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WO1999038989A1 - Genes et polypeptides de resistance aux phytopathies constitutivement actifs - Google Patents

Genes et polypeptides de resistance aux phytopathies constitutivement actifs Download PDF

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Publication number
WO1999038989A1
WO1999038989A1 PCT/US1999/001970 US9901970W WO9938989A1 WO 1999038989 A1 WO1999038989 A1 WO 1999038989A1 US 9901970 W US9901970 W US 9901970W WO 9938989 A1 WO9938989 A1 WO 9938989A1
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Prior art keywords
disease resistance
plant
polypeptide
nucleic acid
plant disease
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PCT/US1999/001970
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English (en)
Inventor
Richard W. Michelmore
John P. Rathjen
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The Regents Of The University Of California
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Priority to AU24845/99A priority Critical patent/AU2484599A/en
Publication of WO1999038989A1 publication Critical patent/WO1999038989A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance

Definitions

  • This invention pertains generally to disease resistance in plants.
  • this invention pertains to a novel family of genetically engineered, constitutively active, disease resistance genes and polypeptides. When expressed in a plant cell, these polypeptides can induce a hypersensitive reaction.
  • Plants are the target of a variety of pathogens, including viruses, bacteria, fungi, nematodes and insects (Staskawicz (1995) Science 268:661-667; Baker (1997) Science 276:726-733).
  • pathogens include viruses, bacteria, fungi, nematodes and insects (Staskawicz (1995) Science 268:661-667; Baker (1997) Science 276:726-733).
  • One particularly effective means to control or limit the extent of pathogenic infection is by containment of the pathogen at the site of invasion.
  • the pathogen often enters the plant through a natural opening, e.g., stomata, or hydathodes, or wounds.
  • the pathogen multiplies in the intercellular spaces of the host plant, as in the xylem or leaf mesophyll.
  • ability of the plant to contain the pathogen to a limited area near its source is an effective means of preventing further spread of the infection.
  • HR hypersensitive reaction
  • the HR reaction is commonly referred to as a "gene for gene” disease resistance phenomenon.
  • a number of plant resistance genes have been characterized at the molecular level (see, e.g., Staskawicz (1995) supra).
  • the derived amino acid sequences of the most common class all contain leucine-rich repeats (LRR) and nucleotide binding sites (NBS). Examples included RPS2, RPM1 (bacterial resistances in Arabidopsis; Mindrinos et al. Cell 78: 1089-1099 (1994)); Bent et al.
  • NBS is a common motif in several mammalian gene families encoding signal transduction components (e.g., Ras) and is associated with ATP/GTP-binding sites.
  • LRRs are leucine rich regions often comprising 20-30 amino acid repeats where leucine and other aliphatic residues occur periodically. LRR domains can mediate protein-protein interactions and are found in a variety of proteins involved in signal transduction, cell adhesion and various other functions. LRRs can function extracellularly or intracellularly.
  • Another class of plant resistance genes contain kinase domains, as the tomato "Rto” resistance gene which confers resistance to bacterial speck disease caused by
  • Pseudomonas syringae pathovar tomato The plant Pto locus encodes a family of related serine-threonine kinases (Staskawicz (1995) supra). The Pto kinase binds to the P. syringae p. tomato avirulence (" ⁇ vr") gene product designated " ⁇ vrRt ⁇ .” Intracytoplasmic interaction of the tomato's Pto kinase with the P. syringae " ⁇ vrRto" gene product triggers plant disease resistance response, specifically, an HR response. This interaction is a proteimprotein binding event. Inactive Pto resistance alleles, unable to initiate an HR response, cannot bind to the avr gene product.
  • Resistance-conferring Pto alleles bind to avoPto gene product (Sco field (1996) Science 274:2063-2065). Thus, a specific a protein:protein binding event must occur to trigger the HR reaction in the plant.
  • Structural analysis of plant disease resistance genes have identified regions associated with binding to the pathogen ⁇ vr gene product. For example, in the tomato Pto resistance gene product ("Pto") the carboxy terminal amino acids residues 190 to 213 are necessary to bind the bacterial ⁇ vr gene product. This area corresponds to a kinase "subdomain VIII" region, which has been implicated in substrate binding in other kinases (Hanks (1995) FASEB J. 9:577).
  • This invention provides for a genus of genetically engineered plant disease resistance (“PDR”) polypeptides capable of constitutive activity and genes encoding these proteins.
  • PDR plant disease resistance
  • a variety of means to express these polypeptides are also provided, including expression cassettes, vectors, transformed cells and transgenic plants.
  • the invention further provides a novel means to control plant cell growth and plant cell death, as well as novel reagents complementing this significant achievement.
  • the invention provides for an isolated nucleic acid encoding a constitutively active PDR polypeptide, wherein the polypeptide has a leucine rich repeat, a nucleotide binding site, or a kinase domain, or any combination thereof, and, the PDR polypeptide, upon expression in a plant cell, is capable of inducing a disease resistance response, such as cell death, in the absence of a pathogen derived ligand.
  • the PDR polypeptide has a kinase domain.
  • the kinase domain can comprises at least one activation segment, which can have at least one negatively charged residue or other amino acid substitution (as compared to wild type polypeptide) that has altered the conformation of the activation segment to confer constitutive activation.
  • the negatively charged residue can be, e.g., aspartate or glutamate.
  • the activation segment can further comprise a T loop and/or a P+l loop. In one embodiment, the activation segment comprises a constitutively active plant kinase domain.
  • the isolated nucleic acid encoding a constitutively active PDR comprises a sequence with at least 50%, or at least 70%, or at least 90%, nucleic acid sequence identity to SEQ LD NO: 1 , or, can be the sequence as set forth in SEQ ED NO: 1 ,
  • SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.
  • This isolated nucleic acid can encode a protein having at least 60%, at least 80%, or at least 90%, amino acid sequence identity to the polypeptide of SEQ ID NO:2, or, can encode for a polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10.
  • the isolated nucleic acid of the invention can be from a plant cell, such as a tomato plant cell.
  • the isolated nucleic acid can specifically hybridizes to SEQ ID NO: 1 under stringent conditions, which are defined below.
  • the invention further provides for an expression cassette comprising a nucleic acid encoding a constitutively active PDR polypeptide of the invention, wherein the nucleic acid is operably linked to a promoter.
  • the expression cassette's promoter can be a plant promoter, such as a tomato promoter or a disease resistance promoter.
  • the plant promoter can be a constitutive promoter.
  • the plant promoter can be an inducible promoter, such as a pathogen-induced promoter, or a developmentally-induced (e.g., abscission-induced) promoter.
  • the plant promoter can also be a tissue-specific promoter,
  • the invention also provides for a transfected cell comprising a nucleic acid encoding a constitutively active PDR polypeptide of the invention and a non-naturally occurring nucleic acid sequence, wherein the nucleic acid is any one of the species of PDR polypeptides described above.
  • the transfected cell of the invention can further comprise the expression cassette described above, or, can be a plant cell, such as a tomato cell.
  • One embodiment of the invention provides for a transgenic plant, or progeny thereof, into which an exogenous nucleic acid sequence which specifically hybridizes under stringent conditions to the PDR polypeptide-encoding nucleic acid of the invention has been introduced.
  • the transgenic plant is a a tomato plant or a Nicotiana plant.
  • the invention provides for an isolated, constitutively active PDR polypeptide, wherein the polypeptide has a leucine rich repeat, a nucleotide binding site, or a kinase domain, or any combination thereof, and, the PDR polypeptide, upon expression in a plant cell, is capable of inducing a disease resistance response ("DRR"), which can be a rapid cell death response.
  • the kinase domain can comprises at least one activation segment, which can have at least one negatively charged residue or other amino acid substitution (as compared to wild type polypeptide) that has altered the conformation of the activation segment to confer constitutive activation.
  • the negatively charged residue can be an aspartate or a glutamate.
  • the isolated PDR polypeptide of the invention is encoded by a nucleic acid comprising a sequence with at least 50%, at least 70%, or at least 90%, sequence identity to SEQ ID NO: 1 , or, is encoded by a nucleic acid comprising SEQ ID NO: 1 , SEQ ID NO:3, SEQ ED NO:5, SEQ LD NO:7, or SEQ LD NO:9.
  • the isolated PDR polypeptide of the invention has at least 60% amino acid sequence identity to the polypeptide of SEQ ID NO:2, or, has at least 80% amino acid sequence identity to the polypeptide of SEQ ID NO:2, or, has at least 90% amino acid sequence identity to the polypeptide of SEQ ED NO:2, or, has a sequence as set forth in SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ED NO:8, or SEQ ID NO:10.
  • the isolated PDR polypeptide of the invention can be from a plant cell, such as a tomato plant cell.
  • the invention also provides for a method for screening for a compound that binds to a PDR polypeptide comprising the following steps: a) providing a composition comprising a nucleic acid encoding a PDR polypeptide of the invention, or a PDR polypeptide; b) contacting the composition with a test compound; and, c) measuring the ability of the test compound to bind the PDR polypeptide.
  • the test compound can comprise a pathogen polypeptide, a plant polypeptide, or an animal polypeptide.
  • the plant cell in which the PDR polypeptide is expressed is a tomato plant cell.
  • the invention also provides for a method for inducing a disease resistance response in a plant cell comprising the following steps: a) providing a composition comprising a PDR polypeptide of the invention or a nucleic acid sequence encoding a PDR polypeptide of the invention; and b) expressing the PDR polypeptide inside the plant cell, thereby inducing a hypersensitive reaction.
  • FIGS 1A and IB show the three dimensional structure of two kinases: cdk7 and cyclic adenosine monophosphate-dependent protein serine/threonine kinase, respectively.
  • T Loop subdomains is labeled.
  • T Loop kinase subdomain is indicated by its phosphorylated residues serines S409 and S416, shown bound to a polypeptide substrate.
  • This invention is a first description of a genus of genetically engineered plant disease resistance (PDR) genes and polypeptides capable of constitutively inducing a disease resistance response (DRR), which can be a rapid cell death reaction, mediated by a plant in response to pathogenic infection, such as the hypersensitive reaction (HR) described herein.
  • PDR plant disease resistance
  • the invention further provides a means to control plant cell growth and plant cell death, as well as novel reagents for implementing this significant achievement.
  • the PDR polypeptides of the invention can comprise either a leucine rich repeat, a nucleotide binding site, a kinase 7 domain, or a combination thereof, which, together with other unique structural characteristics give the polypeptide constitutive activity in plant cells, thus conferring upon the plant the DRR phenotype.
