 of Figure3.include, for example, amino acids 2 to 7,The amino acid sequence of the polypeptide caninclude the deduced amino acid sequence of Fig. 3. Inother embodiments, a polypeptide of the inventionincludes an amino acid sequence substantially identicalto that of Fig. 3, e.g., about 80% or greater sequenceidentity, or about 90% or greater sequence identity, or?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-14-about 95% or greater sequence identity. Generally,conservative amino acid substitutions or substitutions ofsimilar amino acids are tolerated without affectingprotein function. Similar amino acids are those that aresimilar in size and/or charge properties. For example,isoleucine and valine are similar amino acids.Similarity between amino acid pairs has been assessed inthe art in a number of ways.(1978)5, Suppl. 3,For example, Dayhoff et al.in Atlas of Protein Sequence and Structure, Vol.PP-amino acid substitutions which can be employed as a345-352, provides frequency tables formeasure of amino acid similarity. Protein kinase domainsand calcium—binding site domains may be altered byconservative substitutions, but generally are retainedwithout alterations in amino acid sequence.An "isolated" polypeptide is expressed andproduced in a manner or environment other than the manneror environment in which the polypeptide is naturallyexpressed and produced. For example, a polypeptide isisolated when expressed and produced in bacteria orfungi.Similarly, a polypeptide is isolated when a geneencoding it is operably linked to a chimeric regulatoryelement and expressed in a tissue or species where thepolypeptide is not naturally expressed. In addition, apolypeptide is isolated when a gene encoding it isoperably linked to a chimeric regulatory element and isexpressed in a tissue where the polypeptide is naturallyexpressed, but at higher levels. A polypeptide of theinvention can also be isolated by standard purificationmethods to obtain it in about 80% or greater purity, orabout 90% or greater purity or about 95% or greaterpurity.In some embodiments, a polypeptide of theinvention is an analog or variant of a polypeptideincluding the deduced amino acid sequence of Fig. 3.?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-15..Such analogs or variants include, for example, naturallyoccurring allelic variants, non—naturally occurringallelic variants, deletion variants, and insertionvariants, that do not substantially alter the function ofthe polypeptide.A polypeptide of the invention may comprise thesequence shown in Fig. 3 as well as the flanking aminoterminal and carboxy terminal sequences encoded by thesame gene as that comprising the nucleotide sequence ofSEQ ID NO:l.be produced from a gene that links, in—frame, nucleotidesAlternatively, a chimeric polypeptide mayfrom the 5’ region of a first CDPK gene to nucleotidesfrom the 3’ region of a second CDPK gene, thereby forminga chimeric gene that encodes the chimeric polypeptide.An illustrative example of a chimeric CDPK polypeptide isa polypeptide expressed by a polynucleotide encodingamino acids 1 to 156 from the amino terminal region of a(Fig. 4),sequence of Fig. 3,soybean CDPK gene followed by the amino acidfollowed by amino acids 465 to 508from the carboxy terminal region of the same soybean CDPKgene, all of which are fused in—frame.A transgenic plant of the invention contains anucleic acid construct as described herein. Such aconstruct is introduced into a plant cell and at leastone transgenic plant is obtained. Seeds produced by atransgenic plant can be grown and selfed (or outcrossedand selfed) to obtain plants homozygous for theconstruct. Seeds can be analyzed to identify thosehomozygotes having the desired expression of theconstruct. Transgenic plants may be entered into abreeding program, e.g., to increase seed, to introgressthe novel construct into other lines or species, or forfurther selection of other desirable traits.Alternatively, transgenic plants may be obtained by?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-16-vegetative propagation of a transformed plant cell, forthose species amenable to such techniques.As used herein, a transgenic plant also refers toprogeny of an initial transgenic plant. Progeny includesdescendants of a particular plant or plant line, e.g.,Progeny of anF2, F3,and subsequent generation plants, or seeds formed on BCMBC2, BC3,In some embodiments,seeds developed on an instant plant.instant plant also includes seeds formed on FMand subsequent generation plants.a transgenic plant contains aconstruct that includes a polynucleotide of the inventionoperably linked in sense orientation to a suitableregulatory element,so that a sense mRNA is produced. Ifdesired, a selectable marker gene can be incorporatedinto the construct in order to facilitate identificationof transformed cells or tissues.Inhibition of the novel CDPK genes in plants isalso useful. For example, inhibition of CDPK geneexpression shortly before harvest of a seed crop canpermit plant pathogens to more readily invade plantvegetative tissues, thereby reducing the amount of plantbiomass that interferes with mechanical harvesting of theseeds. Regulated inhibition of CDPK gene expression canbe accomplished by operably linking, in antisenseorientation, a polynucleotide of the invention to ae.g., U.S.One can achieve the same effect bysuitable inducible regulatory sequence.Patent 5,453,566.cosuppression,See,i.e, expression in the sense orientationof the entire or partial coding sequence of a novel CDPKgene can suppress corresponding endogenous CDPK genes.e.g., WO 94/11516.In some embodiments,See,a nucleic acid constructincludes a polynucleotide disclosed herein, operablylinked to a minimal promoter. Such a construct, whenintroduced into and expressed in a plant, can confer low?