OA10990A - 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

- 010990 ι-

PROTEIN KINASES AND USES THEREOF

Statement as to Federally Sponsored Research5 The research reported herein was performed in part with funding from the National Science Foundation ci theUnited States Government. The United States Governmentmay nave certain rights in this invention.

Field of the Invention 10 This invention relates to nucleic acids enccding calcium dépendent protein kinases, polypeptides procucedfrom such nucleic acids and transgenic plants expressingsucn nucleic acids.

Background of the Invention 15 In plants, disease résistance to fungal, bacterial, and viral pathogens is associated with a plantresponse termed the hypersensitivity response (ER). Inthe HR, the site in the plant where the potentielphvtopathogen invades undergoes localized cell death, and 20 it is postulated that this localized plant cell deathcontains the invading microorganism or virus, therebyprotecting the remainder of the plant. Other plantdefense responses include the production of phvtoalexins,the production of lytic enzymes capable of averting 25 pathogen 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 zabaczun' 30 produces sescruiterpenes in response to microbialinvaders, e.g., Pseudononas lachrymans. A variety of compositions can serve as elicitorso: plant ohvtoalexin svnthesis. These include cr.e cr 010990 - 2 - mcre :ox:c ions, e.g., mercuric ions, other chemicallvdefined compositions, metabolic inhibitors, celi.wallglycans, certain glycoproteins, certain enzymes, fungalspores, chitosans, certain fatty acids, and certain 5 oligosaccharides derived from plant cell walls. See,e.g., Sequeira, L. (1983) Annu. Rev. Microbiol. 3 7 : Ξ1 - 7 9and references cited therein. Cell wall fragments cfcertain Phytophthora species and cellulase fromTrichcdema viride but not Aspergillus japonicum 10 pectolyase 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 the 15 HR in plants. Generally, but not necessarily, localizedcell death is the resuit of the elicitin-induced responsein the infected (or challenged) plant tissue. Theseresponses médiate full or partial résistance to destructive infection by the invading, potentialiy plant 20 pathogenic microorganism. Amino acid and nucléotidecoding sequences for an elicitin of Phytophthoraparasitica hâve been published. Kamoun et al. (1993)

Mol. Plant-Microbe Interactions 6:573-581.

Plant pathogenic viruses including, but not 25 limited to, Tobacco Mosaic Virus (TMV), induce the HR ininfected plants. Bacteria that infect plants also caninduce HR and thereby disease résistance; représentativebacteria eliciting HR include, e.g., Xanthomonas spp. andPseudomonas syringae. Plant pathogenic fungi generally 3 0 do not induce the HR response after attack on a hostplant, e.a., Phytophthora parasitica and Peronosporatabaci on tobacco hosts, but can induce the HR afterattack en a non-nost plant.

The signal transduction rechansms mvolved in 35 exoression of disease résistance are ur.der '..nvecticaticr 010990 - 3 - and scme of the genetic and biochemical features hâvebeen outlmed. Ses, e.g., Staskawicz, B. et al., Science263:651-567 (19 95). However, many aspects of signaltransduction pathways and the rôle of many spécifiecomponents are not well understood.

There is a long felt need in the art for methedsof protecting plants, particularly crop plants, frominfection by plant pathogens. Especially important fromthe standpoint of économie and environmental concerns arebiolcgical or "natural" metheds rather than those whichdépend on the application of Chemicals to crop plants.There is also a need in the art for plant polynuclectidesequences for enhancing and/or improving diseaserésistance in plants.

Summary of the Invention

Nucleic acids of the présent invention are basedon novel calcium dépendent protein kinase (CDPK) ger.esand their corresponding proteins. Induction ofexpression of these novel CDPK genes is surprisinglyrapid, i.e., mRNA transcription of such genes can beobserved as soon as 30 minutes after elicitor-mediatedinduction of plant defense responses. Thus, the novelgenes disclosed herein are among those genes that aremost rapidly induced in response to signais indicating aninvading plant pathogen.

An isolated polynucleotide is disclosed herein,that comprises the nucléotide sequence of SEQ ID NO:1 andits complément, and an RNA analog of SEQ ID N0:l or itscomplément. Such a polynucleotide can also be a nucleicacid fragment of the above that is at least 20 nucléotides in length and that hybridizes under stringer.tconditions to genomic DNA encoding the polypeptide ofFigure 3. The polynucleotide can comprise, for example, 010990 nucléotides 1 to 170, nuciectides 160 co 560, crnuciectides 550 to 920 of Figure 2. A nucleic acid construct as disclosed hereincomprises a polynucieotide of the invention. Ir. such aconstruct, a polynucieotide of the invention car. beoperablv iinked to one or more éléments that regulatetranscription of the polynucieotide, for example, aregulatory element induced in response to a plantpathogen such as a fungus (e.g., Phytophthora), abactenum (e.g., Pseudomonas'i , or a virus (e.g., TobaccoMcsaic Virus) as described herein. In other embodiments,such induction is mediated by an elicitor (e.g., byfungal or bacterial elicitors).

Further aspects of the présent invention aretransgenic plant cells, plant tissues, and plants thathâve been genetically engineered to contain and express apolynucieotide of the invention, for example, a codingsequence, or an antisense sequence. The construct car.further comprises a regulatory element operably Iinked tothe polynucieotide, e.g., an inducible regulatoryelement. The plant can be a dicotyledonous plant, e.g.,a member of the Solanaceae family such as Nicotianatabacum. The plant can also be a monocotyledonous plant,a gymnosperm, or a conifer. A transgenic plant is disclosed herein thatcontains a polynucieotide expressing a polypeptide havir.gfrom about 250 to about 550 amino acids. The polypeptidecomprises an amino acid sequence substantiallv identicalto the amino acid sequence of Figure 3. A method of using a polynucieotide is disclosedherein. The method comprises the step of hybridizing thepolynucieotide 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 7C^ or greater seouence ider.titv to the 010990 - 5 - polynucleotide, and the step of cloning at least aportion cf the DNA or RNA segment. The cicned portionmay further comprise DNA flankmg the segment haying 70%or greater sequence identity. 5 In another aspect, the invention features a methcd cf altering disease résistance in a plant. The methodcomprises the steps of introducing a polynucleotide ofthe invention into a plant cell; and producing a plantcontaining the polynucleotide from the plant cell. 10 Expression of the polynucleotide alters disease résistance 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 upon 1Ξ exposure of the plant to an elicitor or plant pathogen.

In another aspect, the invention features an isolated polypeptide, having from about 250 to about 350amino acids and comprising an ammo acid sequencesubstantially identical to Figure 3. 20 An inducible regulatory element is a DNA sequence effective 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 hypersensitive 25 response) can be operably linked to a developmentally- regulated regulatory element. Also included in this termare regulatory éléments that are sufficient to rendergene expression inducible in response to disease-associated external signais or agents (e.g., pathogen- or 3 0 elicitor-induced signais or agents as described nerein) .Also included in this term are those regulatory élémentsflankmg a navel CDPK gene and involved in rapidinduction of transcription of such a novel gene. Ingeneral, defense response regulatory éléments are located daàsfciè*-* 010990 5' cc the coding regicn of a gene, but are net solimited.

By "tissue-specific" is meant capable cfpreferentially increasing expression of a gene product(e.g., an mRNA molécule or polypeptide) in cne “issue(e.g., xylem tissue) as compared to another tissue (e.g.,phloem) . 3y "cell-spécifie1' is meant capable ofpreferentially increasing expression of a gene product(e.g., an mRNA molécule or polypeptide) in one cell(e.g., a parenchyma cell) as compared to another cell(e.g., an epidermal cell). A "pathogen" is an organism whose infection of, orassociation with, cells of viable plant tissue car. resuitin a disease. An "elicitor" is any molécule that iscapable of initiating a plant defense response. Exampiesof elicitors include, without limitation, one or moretoxic ions, e.g., mercuric ions, other chemically defir.edcompositions, metabolic inhibitors, cell wall glycans,certain glycoproteins, certain enzymes, fungal spores,chitosans, certain fatty acids, and certain oligosaccharides derived from plant cell walls, andelicitins (e.g., harpin, cryptogein, and parasiticein).