  • the PDR polypeptide is expressed or introduced intracellularly, it is capable of inducing a DRR reaction.
  • the genus of genes or polypeptides of the invention can be produced by any means, including, e.g., genetic engineering, wherein the polypeptides are expressed recombinantly, e.g., in vitro, in cell lines and transgenic plants. Alternatively, they can be the product of chemical synthesis in vitro, either completely, or partially.
  • the constitutively active genes and polypeptides of the invention are derived from a PDR gene initially isolated from a tomato plant, the so-called "Pto” resistance gene, or its homologs.
  • the tomato "Pto” disease resistance gene confers resistance to bacterial speck disease caused by the Pseudomonas syringae pathovar tomato.
  • the Pto gene product specifically interacts with the P. syringae p. tomato avirulence " ⁇ vr” or "avrPto” bacterial gene product. This interaction is a specific proteimprotein binding event: plant Pto resistance alleles which cannot bind to the bacterial avrPto gene product are inactive, i.e., they cannot induce DRR and confer resistance.
  • the plant Pto gene product's carboxy terminal amino acids residues 190 to 213 are necessary to bind to the bacterial avrPto gene product.
  • This area corresponds to a kinase "subdomain VIII" region found in kinases (Hanks (1995) supra).
  • Four residues in this subdomain region of Pto are essential for binding to the bacterial avrPto gene product (see detailed discussion, infra).
  • the altered Pto resistance gene product to can no longer bind the bacterial avrPto gene product, and the Pto's ability to initiate the PDR reaction is abrogated.
  • the bacterial ⁇ vr gene product must be present inside the plant cell and the Pto must bind to the ⁇ vr gene product.
  • Alteration of the confirmation of the kinase activation segment can confer constitutive DRR-inducing activity.
  • the invention provides for a novel modification of the kinase sequences in PDR polypeptides, wherein the wild-type activation segment residues are replaced with residues that result in conformational changes that confer constitutive activity, such as replacement of some residues with negatively charged residues.
  • position 207 can be replaced by an amino acid residue with a negative charge, such as an aspartate (SEQ ID NO:2) or a glutamate (Pto-glu 207 or Pto-asp 207 ).
  • a negative charge such as an aspartate (SEQ ID NO:2) or a glutamate (Pto-glu 207 or Pto-asp 207 ).
  • the invention is not limited by the mechanistics of the constitutive activity created by these mutations, and other amino acid substitutions in the putative DRR activation segment that mimic the effects of phosphorylation or ligand binding (such as binding to bacterial AvrPto) are provided for by the invention.
  • This invention provides plant pathogen resistance genes encoding polypeptides that can constitutively induce a DRR response, such as an HR response.
  • a DRR response such as an HR response.
  • the genes can be expressed in vitro or in vivo, the invention provides for a variety of means of expressing these genes, including expression cassettes, vectors, cell lines, transgenic plants, and the like.
  • the constitutively active plant resistance genes and nucleic acids of this invention may be isolated from a variety of sources and genetically engineered, expressed recombinantly, or may be synthesized in vitro.
  • Nucleic acids encoding for the plant resistance polypeptides of the invention can be expressed in transgenic plants and animals, transformed cells and cell lines, in a transformed cell lysate, or in a partially purified or a substantially pure form.
  • Nucleic acids and proteins are detected and quantified in accordance with the teachings and methods of the invention described herein by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno- fluorescent assays, and the like, Southern analysis, Northern analysis, Dot-blot analysis, gel electrophoresis, RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography, to name only a few.
  • the invention provides for a genus of PDR genes encoding polypeptides capable of constitutively inducing the DRR reaction in plants.
  • the invention provides examples of a representative number of constitutively acting PDR genes and a recitation of structural features common to the members of the genus, i.e., an activation segment in a kinase domain, such as a negatively charged amino acid in a T loop motif, or, a leucine rich repeat, or, a nucleotide binding site, or any combination of these domains.
  • conserved PDR polypeptide structural domains activation segments, kinase motifs, including T loop domains, leucine rich repeats, nucleotide binding sites
  • plant resistance mRNA, cDNA and genes of numerous organisms may be obtained or identified using primers, nucleic acid probes, and 10 antibodies to one or more of the structural domain (motif) sequences.
  • motif regions can be recognized in and by certain reagents of the invention useful to construct additional constitutively active PDR genes and polypeptides.
  • Additional plant resistance genes can also be identified and characterized using various methods, including: i) computer searches of DNA databases for DNAs containing sequences conserved with PDR genes and having sequence identity with conserved plant resistance polypeptide structural domains (motifs) described above, ii) hybridization with a probe from a known PDR gene sequence to mRNA, cDNA or DNA sequence or libraries from a target plant, and, iii) by PCR or other signal or target amplification technologies using primers complementary to regions highly conserved among different plant resistance genes or their structurally similar domains, such as the kinase, leucine-rich or nucleotide binding motifs.
  • PDR genes can be modified as described herein to be a constitutively active plant disease resistant species of the invention.
  • Nucleic acid amplification methods such as PCR are illustrated as an exemplary means used to identify, isolate and generate members of the plant resistance gene genus of the invention.
  • Amino acid sequences can be conserved, but, because of the degeneracy of the genetic code, codon usage bias, or amino acid changes, the DNA sequences corresponding to conserved plant resistance polypeptide structural domain (motif) regions can be different between organisms. For this reason, one can employ in the methods nucleotides at the positions in the primers that are degenerate for a particular amino acid to ensure that one or more of the different primers can hybridize to a plant resistance specie whose nucleotide sequence is not completely known.
  • the present invention further provides illustrative and representative members of the genus of constitutively active PDR species of the instant invention, including the exemplary tomato Pto gene and gene product, SEQ ID NO:l and SEQ ID NO:2, respectively.
  • the invention also provides methods and reagents for isolating, characterizing and expressing this and additional members of the genus. While methods for isolating total DNA or RNA are well known to those of skill in the art, as for example in Ausubel, Tijssen and Sambrook, provided below are illustrative example of methods for identifying, characterizing and isolating the nucleic acids of the PDR genus of the invention.
  • PDR polypeptide-encoding DNA can be identified by stringent hybridization and isolated from a genomic or cDNA library using oligonucleotide probes, typically labeled, having sequences complementary to PDR sequences or subsequences, such the conserved structural domain motifs, as disclosed herein.
  • the tomato PDR gene encoded by the cDNA of SEQ ID NO:l can be used to construct such probes or primers.
  • probes can be used directly in hybridization assays to isolate DNA encoding plant disease resistant species.
  • probes can be designed for use in a variety of amplification techniques, such as PCR, as discussed herein.
  • the invention provides compositions and methods to screen both genomic and cDNA libraries for PDR sequences. Screening cDNA libraries for coding sequences has certain advantages in that no intronic sequences are present. Screening genomic libraries can have an advantage in that upstream and downstream c/s-acting transcriptional regulatory elements (e.g., promoters and enhancers) can be identified and isolated, as well as introns, which may be beneficial to include in some expression cassettes and vectors. Furthermore, in some species, the intronic or untranscribed PDR sequences may be the most conserved.
  • the invention provides for PDR genomic nucleic acid, including introns, protein-encoding exons. and transcribed and non-transcribed genomic sequences as additional reagents and means to identify and screen for PDR species.
  • these newly identified plant disease resistant species can be modified using the novel reagents of the 12 invention and according to the teachings set forth herein to be constitutively active plant disease resistant polypeptide species of the invention.
  • mRNA is isolated and reverse transcribed from the mRNA and inserted into vectors in accordance with general procedures well known in the art.
  • the vectors are transfected into a host for propagation, screening and other applications.
  • DNA of appropriate size can be produced by known methods, such as mechanical shearing or enzymatic digestion, to yield DNA fragments, e.g., of about 12 to 20 kb. The fragments are then separated, as for example, by gradient centrifugation, or gel electrophoresis, from undesired sizes. Selected fragments can be inserted in bacteriophage or other vectors. These vectors and phage can be packaged in vitro, (see, e.g., Ausubel, Sambrook). Recombinant phage can be analyzed by plaque hybridization described, e.g., in Benton (1977) Science 196:180; Chen (1997) Methods Mol. Biol.
  • Colony hybridization can be carried out as generally described in the scientific literature, e.g., as in Grunstein (1975) Proc. Natl. Acad. Sci. USA 72:3961-3965;Yoshioka (1997) J. Immunol Methods 201:145-155; Palkova (1996) Biotechniques 21 :982.
  • DNA encoding an plant resistance gene specie such as a variant, an allele, an isoform of SEQ ID NO:l
  • DNA encoding an plant resistance gene specie can be identified in either cDNA or genomic libraries by hybridization with nucleic acid probes of the invention, for example, probe containing 10 to 20 to 50 or more contiguous nucleotides of SEQ ID NO:l, on Southern blots. Once identified, these DNA regions are isolated by standard methods familiar to those of skill in the art.
  • RNA encoding plant resistance, DRR-inducing polypeptides may be identified by hybridization to nucleic acid probes in Northern blots or other formats; e.g. , see Sambrook, Ausubel, Tijssen for general procedures relating to such formats.
  • Oligonucleotides for use in expression systems or as probes can be chemically synthesized, as described below.
  • Synthetic nucleic acids including oligonucleotide probes and primers, PDR, DRR-inducing polypeptide encoding sequences, antisense.
  • ribozymes and the like can be prepared by a variety of solution or solid phase methods. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, 13 phosphotriester, and H-phosphonate chemistries are widely available. For example, the solid phase phosphoramidite triester method of Beaucage and Carruthers using an automated synthesizer is described in Itakura, U.S. Pat. No.
  • oligonucleotides 14:5399-5407 (1986); Sinha, Tetrahedron Lett. 24:5843-5846 (1983); and Sinha (1984) Nucl. Acids Res. 12:4539-4557.
  • Methods to purify oligonucleotides include, e.g., native acrylamide gel electrophoresis, anion-exchange HPLC, as described in, e.g., Pearson (1983) J. Chrom.
  • the present invention provides oligonucleotide primers and probes that can hybridize specifically to and amplify nucleic acids having protein-encoding (cDNA) or genomic nucleic acid, such as the sequence of SEQ ID NO:l, encoding the polypeptide of SEQ ID NO:2.
  • cDNA protein-encoding
  • genomic nucleic acid such as the sequence of SEQ ID NO:l, encoding the polypeptide of SEQ ID NO:2.