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/ 14109-17..level constitutive expression of the polynucleotide,resulting in an enhanced systemic defense response by theplant. A minimal promoter contains the DNA sequencesignals necessary for RNA polymerase binding andinitiation of transcription. Generally, transcriptiondirected by a minimal promoter is low and does notrespond either positively or negatively to environmentalor developmental signals in plant tissue. An exemplaryminimal promoter suitable for use in plants is thetruncated CaMV 35S promoter, which contains the regionfrom —90 to +8 of the 35S transcription unit.Transcriptional regulatory sequences can be usedto control gene expression in suspension cultures. Forexample, the EAS4 promoter including the transcriptioninitiation signals, the inducible transcriptionregulatory element and the transcription-enhancingelement, can be used to mediate the inducible expressionof the disclosed coding sequence in transgenic plants orsuspension cell cultures.No. 08/577,483.sequence of interest is induced by the application of anSee U.S. Application SerialWhen desired, expression of the codingelicitor or other inducing signal.Transgenic techniques for use in the inventioninclude, without limitation, Agrobacterium—mediatedtransformation, electroporation and particle guntransformation. Illustrative examples of transformationtechniques are described in U.S. Patent 5,204,253,(particle gun) and U.S. Patent 5,188,958 (Agrobacterium).Transformation methods utilizing the Ti and Ri plasmidsof Agrobacterium spp. typically use binary type vectors.Walkerpeach, C. et al., in Plant Molecular BiologyManual, S.Dordrecht,Gelvin and R. Schilperoort, eds., KluwerCl 1-19 (1994).In some embodiments, an inducible transcriptionregulatory sequence can be coupled to a promoter sequence?WO 99/02655l01520253035CA 02265441 1999-03-05PCT/US98/14109-18-functional in plants, both of which are operably linkedto a polynucleotide of the invention. When such aregulatory element is coupled to a promoter, a truncated(or minimal) promoter generally is used, thefor example,truncated 35S promoter of Cauliflower Mosaic Virus(CaMV).promoters can also be used,Truncated versions of other constitutivee.g., A.and mas,such as the CaMV 19S gene.tumefaciens T—DNAgenes such as nos, ocs, and plant virus genesTechniques are well-known to the art for theintroduction of DNA into monocots as well as dicots, asare the techniques for culturing plant tissues andregenerating those tissues. Monocots which have beensuccessfully transformed and regenerated include wheat,e.g., U.S.Transgenic aspen tissuecorn, rye, rice and asparagus.5,484,956 and 5,550,318.has been prepared and transgenic plants have beenSee, PatentNos.regenerated. Poplars have also been transformed.Technology is also available for the manipulation,transformation, and regeneration of Gymnosperm plants.See, e.g., U.S. Patent No. 5,122,466 and U.S. Patent No.5,041,382.A method according to the invention includes theintroduction of a nucleic acid construct into a plantcell and the production of a plant from such atransformed cell. Expression of the polynucleotidepresent in the construct alters the disease resistancee.g.,phenotype is conferred on the plant or an existingphenotype of the plant, a novel disease resistancedisease resistance phenotype is enhanced.Disease resistance phenotype involves the leveland timing of host defensive responses in the transgenicplant. Assays to indicate that disease resistance hasbeen altered typically include the application of acompound that ordinarily elicits a defensive response to?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-19-a transgenic plant and, in parallel, the application ofthe same compound to a control plant. A control planttypically is from the same parental line as the one intowhich a new nucleic acid construct was introduced.Disease resistance is enhanced or conferred on a plant byexpression of a polynucleotide of the invention whenthere is a higher level of resistance in the transgenicplant than the corresponding resistance in the controlplant. Disease resistance can be measured with referenceto a specific pathogen, e.g., a Phythophthora spp..Disease resistance can also be measured with reference toseveral pathogens, to identify an enhanced systemicdefense response.Where transgenic plants are to be induced forexpression of a CDPK coding sequence operably linked toan elicitor—mediated regulatory element, the elicitortypically must penetrate the cuticle of the plant to havean inductive effect. Plant tissue can be wounded tofacilitate or allow the uptake of the elicitor into theplant tissue. A wide variety of inducing compositions,including elicitors and other chemical signals, such asthe combination of ethylene and methyl jasmonate, can beeffectively used to induce expression.A method of using a polynucleotide of theinvention comprises the step of hybridizing thepolynucleotide to DNA or RNA from a plant. Hybridizationcan be carried out, for example, as describedhereinabove. The method can further comprise the step ofidentifying a segment of the plant DNA or RNA that has asignificant degree of sequence identity to thepolynucleotide, e.g., 70% sequence identity, preferably80% sequence identity, 90% sequence identity, or 95%sequence identity. The segment can be identified byelectrophoretic separation of the plant DNA or RNA andthe use of labeled polynucleotide probe, which results in?