By "operably linked" is meant that twopolynucleotides 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 molécules are présent.

Unless otherwise defined, ail technical andscientific terms used herein hâve the same meaning ascommonly understood by one of ordinary skill in the artto which this invention belongs. Although methods andmaterials similar or équivalent to those described hereincan be used in the practice or testinc of the oresentinvention, suitable methods and materials are deutribed y -· v. 010990 - 7 - below. Ail publications, patent applications, patents,and other reierences mentioned herein are incorporated byreference in their entirety. In case of confiiez, theprésent spécification, including définitions, will 5 contrcl. In addition, the matériels, methods, andexamples are illustrative only and not intended to belimiting.

Other features and advantages of the inventionwill be apparent from the following description of the C preferred embodiments thereof, and from the daims.

Brief Description of the DrawingsFigure 1 is a représentation of the nucléotide sequences of the primers FokinB and RecalIV.

Figure 2 is a représentation of the DNA sequence 5 (SEQ ID NO : 1 ) of a partial cDNA clone isolated from acell suspension culture derived from a tobacco cultivarKY14 expiant, after growth in the presence of theelicitin parasiticein.

Figure 3 is a représentation of the deduced amino 0 acid 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 Invention 5 The présent invention relates to isolated polynucleotides (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 signais. Such nucleic acids typically 0 encode a calcium dépendent protein kinase (CDPK) polypeptide or CDPK-related polypeptide. Induction ofthe novel polynucleotides disclosed herein corresponds intime to that of plant defense response genes, whereas 01 0990 - 3 - other CDPK aenes appear to be induced less rapidly.Induction of gene expression for such novel genejs is morerapid than that of genes involved m developmentailyregulated processes in plants, e.g., develcpmentallyregulated processes such as floral development.

Induction of the novel CDPK genes disclosed herein isalso more rapid than that of many genes involved inresponses to abiotic stress, such as sait or waterstress. A polynucleotide of the présent 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., can hâve adifferent nucléotide sequence that, due to the degeneracyof 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 disclosedherein. See, e.g., U.S. 5,629,193. A polynucleotide canfurther include one or more regulatcry éléments operably-mked to a CDPK polynucleotide disclosed herein. 010990 - 9 -

The présent invention also includespolynuclectides that selectively hybridize to a CDPKpolynuclectide sequence disclosed herein. Hybridizationmav involve Southern analysis (Southern blotting), a

5 method 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 séparation ofDNA digests on agarose gels, dénaturation of the DNA 10 after electrophoretic séparation, 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., (1989) Molecular Cloning, second 15 édition, Cold Spring Harbor Laboratory, Plainview, NY. A pclynucleotide can hybridize under moderate stringency conditions or under high stringency conditionsto a polynucleotide disclosed herein. High stringencyconditions are used to identify nucleic acids that hâve a 20 high degree of homology or sequence identity to the probe. High stringency conditions can include the use ofa denaturing agent such as formamide during

hybridization, e.g., 50% formamide with 0.1% bovine sérumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM 25 sodium phosphate buffer at pH 6.5 with 750 mM NaCl, and75 mM sodium citrate at 42°C. Another example is the useof 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5 x Denhardt’s solution, sonicated salmcn 30 sperm DNA (50 /zg/ml), 0.1% SDS, and 10% dextran sulfateat 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.Alternatively, low ionic strength and high températurecan be employed for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate (0.1X SSC); 0.1% sodium

Pt 010990 - 10 -

Moderate stringency conditions are hybridizationconditions used to identify nucleic acids that hâve iesshcmology or identity to the probe than do nucleic acidsidentifiée under high stringency conditions. Moderatestringency conditions can include the use of higher ionicstrength and/cr lower températures for washing cf thehybridization membrane, compared to the ionic strengthand températures 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 IX SSC, at 65°C. Alternatively, a hybridizationwash in IX 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 32P, by biotinylaticn or withan enzyme. The RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon,or other suitable membrane, and hybridized with theprobe, using standard techniques well known in the artsuch as those described in sections 7.39-7.52 cf Sambrcoket al., supra.

It is generally preferred that a probe of at leastabout 20 nucléotides in length be used, preferably atleast about 50 nucléotides, more preferably at leastabout 100 nucléotides. If a relatively short probe is tobe used, the nucléotide sequence of the probe preferablyavoids régions conserved among plant CDPK genes (proteinkinase domains and calcium-binding domains), to morereaaily distinguish the rapidly induced CDPK genesdisclosed'herein from more slowly induced CDPK genes,constitutive CDPK genes or low-level constitutive CDPKgenes. Nevertheless, probes containmg such conserves 7 010990 - Il - régions can be used, provided that there are sufficientr.on-conssrved régions présent in the probe that are morespécifie for the novel polynucleotides discicsed. herein. A polynucleotide of the invention has at least5 about 70% sequence identity, preferably at least about 80% sequence identity, more preferably at least about 9C%sequence identity to SEQ ID NO:1. Sequence identity canbe determined, for example, by computer programs désignéeto perform single and multiple sequence alignments. 10 Polynucleotides having at least about 70% nuclectidesequence identity to the polynucleotide of SEQ ID NC : 1are included in the invention and can be identified byhybridization under conditions of moderate stringency.Polynucleotides having at least about 80% sequence 15 identity, or at least about 90% sequence identity, or atleast about 95% sequence identity to the polynucleotideof SEQ ID NO:1 can be identified by high stringencyhybridization. A polynucleotide of the invention can be obtamed 20 by Chemical synthesis, isolation and cloning from plantgenomic DNA, or other means known te the art, inciudingthe use of polymerase chain reaction (PCR) technologycarried out using oligonucleotides corresponding topartions of SEQ ID NO:1. PCR refers to a procedure or 25 technique in which target nucleic acid is amplified in amanner similar to that described in U. S. Patent Ne.4,683,195, incorporated herein by reference, andsubséquent modifications of the procedure describedtherein. Generally, sequence information from the ends 30 of the région 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 spécifie RNAsequences, spécifie DNA sequences from total genomic -NA, 35 and cDNA trar.scnbed from total cellular RNA, T.

010990 - 12 - bactériophage or plasmid sequences, and the like.Alternative]//, it is contemplated that a cDNA library (inan expression vector) can be screened with CDPK-specificantibcdy prepared using peptide sequence(s) from 5 hydrophilic régions of the CDPK sequence of Figure 3 andtechnology known in the art.

The novel polynuclectides of the invention can befound in substantially ail plants, including members ofthe Leguminaceae (e.g., soybean), members of the 10 Solanaceae (e.g., N. tabacum) , members of the

Brassicaceae family (e.g., Arabidopsïs thaliana) andmembers of the Graminaceae (e.g., Zea mays) . Preferably,polynucleotides of the invention are selected from theSolanaceae family. 15 In some embodiments, a polynucleotide of the invention is identified and isolated from a plant basedon nucléotide sequence homology and on the rapidinduction of expression after elicitor or pathogentreatment. For example, DNA:DNA hybridization under 20 conditions of moderate to high stringency with apolynucleotide probe disclosed herein allows theidentification of corresponding genes from other plantspecies. Use of a target nucleic acid (e.g., cDNA)prepared from a tissue shortly after induction of defense 25 responses facilitâtes the isolation of the novelpolynucleotides disclosed herein, because suchpolynucleotides typically are more rapidly induced thanother CDPK genes. A nucleic acid construct comprises a 3 0 polynucleotide 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 01 0990 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 msense or antisense orientation. Regulatory sequencestypically do net themselves code for a gene product.Instead, regulatory sequences affect the expression levelof the coding sequence. Examples of regulatory sequencesare known m the art and include, without limitation,minimal premoters and promoters of genes induced inresponse to elicitors. Native regulatory sequences cfthe 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-likeéléments, introns, 3' non-coding régions such as poly Asequences and other regulatory sequences discussedherein. Molecular biology techniques for preparmg suchchimeric genes are known in the art.