  • Such reagents can be used to identify the invention's genus of plant resistance, DRR-inducing protein-encoding and genomic sequences. Included in the invention's genomic sequences are intronic and genomic, non-transcribed sequences, promoters, and enhancers which can also be amplified using the PCR primers of the invention to identify new species. Illustrative amplification methods are described below.
  • oligonucleotides that are preferred reagents for such amplifications. These reagents are also used as hybridization probes to identify and isolate additional PDR, DRR-inducing species from other plants. These oligonucleotides can also be used as primers to directly amplify additional such species, using any amplification technique, such as, for example RACE, as described below.
  • RACE reverse transcriptase
  • Oligonucleotides can be used to identify and detect additional PDR, DRR- inducing species using a variety of hybridization techniques and conditions.
  • amplification method include, but are not limited to: polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.
  • RNA polymerase mediated techniques e.g., NASBA, Cangene, Mississauga, Ontario
  • Methods for cloning in vitro amplified nucleic acids are described in Wallace, U.S. Pat. No. 5,426,039.
  • the invention provides for amplification, manipulation and detection of the products from each of the above methods to prepare and modify DNA encoding PDR, DRR- inducing polypeptides.
  • oligonucleotide primers complementary to the two borders of the DNA region to be amplified are synthesized and used (see, e.g, Innis).
  • PCR can be used in a variety of protocols to amplify, identify, isolate and manipulate nucleic acids 15 encoding PDR, DRR-inducing polypeptides.
  • appropriate primers and probes for identifying and amplifying such DNA are generated that comprise all or a portion of any of the DNA sequences listed herein, such as SEQ ID NO:l.
  • PCR-amplified sequences can also be labeled and used as detectable oligonucleotide probes, but such nucleic acid probes can be generated using any synthetic or other technique well known in the art, as described herein.
  • the labeled amplified DNA or other oligonucleotide or nucleic acid of the invention can be used as probes to further identify and isolate PDR species from various cDNA or genomic libraries.
  • the present invention provides RACE-based methods for isolating nucleic acids from any organism (RACE is another PCR-based approach for DNA amplification).
  • this technique involves using PCR to amplify a DNA sequence using a random 5' primer and a defined 3' primer (5' RACE) or a random 3' primer and a defined 5' primer (3' RACE).
  • the amplified sequence is then subcloned into a vector where can be sequenced and manipulated using standard techniques.
  • the RACE method is well known to those of skill in the art and kits to perform RACE are commercially available, e.g. Gibco BRL,
  • the genus of PDR nucleic acid sequences of the invention includes genes and gene products identified and characterized by analysis using the sequences nucleic acid and protein sequences of the invention, including SEQ ED NO:l and SEQ ID NO:2, respectively.
  • Optimal alignment of sequences for comparison can use any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method of termed "PELEUP" (see below); by the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2: 482; by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol.
  • BLAST algorithm Another example of algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul (1990) J. Mol. Biol. 215: 403-410.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/ ; see also Zhang (1997) Genome Res. 7:649-656 (1997) for the "PowerBLAST" variation.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra.).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad. Sci.
  • BLAST refers to the BLAST algorithm which performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787.
  • P(N) the smallest sum probability
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat l Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • PDR-encoding sequences can be sequenced as inserts in vectors, as inserts released and isolated from the vectors or in any of a variety of other forms (i.e., as amplification products). PDR-encoding inserts can be released from the vectors by restriction enzymes or amplified by PCR or transcribed by a polymerase. For sequencing of the inserts to identify full length coding sequences, primers based on the N- or C- terminus, or based on insertion points in the original phage or other vector, can be used.
  • Additional primers can be synthesized to provide overlapping sequences.
  • a variety of nucleic acid sequencing techniques are well known and described in the scientific and patent literature, see, e.g. Rosenthal (1987) supra; Arlinghaus (1997) Anal. Chem. 69:3747-3753; Dubiley (1997) Nucleic Acids Res. 25:2259-2265; for use of biosensor chips for identification and sequencing of nucleic acids.
  • nucleic Acid Hybridization Techniques can be utilized to identify, isolate and characterize genes and gene products (i.e., mRNA) encoding for the genus of PDR sequences of the invention.
  • a variety of methods for specific DNA and RNA detection and measurement using nucleic acid hybridization techniques are known to those of skill in the art. See. e.g., NUCLEIC ACID HYBRIDIZATION, A PRAC ⁇ CAL APPROACH, Ed. Hames et al, IRL Press, 1985; Gall (1989) Proc. Natl. Acad. Sci. USA 63:378; and Sambrook.
  • the selection of a DNA hybridization format is often optional.
  • nucleic acid sample such as digested DNA or mRNA
  • the nucleic acid probes may comprise nucleic acid sequences encoding structurally conserved regions amongst the genus, such as kinase domains (e.g., activation segments, T Loop motifs), leucine-rich regions, nucleic acid binding site domains. See, e.g., Sambrook for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization. Both quantitative and qualitative determination of the presence or absence of DNA or RNA encoding PDR genes can be performed in accordance with the present methods.
  • a Northern transfer can be used for the detection of mRNA encoding PDR polypeptides.
  • probes such as labeled probes or PCR amplification products can be used to identify the presence or absence of PDR, DRR-inducing, polypeptide-encoding nucleic acid.
  • Another means for determining the level of expression of a gene encoding a protein is in situ hybridization. In situ hybridization assays are well known and are generally described in
  • the invention provides for a genus of genetically engineered PDR polypeptides derived from wild-type PDR polypeptides that have been modified to confer a gain of function phenotype comprising a constitutively active DRR, such as an HR.
  • the new conformation is effected by recombinant engineering by mutagenesis, as site-specific mutagenesis, resulting in amino acid substitutions, additions, or deletions. Exemplary site-specific mutations resulting in PDR polypeptides capable of conferring a constitutive DRR are described below.
  • a correctly folded, complete protein and its mutagenized encoding mRNA both remain attached to a ribosome and can be assessed for alterations in ligand-binding properties of the native protein.
  • Libraries of native folded proteins with engineered site-specific mutations can now be screened while "evolving" in a cell-free system without the transformation or other constraints imposed when using a host cell (Hanes ( 1997) Proc. Natl. Acad. Sci. USA 94:4937-4942).
  • Modified PDR polypeptides of the invention can also be produced by site-directed mutagenesis and/or chemical modification methods to introduce unnatural amino acid side chains (see Paetzel (1997) J. Biol. Chem. 272:9994-10003 for general methodology) .
  • the invention provides for a genus of genetically engineered, constitutively active PDR polypeptides derived from (constructed from) wild-type PDR polypeptides that have been modified, e.g., recombinantly engineered by mutagenesis, additions, or deletions, as described herein. These genetic manipulations generate polypeptides which constitutively induce a DRR reaction through the modification of kinase domains, leucine rich repeat domains and or a nucleotide binding site domains.
  • Such constitutively active PDR polypeptides can be employed in the screens of the invention to discover new binding partners, or alternatively, as antagonists of the constitutive activity.
  • a PDR polypeptide comprising a nucleotide binding site can further engineered to lose its ability to bind substrate nucleotide, such as small nucleotide 20 phosphates.
  • a PDR polypeptide comprising a leucine-rich region can be further engineered to lose its ability to associate with another binding partner.
  • the resulting "mutant proteins” or “muteins” can be used to identify compounds that specifically modulate one, several, or all functions or activities of the constitutively active PDR polypeptides of the invention.
  • Mutations can be introduced into a nucleic acid by a variety of conventional techniques, well described in the scientific and patent literature. For example, one rapid method to perform site-directed mutagenesis efficiently is the overlap extension polymerase chain reaction (OE-PCR) (Urban (1997) Nucleic Acids Res. 25:2227-2228).
  • OE-PCR overlap extension polymerase chain reaction
  • a correctly folded, complete protein and its mutagenized encoding mRNA both remain attached to a ribosome and can be assessed for alterations in ligand-binding properties of the native protein. Libraries of native folded proteins with engineered site-specific mutations can now be screened while "evolving" in a cell-free system without the transformation or other constraints imposed when using a host cell (Hanes (1997) Proc. Natl. Acad.
  • Modified proteins of the invention can also be produced by site-directed mutagenesis and/or chemical modification methods to introduce unnatural amino acid side chains (see Paetzel (1997) J. Biol. Chem. 272:9994-10003 for general methodology).
  • site-directed mutagenesis and/or chemical modification methods to introduce unnatural amino acid side chains (see Paetzel (1997) J. Biol. Chem. 272:9994-10003 for general methodology).
  • biotin-containing amino acid biocytin see Gallivan (1997) Chem. Biol. 4:739-749
  • site-specific incorporation of unnatural amino acids into proteins in vivo see Liu (1997) Proc. Natl. Acad. Sci. USA 94:10092-10097; see also, e.g., Koh (1997) Biochemistry 36:11314-11322. Characterization of Kinase Domains
  • the invention provides for nucleic acids and polypeptides encoding genetically engineered, constitutively active PDR polypeptides, wherein the polypeptide has a kinase domain, or a combination of a kinase domain and a leucine rich repeat and/or a nucleotide binding site, which, upon expression in a plant cell, is capable of inducing a DRR.
  • the mutated PDR polypeptide has a kinase domain
  • that domain comprises at least one activation segment, such as a T loop with at least one negatively charged residue, or other substitution capable of conferring constitutive PDR activity.
  • the negatively charged residue can be aspartate or glutamate.
  • Protein kinases make up a large superfamily of homologous proteins.
  • the kinase domain consists of about 250 to 300 amino acid residues that fold into 21 a common catalytic core.
  • tyrosine kinases There are two main subdivisions within this superfamily: tyrosine kinases and serine/threonine kinases. All of these enzymes use a gamma-phosphate of ATP or GTP to generate phosphate monoesters using either protein alcohol groups (for serine and threonine) or protein phenolic groups (as on tyrosine) as phosphate acceptors.
  • conserveed features of kinase domain primary structure have been identified by aligning and comparing large numbers of known kinase sequences. For a general review, see, e.g., Hanks (1995) FASEB J. 9:576-596.
  • kinases comprise a structurally homologous kinase domain, which itself can be further characterized as comprising twelve kinase subdomains. These subdomains are recognized as being invariant or relatively invariant throughout the superfamily, (see Hanks (1995) supra, illustrating alignments of 60 kinase domains representative of members of the eukaryotic protein kinase superfamily), the subdomains designated as Roman numerals I, II, IEI, EV, V, VIA, VLB, VII, VIA, IX, X, and XI. The homologous nature of the kinase domains indicates that they all fold into similar three dimensional core structures and impart phosphotransfer according to a common mechanism.