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-20-a visible band at the position of the homologous segment.Segments can be generated, for example, by physicalshearing or by restriction endonuclease digestion. Asegment can be as short as 100 bp (nucleotides) inlength, but typical segments are at least 1000 bp, andcan be 10,000 bp or greater.Such a method can further comprise the step ofcloning at least a portion of the DNA or RNA segment,including, but not limited to, DNA flanking thehomologous segment. Such flanking DNA can includepromoters, enhancers, transcriptional regulatory elementsand poly A sequences. Flanking DNA can be either 5’ toor 3' to the homologous segment and preferably includes300, or 600, or 1,000 bp of DNA beyond the codingsequence, because regulatory elements generally are foundwithin this span.Promoters and other elicitor or pathogen-responsive regulatory elements flanking the novelpolynucleotides disclosed herein are particularly useful,because such elements confer very rapid induction of geneexpression after treatment with pathogen or elicitor.Such regulatory elements can be operably linked to usefulgenes to allow rapid production of desirable compounds.For example, such regulatory elements can be used todrive expression of genes encoding antibodies, bloodclotting factors, antigenic peptides, viral replicases orcoat proteins, and enzymes involved in secondarymetabolite synthesis (such as isoprenoid biosynthesis).e.g., U.S. Patent 5,612,487; U.S. Patent 5,484,719;and U.S. Application Ser. No. 08/577,483, filed December22, 1995fAfter introducing a chimeric gene having anSee,elicitor or pathogen—responsive element into a plant,expression of the chimeric gene product can be inducedwith an appropriate pathogen or elicitor. Production of?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-21-the desired gene product (or its enzymatic end product)rapidly ensues and the desired product can then beobtained.The invention will be further described in thefollowing examples, which do not limit the scope of theinvention described in the claims.EXAMPLESThe following examples use many techniqueswell-known and accessible to those skilled in the arts ofmolecular biology, in the manipulation of recombinant DNAin plant tissue and in the culture and regeneration oftransgenic plants. Enzymes are obtained from commercialsources and are used according to the vendors’recommendations or other variations known to the art.Reagents, buffers, and culture conditions are also knownto the art. Abbreviations and nomenclature, whereemployed, are deemed standard in the field and arecommonly used in professional journals such as thosecited herein.Example 1.Cloning of a Tobacco CDPK cDNAThe elicitor parasiticein was prepared byexpression of the Phytophthora_parA1 gene in E. colicells and isolation of the gene product from theperiplasmic space. IGenomic DNA of Phytophthora Race 0 was isolatedfrom mycelium essentially as described et al.Trends in Genetics 10:226-227 (1994).sheared and used as a template for PCRin Xu, J.,The DNA wasamplification ofthe parAl gene, using primers designed according to theparAl sequence reported in Kamoun, S., et al. Mol. Plant-6:573—58l (1993) . The parAl PCRproduct was cloned into pBluescript (Stratagene, SanMicrobe Interact.?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-22-CA)double—stranded DNA sequencing using the dideoxy chainDiego, and the sequence of the product determined bytermination method.The parA1 insert in pBluescript was amplified byPCR, using primers that created an N—terminal histidinetag and a protein kinase site at the 5' end of the gene.The PCR product was ligated into the expression vectorpET28b (Novagen, Madison, WI) and, after confirming theDNA sequence of the parA1 fusion, the pET28b constructcoli BL21.A BL2l culture containing the parA1 fusion waswas transformed into E.grown at 37° C in the presence of kanamycin to an ODwo of0.3. IPTG (1mM) was added and the culture was incubatedfor 5 hours at 27° C.Periplasmic proteins were prepared by osmoticshock essentially as described in Ausubel, F., et al. inCurrent Protocols in Molecular Biology, John Wiley &New York (1989). Cells (1.5 ml) were harvested bycentrifugation, resuspended in 500 pl of 50 mM Tris—HCl,Sons,pH 8.0, 20% sucrose, 1 mM EDTA and incubated with shakingfor 10 minutes at room temperature. Aftercentrifugation, the pellet was resuspended in 200 pl icecold MgSO4 (5 mM)minutes at 4° C.and incubated with shaking for 10The mixture was centrifuged and theresulting supernatant (containing periplasmic proteins)was applied to a Ni“ column. The parA1 protein waspurified from the column according to the manufacturer'sdirections. The protein concentration in the parA1extract was determined by the Bradford method.Nicotiana tabacum L. cv. KY14 cell suspensioncultures were treated with parasiticein at a finalconcentration of 2 pg/ml during rapid growth phase toinduce stress response genes. Parallel suspension cellcultures which were not treated with parasiticein servedas controls. Cells were collected by gentle vacuum?WO 99/02655101520253035CA 02265441 1999-03-05PCT/US98/14109-23-filtration O, 30, 60 and 120 minutes after the additionof elicitor.Total RNA was isolated from treated and untreatedtobacco cells and used as template for targeteddifferential display reverse transcriptase PCR (TDDRT—PCR) .kit from Invitrogen (San Diego, CA).First strand CDNA was generated using a CDNA cycleThe first strandcDNAs were then used as templates for PCR. The PCRreaction was carried out using typical conditions asdescribed in PCR Protocols: A Guide to Methods andGelfand, D.,T., eds. Academic Press Inc.,Applications, Innis, M.,White,Sminsky, J. andCA(1990), except that the annealing temperature was 58°C.