Polypeptides of the invention hâve from about 250to about 550 amino acids, e.g., from about 300 ammoacids to about 508 amino acids, or from about 30S ammoacids to about 500 amino acids. A polypeptide of theinvention typically contains protein kinase domains aswell as calcium-binding site domains. Such domainsinclude, for example, amino acids 2 to 7, 42 to 45, 191to 202, 227 to 238, 264 to 274, and 297 to 307 of Figure3 .

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 010990 - 14 - about 95% or greater sequence identity. Generally,conservative amino acid substitutions or substitutions cfsimilar amino acids are tolerated without affectingprotein fonction. Similar amino acids are those that are 5 similar in size and/or charge properties. For exampie,isoleucine and valine are similar amino acids.

Similarity between amino acid pairs has been assessed ;nthe art in a number of ways. For example, Dayhoff et al.(1978) in Atlas of Protein Sequence and Structure, Vol. 10 5, Suppl. 3, pp. 345-352, provides frequency tables for amino acid substitutions which can be employed as ameasure of amino acid similarity. Protein kinase domainsand calcium-binding site domains may be altered byconservative substitutions, but generally are retained 15 without alterations in amino acid sequence.

An "isolated" polypeptide is expressed and prcduced in a manner or environment other than the marneror environment in which the polypeptide is naturallyexpressed and produced. For example, a polypeptide is 20 isolated when expressed and produced in bacteria or fungi. 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, a 25 polypeptide is isolated when a gene encoding it is operably linked to a chimeric regulatory element and isexpressed in a tissue where the polypeptide is naturallyexpressed, but at higher levels. A polypeptide of theinvention car also be isolated by standard purification 30 methods to obtain it in about 80% or greater purity, crabout 90% or greater purity or about 95% or greaterpurity.

In sorte embodiments, a polypeptide of theinvention is an analog or variant of a polypeptide 2 5 tncluding the deduced amino acid sequence of Fig. 3. p '* ;. A jL X# ÜίΛΑΜ-^Λ- 15 - 010990

Such analogs or variants include, for example, naturallycccurring allelic variants, non-naturally occurrxngallelic variants, délétion variants, and insertionvariants, that do not substantially alter the fonction ofthe polypeptide. A polypeptide of the invention may comprise thesequence snown in Fig. 3 as well as the flanking aminoterminal and carboxy terminal sequences encoded by thesanie gene as that comprising the nucléotide sequence ofSEQ ID NO : 1. Alternatively, a chimeric polypeptide maybe produced frcm a gene that links, in-frame, nucléotidesfrom the 5' région of a first CDPK gene to nucléotidesfrom the 3' région of a second CDPK gene, therefcy 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 région of asoybean CDPK gene (Fig. 4) , followed by the amino acidsequence of Fig. 3, followed by amino acids 465 to 50Ξfrom the carboxy terminal région of the same soybean CDPKgene, ail of whioh are fused in-frame. A transgenic plant of the invention contams 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 intrcgressthe novel construct into other Unes or species, cr forfurther sélection of other désirable traits.Alternatively, transgenic plants may be obtained by .. " · ·ΛΛ II. »1 _ . 010990 - 1S - végétative propagation of a transformée! plant cell, forthose speoies amenable to such techniques.

As used herein, a transgenic plant also befers teprogeny of an initial transgenic plant. Progeny includesdescendants of a particular plant or plant line, e.g.,seeds developed on an instant plant. Progeny of aninstant plant also includes seeds formed on F., F:, F3,and subséquent génération plants, or seeds formed on BC1ZBC2, BC3, and subséquent génération plants.

In sortie embodiments, 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 plantvégétative tissues, thereby reducing the amount of plantbiomass that interfères with mechanical harvesting of theseeds. Regulated inhibition of CDPK gene expression canbe acccmplished by operably linking, in antisenseorientation, a polynucleotide of the invention to asuitable inducible regulatory sequence. See, e.g., U.S.Patent 5,453,566. One can achieve the same effect bycosuppression, i.e, expression in the sense orientationof the entire or partial coding sequence of a novel CDPKgene can suppress corresponding endegenous CDPK genes.

See, e.g., WO 94/11516.

In some embodiments, a nucleic acid constructincludes a polynucleotide disclosed herein, operablylinked to a minimal promoter. Such a construct, wher.mtroduced mtc and expressed m a plant, can conter low 010990 - 17 - level constitutive expression of the polynuclectide,resulting in an enhanced systemic defense response by theplant. A minimal promoter contains the DNA sequéncesignais necessary for RNA polymerase binding andinitiation of transcription. Generally, transcriptiondirected by a minimal promoter is low and does notrespond either positively or negatively to environmentaior developmental signais in plant tissue. An exemplaryminimal promoter suitable for use in plants is thetruncated CaMV 35S promoter, wnich contains the régionfrom -90 to +8 of the 35S transcription unit.

Transcriptional regulatory sequences can be uséeto control gene expression in suspension cultures. Forexample, the EAS4 promoter including the transcriptioninitiation signais, the inducible transcriptionregulatory element and the transcription-enhancingelement, can be used to médiate the inducible expressionof the disclosed coding sequence in transgenic plants orsuspension cell cultures. See U.S. Application SerialNo. 08/577,483. When desired, expression of the codingsequence of interest is induced by the application of anelicitor 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 Agrobac ter ium spp. typically use binary type vectors.Walkerpeach, C. et al., in Plant Molecular BiologyManual, S. Gelvm and R. Schilperoort, eds., KluwerDordrecht, Cl:l-19 (1994).

In some embodiments, an inducible transcriptionregulatory sequence can be coupled to a promoter sequence 010990 - 18 - functional in planes, both of which are operably linkedco a polynucleotide of the invention. When suefr aregulatory element is coupled to a promoter, a truncated(or minimal) promoter generally is used, for example, thetruncated 35S promoter of Cauliflower Mosaïc Virus(CaMV). Truncated versions of other constitutivepromoters can also be used, e.g., A. tiunefaciens T-DNAgenes such as nos, ocs, and mas, and plant virus genessuch as the CaMV 19S gene.

Techniques 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 hâve beensuccessfully transformed and regenerated include wheat,corn, rye, rice and asparagus. See, e.g., U.S. PatentNos. 5,484,956 and 5,550,318. Transgenic aspen tissuehas been prepared and transgenic plants hâve beenregenerated. Poplars hâve also been transformed.Technology is also available for the manipulation,transformation, and régénération 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 polynuclectideprésent in the construct alters the disease résistancephenotype of the plant, e.g., a novel disease résistancephenotype is conferred on the plant or an existmgdisease résistance phenotype is enhanced.

Disease résistance phenotype involves the leveland timing of host défensive responses in the transgenicplant. Assays to indicate that disease résistance hasbeen altered typicaily include the aoplication et acompound that criitarilv elicits a défensive ressens^ tt 19 ί...*''1, 'îiv.’1 : «ί *.,> k «4·ΧίMaLA ν*Ι3ΚΛ» 010990 a transgenic plant: and, in paraliei,the same ccmccund to a centre! niant :he annlicati: A centre! uant typically is from the same parental line as the ene entewhich a nsw nucleic acid ccnstruct was introduced. 5 Disease résistance is enhanced or conferred on a plant byexpression of a polynucleotide of the invention whenthere is a higher level of résistance in the transgenicplant than the corresponding résistance in the contre!plant. Disease résistance can be measured with refersnce 10 te a spécifie patnogen, e.g., a Phythophzhora spp. .