  • the smaller, amino terminal lobe includes subdomains I to IV, and is primarily involved in anchoring and orienting the nucleotide. This lobe is predominantly an anti-parallel beta sheet that is unique amongst nucleotide binding proteins.
  • the larger carboxy terminal lobe includes subdomains VIA to XI, and is largely responsible for binding the peptide substrate and initiating phosphotransfer. This lobe is predominantly alpha helical in structure.
  • Subdomain V spans the two lobes. A deep cleft between the lobes is the site of catalysis.
  • Kinase subdomain I at the amino terminus, contains the consensus sequence: Gly-x-Gly-x-x-Gly-x-Val Subdomain residues fold into a beta-strand-tum-beta strand structure, acting as a flexible flap or clamp that covers and anchors the non-transferable phosphates of ATP. 22
  • Kinase subdomain II contains an invariant lysine (lys72 in PKA-C alpha), which has long been recognized as being essential for maximal activity. This lys lies within the beta strand 3 of the small lobe to help anchor and orient ATP by interacting with the alpha and beta phosphates. This lysine also forms a salt bridge with the carboxyl group of the nearly invariant glutamic acid (glu91 in PKA-C alpha) in subdomain III.
  • Kinase subdomain III contains a large alpha helix in the small lobe. It has a nearly invariant glu residue centrally located in its helix and helps stabilize the interaction between the invariant lysine of domain II and the alpha and beta phosphates of ATP.
  • Kinase subdomain IV corresponds to the hydrophobic beta strand 4 in the small lobe. This domain contains nearly no invariant residues and may not be directly involved in catalysis or substrate recognition.
  • Kinase subdomain V links the small and large lobes of the catalytic subunit and consists of the very hydrophobic beta strand 5 in the small lobe, the small alpha helix D in the large lobe and an extended, connecting chain that connects them.
  • glul21, vail 23 and glul 27 in the connecting chain help anchor ATP by forming hydrogen bonds with either the adenine or the ribose ring; metl20, tyrl22 and vall23 (PKA-C alpha) contribute to the hydrophobic pocket surrounding the adenine ring of ATP.
  • Kinase subdomain VIA folds into the large hydrophobic alpha-helix E that extends through the large lobe. This part of the kinase molecule appears to act mainly as support structure.
  • Kinase subdomain VLB folds into the small hydrophobic beta-strands 6 and 7 with an intervening loop.
  • the loop has two invariant residues, aspartic acid and asparagine (asp 166 and asn 171 in PKA-C alpha) that lie within the consensus kinase motif:
  • the loop has been termed the "catalytic loop" because Asp 166 may be a catalytic base.
  • Kinase subdomain VII folds into a beta-strand-loop-beta-strand structure, a highly conserved Asp-Phe-Gly triplet (Aspl84-Phel85-Glyl86 of PKA-C alpha) lies in the loop. It is stabilized by a hydrogen bond between the Asp and the Gly. The Asp chelates the primary activating magnesium ions that bridge the beta and gamma phosphates of the ATP.
  • Kinase subdomain VIFI folds into a chain that faces the cleft between the kinase lobes. It includes the highly conserved Ala-Pro-Glu motif (Ala206-Pro207-Glu208 of PKA- 23
  • the nearly invariant Glu forms an ion pair with an invariant Arg in subdomain XI (Arg280 of PKA-C alpha) helping to stabilize the large lobe.
  • This domain plays a major role in recognizing peptide substrates. Many protein kinases are known to be activated by phosphorylation of this domain, in particular, at a Thr residue (corresponding to Thrl97 of PKA-C alpha), probably through an intramolecular autophosphorylation mechanism.
  • subdomain VIII This portion of subdomain VIII is also called the "activation segment,” or “activation loop,” or, “T Loop,” domain, as discussed in further detail below (see also, Johnson (1996) Cell 85:149- 158); Martinez (1997) E ROJ 16:343-354; Jeffrey (1995) Nature 376:313-320).
  • activation segment or “activation loop” or, “T Loop” domain
  • phosphorylation of both a Thr and a Tyr is required for activation.
  • the peptide binding cleft is sterically blocked.
  • Subdomain VIII also plays a major role in recognition of peptide substrates.
  • Residues important in this peptide binding include residues corresponding to Leul98, Cysl99, Pro202, Leu205, of PKA-C alpha, which provide a hydrophobic pocket that accommodates the side chain of a hydrophobic residue in the peptide substrate.
  • Kinase subdomain IX corresponds to the large alpha helix F of the large lobe, a nearly invariant Asp (corresponding to Asp220 of PKA-C alpha) lies in the amino terminal region of the helix and acts to stabilize the catalytic loop by hydrogen binding to the backbone amides that precede the loop.
  • Kinase subdomain X is the most poorly conserved domain. Its function is unclear. In PKA-C alpha it corresponds to a small alpha helix G that occupies the base of the large lobe. Large inserts have been found between domains X and XI.
  • Kinase subdomain XI extends to the carboxy terminus of the kinase domain, a nearly invariant Arg (corresponding to Arg280 of PKA-C alpha) lies between alpha helices H and I.
  • the consensus motif His-X- Aromatic residue-Hydrophobic residue is found downstream of the invariant Arg.
  • a hydrophobic amino acid residue lies ten residues downstream of the invariant Arg.
  • the constitutively active polypeptides of the invention can also comprise all plant kinase domains, including those that cannot be categorized by the kinase superfamily and domains described above.
  • the polypeptides of the invention can also comprise a kinase domain from the plant calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural 24 visinin-like domain (Paul (1995) Proc. Natl. Acad. Sci. USA 92:4797-4801; Takezawa (1996) J. Biol. Chem. 271 :8126-8132; Ramachandiran (1997) J. Biochem.
  • CaMK plant calcium-binding calcium/calmodulin-dependent protein kinase
  • polypeptides of the invention can also comprise, e.g., kinase domains from a family of calcium-responsive protein kinases abundant in plant cell extracts (but not identified in animals and fungi), as described by Hrabak (1996) Plant Mol Biol. 31:405-412; domains from calcium-dependent protein kinases (CDPKs) as described by Lindzen (1995) Plant Mol. Biol. 28:785-797.
  • CDPKs calcium-dependent protein kinases
  • the invention also provides for constitutively active plant resistance genes comprising "activation segment” domains, also referred to as “activation loops.” They are subportions of kinase subdomains VII and/or VIII, or homologs thereof (see above discussion), and can include “T loops” and/or “P+l loops.”
  • the invention provides for PDR polypeptides, and their encoding nucleic acids, comprising activation segments which induce a DRR and are constitutively active.
  • the constitutive activity is a "gain of function" phenotype effected by genetic engineering of the nucleotide sequence encoding a PDR polypeptide to alter the primary structure (sequence) or secondary or tertiary structure (two and three-dimensional conformation) of the activation domain of the PDR polypeptide to confer a constitutive DRR.
  • activation segment conformation is altered by at least one amino acid residue modification, including the substitution of a tyrosine by a negatively charged amino acid.
  • Exemplary amino acid modifications which confer constitutive PDR activity include: a tyr residue to an asp residue, as in SEQ ID NO:2 , encoded by SEQ ID NO:l (e.g., Pto-tyr 207 , numbering based on SEQ ID NO:2, is replaced by Pto-asp 207 ); a tyr residue to a glu residue (Pto-tyr 207 replaced by Pto- glu 207 ); a thr residue to an asp residue, as in the polypeptide encoded by the sequence set forth in SEQ ID NO:3, encoded by the nucleic acid of SEQ ID NO:4 (e.g., Pto-thr 204 is replaced by Pto-asp 204 ); a tyr residue to an ala residue, as in the polypeptide, encoded by the sequence set forth in SEQ ID NO:5, encoded by the nucleic acid of SEQ ID NO:6 (e.g., Pto
  • Another embodiment provides for a constitutively active PDR as set forth by the sequence of SEQ ID NO: 10, encoded by the nucleic acid of SEQ ID NO:9.
  • This PDR polypeptide of the invention is a modified fen polypeptide in which the wild-type tyr residue has been replaced by an asp residue, i.e., fen-tyr 205 (numbering based on SEQ ID NO: 10) is replaced by fen-asp 205 or fen-glu 205 .
  • the Fen gene is a homolog of Pto (SEQ ID NO:2) that is 80% identical at the amino acid sequence level. Fen activates a DRR, specifically, an HR, in response to application of an organophosphate insecticide, fenthion (see Martin (1994) Plant Cell 6:1543-1552; Salmeron (1994) Plant Cell 6:511-520).
  • the subdomain VIII "T loop” is an exemplary activation segment, plays a major role in substrate recognition.
  • T loop must be phosphorylated for cdk7 to bind cyclin (Martinez (1997) supra).
  • Many protein kinases are known to be activated by phosphorylation of the T Loop subdomain.
  • one or more Thr residues are phosphorylated.
  • TGF-beta type II transforming growth factor-beta receptor ser/thr kinase TbetaRII kinase
  • TGF-beta type II transforming growth factor-beta receptor ser/thr kinase TbetaRII kinase
  • P+l loop refers to a subportion of kinase subdomain VII.
  • the P+l loop can be an element of an activation segment.
  • a P+l loop is a highly recognizable substructure of the activation segment (see, e.g., Taylor (1995) FASEB J. 9:1255-1266).
  • P+l loops, as T loops can be readily identified through sequence homology with other kinases or by analysis of the three dimensional structure of a kinase. T loops and P+l loop were initially defined on the basis by analysis of the three dimensional (3D) structures of kinase polypeptides.
  • the P+l loop can be recognized by different consensus sequences in serine/threonine (S/T) and tyrosine kinases.
  • S/T kinases the consensus is G(S/T)xx(F/Y)xAPE, where x is any amino acid.
  • tyrosine kinases it is xPxxWxAPE.
  • the T loop controls kinase activation and the P+l loop controls peptide substrate binding.
  • Activation segments such as T loop kinase subdomains, are well characterized in known kinases.
  • T loops are found in the type II transforming growth 26 factor-beta (TGF-beta) receptor ser/thr kinase TbetaRII (Luo ( 1997) supra).
  • Cyclin-dependent kinases are another example of T- Loop subdomain- containing kinases, see, e.g., Labbe (1994) EMBOJ 13:5155-5164; Poon (1994) J. Cell. Sci. 107:2789-2799; Diehl (1997) Mol. Cell. Biol. 17:7362-7374; Russo (1996) Nat. Struct. Biol. 3:696-700; Fesquet (1997) Oncogene 15 : 1303- 1307.