(GTTGACTCCCTACCCTCTT) and(GGTACTTAGGAAGTGTTACGGG) . PCRproducts were separated by electrophoresis on a 1% (w/v)San Diego,The PCR primers were FokinBReca1IV See Figure 1.agarose gel and products of greater than about 800 basepairs (bp) from the 60 minute treated culture werepurified by electroelution onto DE-81 paper (Whatman).Ends of the purified PCR products were filled in withKlenow polymerase, ligated to the EcoRV site ofcoli TB1.Ampicillin resistant TB1 colonies were screenedpBluescript, and transformed into E.for the presence of a 3800 bp DNA fragment inserted intopBluescript, The sequence of one such insert wasdetermined by the dideoxynucleotide chain termination(1977) Proc. Natl. Acad. Sci.USA _Zg:8073—8077, with a sequenase® kit from UnitedStates Biochemical Corp.,procedure of Sanger et al.Cleveland, OH) or an automatedfluorescence based system (Applied Biosystems, FosterCA) .determined on both strands.City, The sequence of the insert in the vector wasThe plasmid containing thisinsert was designated pCDPK—1.The nucleotide sequence of the insert in pCDPK—1is shown in Figure 2 and the deduced amino acid sequence?WO 99/02655101520253035CA 02265441 1999-03-05PCT/U S98/ 14109-24-of the insert is shown in Figure 3. The deduced aminoacid sequence was compared to amino acid sequences ofplant genes in the GenBank, EMBL, and Swiss Protdatabases. Homology was found to plant CDPKpolypeptides, including polypeptides from Glycine max,Arabidopsis thaliana, Vigna radiata, Zea mays andCucurbita pepo.Using the BLASTP program and a BLOSUM62 scoringmatrix, two regions of homology to serine/threonineprotein kinase domains were identified in the aminoterminal portion of the polypeptide and four regions ofhomology to Ca“ binding domains were identified in thecarboxyl terminal portion of the polypeptide. Figure 4shows a comparison of the amino acid sequence of Fig. 3and a soybean CDPK amino acid sequence (Genbank AccessionNo:M64987). The amino acid sequence of the tobaccocalcium binding sites were similar to the amino acidsequence of corresponding sites in the soybean CDPK.However, there were significant differences in otherparts of the sequence. The comparison indicates thatthere is about 78% overall sequence identity between thesoybean CDPK and CDPK-1.The BLASTN program was used to compare the pCDPK—1nucleotide sequence to nucleic acid sequences on variousdatabases. Based on the nucleotide sequence of otherplant CDPK genes and the length of the polypeptidesencoded thereby, the nucleic acid insert present inpCDPK—1 is estimated to lack about 560 bp of 5’ CDPK—lcoding sequence and about 130 bp of 3' CDPK—l codingsequence.Example 2.Isolation of a full-length cDNA clonea RACE (Rapidapproach is used, with polyA+To obtain a full-length clone,Amplification of cDNA Ends)RNA prepared from tobacco cells after induction with?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/ 14109-25-elicitor being the template. PolyA+ RNA is prepared asdescribed in Example 1.A primer having the sequence GAC AAG GAC GGG AGTGGG TAT internal to CDPK-l) and a primerhaving the sequence GAC TCG AGT CGA CAT CGA TTT TTT TTTTTT TTT TT (dTu adapter—primer) are used to amplify the3’ end of the CDPK coding sequence.(Primer A,The reversetranscriptase reaction is carried out in 2 pl 10X RTCbuffer,dTU adapter—primer and 10 Units of AMV reverse10 units of RNasin (Promega Biotech), 0.5 pg oftranscriptase (Life Sciences) in a total volume of 3.5pl, as described in Frohman, M. in PCR Protocols: A Guideto Methods and Applications, supra, pp. 28-38. The PCRamplification reaction is carried out in 5 pl 10X PCRbuffer, 5 pl DMSO, 5 pl 10X dNTPs (15 mM each), 30 pl H§L1 pl adapter—primer (25 pmol, GAC TCG AGT CGA CAT CG), 1pl primer A and l—5pl CDNA. Cycle times are as indicatedin Frohman, supra.The 5’ end of the CDPK coding sequence is clonedby carrying out reverse transcription as described above,using 10 pmole of primer B (AGG GGC TAC GTA GTA AGG ACT)instead of dTN adapter—primer. The CDNA product isextended using terminal transferase and dATP as describedin Frohman, supra, and then amplified by PCR as describedabove with 10 pmole of dTn adapter—primer, 10 pmole ofadapter—primer and 10 pmole of primer C (ATT CTC AGG CTTAAG GTC CCT). PCR is carried out under standardconditions. Back et al. (1994) Arch. Biochem. Biophys.;;§:523—532. The amplified 3’ and 5’ products are blunt-end cloned into pBluescript SK (Stratagene) and combinedwith the pCDPK-1 insert by routine molecular biologytechniques to form a full-length cDNA of the tobacco CDPKcoding sequence.?