Disease résistance can also be measured with reference teseveral pathogens, to identify an enhanced systemicdefense response.

Where transgenic plants are to be induced for 15 expression of a CDPK coding sequence operably linked toan elicitor-mediated regulatory element, the elicitortypically must penetrate the cuticle of the plant to hâvean inductive effect. Plant tissue can be wounded tofacilitate cr allow the uptake of the elicitor mto the 20 plant tissue. A wide variety of inducing compositions,inciuding elicitors and other Chemical signais, such asthe combination of ethylene and methyl jasmonate, can beeffectively used to induce expression. A method of using a polynucleotide of the 25 invention comprises the step of hybridizing the polynucleotide to DNA or RNA from a plant. Hybridizationcan be carried out, for example, as described hereinabove. The method can further comprise the step ofidentifying a segment of the plant DNA or RNA that has a 20 significant degree of sequence identity to the polynucleotide, e.g., 70% sequence identity, preferably80% sequence identity, 90% secuence identity, or 55%sequence identity. The segment can be identified byelectrophoretic seoaration cf the niant DNA or RNA anc ne use

Ecuvnmn -Όίδ, wmon resu_:

Mi-V - >-i_ v* AZ*kw4. ^XliwéMlrtdll 010990 - 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 (nucléotides) 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 élémentsand poly A sequences. Flanking DNA can be either 5' toor 3 ' to the homologous segment and preferably includes3 00, or 600, or 1,000 bp of DNA beyond the codingsequence, because regulatory éléments generally are foundwithin this span.

Promoters and other elicitor or pathogen-responsive regulatory éléments flanking the novelpolynuclectides disclosed herein are particularly useful,because such éléments ccnfer very rapid induction of geneexpression after treatment with pathogen or elicitor.

Such regulatory éléments can be operably linked to usefulgenes to allow rapid production of désirable compounds.For example, such regulatory éléments can be used todrive expression of genes encoding antibodies, bloodclotting factors, antigenic peptides, viral replicases orcoat proteins, and enzymes involved in secondarymétabolite synthesis (such as isoprenoid biosynthesis) .See, e.g., U.S. Patent 5,612,487; U.S. Patent 5,484,715;and U.S. Application Ser. No. 08/577,483, filed December22, 1995.'

After introducing a chimeric gene having anelicitor or pathogen-responsive element into a plant,expression of the chimeric gene product can be inducedwith an appropriate pathogen or elicitor. Production of û 1 Ο 9 9 Ο 21 the desired gene product (or its er.zymaoic enc croduc:!rapidly ensues and the desired product oan cher. becbtained.

The invention will be further descrioed m the5 foilowing examples, which do not limit the scope of the invention described in the daims.

EXAMPLES

The foilowing examples use many techniqueswell-known and accessible to those skilled in the arts cf 10 mclecular biology, in the manipulation of recombinant DNAin plant tissue and in the culture and régénération oftransgenic plants. Enzymes are obtamed frcm commercialsources and are used according to the vendors'recommendations or other variations known te the art. 15 Reagents, buffers, and culture conditions are also knownte the art. Abbreviations and nomenclature, whereemployed, are deemed standard in the field and areccm.monly used in professional tournais such as tnosecited herein. 2 0 Example 1.

Cloning of a Tobacco CDPK cDNA

The elicitor parasiticein was préparée byexpression of the Phytophthora parAl gene in s. edicells and isolation of the gene product from the 2Ξ penplasmic space.

Genomic DNA of Phytophthora Race O was isolatedfrcm mycélium essentially as described in Xu, J., et al.Trer.ds in Genetics 10:226-227 (1994) . The DMA was sheared and used as a template fer FTP. amplification cf 30 the parAl gene, usina primers désignée according te tnepar.'.l sequence reported in Kair.oun, S., et al. Mol. Fiant -Microbe Interact. 6:573-531 (1993) . The parAl PC?,prcduct was cloned into pBluescript (Stratager.e, San '-yr- τ’»τι*·λϊ.*τγ·«ϊ*τ·’'ϊ' 01Ü990 22

Diego, CA) and the sequence of the product determined bvdouble-stranded DNA sequencing usina the dideozy chainterminâtion method.

The parAl insert in pBiuescript was amplified by5 PCR, using primers that created an N-terminal histidine tag and a protein kinase site at the 5' end of the gene.The PCR product was ligated into the expression vectcrpET28b (Novagen, Madison, WI) and, after confirminc theDNA sequence of the parAl fusion, the pET28b construct 10 was transformed into E. coli BL21. A BL21 culture containing the parAl fusion was grown at 37° C in the presence of kanamvcin to an OD6=. o0.3. IPTG (ImM) was added and the culture was incubatedfor 5 hours at 27° C. 15 Periplasmic proteins were prepared by osmotic shock essentially as described in Ausubel, F., e al. inCurrent Protocols in Molecular Biology, John Wiley &Sons, New York (1989). Cells (1.5 ml) were harvested bvcentrifugation, resuspended in 500 μΐ of 50 mM Tris-HCl, 20 pH 8.0, 20% sucrose, 1 mM EDTA and incubated with shakir.for 10 minutes at room température. Aftercentrifugation, the pellet was resuspended m 200 μΐ icecold MgSO4 (5 mM) and incubated with shaking for 10minutes at 4° C. The mixture was centrifugea and the 25 resulting supernatant (containing periplasmic proteins)was applied to a Ni” column. The parAl protein waspurified from the column accordina to the manufacturer'sdirections. The protein concentration in the parAlextract was determined by the Bradford method. 30 Nicoziana Zabacum D. cv. KY14 cell suspension cultures were treated with parasiticein at a finalconcentration of 2 pg/ml during rapid growth phase ttinduce stress response genes. Parallel suspension cellcultures which were not treated with parasiticein serzed 35 as Controls. Cells were collected by gentle vacuum 23 010990 filtration 0, 30, 60 and 120 minutes after the addition of elicitor.

Total RNA was isolated from treated and -untreatedtobacco cells and used as template for targeted 5 differential display reverse transcriptase PCR (TDDRT-PCR). First strand cDNA was generated using a cDNA cyclekit from Invitrogen (San Diego, CA). The first strandcDNAs were then used as templates for PCR. The PCRreaction was carried out using typical conditions as 10 described in PCR Protocols: A Guide to Methods and

Applications, Innis, M. , Gelfand, D., Sminsky, J. andWnite, T., eds. Academie Press Inc., San Diego, CA(1990), except that the annealing température was 58°C.The PCR primers were FokinB (GTTGACTCCCTACCCTCTT) and 15 RecalIV (GGTACTTAGGAAGTGTTACGGG) . See Figure 1. PCRproducts were separated by electrophoresis on a 1% (w/'v)agarose gel and products of greater than about £00 basepairs (bp) from the 60 minute treated culture werepurified by electroelution onto DF-61 paper (Whatman). 20 Ends of the purified PCR products were filled ir. withKlenow polymerase, ligated to the EcoRV site ofpEIuescript, and transfermed into E. coli TRI.

Ampicillin résistant TB1 colonies were screenedfor the presence of a .>800 bp DNA fragment mserted mto 25 pBluescript. The sequence of cne such insère was determined by the dideoxynucleotide chain terminaticnprocedure of Sanger et al. (1977) Proc. Natl. Acad. Soi.USA 74.:8073-8077, with a Sequenase1 kit from UnitedStates Biochemical Corp., Cleveland, OH) or an automates 32 fluorescence based System (Applied Biosystems, Foster

City, CA). The sequence of the msert m the vectcr wasdetermined on both strands. The plasmid contaimng thismsert was designated pCD?K-l.