  • cyclin dependent kinase formation of active cyclin dependent kinase (cdk)/cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic-subunit by CAK (Cdk Activating Kinase) (Fesquet (1997) supra).
  • Xenopus cdk7 is phosphorylated in vivo on two residues of the T loop (Martinez (1997) supra; Labbe (1994) supra; Poon (1994) supra).
  • Activation segments, as T loop subdomains can be readily identified through sequence homology with other kinases or by analysis of the three dimensional structure of a kinase.
  • FIGS 1 A and IB clearly identify the T loop subdomain in the three dimensional structure of two kinases: cdk7 (Martinez (1997) supra) and cyclic adenosine monophosphate-dependent protein serine/threonine kinase (Luo (1997) supra; Knighton (1991) Science 253:414-420), respectively.
  • the T Loop subdomains is labeled.
  • the T Loop kinase subdomain is indicated by its phosphorylated residues serines S409 and S416, shown bound to a polypeptide substrate.
  • Exemplary T loops which can be incorporated in the instant invention or used to find additional activation segments by homology can be found in (GenBank Accession No.
  • the invention provides for nucleic acids and polypeptides encoding a genetically engineered, constitutively active PDR polypeptide, wherein the polypeptide has a leucine rich repeat ("LRR"), or a combination of a leucine rich repeat and a kinase domain and/or a nucleotide binding site, which, upon expression in a plant cell, is capable of inducing a DRR, which can be a rapid cell death reaction.
  • LRR leucine rich repeat
  • a leucine rich region is a region of a polypeptide that is rich in the amino acid leucine and chemically related aliphatic amino acids, such as isoleucine, valine, and alanine.
  • the sequence usually, but not always, has a periodicity of 15 to 30 amino acids, with part of the repeated sequence having the approximate consensus "axxaxa,” where "a” is an aliphatic residue and "x" is any amino acid.
  • the constitutively active polypeptides of the invention can comprise all leucine rich domains found in plants, and especially those characterized in plant resistance genes.
  • Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica encodes a polypeptide with a putative nucleotide binding site and leucine-rich repeats, as described by Parker (1997) Plant Cell. 9:879-894.
  • the invention also includes, e.g., the receptor-like protein kinase gene (RPK1) isolated from Arabidopsis thaliana encoding for a polypeptide comprising a domain with five extracellular leucine-rich repeat sequences and a membrane-spanning domain; and a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases, as describe above.
  • This protein kinase gene is rapidly induced by abscisic acid, dehydration, high salt, and cold (Hong (1997) Plant Physiol. 113:1203-1212).
  • the leucine-rich domain of the invention can also comprise a member of the basic region-leucine zipper (bZEP) class of DNA-binding proteins.
  • the maize (Zea mays L.) endosperm specific transcription factor encoded by the Opaque-2(O2) locus, is a DNA-binding protein comprising a leucine zipper and contains a number of regions rich in either pro line or acidic residues, which are candidates for activation domains (Schmitz (1997) Nucleic Acids Res. 25:756-763).
  • leucine-rich domains can be found in PDR genes, such as the sorghum mRNA that accumulates rapidly in mesocotyls and juvenile leaves infected with the fungus Colletotricum graminicola. This message encodes a putative protein 28 containing six imperfect leucine-rich repeats, of approximately 22 amino acids in length, with significant homology to some known PDR genes (Hipskind (1996) Mol. Plant Microbe Interact. 9:819-825).
  • Another example is a tomato gene whose expression is under developmental regulation and exhibits tissue-specificity, particularly in certain cell types of the stele, like phloem fibers, parenchyma cells of the protoxylem, and in the cell files that constitute the rays of the secondary xylem.
  • the gene is upregulated in diseased tomato plants infected with citrus exocortis viroid.
  • the polypeptide contains four tandem repeats of a canonical 24 amino acid leucine-rich repeat (LRR) sequence present in different proteins that mediates molecular recognition and or interaction processes, as described by Tornero (1996) Plant J. 10:315-330.
  • LRR leucine-rich repeat
  • Another leucine rich domain example is the tomato resistance locus Cf-2 encoding a polypeptide which confers resistance to tomato leaf mould C.fulvum by binding to the pathogen-encoded avirulence gene (Avr) product.
  • Two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat motifs (Dixon (1996) Cell 84:451-459).
  • the tomato Cf-9 gene product which confers resistance to infection by the fungus Cladosporium fulvum that carry the avirulence gene Avr9, is also homologous to members of the leucine-rich repeat family of proteins (Jones (1994) Science 266:789-793).
  • the A is also homologous to members of the leucine-rich repeat family of proteins.
  • thaliana RPS2 gene which confers resistance to the bacterial pathogen P. syringae carrying the avirulence gene avrRpt2, encodes a novel 105 kDa protein containing a leucine zipper, a nucleotide-binding site, and 14 imperfect leucine-rich repeats (Mindrinos (1994) Cell 78:1089-1099).
  • Polypeptides of the invention can also comprise leucine rich domains from the flax L6 rust resistance gene, which encodes two products of 1294 and 705 amino acids that result from alternatively spliced transcripts.
  • the longer product is similar to the products of two other PDR genes, the tobacco mosaic virus resistance gene N of tobacco and the bacterial resistance gene RPS2 of Arabidopsis.
  • the similarity involves the presence of a nucleotide (ATP/GTP) binding site and several other amino acid motifs of unknown function in the N-terminal half of the polypeptides and a leucine-rich region in the C-terminal half (Lawrence (1995) Plant Cell 7:1195-1206).
  • a further example includes the rice Xa21 gene, which confers resistance to Xanthomonas oryzaepv.
  • oryzae race 6 encodes a protein with both a leucine-rich repeat motif and a serine-threonine kinase-like domain (Song (1995) Science 270:1804-1806).
  • wheat Cre3 gene confers a high level of resistance to the root 29 endoparasitic nematode Heterodera avenae, and encodes a polypeptide with a nucleotide binding site (NBS) and a leucine-rich region; this member of the PDR gene family also displays tissue-specificity, being expressed only in roots (Lagudah (1997) Genome. 40:659-665). Characterization of Nucleotide Binding Site Domains
  • the invention also provides for nucleic acids and polypeptides encoding constitutively active PDR polypeptides, wherein the polypeptides have a nucleotide binding site, or a combination of a nucleotide binding site, a leucine-rich repeat and/or a kinase domain, which, upon expression in a plant cell, is capable of inducing a PDR response, such as a hypersensitive reaction.
  • a nucleotide binding site is a region of a polypeptide that has homology to sequences in the G protein superfamily that are responsible for binding and hydrolyzing small nucleotide phosphates.
  • the kinase domains in the NBS are not related to kinase domains of protein kinases like Pto.
  • NBS domains of PDR genes typically include three subdomain motifs: the P-loop, kinase-2, and kinase-3a subdomains, amongst others; see, e.g., Yu (1996) Proc. Natl.
  • the constitutively active polypeptides of the invention can comprise all nucleotide (e.g., ATP/GTP) binding domains found in plants, especially those found in PDR polypeptides.
  • the Arabidopsis RPP5 gene specifying resistance to Peronospora parasitica encodes a polypeptide with a putative nucleotide binding site (Parker (1997) supra); the A. thaliana RPS2 gene, which confers resistance to the bacterial pathogen P.
  • syringae carrying the avirulence gene avrRpt2 encodes a novel 105 kDa protein containing a nucleotide-binding site (Mindrinos (1997) supra); the flax L6 rust resistance gene encodes a polypeptide with a nucleotide (ATP/GTP) binding site (Lawrence (1995) supra); the wheat PDR Cre3 gene which encodes a polypeptide with a nucleotide binding site (NBS) and a leucine-rich region (Lagudah (1997) supra).
  • the invention provides for methods and reagents the expression of the novel constitutively active PDR nucleic acids of the invention in any prokaryotic. eukaryotic, yeast, 30 fungal, plant, insect, or animal cell.
  • the constitutively active PDR-expressing nucleic acids of the invention may be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature. See, for example Roberts (1987) Nature 328:731; Berger (1987) supra; Schneider (1995) Protein Expr. Purif. 6435:10; Sambrook and Ausubel.
  • the invention providing methods and reagents for making the novel genus of constitutively active PDR nucleic acids described herein, further provides methods and reagents for expressing these nucleic acids using novel expression cassettes, vectors, transgenic plants and animals, using constitutive and inducible transcriptional and translational cis- (e.g., promoters and enhancers) and tr ⁇ ns-acting control elements.
  • constitutive and inducible transcriptional and translational cis- e.g., promoters and enhancers
  • tr ⁇ ns-acting control elements e.g., promoter ⁇ ns-acting control elements.
  • the expression of natural, recombinant or synthetic PDR polypep tide-encoding or other (i.e., antisense, ribozyme) nucleic acids can be achieved by operably linking the coding region a promoter (that can be plant-specific or not, constitutive or inducible), incorporating the construct into an expression cassette (such as an expression vector), and introducing the resultant construct into an in vitro reaction system or a suitable host cell or organism. Synthetic procedures may also be used.
  • Typical expression systems contain, in addition to coding or antisense sequence, transcription and translation terminators, polyadenylafion sequences, transcription and translation initiation sequences, and promoters useful for transcribing DNA into RNA.
  • the expression systems optionally at least one 31 independent terminator sequence, sequences permitting replication of the cassette in vivo, e.g., plants, eukaryotes, or prokaryotes, or a combination thereof, (e.g., shuttle vectors) and selection markers for the selected expression system, e.g., plant, prokaryotic or eukaryotic systems.
  • a polyadenylafion region at the 3'-end of the coding region can be included (see Li (1997) Plant
  • the polyadenylafion region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA (e.g., using Agrobacterium tumefaciens T-DNA replacement vectors, see e.g., Thykjaer (1997) Plant Mol Biol. 35:523-530; using a plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences; Hansen (1997) "T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes," Proc. Natl Acad. Sci. USA 94:11726-11730.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses which are transiently expressed in cells using, for example, episomal expression systems (e.g., cauliflower mosaic virus (CaMV) viral RNA is generated in the nucleus by transcription of an episomal minichromosome containing supercoiled DNA, Covey (1990) Proc. Natl. Acad. Sci. USA 87: 1633-1637).
  • episomal expression systems e.g., cauliflower mosaic virus (CaMV) viral RNA is generated in the nucleus by transcription of an episomal minichromosome containing supercoiled DNA, Covey (1990) Proc. Natl. Acad. Sci. USA 87: 1633-1637.