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-25-The DNA sequence of the full—length CDNA isdetermined by a dideoxynucleotide chain terminationprocedure, as described in Example 1.Example 3.Induction of CDPK-Homologous RNAin Tobacco Suspension CulturesThe DNA insert in pCDPK—l was used as a probe tofollow the induction of gene expression in response toelicitor. Nicotiana tabacum L.cv. KY14 cell suspensioncultures were treated with parasiticein for O, X, 1, 2, 6Total RNA wasTheand 12 hours as described in Example 1.isolated and electrophoresed on a 1% agarose gel.insert from pCDPK—l was radiolabeled by the randompriming method and hybridized to the gel—separated RNA aset al., No mRNAhybridizing to CDPK—1 was detected prior to elicitordescribed in Sambrook, J. supra.treatment, whereas mRNA hybridizing to CDPK-1 was readilydetected at 1/2,At 6 and 12 hours after elicitor treatment,hybridizing to CDPK-1 could be detected,1 and 2 hours after elicitor treatment.no mRNAindicating thatCDPK—1 gene expression had decreased to undetectablelevels by about 6 hours.Example 4.Construction of a Chimeric CDPK GeneA CDPK gene is constructed from: a chemicallysynthesized DNA encoding amino acids 1 to 156 of thesoybean CDPK of Figure 6, a chemically synthesized DNAencoding amino acids 465 to 508 of the soybean CDPK ofand the CDPK insert of pCDPK—l. The three DNAsare ligated by routine molecular biology techniques toFigure 6,form a chimeric CDPK coding sequence having amino acids 1to 156 of soybean CDPK at the amino terminal end, fused?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-27-in—frame to amino acids 1 to 307 of tobacco CDPK (Fig.3), which in turn is fused in-frame to amino acids 465 to508 of soybean CDPK at the carboxyl terminal end.The chimeric coding sequence is inserted in senseorientation into an Agrobacterium binary vectorcontaining a minimal 35S and EAS4 inducible regulatoryelement. Operable linkage of the regulatory element,promoter, and coding sequence is confirmed by determiningthe DNA sequence of the junction regions and byexpression in transgenic plants.Example 5.Generation of Transgenic PlantsTransformed plant cell lines are produced using amodified Agrobacterium tumefaciens transformationprotocol. Nucleic acid constructs are prepared thatcontain the full-length CDPK cDNA of Example 3 or theTherecombinant constructs containing the sequences to bechimeric CDPK coding sequence of Example 4.introduced into plants are transferred into A.tumefaciens strain GV385O by triparental mating with E.coli TBl (pRK2013). N.stages of growth are cut into 1 cm? pieces, and dipped in(about 10‘ to 105After 3 to 10 minutes, the leaf segments aretabacum leaves at a variety ofa suspension of Agrobacterium cellscells/ml).then washed in sterile water to remove excess bacterialcells and to reduce problems with excess bacterial growthon the treated leaf segments. After a short drying time(30 to 60 seconds), the treated leaf segments are placedon the surface of Plant Tissue Culture Medium withoutantibiotics to promote tissue infection and DNA transferfrom the bacteria to the plant tissue. Plant TissueCulture Medium contains per liter: 4.31 g of Murashigeand Skoog Basal Salts Mixture (Sigma Chemical Company,St.Louis, M0), 2.5 mg of benzylaminopurine (dissolved in?WO 99/026551015202530CA 02265441 1999-03-05PCT/US98/14109-28-1 N NaOH) ,30 g sucrose,10 ml of 0.1 mg/ml indoleacetic acid solution,2 ml of Gamborg's Vitamin Solution (SigmaSt. MO) and 8 g of agar.adjusted between pH 5.5 and 5.9 with NaOH.Chemical Co., Louis, The pH isAfter 2 days,the leaf segments are transferred to Plant Tissue Culture500 pg/ml ofKanamycin selects forMedium containing 300 pg/ml of kanamycin,NJ).transformed plant tissue,mefoxin (Merck, Rahway,and mefoxin selects againstAgrobacterium.It may be necessary to minimize the exposure ofthe explant tissue to Agrobacterium cells during thetransformation procedure if a pathogen—inducibleregulating element is used, because Agrobacterium cellsmay themselves induce the element after introduction intothe plant cells. Accordingly, the biolistic techniquefor the introduction of DNA containing cell suicide genesunder the regulatory control of the inducibletranscriptional regulatory element is a usefulalternative transformation technique because it does notentail the use of Agrobacterium cells or fungal cell walldigestive enzymes (as necessary for the generation ofprotoplasts for electroporation), both of which can leadto induction of the coding sequences under the control ofthat regulatory element.Transgenic plants are regenerated essentially asdescribed by Horsch et al. (1985) Science ;g1:1229—1231.Example 6.E1icitor- and Pathogen-inducible Expressionof a Chimeric CDPK Gene in Transgenic TobaccoThe activity of the CDPK constructs of Example 7are measured in transgenic tobacco plants treated witheither an elicitor or pathogen. As controls, transgenictobacco plants expressing the GUS reporter gene under thecontrol of the cauliflower mosaic virus (CaMV) 35S?WO 99/0265510152O2530CA 02265441 1999-03-05PCT/US98/14109-29-promoter are also produced. 13 seeds from regeneratedtransgenic tobacco plants are germinated on mediumcontaining 100 mg/L kanamycin. The resultingkanamycin-resistant plants are subsequently transferredHalf of the plantsare tested for the expression of the CDPK gene underinto soil and grown in a greenhouse.inducing conditions, e.g., by intercellular applicationof elicitor or cellulase to the transgenic plants.Elicitor or cellulase is applied with a mechanicalpipetter. As a control, remaining plants aremock—treated with a solution lacking cellulase orelicitor. Tobacco tissue is wounded with a scalpel insome experiments to facilitate exposure to the inducingcompound.Example 7.Identification of CDPK Homologous SequencesTobacco leaf genomic DNA is isolated as describedin Murray and Thompson (1980) Nucleic Acids Research§:4321—4325. After digestion of aliquots with desiredrestriction enzymes, the digested DNA samples areelectrophoresed on 0.8% agarose gels and thesize—separated DNAS are transferred to nylon membranes.DNA blots are hybridized with the 900 bp CDPK cDNA insertof Example 1 that is