The nucléotide sequence of the msert in pCDPR-1 35 is snown m Figure 2 and the deduced amino acid sequence Q10990 24 of the înser: is shown m Figure 2 . The deduced amine acid sequence was compared to amino acid sequences of plant aenes in the GenBank, EM3L, and Ewiss Profe

databases. Homology was found to plant CDPK 5 polypeptides, including polypeptides from Glycine max,G.rabidcpsis thaliana, Vigna radiata, Zea mays andGucurbi ta pepo.

Using the BLASTP program and a BLOSDM62 scorir.çmatrix, two régions of homology to senne/threonine IG pretein kinase domains were identified m the amino terminal portion of the polypeptide and four régions cihomology to Ca" binding domains were identified in thecarboxyl terminal portion of the polypeptide. Figure 4shows a comparison of the amino acid sequence of Fig. 2 15 and a soybean CDPK amino acid sequence ÎGenoank AccessionNc: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 différences in other 21 parts of the sequence. The comparison mdicates that tnere is about 78% overall sequence identity between thesoyoean CDPK and CDPK-1.

The BLASTN program was used to compare the pCD2K-2nucléotide sequence to nucleic acid sequences on varions 25 databases. Based on the nucieotiae sequence of otherplant CDPK genes and the length of the polypeptidesencoded thereby, the nucleio acid insert présent inpCDPK-1 is estimated te lack about 5£C bp of 5' CDPK-1ccdir.g sequence and about 13C bp of 2' CDPK-1 codir.g 2 G secuence.

Example 2.

Isolation of a full-length. cDNA clone

To obtam a full-length clone, a .LACE (RapidAmplification of cDNA Ends) approach is used, with polyA- 25 RNA prepared from tcbacco cells after induction with ’r « ,*· ? f*-*. r 25 01 0990 elicitor being the tempiate. PolyA-r PUA is préparai as described in Example 1.

A primer having the sequence GAC AAG GAC'GGG AGT GGG TAT (Primer A, internai to CDPK-1) and a primer 5 havina the sequence GAC TCG AGT CGA CAT CGA TTT TTT TTTTTT TTT TT (dT.7 adapter-primer) are used te amplify the3 ' end of the CDPK coding sequence. The reversetranscriptase reaction is carried out in 2 μΐ 10X RTCbuffer, 10 units of RNasin (Promega Biotech), 0.5 μο cf 10 dTK adapter-pnmer and 10 Units of AMV reverse

transcriptase (Life Sciences) in a total volume of 2.5μΐ, as described m Frohman, M. in PCR Protocole: A Guideto Methods and Applications, supra., pp. 28-35. The PCRamplification reaction is carried out in 5 μΐ 10X PCR 15 buffer, 5 μΐ DMSO, 5 μΐ 10X dNTPs (15 mM each), 30 μΐ H?O1 μΐ adapter-primer (25 pmol, GAC TCG AGT CGA CAT CG), 1μΐ primer A and 1-5μ1 cDNA. Cycle times are as mdicatedin Frohman, supra.

The 5' end of the CDPK coding sequence is cicned 20 by carrying out reverse transcription as described above,usine 10 pmole of primer B (AGG GGC TAC GTA GTA AGG ACT)instead of dT17 adapter-primer. The cDNA prcduct isextended using terminal transferase and dATP as describedin Frohman, supra, and then amplified by PCR as described 25 above with 10 pmole of dTr adapter-pnmer, 10 pmole of adapter-primer and 10 pmole of primer C (ATT CTC AGG CTTAAG GTC CCT). PCR is carried out under standardconditions. Back et al. (1954) Arcs. Eicches. Eicpsys.315:523-532. The amplified 2' and 5' prcducts are blunt- 30 end cloned mto pBluescnpt SR (Stratagene) and combinéewith the pCDPK-1 insert by routine molecuiar biolcgytechniques to form a full-length cDNA of the tcbaccc CDPKcoding sequence. •rsnr 01 0990 - ZO "

The DNA seauence of the full-ler.gth cDNA isdetermmed by a dideoxvnucieotide chair, terminatior.procedure, as described in Exemple 1.

Example 3.

Induction of CDPK-Homologous RNAin Tobacco Suspension Cultures

The DNA insert in pCDPK-1 was used as a crooe t:foilow the induction of gene expression in response to elicitor. Nicotiana tabacum cv. KY14 ce: sustensicn cultures were treated with parasiticein for C, 1, 2and 12 hours as described in Example 1. Total RNA wasisolated and electrophoresed on a 1% agarose gel. Theinsert from pCDPK-1 was radiolabeled by the randomprirnmg method and hybridized to the gel-separated RNAdescribed in Sambrook, J. et al., supra. No mRNAhybridizing to CDPK-l was detected prior to elicitortreatment, whereas mRNA hybridizing te CDPK-l was read:detected at 1/2, 1 and 2 hours after elicitor treatmen:At z and 12 hours after elicitor treatment, no mRNA.hybridizing to CDPK-l could be detected, indicatmg th=CDPK-l gene expression had decreased to undetectablelevels bv about 6 hours.

Example 4.

Construction of a Chimeric CDPK Gene A CDFK gene is constructed from: a Chemicallysynthesized DNA encodmg amino acids 1 to 15€ of thescyhean CDPK of Figure 6, a chemically synthesized DNA.enccding amino acids 4-ΞΞ to 50£ cf the soybear. CDPK ofFigure 6, and the CDPK insert cf pCDPK-1. The tnree DNAsare ligated by routine molecular bioicgy techniques tofcrm a chimeric CDPK coding seçuence r.aving amino aoics ito 1Ξ5 of soybean CDPK at the amino terminai end, fused **-*·>** rr·' Ο’** î Ί.” ' ’ ' h' 27 010990 10 in-frame to amino acids 1 to 307 of tobacco CDPK (Fig. 3) , which in turn is fused in-frame te amino acids 465 to50£ cf soybean CDPK at the carboxyl terminal end.

The chimeric ccdmg sequence is inserted in senseorientation into an Agrobaccérium binary vectorcontaining a minimal 35£ and EAS4 inducible recuiatoryelement. Opérable linkage of the regulatory element,promoter, and codinc sequence is confirmed by determimnathe DNA sequence of the junction régions and byexpression in transgenic plants.

Example 5. Génération of Transgenic Plants

Transformée plant oeil Unes are produced using amodified Agrobac terium tumetaciens transformation 15 protocol. Nucleic acid ccnstructs are préparée thatcontain the full-length CDPK cDNA of Example 3 cr thechimeric CDPK codinc sequence of Example 4. Therecombinant constructs containing the sequences to beintroduced into plants are transferred into A. 20 tumefaciens strain GV3S50 by triparental tnating with Σ.coli T31 (pRK2013) . N. tabacum leaves at a vanetv cfstages of growth are eut mto 1 cm* pièces, and dioped ina suspension of Agrobactenum cells (about 10" to 10"cells/ml). After 3 to 10 minutes, the leaf segments are 25 then washed in stenle water to remove excess bactenalcells and to reduce problems with excess bactenal growthon the treated leaf segments. After a short drying time(30 to 60 seconds), the treated leaf segments are placéeon the surface of Plant Tissue Culture Medium without 30 antibiotics to promets tissue infection and DNA transferfrom the bacteria te the plant tissue. Plant TissueCulture Medium contans per liter: 4.31 g of Murashigeand Skocg Basai Salts Mixture (Sigma Chemical Company,

St. Louis, MO), 2.Ξ m.g of benzylam.mopurine (dissclved m ’F***’ F’p'"'·?· • * » Ai'·*·· e ! -i,k

»»··· Λ '· 'SV'·· ’ 010990 10 20 25 1 N NaOH), 10 ml cf 0.1 mg/ml indoleaoetio acid solution,30 g sucrose, 2 ml cf Gamborg's Vitandn Solution (Siarr.aChemical Co. , St. Louis, MO) and 6 g cf agar. The pH isadjusted between pH 5.5 and 5.5 with NaOH. After 2 davs,the leaf segments are transferred to Plant Tissue CultureMedium containing 300 Mg/ml of kanamycin, 500 Mg/ml cfmefoxin (Merck, Rahway, NJ). Kanamycin selects fortransforme! plant tissue, and mefoxin selects aqamstAgrobacterium.