  • Expression vectors capable of expressing proteins in plants are well known in the art, and can include, e.g., vectors from Agrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBOJ.
  • Suppressor-mutator (Spm) transposable element see, e.g., Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.
  • coding sequences can be inserted into the host cell genome becoming an integral part of the host chromosomal DNA.
  • Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences coding for episomal maintenance and replication such that integration into the host genome is not required.
  • the marker may encode antibiotic resistance, particularly resistance to chloramphemcol, kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta, to permit selection of those cells transformed with the desired DNA sequences, see for example, Blondelet-Rouault (1997) Gene 190:315-317; Aubrecht (1997) J. Pharmacol. Exp. Ther. 281 :992-997 .
  • chemoresistance genes are also used as selectable markers in vitro and in vivo. See also, Mengiste (1997) "High-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1' promoter," Plant J. 12:945-948, showing that the 1' promoter is an attractive alternative to the cauliflower mosaic virus (CaMV) 35S promoter for the generation of T-DNA insertion lines, the 1' promoter may be especially beneficial for the secondary transformation of transgenic strains containing the 35S promoter to exclude homology-mediated gene silencing.
  • CaMV cauliflower mosaic virus
  • Constitutive Promoters In construction of recombinant expression cassettes, vectors, transgenics, of the invention, a promoter fragment can be employed to direct expression of the desired gene in all tissues of a plant or animal. Promoters that drive expression continuously under physiological conditions are referred to as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include those from viruses which infect plants, such as the cauliflower mosaic virus (CaMV) 35S transcription initiation region (see, e.g., Dagless (1997) Arch. Virol.
  • CaMV cauliflower mosaic virus
  • a plant promoter may direct expression of the PDR nucleic acid of the invention under the influence of changing environmental conditions or developmental conditions.
  • environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.
  • inducible promoters are referred to herein as "inducible" promoters.
  • the invention incorporates the drought-inducible promoter of maize (Busk (1997) supra); the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant Mol. Biol. 33:897-909).
  • Embodiments of the invention also incorporate use of plant promoters which are inducible upon injury or infection to express the invention's constitutively active PDR polypeptides.
  • Various embodiments include use of, e.g., the promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene (EAS4 promoter), which is expressed in wounded leafs, roots, and stem tissues, and upon infection with microbial pathogens (Yin (1997) Plant Physiol 115(2):437-451); the ORF13 promoter from Agrobacterium rhizogenes 8196, which is wound inducible in a limited area adjacent to the wound site (Hansen (1997) Mol.
  • EAS4 promoter the promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene
  • Shpx ⁇ b gene promoter which is a plant peroxidase gene promoter induced by microbial pathogens (demonstrated using a fungal pathogen, see Curtis (1997) Mol. Plant Microbe Interact. 10:326-338); the wound-inducible gene promoter wunl, derived from potato (Siebertz (1989) Plant Cell 1 :961-968); the wound-inducible Agrobacterium pmas gene (mannopine synthesis gene) promoter (Guevara-Garcia (1993) Plant J.
  • auxins which are inducible upon exposure to plant hormones, such as auxins
  • the invention can use the auxin-response elements El promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol Plant Microbe Interact. 34
  • Plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics, are also used to express the nucleic acids of the invention. As with the tissue-specific or developmentally specific promoters, harvesting of fruits and plant parts would be greatly facilitated.
  • the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem.
  • Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11 :465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11 :1315-1324.
  • a tetracycline-inducible promoter e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11 :465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11 :1315-1324.
  • a tetracycline-inducible promoter e.g., as described with transgenic tobacco plants containing the Avena
  • a chemical which can be applied to the transgenic plant in the field and induce expression of a polypeptide of the invention throughout all or most of the plant would make a environmentally safe defoliant or herbicide.
  • the invention also provides for transgenic plants containing an inducible gene encoding for the constitutively active polypeptide of the invention whose host range is limited to target plant species, such as weeds or crops before, during or after harvesting.
  • Abcission promoters are activated upon plant ripening, such as fruit ripening, and are especially useful incorporated in the expression systems (e.g., expression cassettes, vectors) of the invention.
  • the plant disease resistant polypeptide-encoding nucleic acid is under the control of such a promoter, rapid cell death, induced by expression of the invention's polypeptide, accelerates and/or accentuates abcission, increasing the efficiency of the harvesting of fruits or other plant parts, such as cotton, and the like. Induction of rapid cell death at this time would accelerate separation of the fruit from the plant, greatly augmenting harvesting procedures, increasing the efficiency of the harvesting of fruits or other plant parts, such as cotton.
  • Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues. Plant promoters which are active only in specific tissues or at specific times during plant development are used to express the nucleic acids of the invention. Examples of promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as roots, leaves, fruit, ovules, seeds, pollen, pistols, or flowers. A seed-specific promoter directs expression in seed tissues. Such promoters may be, for example, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, or some combination thereof. A leaf-specific promoter has been identified in maize, Busk (1997) Plant J. 11:1285-1295.
  • the ORF13 promoter from Agrobacterium rhizogenes exhibits high activity in roots (Hansen (1997) supra).
  • a maize pollen-specific promoter has been identified in maize (Guerrero (1990) Mol. Gen. Genet. 224:161-168).
  • a tomato promoter active during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers can be used (Blume (1997) Plant J. 12:731-746).
  • a pistol specific promoter has been identified in the potato (Solanum tuberosum
  • the Blec4 gene from pea (Pisum sativum cv. Alaska) is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa, making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots.
  • the activity of the Blec4 promoter in the epidermis of the shoot apex makes it particularly suitable for genetically engineering defense against insects and diseases that attack the growing shoot apex (Mandaci (1997) Plant Mol Biol. 34:961-965).
  • tissue-specific plant promoters include a promoter from the ovule-specific BELI gene described in Reiser (1995) Cell 83:735-742, GenBank No. U39944.
  • Suitable seed specific promoters are derived from the following genes: MACI from maize, Sheridan (1996) Genetics 142:1009-1020; Ca ⁇ from maize, GenBank No. 36
  • tissue specific E8 promoter from tomato is particularly useful for directing gene expression so that a desired gene product is located in fruits.
  • suitable promoters include those from genes encoding embryonic storage proteins.
  • a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
  • a plant disease resistant polypeptide-expressing nucleic acid of the invention is expressed through a transposable element. This allows for constitutive, yet periodic and infrequent expression of the constitutively active polypeptide.
  • tissue-specific promoters derived from viruses which can include, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc.
  • RTBV rice tungro bacilliform virus
  • CVMV cassava vein mosaic virus
  • the invention provides for a variety of in vivo systems expressing the constitutively active PDR polypeptides of the invention, including transformed cells and transgenic plants and animals.
  • the polypeptides of the invention are expressed in, in addition to plants cells, a variety of additional expression systems to generate large amounts of protein for, e.g., in vitro functional testing, such as screening for compounds that bind to a PDR polypeptide of the invention, to generate antibodies, structural studies (i.e., crystallization), to generate sufficient protein to apply to a plant to induce a DRR reaction, and the like.
  • the present invention also provides methods and reagents for recombinant, genetically engineered PDR genes in a variety of plant cell systems.
  • these can include fusion of the recipient cells with bacterial protoplasts containing DNA, use of DEAE dextran, polyethylene glycol precipitation (described in Paszkowski (1984) Embo J. 3:2717- 2722), infection with viral vectors, and the like.
  • the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation (described in Fromm (1985) Proc. Natl. Acad. Sci. USA 82:5824) and microinjection of plant cell protoplasts (Schnorf (1991) Transgenic Res. 1 :23-30), or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment, or DNA can be introduced using viruses.
  • Plants can be transformed using viral vectors, such as, for example, tobacco mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) "Use of viral replicons for the expression of genes in plants," Mol. Biotechnol. 5:209-221. Selection and construction of vectors and techniques for transforming a wide variety of plant cells are well known, for example, see Hamamoto, U.S. Patent No. 5,618,699.
  • viral vectors such as, for example, tobacco mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) "Use of viral replicons for the expression of genes in plants," Mol. Biotechnol. 5:209-221. Selection and construction of vectors and techniques for transforming a wide variety of plant cells are well known, for example, see Hamamoto, U.S. Patent No. 5,618,699.
  • Agrobacterium tumefaciens host vector DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.
  • Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. Agrobacterium tumefaciens is routinely utilized in gene transfer to dicotyledonous plants.
  • transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions (Katavic (1994) Mol. Gen. Genet. 245:363-370).
  • Agrobacterium tumefaciens see, e.g., den
  • Bombardment-based (ballistic) methodology is another effective means of transforming plant and other cells.
  • Microprojectile bombardment to deliver DNA into plant cells is an alternative means of transformation for the numerous species considered recalcitrant to Agrobacterium- or protoplast-mediated transformation methods. For example, see, e.g.,
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans, PROTOPLASTS ISOLATION AND CULTURE, HANDBOOK OF PLANT CELL CULTURE, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, REGENERATION OF PLANTS, PLANT PROTOPLASTS, pp. 21-73, CRC Press, Boca Raton, 1985.
  • Regeneration can also be obtained from plant callus, explants, organs, or parts thereof.
  • Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467. See also, e.g., Cheng (1995) "Protoplast electrofusion and regeneration in potato," Methods Mol. Biol.
  • constitutively active plant resistance polypeptides of this invention can be expressed in other systems, such as bacterial, yeast, insect (baculovirus) and mammalian cells. The system used will depend on a variety of factors, including activities and amounts desired.
  • the invention also provides for transgenic plants to be used for producing large amounts of the polypeptides of the invention.
  • transgenic plants For example, see Palmgren (1997) Trends Genet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing human milk protein beta-casein in transgenic potato plants using an auxin-inducible, bidirectional mannopine synthase (masl',2') promoter with Agrobacterium tumefaciens-mzdiated leaf disc transformation methods).
  • the nucleic acids and polypeptides of the invention are expressed in or inserted in plant cells to confer desired traits (e.g., conference of the ability to express a constitutively active plant disease resistant polypeptide) on essentially any plant.
  • the invention has use over a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine,
  • transgenic expression of the nucleic acids of the invention leads to phenotypic changes in seeds and fruit
  • plants comprising the expression cassettes discussed above can be sexually crossed with a second plant to obtain a final product.
  • the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a plant of the invention and another plant.
  • the desired effects e.g., expression of the polypeptides of the invention to induce a DRR reaction
  • both parental plants contain expression cassettes of the invention can be enhanced when both parental plants contain expression cassettes of the invention.