radiolabeled by the random primermethod.sodium phosphate buffer, pH 8.0, 0.7% SDS,serum albumin, 1 mM EDTA.at 45°C with 2X SSC,0.1% SDSpH 7.0).various bands on the membrane are estimated fromHybridization is performed at 60°C in 0.25 M1% bovineThe blot is then washed twice0.1% SDS and twice with 0.2X SSC,(lX SSC is 0.15 M NaCl,Relative hybridization intensities of the0.015 M sodium citrate,autoradiograms using a video densitometer(MilliGen/Biosearch, Ann Arbor, MI).?WO 99/02655l0l5202530CA 02265441 1999-03-05PCT/US98/14109-30-To identify polynucleotides having homologoussequences to tobacco CDPK and to determine the apparentnumber of copies per genome of those sequences, Southernhybridization experiments are carried out using targetDNA isolated from other plant species and tobacco CDPKprobes. Restriction endonuclease—digested genomic DNASof various plant species are separated by agarose gelelectrophoresis (0.8% agarose), and then transferred to aHybond-N*nembraneIL). Radiolabeled probe fragments comprising codingsequences of pCDPK—l are hybridized to the digested(Amersham Corp., Arlington Heights,genomic DNA essentially as described in Sambrook et al.(1989), supra. Moderate stringency conditions are used(hybridization in 4X SSC, at 65°C with the last wash in1X SSC, at 65°C) .Alternatively, PCR is carried out using targetgenomic DNA as a template and primers derived from highlyconserved regions of the pCDPK—l coding sequence.Example 8.Genomic DNA Flanking a CDPK Coding SequenceThe cDNA clone described in Example 1 is used as ahybridization probe for screening a N. tabacum cv. NK326(Clontech, PaloGenomic DNA clones having 70% or greatergenomic library in the AEMBL3 vectorAlto, CA) .sequence identity to the tobacco CDPK of Example 1 areThenucleotide sequences of the cloned nucleic acid insertsidentified using routine subcloning protocols.are determined using routine DNA sequencing protocols.One of the genomic DNA clones has a full-lengthcoding sequence that comprises the tobacco CDPK codingThe clone also contains DNAto,sequence of Example 1.contiguous with, and 5' the coding sequence ofExamination5’ flanking DNA in thisExample 1. of the nucleotide sequence of theclone reveals a putative ATG?WO 99/0265510CA 02265441 1999-03-05PCT/US98/ 14109-31-start codon as well as one or more putative regulatoryelements upstream of the start codon and within about1000 bp of the start codon.Other EmbodimentsIt is to be understood that while the inventionhas been described in conjunction with the DetailedDescription thereof, that the foregoing description isintended to illustrate, and not limit the scope of theinvention, which is defined by the scope of the appendedclaims. Other aspects, advantages, and modifications arewithin the scope of the following claims.?CA02265441 1999-03-0560120180240300360WO 99/02655 PCT/US98/14109- 32 _SEQUENCE LISTING(1) GENERAL INFORMATION(i) APPLICANT: University of Kentucky Research Foundation(ii) TITLE OF THE INVENTION: PROTEIN KINASES AND USESTHEREOF(iii) NUMER OF SEQUENCES: 11(iv) CORRESPONDENCE ADDRESS:(A) ADDRESSEE: Fish & Richardson P.C., P.A.(B) STREET: 60 South Sixth Street, Suite 3300(C) CITY: Minneapolis(D) STATE: M(E) COUNTRY: USA(F) ZIP: 55402(V) COMPUTER READABLE FORM:(A) MEDIUM TYPE: Diskette(B) COMPUTER: IBM Compatible(C) OPERATING SYSTEM: DOS(D) SOFTWARE: FastSEQ for Windows Version 2.0(Vi) CURRENT APPLICATION DATA:(A) APPLICATION NUMBER:(B) FILING DATE: O7-JUL-1998(C) CLASSIFICATION:(Vii) PRIOR APPLICATION DATA:(A) APPLICATION NUMBER: O8/889,655(B) FILING DATE: 08—JUL-1997(viii) ATTORNEY/AGENT INFORMATION:(A) NAME: Lundquist, Ronald C(B) REGISTRATION NUMBER: 37,875(C) REFERENCE/DOCKET NUMBER: 07678/O2OWOl(ix) TELECOMUNICATION INFORMATION:(A) TELEPHONE: 612-335-5050(B) TELEFAX: 612-288-9696(C) TELEX:(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 921 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AGGGACCTTA AGCCTGAGAA TTTCCTTTTC AGTGCCGACG ACTTCATGGT AAAGAGTAAGGCCACCGACT TCGGGCTTAG TGTATTCTAT AAGCCTGGGC AAAAGTTCAC GGACATAGTAGGGAGTCCTT ACTACGTAGC CCCTGAGGTA CTTAGGAAGT GTTACGGGCC TGGGAGTGACGTATGGAGTG CCGGGGTAAT ACTTTACACC CTTCTTTGTG GGGCCCCTCC TTTCATGGCCGACAGTGAGC CTGGGGTAGC CCTTCAAATA CTTCATGGGG ACCTTGACTT CAAGAGTGACCCTTGGCCTA CCATAAGTGA GAGTGCCAAG GACCTTATAA GGAAGATGCT TGAGCAAGACCCTAAGAGGA GGCTTACCGC CCATGAGGTA CTTAGGCATC CTTGGATAGT AGACGAGAAT420?WO 99/02655ATAGCCCCTGATGAATAAGAATAGTAGGGCTTCCATCTTAGACCTTATGGACCGCCGCCATATCATGACAGAGTTCGGGGGATGGGCAAACAACAAGCCTCTTAAAGAAGATTTAAGGAGATAGCAAGGGCTACGCCGCCGATGCATCTTAAAGGACGGGAGTACCTGACACTAGATTATGG(2)TGGGCCTGCCGGCCCTTAGGGTTCAAGATGTAAGAGGGTACGTAGACAATTAAGATAAAGTGGGTATATACAGTCTTGAGG02265441 1999-03-0533 —GTACTTAGTAGTAATAGCCGGACACCGACAGGGAGTCAACAGTGGGACCAAGGGAGGACCGAGGTAGACGGACATGATAAINFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH:(B)TYPE: am307 amino acidsino acid(D) TOPOLOGY: linear(ii) MOLECULE(xi)ArgVal LysGly GlnAsp LeuSerLysSEQUENCEPro5AlaLysLys20Phe Thr35Glu Val50Gly Val65Asp SerPhe LysIle ArgLeuIleGluSerLysArg LysLeu TyrPro Gly85Asp Pro100Met Leu115Glu Val130Lys Pro145Met AsnSer GluAsp AsnLeuLeuLysGluSerArg HisGly ProIle Lys165Glu Ile180Gly Thr195Val210AlaArgAla225Thr AlaSer AlaAsp GluGlyAspAlaPheLeuSer GlnVal AspMet His245Ser Tyr260Arg Gln275Glu290TyrLeuAsp305AspMet IleGlyGluThrAspCysThrValTrpGluAlaLysValValLeuAsnLeuHisAlaLysDESCRIPTION: SEQ IDAsn PheAsp PheIle Val40Tyr Gly55Leu Leu70Ala LeuPro ThrGln Asp120Pro Trp Ile135Val Leu150Met AlaGly LeuThr Phe200Gly Glu215Ser Gly230Asn LysAsp LysGlu280ValLeuGlu295TYPE: proteinNO:Leu Phe10LeuSerGly Ser25GlySer ProPro Gly SerAla75LeuCys GlyGln