It may be necessary to minimize the exposure cfthe expiant tissue to Agrobac ter ium cells durmç thetransformation procedure if a pathogen-inducibleregulatmg element is used, because Agrobacterium cellsmay themselves induce the element after introduction mtothe plant cells. Accordingly, the biolistic techniquefor the introduction of DNA containing cell suicide genesunder the regulatory control of the inducibletranscriptional regulatory element is a usefuialternative transformation technique because it does notentail the use of Agrobacterium cells or funqal cell walldigestive enzymes (as necessary for the génération orprotoplasts for electroporation), both of whicr. car leadte induction of the coding sequenoes under the contrcl ofthat regulatory element.

Transgenic plants are regenerated essentially asdescribed by Horscn et al. (1585) Science 227:1225-123 1.

Example 6 .

Elicitor- and Pathogen-inducible Expressionof a Chimeric CDPK Gene in Transgenic Tobacco

The activity of the CDPK constructs of Example ~are measured in transgenic tobacco plants treated witheither an elicitor or pathogen. As cor.trois, transgenictobacco plants expressing the GUS reporter gene under theccntrcl of the cauliflower mosaïc virus (CaMY 25Ξ ‘TFl*·*#·*· v»* rr f~ f--·- ·fc 1»»·· '•« ί-, . *·. x i » 29 Ü1 U 9 9 ο cromoter are aise produced. F- seeds from regenerated transgenic tobaccc planés are germmated or. medium containing 100 mg/L kanamycin. The resultmg kanamycin-résistant plants are subsequently transferred 5 intc? soil and grown in a greenhouse. Half of the plant,are tested for the expression of the CD??C gene underinducing conditions, e.g., by intercellular applicationof elicitor or cellulase to the transgenic plants.Elicitor or cellulase is applied with a mechanical 10 ripetter. As a control, remaining plants are mcck-treated with a solution lackmg cellulase orelicitor. Tobacco tissue is wounded with a scalpel insome experiments te facilitate exposure to the inducingcompound. 15 Example 7.

Identification of CDPK Homologous Seçuencea

Tobacco leaf aenomic DNA is tsolatea as describe in Murray and Thompson (1980) Nucleic Acids Research8,:4321-4325. After digestion of aliquots with desired 20 restriction 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 90 0 bp CDPH cDNA mse:of Example 1 that is radiolabeled by the ranoom primer 25 method. Hybridization is performed at 6C°C m 0.25 Msodium phosphate buffer, pK 8.0, 0.7% SDS, 1î bovinesérum albumin, 1 mM EDTA. The blot is then wgshec twiceat 45°C with 2X SSC, 0.1% SDS and twice with 2.2X SSC,0.1% SDS (IX SSC is 0.15 M NaCl, 0.015 M sodium, citrate, 30 pH 7.0). Relative hybridization mtensities tf thevanous bands on the membrane are estimâtes fromautoradiograms usina a video densitometer(MilIiGen/Biosearch, Ann Arbor, MI) . m ''· r » U109 90

To identify polynucleotiàes having homoicgoussequences to tobacco CDPK and te détermine trie aooarer.tr.umber of copies per genome cf Chose sequences, Southernhybridization experiments are carried eut using target Ξ DNA isolated from other plant speoies and tobacco CDPKprobes. Restriction endonuclease-digested genome DNA.sci various plant species are separated bv agarose aeielectrophoresis (0.8% agarose), and then transferred toHybond-N* membrane (Amersham Corp., Arlington Heignts, 10 IL). Radiolabeled probe fragments comprising codingsequences of pCDPK-1 are nybridized to the digestedgenomie DNA essentially as described in Sambrook et al.(1989) , suora. Moderate strmgency conditions are used(hybridization in 4X SSC, at c5°C with the last wash m 1Ξ IX SSC, at 65°C).

Alternatively, PCR îs carried out usmg targetgenomie DNA as a template and primers derived from highlccnserved régions of the pCDPK-1 coding sequence.

Example 8. 11 Genomie DNA Flanking a CDPK Coding Sequence

The cDNA clone described m Example 1 is used ashybridization probe for screening a N. zabacum ον. MK326genomie library m the XEMBL3 vector (Clontech, PaleAlto, CA). Genomie DNA clones having 70% or greater 2Ξ sequence identity to the tobacco CDPK of Example 1 areidentified using routine subclomng protocols. Thenucléotide sequences of the cloned nucleic acid msertsare determined using routine DNA sequencmg protocols.

One of the genomie DNA clones has a fuli-length

21 coding semence that comprises the tobacco CDPK codingsequence of Example 1. The clone also certains DNAcontiguous with, and 5' te, the ccdmg sequence ofExample 1. Examination of the nucléotide sequence cf te5' flanking DNA in this clone reveals a putative ATG 31 Ü1 0990 start codon as well as one or more putative reguiatoryéléments upstream of the start ccdon and within about1000 bp of the start codon.

Other Embodiments 5 It is to be understood that while the invention has 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 appended 10 claims. Other aspects, advantages, and modifications arewithin the scope of the following claims. 32 01 0990

SEQUENCE LISTING

(1) GENERAL INFORMATION (i) APPLICANT: University of Kentucky Research Foundation

(ii) TITLE OF THE INVENTION: PROTEIN KINASES AND USES

THEREOF (iii) NUMBER 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: MN

(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: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: PCT/US98/14109 (B) FILING DATE: 07-JUL-1998 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/889,655 (B) FILING DATE: 08-JUL-1997 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Lundquist, Ronald C (B) REGISTRATION NUMBER: 37,875 (C) REFERENCE/DOCKET NUMBER: 07678/0200A1 (ix) TELECOMMUNICATION 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 33 01 0990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : AGGGACCTTA AGCCTGAGAA TTTCCTTTTC AGTGCCGACG ACTTCATGGT AAAGAG'I'AAG 6 0GCCACCGACT TCGGGCTTAG TGTATTCTAT AAGCCTGGGC AAAAGTTCAC GGAC’ATAGTA 12 CGGGAGTCCTT ACTACGTAGC CCCTGAGGTA CTTAGGAAGT GTTACGGGCC TGGGAGTGAC 18CGTATGGAGTG CCGGGGTAAT ACTTTACACC CTTCTTTGTG GGGCCCCTCC TTTCATGGCC 24CGACAGTGAGC CTGGGGTAGC CCTTCAAATA CTTCATGGGG ACCTTGACTT CAAGAGTGAC 300CCTTGGCCTA CCATAAGTGA GAGTGCC'AAG GACCTTATAA GGAAGATGCT TGAGCAAGAC 3 6CCCTAAGAGGA GGCTTACCGC CCATGAGGTA CTTAGGCATC CTTCGATAGT AGACGAGAAT 42GATAGCCCCTG ACAAGCCTCT TGGGCCTGCC GTACTTAGTA GGCTTAAGCA ATTCAGTGCC 48GATGAATAAGA TAAAGAAGAT GGCCCTTAGG GTAATAGCCG AGAGGCTTAG TGAGGAGGAG 54 0 5 ATAGTAGGGC TTAAGGAGAT GTTCAAGATG GACACCGAC’A ATAGTGGGAC CGTAACCTTC 600TTCCATCTTA AGCAAGGGCT TAAGAGGGTA GGGAGTCAAC TTGGGGAGAG TGAGATAAAG 660GACCTTATGG ACGCCGCCGA CGTAGACAAT AGTGGGACCA TAGACTATGG GGAGTTCGTA 720ACCGCCGCCA TGCATCTTAA TAAGATAAAG AGGGAGGACC ATCTTGTAAG TGCCTTCAGT 75 0TATCATGACA AGGACGGGAG TGGGTATATA GAGGTAGACG AGCTTAGGCA AGCCCTTC-AG 840GAGTTCGGGG TACCTGACAC CAGTCTTGAG GACATGATAA AGGAGGTACA CACCGACAAT 900GATGGGCAAA TAGATTATGG G 921 (2) INFORMATION FOR SEQ ID NO : 2 : n (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 307 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Arg 1 Asp Leu Lys Pro 5 Glu Asn Plie Leu Plie 1 0 Ser. Ala Asp Asp Plie 15 Met Val Lys Ser Lys 20 Al a Thr Asp Phe Gly 25 Leu Ser Va 1. Plie Tyr 30 Lys Pro Gly Gin Lys 35 Plie Thr Asp Ile Val 40 Gly Ser Pro Tyr Tyr 45 Va 1. Ala Pro Glu Val 50 Leu Arg Lys Cys Tyr 55 Gly Pro Gly Ser Asp 60 Val T rp Ser Ala Gly 65 Val Ile Leu Tyr Thr 70 Leu Leu Cys Gly Ala7 5 Pro Pro Plie Met Ala 80 Asp Ser Glu Pro Gly 85 Val Ala Leu Gin Ile 90 Leu His Gly Asp Leu 95 Asp Phe Lys Ser Asp 100 Pro Trp Pro Thr Ile 105 S»r Glu Se r Ala Lys 110 Asp Leu Ile Arg Lys 115 Met Leu Glu Gin Asp 120 Pro Lys Arg Arg Leu 125 Thr Ala His Glu Val 130 Leu Arg His Pro Trp 135 Ile Val Asp Glu Asn 140 Ile Al a Pro Asp Lys 145 Pro Leu Gly Pro Al a150 Val Leu Ser Arg Leu 155 Lys Gin Phe Ser Ala16 0 Met Asn Lys Ile Lys 165 Lys Met Ala Leu Arg17 0 Va 1 Il e Ala G J u Arg 175 Leu Ser Glu Glu Glu 180 Ile Va 1 Gly Leu Lys 185 GJ u Met; Phe Lys Met: 1 9 0 Asp Thr Asp Asn Ser 195 Gly Thr Val Thr Phe 200 Phe His Leu Lys Gin 2 05 Gly Leu Lys Arg Val 210 Gly Ser Gln Leu Gly 215 Glu Ser Glu 11« Lys22 0 Asp L°u Met Asp 010990 34