  • the invention also provides methods and reagents for detecting or quantitating the polypeptides of the invention by a variety of methods.
  • plant disease resistant polypeptide can be detected and quantified by incorporating functional activity assays of the invention, by immunological assays utilizing a variety of anti-plant disease resistant polypeptide antibodies provided by the invention, and by nucleic acid-based methodologies, examples of which are also described in detail below.
  • the invention provides antibodies that bind a specific plant disease resistant polypeptide specie or bind a subgenus of polypeptides of the invention, e.g., a kinase domain comprising, or, a leucine-rich domain comprising, or, a nucleotide binding site comprising a plant disease resistant polypeptide, and so can be used to identify and isolate any member of the genus of polypeptides provided for in the invention or to identify a single specie, as described above.
  • Antibodies which can identify any member of the genus or subgenus can be generated by using as antigens peptides containing structural features common to all members of the genus or subgenus. The common structural features of constitutively active plant disease resistant polypeptide are described above.
  • the antibodies of the invention can be used to identify, purify, or inhibit any or all activity of the constitutively active plant disease resistant polypeptides of the invention.
  • Such techniques include selection of antibodies from libraries of recombinant antibodies displayed in phage ("phage display libraries") or similar on cells. See, Huse (1989) Science 246:1275; Ward (1989) Nature 341 :544; Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997)
  • Recombinant antibodies can be expressed by transient or stable expression vectors in mammalian cells, as in Norderhaug (1997) J. Immunol Methods 204:77-87; Boder (1997) "Yeast surface display for screening combinatorial polypeptide libraries," Nat. Biotechnol. 15:553-557. Purification of Constitutively Active Plant Disease Resistant Proteins
  • the methods and reagents of the invention enable one to purify the polypeptides of the invention from a variety of sources, depending on which natural source, synthetic or recombinant expression system selected, such as plant cells, larval homogenates, bacterial cells, yeast, mammalian cells, human cells, tissue culture media, transgenic plants and animals, to substantial purity.
  • natural source, synthetic or recombinant expression system selected such as plant cells, larval homogenates, bacterial cells, yeast, mammalian cells, human cells, tissue culture media, transgenic plants and animals, to substantial purity.
  • Plant disease resistant polypeptides can also be expressed as recombinant proteins with one or more additional polypeptide domains linked thereto to facilitate protein detection, purification, or other applications.
  • detection and purification facilitating domains include, but are not limited to, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein a domains that allow purification on immobilized immunoglobulin, and the domain utilized in 42 the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between the purification domain and plant disease resistant polypeptide may be useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein comprising the sequence encoding a plant disease resistant polypeptide of the invention and nucleic acid sequence encoding six histidine residues followed by thioredoxin and an enterokinase cleavage site (e.g., see Williams (1995) Biochemistry 34:1787-1797). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the desired protein(s) from the remainder of the fusion protein.
  • Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described, see e.g., Kroll (1993) DNA Cell. Biol, 12:441-53.
  • the functional assays described below can be used to identify a constitutively active plant disease resistant polypeptide within the scope of this invention.
  • the assays can also be used to detect, assess the purity of and quantify synthetically produced or recombinant proteins produced in plant, bacteria, insect and yeast, tissue culture fluid and animal tissues.
  • the biological assays can also be used to determine the level of purification of isolated polypeptide.
  • the activity assays described below and provided by the present invention can be used to identify compositions which modulate, i.e., modify, activate or inhibit, the activity of a constitutively active plant disease resistant polypeptide.
  • One exemplary assay to demonstrate the constitutive biological activity of a polypeptide of the invention is by transient transformation of Nicotiana spp. and the tomato Lycopersicon esculentum cv. Rio Grande 76S. Leaves of each species are infiltrated with suspensions of a Agrobacterium tumefaciens gene transfer vector capable of expressing the genetically engineered Pto kinase. The expression vectors are under the control of a promoter which is constitutively expressed in plant cells. Activation of the DRR response in leaves expressing a polypeptide of the invention is visualized.
  • RNA Quantitation of message
  • Quantitation of message is useful for determining the transcriptional efficiency of recombinant DNA in expression systems, such as with in vitro transcription, antisense RNA expression, transfection of mortal or immortal cells and transgenic plants and animals. Evaluating levels of RNA is also useful in evaluating cis- or trans- transcriptional regulators.
  • the invention provides for a method for screening for a compound that binds to a PDR polypeptide comprising contacting the polypeptide of the invention with a test compound measuring the ability of the test compound to bind the PDR polypeptide.
  • the test compound can comprise a pathogen polypeptide, a plant polypeptide, or an animal polypeptide.
  • phage display Keratz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45
  • two hybrid system as in James (1996) Genetics 144:1425-1436; Adey (1997) Biochem. J.
  • kinase domain refers to a region common to all protein kinases, including tyrosine kinases and serine/threonine kinases, which make up a large superfamily of homologous proteins. All of these enzymes use a gamma-phosphate of ATP or GTP to generate phosphate monoesters using either protein alcohol groups (for serine and threonine) or protein phenolic groups (as on tyrosine) as phosphate acceptors. Typically, the "kinase domain” consists of about 250 to 300 amino acid residues that fold into a common catalytic core.
  • kinase domain can be further characterized as comprising twelve kinase subdomains, designated as Roman numerals I, II, III, IV, V, VIA, VIB, VII, VIII, IX, X, and XI, as reviewed by, e.g., Hanks (1995) supra, and discussed in detail above.
  • kinase domains including any member of the genus of kinase-containing plant disease resistant polypeptides of the invention.
  • activation segment refers to a kinase subdomain which is a subportion of kinase subdomain VIEI and or subdomain VII, described above, or a related homolog or allele thereof.
  • the activation segment can be described as being located in the region between the DFG and APE kinase domain sequence motifs (see, e.g., Hanks (1995) FASEB J. 9:576-596).
  • the activation segment plays a major role in kinase substrate recognition.
  • the invention provides for the modification of activation segments within PDR polypeptides, and their encoding nucleic acids, that alter the confirmation of this domain to confer constitutive activation of a DRR.
  • the modification typically comprises at least one amino acid substitution, as described above.
  • T loop refers to a subportion of kinase subdomain VIII.
  • the T loop can be an element of an activation segment.
  • T loops can be readily identified through sequence homology with other kinases or by analysis of the three dimensional structure of a kinase.
  • the T loop was initially defined on the basis of three dimensional (3D) structural information.
  • Figures 1 A and IB clearly identify a T loop subdomain in the three dimensional structure of two kinases; see detailed discussion, supra. 45
  • P+l loop refers to a subportion of kinase subdomain VII.
  • the P+l loop can be an element of an activation segment.
  • P+l loops, as T loops can be readily identified through sequence homology with other kinases or by analysis of the three dimensional structure of a kinase.
  • the P+l loop was also initially defined on the basis of three dimensional (3D) structural information.
  • the P+l loop can be recognized by different consensus sequences in serine/threonine and tyrosine kinases. In S/T kinases the consensus is G[S/T]xx[F/Y]xAPE. In tyrosine kinases it is xPxxWxAPE.
  • nucleotide binding site or “nucleotide binding domain” (“NBS”) includes reference to regions of a polypeptide that has homology to sequences in the G protein superfamily that are responsible for binding and hydrolyzing small nucleotide phosphates, i.e., ATP and GTP. While ATP and GTP binding domains are typically included in the "kinase domain" of kinase polypeptides, as described above, the kinase domains in the NBS are not related to kinase domains of protein kinases like Pto.
  • kinase NBS subdomains of resistance genes can include three subdomain motifs: the P-loop, kinase-2, and kinase-3a subdomains (Yu (1996) supra).
  • examples include the Arabidopsis RPP5 gene (Parker (1997) supra), the A thaliana RPS2 gene (Mindrinos (1997) supra), and the flax L6 rust resistance gene (Lawrence (1995) supra) which all encode proteins containing an NBS; and Mindrinos (1994) Cell 78:1089-1099; and Shen (1993) EERS 335:380-385.
  • the teachings disclosed and incorporated herein and standard nucleic acid hybridization and/or amplification techniques one of skill can identify members having NBS domains, including any of the genus of NBS-containing plant disease resistant polypeptides of the invention.
  • leucine rich region includes a region of a polypeptide that is rich in the amino acid leucine and chemically related aliphatic amino acids, such as isoleucine, valine, and alanine.
  • a leucine rich region typically has a leucine content of at least 20% leucine or isoleucine, or 30% of the aliphatic residues: leucine, isoleucine, methionine, valine, and phenylalanine, and arranged with approximate repeated periodicity.
  • the sequence usually, but not always, has a periodicity of 15 to 30 amino acids, with part of the repeated sequence having the approximate consensus "axxaxa,” where "a” is an aliphatic residue and "x” is any amino acid.
  • the length of the repeat may vary in length, ranging 46 typically from about 15 to 30 amino acids.
  • An LRR-containing polypeptide typically will have a leucine-rich repeat (LRR) amino acid sequence which can, in some proteins, mediate molecular recognition and/or interaction processes; as described, e.g., in Bent (1994) Science
  • negatively charged residue includes any negatively charged residue in a polypeptide, including, e.g., natural amino acids (e.g., glutamate or aspartate) or synthetic negatively charged residues.
  • the negative charge can be incorporated in an otherwise uncharged residue after the polypeptide has been synthesized (which can be, as described above, synthesis by DNA polymerase, as in recombinantly produced polypeptides, or, synthetically produced polypeptides).
  • promoter refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating and/or regulating transcription in plant cells; see also discussion on plant promoters, supra.
  • constitutive promoter refers to a promoter that initiates and helps control transcription in all tissues. Promoters that drive expression continuously under physiological conditions are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation; see also detailed discussion, supra.
  • inducible promoter refers to a promoter which directs transcription under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may effect transcription by inducible promoters 47 include anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as “inducible” promoters; see also detailed discussion, supra.
  • abcission-induced promoter or “abcission promoter” refers to a class of promoters which are activated upon plant ripening, such as fruit ripening, and are especially useful incorporated in the expression systems (e.g., expression cassettes, vectors) of the invention.
  • tissue-specific promoter refers to a class of transcriptional control elements that are only active in particular cells or tissues. Examples of plant promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as roots, leaves, fruit, ovules, seeds, pollen, pistols, or flowers; see also detailed discussion, supra.
  • DRR plant disease resistance
  • An exemplary DRR includes a rapid cell death reaction mediated by a plant in response to pathogenic infection; which can include the hypersensitive reactive (HR) described herein.