Ile90Ile Ser105ProGluLys ArgVal Asp GluSer Leu155ValArgLeu Arg170Lys Glu Met185Phe His LeuSer Glu IleThr Ile Asp235Ile Lys Arg250Asp Gly Ser265Glu Phe GlyAsp Thr AspGGCTTAAGCAAGAGGCTTAGATAGTGGGACTTGGGGAGAGTAGACTATGGATCTTGTAAGAGCTTAGGCAAGGAGGTAGAAla AspVal PheAspTyr LysPCT/U S98/ 14109ATTCAGTGCCTGAGGAGGAGCGTAACCTTCTGAGATAAAGGGAGTTCGTATGCCTTCAGTAGCCCTTGAGCACCGACAATPhe15MetPro30Tyr Tyr45Asp Val60Pro ProHis GlySer AlaValTIPPheAspLysAla ProSer AlaMet AlaLeu95AspAspLeu110Leu125IleArgAsn140Lys GlnIle AlaPhe LysThrAlaPheGluMetAla HisPro AspAla160LeuSerArg175Asp Thr190Gln205AspLysLys220Tyr GlyGlu AspGly TyrGlyLeuGluHisIleLeu LysMet AspPhe Val240Leu Val255Glu Val270Val Pro285Asn Asp300AspGlyThr SerGln Ile480540600660720780840900921?CA 02265441 1999-03-05WO 99/02055 PCT/U S98/ 14109_ 34 _(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GGTACTTAGG AAGTGTTACG GG 22(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GTTGACTCCC TACCCTCTT 19(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 512 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:Met Ala Ala Lys Ser Ser Ser Ser Ser Thr Thr Thr Asn Val Val Thr1 5 10 15Leu Lys Ala Ala Trp Val Leu Pro Gln Arg Thr Gln Asn Ile Arg Glu20 25 30Val Tyr Glu Val Gly Arg Lys Leu Gly Gln Gly Gln Phe Gly Thr Thr35 40 45Phe Glu Cys Thr Arg Arg Ala Ser Gly Gly Lys Phe Ala Cys Lys Ser50 55 60Ile Pro Lys Arg Lys Leu Leu Cys Lys Glu Asp Tyr Glu Asp Val Trp65 70 75 80Arg Glu Ile Gln Ile Met His His Leu Ser Glu His Ala Asn Val Val85 90 95Arg Ile Glu Gly Thr Tyr Glu Asp Ser Thr Ala Val His Leu Val Met100 105 110Glu Leu Cys Glu Gly Gly Glu Leu Phe Asp Arg Ile Val Gln Lys Gly115 120 125His Tyr Ser Glu Arg Gln Ala Ala Arg Leu Ile Lys Thr Ile Val Glu130 135 140Val Val Glu Ala Cys His Ser Leu Gly Val Met His Arg Asp Leu Lys145 150 155 160Pro Glu Asn Phe Leu Phe Asp Thr Ile Asp Glu Asp Ala Lys Leu Lys165 170 175Ala Thr Asp Phe Gly Leu Ser Val Phe Tyr Lys Pro Gly Glu Ser Phe180 185 190Cys Asp Val Val Gly Ser Pro Tyr Tyr Val Ala Pro Glu Val Leu Arg195 200 205Lys Leu Tyr Gly Pro Glu Ser Asp Val Trp Ser Ala Gly Val Ile Leu?WO 99/02655Tyr225GlyProLeuHisSer305LysIleGlyLysAsp385IleValLeuHis210IleIleTrpAspPro290AlaLysGlyThrArg370AlaAlaSerAspIleLeuPheProGln275TrpValMetGlyIle355ValAlaAla"AlaGlu435Asp450Ile Asp Tyr Gly465Ile Gly Arg ArgLeuArgSer260AsnIleLeuAlaLeu340ThrGlyAspThrPhe420IleAspGly Leu Val Asp(i)(2)(A)(B)(C)(D)500INFORMATION FOR SEQ IDSerGln245IleProValSerLeu325LysPheSerIleValCAGly230IleSerLysAspArg310ArgGluAspGluAsp390His405SerGlnMetGluThr485AsnTyr215ValLeuAspThrAsp295LeuValLeuGluLeu375LysProLeuSerArg280AsnLysIlePheLeu360MetSer35ProGlyAla265Leu.IleGlnAlaLys345LysGluGlyLeu Asn LysPheAspGln Ala CysIlePhe470Lys455Ala440GluAlaMet Arg LysGly Ser AsnLys425LysIleMetThrGln505SEQUENCE CHARACTERISTICS:LENGTH: 21 base pairsTYPE: nucleic acidSTRANDEDNESS:TOPOLOGY: linearsinglePheLys250LysThrAlaPheGlu330MetAspSerThrLeu410AspAspAspMet02265441 1999-03-05Trp235LeuAspAlaProSer315ArgIleGlyGlu220AlaAspLeuHisAsp300AlaLeuAspLeuIle380GluPheIleGln285LysMetSerThrLys365LysIle Asp Tyr395Glu Arg GluGly Ser GlyPhe Gly Leu445PCT7US98?4l09SerHisArg270ValProAsnGluAsp350AspAspGlyGluTyr430AspGln Asp Asn Asp460Arg Lys Gly Asn475Leu Asn Leu Arg Asp490ValNO:Ile Glu Gly Tyr6:(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:GACAAGGACG GGAGTGGGTA T(2)INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:LENGTH: 35 base pairsTYPE: nucleic acid(A)(B)(C)(D)STRANDEDNESS:TOPOLOGY: linearsingle(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GACTCGAGTC GACATCGATT TTTTTTTTTT TTTTT-(2) INFORMATION FOR SEQ ID NO:8:510GluSer255LysLeuLeuLysGlu335AsnGlyLeuGluASH415IleAspGlyGlyAla495PhePro240GluMetArgAspLeu320GluSerLeuMetPhe400LeuThrIleGlnGly480LeuLys2135?CA 02265441 1999-03-05WO 99/02655 PCT/U S98/ 14109._36_(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GACTCGAGTC GACATCG 17(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:AGGGGCTACG TAGTAAGGAC T 21(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:ATTCTCAGGC TTAAGGTCCC T 21(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 308 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:Arg Asp Leu Lys Pro Glu Asn Phe Leu Phe Ser Ala Asp Asp Phe Met1 5 10 15Val Lys Ser Lys Ala Thr Asp Phe Gly Leu Ser Val Phe Tyr Lys Pro20 25 30Gly Gln Lys Phe Thr Asp Ile Val Gly Ser Pro Tyr Tyr Val Ala Pro35 40 45Glu Val Leu Arg Lys Cys Tyr Gly Pro Gly Ser Asp Val Trp Ser Ala50 55 60Gly Val Ile Leu Tyr Thr Leu Leu Cys Gly Ala Pro Pro Phe Met Ala65 70 75 80Asp Ser Glu Pro Gly Val Ala Leu Gln Ile Leu His Gly Asp Leu Asp85 90 95Phe Lys Ser Asp Pro Trp Pro Thr Ile Ser Glu Ser Ala Lys Asp Leu100 105 110Ile Arg Lys Met Leu Glu Gln Asp Pro Lys Arg Arg Leu Thr Ala His115 120 125Glu Val Leu Arg His Pro Trp Ile Val Asp Glu Asn Ile Ala Pro Asp?CA 02265441 1999-03-05WO 99/02655 PC T/U S98/ 1 4109130 135 140Lys Pro Leu Gly Pro Ala Val Leu Ser Arg Leu Lys Gln Phe Ser Ala145 150 155 160Met Asn Lys Ile Lys Lys Met Ala Leu Arg Val Ile Ala Glu Arg Leu165 170 175Ser Glu Glu Glu Ile Val Gly Leu Lys Glu Met Phe Lys Met Ile Asp180 185 190Thr Asp Asn Ser Gly Thr Val Thr Phe Phe His Leu Lys Asp Gly Leu195 200 205Lys Arg Val Gly Ser Gln Leu Gly Glu Ser Glu Ile Lys Asp Leu Met210 215 220Asp Ala Ala Asp Val Asp Asn Ser Gly Thr Ile Asp Tyr Gly Glu Phe225 230 235 240Val Thr Ala Ala Met His Leu Asn Lys Ile Lys Arg Glu Asp His Leu245 250 255Val Ser Ala Phe Ser Tyr His Asp Lys Asp Gly Ser Gly Tyr Ile Glu260 265 270Val Asp Glu Ile Arg Gln Ala Leu Glu Glu Phe Gly Val Pro Asp Thr275 280 285Ser Leu Glu Asp Met Ile Lys Glu Val Asp Thr Asp Asn Asp Gly Gln290 295 300Ile Asp Tyr Gly305