Ala Ala Asp Val Asp Asn Ser Gly Thr Ile Asp Tyr Gly Glu Phe Val 225 230 235 2 40 Thr Ala Ala Met His Leu Asn Lys Ile Lys Arg Glu Asp His Leu Val 245 250 255 Ser Ala Phe Ser Tyr His Asp Lys Asp Gly Ser Gly Tyr Ile Glu Val 260 265 2 70 Asp Glu Leu Arg Gin Ala Leu Glu Glu Plie Gly Va 1 Pi. o Asp Thr Ser 275 280 2 85 Leu Glu Asp Met Ile Lys Glu Val Asp Thr Asp Asn Asp Gly Gin Ile 290 295 300 Asp Tyr Gly 305 lli .e iLi (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 10 (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: linear15 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : GTTGACTCCC TACCCTCTT 1 9 (2) INFORMATION FOR SEQ ID NO : 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 512 amino acids (B) TYPE: amino acid 20 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

Met 1 Ala Ala Lys Ser 5 Ser Ser Ser Ser Thr .10 Thr Thr Asn Va 1 Val· 15 Thr Leu Lys Ala Ala 20 Trp Val Leu Pro Gin 25 Arg Thr Gin Asn 1.1 e3 0 Arg Gl U Val Tyr Glu 35 Val Gly Arg Lys Leu 40 Gly Gin Gly Gl n Phe 4 5 G 1 y Thr Thr Phe Glu 50 Cys Thr Arg Arg Ala 5 5 Ser Gly Gly Lys Phe 6 0 A i a Cys Lys S«r 25 îîWA*

010990 - 35 - 10 15 20 25

Ile Pro Lys Arg Lys65 Leu 70 Leu Cys Lys Glu Asp75 Tyr Glu Asp Val Trp 80 Arg Glu ; Ile Gin Ile Met His His Leu Ser Glu His Ala Asn Val Val 85 90 95 Arg Ile Glu Gly Thr Tyr Glu Asp Ser Thr Ala Val His Leu Val Met 100 105 110 Glu Leu Cys Glu Gly Gly Glu Leu Phe Asp Arg Ile Val Gin Lys Gly His 115 120 125 Tyr Ser Glu Arg Gin Ala Ala Arg Leu Ile Lys Thr Ile Val Glu 130 13 5 140 Val Val Glu Ala Cys His Ser Leu Gly Val Met His Arg Asp Leu Lys 145 150 155 160 Pro Glu Asn Phe Leu Phe Asp Thr Ile Asp Glu Asp Ala Lys Leu Ly3 165 170 175 Ala Thr Asp Phe Gly Leu Ser Val Phe Tyr Lys Pro Gly Glu Ser Phe 180 185 190 Cys Asp Val Val Gly Ser Pro Tyr Tyr Val Ala Pro Glu Val Leu Arg 195 200 2 05 Lys Leu Tyr Gly Pro Glu Ser Asp Val Trp Ser Ala Gly Val Ile Leu 210 215 220 Tyr Ile Leu Leu Ser Gly Val Pro Pro Phe Trp Ala Glu Ser Glu Pro 225 230 235 24 C Gly Ile Phe Arg Gin Ile Leu Leu Gly Lys Leu Asp Phe His Ser Glu 24 5 250 255 Pro Trp Pro Ser Ile Ser Asp Ser Ala Lys Asp Leu I le Arg Lys Met 260 2 65 270 Leu Asp Gin Asn Pro Lys Thr Arg Leu Tln- Ala His Glu Val Leu Arg 2 75 2 80 285 His Pro Trp Ile Val Asp Asp Asn Ile Al a Pro Asp Lys Pro Leu Asp 2 90 295 3 00 Ser Ala Val Leu Ser Arg Leu Lys Gin Plie Ser Ala Met Asn Lys Leu 305 310 3 15 320 Lys Lys Met Ala Leu Arg Val Ile Ala Glu Arg Lieu Ser Glu Glu Glu 325 330 335 Ile Gly Gly Leu Lys Glu Leu Phe Lys Met Ile Asp Thr Asp Asn Ser 340 345 350 Gly Thr Ile Thr Phe Asp Glu Leu Lys Asp Gly Leu Lys Asp Gly lieu 355 360 365 Lys Arg Val Gly Ser Glu Leu Met Glu Ser G1.U I le Lys Asp Leu Met 370 375 380 Asp Ala Ala Asp Ile Asp Lys Ser Gly Thr Ile Asp Tyr Gly Glu Phe 385 3 90 395 400 Ile Ala Ala Thr Val His Leu Asn Lys Leu Glu Arg Glu Glu Asn Leu 405 410 415 Val Ser Ala Phe Ser Tyr Phe Asp Lys Asp Gly Ser Gly Tyr Ile Thr 420 425 4 30 Leu Asp Glu Ile Gin Gin Ala Cys Lys Asp Phe Gly Leu Asp Asp Ile 435 440 445 His Ile Asp Asp Met Ile Lys Glu Ile Asp G.1 n Asp Asn Asp Gly Gin 450 4 55 4 60 Ile Asp Tyr Gly Glu Phe Ala Ala Met Met Arg Lys Gly Asn Gly Glv 465 4 70 4 75 480 Ile Gly Arg Arg Thr Met Arg Lys Thr Leu Asn Leu Arg Asp Ala Leu 485 490 4 95 Gly Leu Val Asp Asn Gly Ser Asn Gin Val lie Glu Gly Tyr Phe Lys 500 505 510 (2) INFORMATION FOR SEQ ID NO : 6 :

010990 - 36 - (i) SEQUENCE CHARACTERISTÏCS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GACAAGGACG GGAGTGGGTA T 21 5 (2) INFORMATION FOR SEQ ID NO : 7 : (i) SEQUENCE CHARACTERISTÏCS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : 10 GACTCGAGTC GACATCGATT ΨΨΨΨΨΤΨΤΤΤ ΨΨΨΨΨ 3 5 (2) INFORMATION FOR SEQ ID NO : 8 : (i) SEQUENCE CHARACTERISTÏCS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : *5 GACTCGAGTC GACATCG 17 (2) INFORMATION FOR SEQ ID NO : 9 : (i) SEQUENCE CHARACTERISTÏCS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linearΌ (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : AGGGGCTACG TAGTAAGGAC T 21 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTÏCS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ATTCTCAGGC TTAAGGTCCC T 21 a*. 010990 - 37 - (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 3 08 arnino 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 Met 1 5 10 15 Val Lys Ser Lys Ala Thr Asp Phe Gly Leu Ser Val Phe Tyr Lys Pro 20 25 3 0 Gly Gin Lys Phe Thr Asp Ile Val Gly Ser Pro Tyr Tyr Val Ala Pro 35 40 4 5 Glu Val Leu Arg Lys Cys Tyr Gly Pro Gly Ser Asp Val Trp Ser Ala 50 55 60 Gly Val Ile Leu Tyr Thr Leu Leu Cys Gly Ala Pro Pro Phe Met Ala 65 70 7 5 8 0 Asp Ser Glu Pro Gly Val Ala Leu Gin Ile Leu His Gly Asp Leu Asp 85 90 95 Phe Lys Ser Asp Pro Trp Pro Thr Ile Ser Glu Ser Ala Lys Asp Leu 100 105 1.10 Ile Arg Lys Met Leu Glu Gin Asp Pro Lys Arg Arg Leu Thr Ala His 115 120 12 5 Glu Val Leu Arg His Pro Trp Ile Va l Asp G.l u Asn 1.1 e Ala Pro Asp 130 135 140 Lys Pro Leu Gly Pro Ala Val Leu Ser Arg Leu Lys Gin Phe Ser Ala 145 150 155 160 Met Asn Lys Ile Lys Lys Met Ala Leu Arg Val I le Ala Glu Arg Leu 165 170 175 Ser Glu Glu Glu Ile Val Gly Leu Lys Glu Met Phe Lys Met Ile Asp 130 185 190 Thr Asp Asn Ser Gly Thr Val Thr Phe Phe His Leu Lys Asp Gly Le vi 195 200 205 Lys Arg Val Gly Ser Gln Leu Gly Glu Ser Glu Ile Lys Asp Leu Met 210 215 220 Asp Ala Ala Asp Val Asp Asn Ser Gly Thr Ile Asp Tyr Gly Glu Phe 225 23 0 235 240 Val Thr Ala Ala Met His Leu Asn Lys Ile Lys Arg Glu Asp His Leu 245 250 255 Val Ser Ala Phe Ser Tyr His Asp Lys Asp Gly Ser Gly Tyr Ile Glu 260 265 270 Val Asp Glu Ile Arg Gin Ala Leu Glu Glu Phe Gly Val Pro Asp Thr 275 2 00 295 Ser Leu Glu Asp Met Ile Lys Glu Val Asp Thr Asp Asn Asp G1 y Gin 290 295 3 00 Ile Asp Tyr Gly 305

Claims (23)

1. .Λ.·..

   • 38 - 01 0990 WHAT IS CLAIMSD IS :

   1. An isolated polynucleotide, saidpolynucleotide comprising: a) the nucléotide sequence of SEQ ID NO:L; 5 b) an RNA analog of SEQ ID NO:1; c) a polynucleotide comprising a nucieic acidsequence complementary to a) or b) ; or d) a nucieic acid fragment of a), b) or c; that îsat least 20 nucléotides in length and that hvbridizes 10 under stringent conditions to genomic DNA encoding thepolypeptide of Figure 3.
2. 2. The polynucleotide of claim 1, wherein saidpolynucleotide comprises nucléotides 1 to 170 of Figure2 -
3. 3. The polynucleotide of claim 1, wherein said polynucleotide comprises nucléotides 160 to 560 of Figure 2 .
4. 4. The polynucleotide of claim 1, wherein saidpolynucleotide comprises nucléotides 550 to 920 of Figure 20 2.
5. 5 . A nucieic acid construct comprising thepolynucleotide of claim l.
6. 6. The nucieic acid construct of claim 5,further comprising a regulatory element operablv linked 25 to said polynucleotide.
7. 7. The nucieic acid construct of claim 6,wherein said regulatory element is an inducibleregulatory element. y. *·*.* - 35 . - 01 0990 S. The nuoieic acid construce of claim ~, wherein said reguiatory element îs induced in restons® ec a plane pathogen.

   5. A transgenic plane coneaining a nucleic acid5 construce comprising the polynucleotide of claim 1.
8. 10. The plane of claim S, wherein said construcefurther comprises a regulacory element op.erablv Imked tosaid polynucleotide.
9. 11. The plane of claim 10, wherein said 10 reguiatory element is an inducible regulaeory element.
10. 12. The plane of claim 11, wherein saidregulaeory element is induced in response to a planepathogen.
11. 13. The plant of claim 11, wherein said 15 regulacory elemene is induced in response to an eliciccr.
12. 14. The plane of claim 9, wherein said plane is adicotyledonous plane.
13. 15 . The plant of claim 14, wherein said plant 1 £ a member of Che Solanaceae family. 16 . The plant of claim 15, wherein said plant iS a îli co ciana plant. 17 . The plant of claim 16, wherein said plane 1 s Wicoeiana tabacum. 18 .' A transgenic plant contaimng , a 25 polynucleotide exnressina a ool-zoentide havinc frem aocue . 010990 250 to about ΞΞΟ amino acids, said polypeptide comprisi:an atnino acid sequence substantially identical te theamine acid sequence of Figure 3.

    15. The plant of claim 18, wnerem saidpolypeptide comprises the amino acid sequence cf Figure
14. 20. The plant of claim 18, wherem said pla:a dicotyledonous plant.
15. 21. The plant of claim 20, wherem said pla:IC a member cf the Solanaceae family.
16. 22. A method of using a polynuclectide, saidmethod comprising the step of hybridizing thepolynucleotide of claim 1 to DNA or RNA from a plant.
17. 23. The method of claim 22, further comprising15 the step of identifying a segment of said plant DNA orRNA that has about 70% or greater sequence identity to said polynucleotide. 20 24 . the step ofsegment. The method of claim 23, further comprisingcloning at least a portion of said DNA or RN1
18. 25. The method of claim 24, wherein said clonedportion further comprises DNA flanking said segmenthaving 70% or greater sequence identity.
19. 26. A method of altering disease résistance m25 plant, said method comprising the steps of: (a) mtrcducing the nucleic acid construct ci:laim 5 mto a plant cell; and n J 4. Λ Λ »ί 7 4 «iZiiSfc' 010990 -41 - (b) prcducmg a plant containing saidpolynucleotide from said tell, wherein expression c: sai:polynucleotide alters disease résistance in said plant.
20. 27. The method of daim 26, wherein said nudeic5 acid construct further comprises an inducible regulatory eiement operably linxed to said polynucleotide and saidexpression is regulated by said regulatory eiement.
21. 28. The method of daim 27, wherein saidexpression is induced by said regulatory eiement upcn 10 exposure of said plant to an elicitor or plant patncgen.
22. 29. An isolâtes polypeptide having from about 25to about 550 amino acids, said polypeptide comprising anamino acid sequence substantially identical to Figure 3.
23. 30. The polypeptide of daim 29, wherein said15 polypeptide comprises the amino acid sequence of Figure 3 .