Claims (30)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide, said polynucleotide comprising:
a) the nucleotide sequence of SEQ ID NO:1;
b) an RNA analog of SEQ ID NO:1;
c) a polynucleotide comprising a nucleic acid sequence complementary to a) or b); or d) a nucleic acid fragment of a), b) or c) that is at least 20 nucleotides in length and that hybridizes under stringent conditions to genomic DNA encoding the polypeptide of Figure 3.

2. The polynucleotide of claim 1, wherein said polynucleotide comprises nucleotides 1 to 170 of Figure 2.

3. The polynucleotide of claim 1, wherein said polynucleotide comprises nucleotides 160 to 560 of Figure 2.

4. The polynucleotide of claim 1, wherein said polynucleotide comprises nucleotides 550 to 920 of Figure 2.

5. A nucleic acid construct comprising the polynucleotide of claim 1.

6. The nucleic acid construct of claim 5, further comprising a regulatory element operably linked to said polynucleotide.

7. The nucleic acid construct of claim 6, wherein said regulatory element is an inducible regulatory element.

8. The nucleic acid construct of claim 7, wherein said regulatory element is induced in response to a plant pathogen.

9. A transgenic plant containing a nucleic acid construct comprising the polynucleotide of claim 1.

10. The plant of claim 9, wherein said construct further comprises a regulatory element operably linked to said polynucleotide.

11. The plant of claim 10, wherein said regulatory element is an inducible regulatory element.

12. The plant of claim 11, wherein said regulatory element is induced in response to a plant pathogen.

13. The plant of claim 11, wherein said regulatory element is induced in response to an elicitor.

14. The plant of claim 9, wherein said plant is a dicotyledonous plant.

15. The plant of claim 14, wherein said plant is a member of the Solanaceae family.

16. The plant of claim 15, wherein said plant is a Nicotiana plant.

17. The plant of claim 16, wherein said plant is Nicotiana tabacum.

18. A transgenic plant containing a polynucleotide expressing a polypeptide having from about 250 to about 550 amino acids, said polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of Figure 3.

19. The plant of claim 18, wherein said polypeptide comprises the amino acid sequence of Figure 3.

20. The plant of claim 18, wherein said plant is a dicotyledonous plant.

21. The plant of claim 20, wherein said plant is a member of the Solanaceae family.

22. A method of using a polynucleotide, said method comprising the step of hybridizing the polynucleotide of claim 1 to DNA or RNA from a plant.

23. The method of claim 22, further comprising the step of identifying a segment of said plant DNA or RNA that has about 70% or greater sequence identity to said polynucleotide.

24. The method of claim 23, further comprising the step of cloning at least a portion of said DNA or RNA
segment.

25. The method of claim 24, wherein said cloned portion further comprises DNA flanking said segment having 70% or greater sequence identity.

26. A method of altering disease resistance in a plant, said method comprising the steps of:
(a) introducing the nucleic acid construct of claim 5 into a plant cell; and (b) producing a plant containing said polynucleotide from said cell, wherein expression of said polynucleotide alters disease resistance in said plant.

27. The method of claim 26, wherein said nucleic acid construct further comprises an inducible regulatory element operably linked to said polynucleotide and said expression is regulated by said regulatory element.

28. The method of claim 27, wherein said expression is induced by said regulatory element upon exposure of said plant to an elicitor or plant pathogen.

29. An isolated polypeptide having from about 250 to about 550 amino acids, said polypeptide comprising an amino acid sequence substantially identical to Figure 3.

30. The polypeptide of claim 29, wherein said polypeptide comprises the amino acid sequence of Figure 3.