  • HR hypersensitive reactive
  • DRR is not limited by any particular biological pathway or mechanism.
  • the “hypersensitive reaction” refers to one mechanism by which a plant can effect a DRR, i.e., a rapid plant cell necrosis, in response to a pathogenic invasion.
  • the HR necrotic reaction is characterized by the appearance of necrotic flecks containing dead plant cell(s) at sites of attempted pathogen ingress.
  • plant cell death is typically seen as soon as a few hours of pathogen contact, but, the onset of the HR reaction can be more rapid or slower.
  • the HR can be phenotypically diverse between different plant species, ranging from the death of a single cell to spreading necrotic areas (Holob (1994) supra).
  • Plant includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and plant cells and progeny of same.
  • the class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous (see discussion, supra).
  • antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments or synthetic or recombinant analogues thereof which specifically bind and recognize analytes and antigens, such as a genus or subgenus of polypeptides of the invention, as described supra.
  • conservative substitution refers to a change in the amino acid composition of a protein, such as the PDR polypeptide of the invention, that does not substantially alter the protein's activity. This includes conservatively modified variations of a particular amino acid sequence, i.e., amino acid substitutions of those amino acids that are not critical for protein activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter activity, a polypeptide sequence of the invention implicitly encompasses conservatively substituted variants thereof. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (a), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see also, Creighton (1984) Proteins, W.H. Freeman and Company).
  • substitutions are not the only possible conservative substitutions.
  • conservative substitutions for each other whether they are positive or negative.
  • individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered “conservatively modified variations.”
  • the term "conservative substitution” also refers to a change in a nucleic acid sequence such that the substitution does not substantially alter the contemplated activity of the nucleic acid, for example, as not changing the activity of the protein encoded by the nucleic acid, a nucleic acid sequence of the invention implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer (1991) Nucleic Acid Res. 19:5081;
  • expression cassette refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell.
  • the term includes linear or circular expression systems.
  • the term includes expression cassettes, e.g., vectors, that remain episomal or integrate into the host cell genome.
  • the expression cassettes can have the ability to self-replicate or not, i.e., drive only transient expression in a cell.
  • the term includes recombinant expression cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
  • isolated when referring to a molecule or composition, such as, for example, a constitutively active plant disease resistant polypeptide or nucleic acid, means that the molecule or composition is separated from at least one other compound, such as a protein, other nucleic acids (e.g., RNAs), or other contaminants with which it is associated in vivo or in its naturally occurring state.
  • a constitutively active plant disease resistant polypeptide or nucleic acid is considered isolated when it has been isolated from any other component with which it is naturally associated, e.g., cell membrane, as in a cell extract.
  • An isolated composition can, however, also be substantially pure.
  • An isolated composition can be in a homogeneous state and can be in a dry or an aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemistry techniques such as 50 polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC).
  • analytical chemistry techniques such as 50 polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC).
  • nucleic acid molecule or “nucleic acid sequence” refers to a deoxyribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form.
  • the term encompasses nucleic acids, i.e., oligonucleotides, containing- known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid.
  • the term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved thereover for the purposes desired.
  • nucleic-acid-like structures with synthetic backbones are examples of synthetic backbones.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem.
  • PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197. Other synthetic backbones encompasses by the term include methylphosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156).
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
  • exogenous nucleic acid refers to a nucleic acid that has been isolated, synthesized, cloned, ligated, excised in conjunction with another nucleic acid, in a manner that is not found in nature, and/or introduced into and/or expressed in a cell or cellular environment other than or at levels or forms different than the cell or cellular environment in which said nucleic acid or protein is be found in nature.
  • the term encompasses both nucleic acids originally obtained from a different organism or cell type than the cell type in which it 51 is expressed, and also nucleic acids that are obtained from the same cell line as the cell line in which it is expressed, invention.
  • recombinant when used with reference to a cell, or to the nucleic acid, protein or vector refers to a material, or a material corresponding to the natural or native form of the material, that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto but produced or derived from synthetic materials.
  • recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise expressed at a different level, typically, under-expressed or not expressed at all.
  • recombinant means encompasses all means of expressing, i.e., transcription or translation of, an isolated and/or cloned nucleic acid in vitro or in vivo.
  • recombinant means encompasses techniques where a recombinant nucleic acid, such as a cDNA encoding a protein, is inserted into an expression vector, the vector is introduced into a cell and the cell expresses the protein.
  • Recombinant means also encompass the ligation of nucleic acids having coding or promoter sequences from different sources into one vector for expression of a fusion protein, constitutive expression of a protein, or inducible expression of a protein, such as the plant disease resistant polypeptides of the invention.
  • sequence identity refers to when two sequences, such as the nucleic acid and amino acid sequences or the polypeptides of the invention, when optimally aligned, as with, for example, the programs PILEUP, BLAST, GAP, FASTA or BESTFIT (see discussion, supra).
  • Percentage amino acid/nucleic acid sequence identity refers to a comparison of the sequences of two polypeptides/nucleic acids which, when optimally aligned, have approximately the designated percentage of the same amino acids/nucleic acids, respectively.
  • nucleic acids encoding constitutively active PDR polypeptides of the invention comprise a sequence with at least 50% nucleic acid sequence identity to SEQ ID NO:l.
  • the constitutively active PDR polypeptides of the invention are encoded by nucleic acids comprising a sequence with at least 50% sequence identity to SEQ ID NO: 1 , 52 or, are encoded by nucleic acids comprising SEQ ID NO:l, or, have at least 60% amino acid sequence identity to the polypeptide of SEQ ID NO:2.
  • the term "specifically hybridizes” refers to a nucleic acid that hybridizes, duplexes or binds to a particular target DNA or RNA sequence.
  • the target sequences can be present in a preparation of total cellular DNA or RNA.
  • Proper annealing conditions depend, for example, upon a nucleic acid's, such as a probe's length, base composition, and the number of mismatches and their position on the probe, and can be readily determined empirically providing the appropriate reagents are available. For discussions of nucleic acid probe design and annealing conditions, see, e.g., Sambrook and Ausubel.
  • stringent hybridization refers to conditions under which an oligonucleotide (when used, for example, as a probe or primer) will hybridize to its target subsequence, such as a plant disease resistant sequence nucleic acid in an expression vector of the invention but not to a non-plant disease resistant sequence.
  • Stringent conditions are sequence-dependent. Thus, in one set of stringent conditions an oligonucleotide probe will hybridize to only one specie of the genus of plant disease resistant nucleic acids of the invention.
  • an oligonucleotide probe will hybridize to all species of the invention's genus but not to non-plant disease resistant nucleic acids. Longer sequences hybridize specifically at higher temperatures. Stringent conditions are selected to be about 5 C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50%) of the probes complementary to the target sequence hybridize to the target sequence at equilibrium (if the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • TJ thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, i.e., about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Often, high stringency wash conditions preceded by low stringency wash conditions to remove background probe signal.
  • An example of medium stringency wash conditions for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C 53 for 15 minutes (see Sambrook for a description of SSC buffer).
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a "specific hybridization.”
  • Nucleic acids which do not hybridize to each other under stringent conditions can still be substantially identical if the polypeptides which they encode are substantially identical.
  • the invention provides for a genus of constitutively active plant disease resistant polypeptides generated by sequence modification of the inducible, wild-type corresponding polypeptide.
  • the following example details the modification of an exemplary PDR sequence.
  • the invention provides for a modification of plant disease resistance kinase domains by engineering at least one negatively charged residue in the kinase domain.
  • the wild-type tyrosine residue at position 207 (tyr 207 , numbering based on SEQ ID NO:2) is replaced by an amino acid residue with a negative charge, such as an aspartate or a glutamate (Pto-glu 207 or Pto-asp 207 ).
  • the presence of a negatively charged amino acid in this position irreversibly turns on the Pto polypeptide's ability to initiate the HR reaction, i.e., the mutated Pto polypeptide becomes constitutively 54 active.
  • the invention is not limited by the mechanistics of the conference of constitutive activity created by these mutations, it may be that the amino acid substitutions in the putative activation segment, such as the presence of a negative charge in the kinase domain, mimics the effects of phosphorylation or the binding of a ligand, such as an AvrPto gene product.
  • Pto-tyr 207 replaced by Pto-asp 207 (SEQ ID NO:2 , encoded by the nucleic acid of SEQ ID NO:l); Pto- thr 204 replaced by Pto-asp 204 (SEQ ID NO:4, encoded by the nucleic acid of SEQ ID NO:3); Pto-tyr 207 replaced by Pto-ala 207 (SEQ ID NO:6, encoded by the nucleic acid of SEQ ID NO:5); Pto-tyr 207 replaced by Pto-trp 207 (SEQ ID NO:8) encoded by the nucleic acid of SEQ ID NO:7); fen-tyr 205 to fen-asp 205 (SEQ ID NO:10).
  • Site-specific mutagenesis was carried out essentially using the method described by Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection," Proc. Natl. Acad. Sci. USA 82:488-492, using single-stranded DNA as a template.
  • the invention provides for a genus of constitutively active plant disease resistant polypeptides.
  • the following example details the induction of a DRR, specifically, an HR response, by an exemplary PDR polypeptide.
  • Pto-tyr 207 replaced by Pto-asp 207 (SEQ ID NO:2 , encoded by the nucleic acid of SEQ ED NO:l); Pto-thr 204 replaced by Pto-asp 204 (SEQ ID NO:4, encoded by the nucleic acid of SEQ ID NO:3); Pto-tyr 207 replaced by Pto-ala 207 (SEQ ID NO:6, encoded 55 by the nucleic acid of SEQ ED NO:5); Pto-tyr 207 replaced by Pto-trp 207 (SEQ ID NO:8) encoded by the nucleic acid of SEQ ID NO:7); fen-tyr 205 to fen-asp 205 (SEQ ED NO:10).
  • Mutant genes were sequenced to confirm the presence of each mutation and cloned into an expression cassette consisting of the cauliflower mosaic virus 35S promoter, the omega fragment leader sequence of tobacco mosaic virus (Gallie (1987) Nucl. Acids Res. 15:3257-3272; Kukla (1979) Ewr. J. of Biochem. 98:1-66; ATCC Accession nos. M24955 and M24992), and the ocs terminator sequence (Jones (1992) Transgenic Res. 1 : 285-297).
  • the entire gene expression cassette thus assembled was cloned into a binary vector, in this example, .TFS-40 (British Sugar), between the left and right borders of the T-DNA.
  • any binary vector for the Agrobacterium-mzdiatcd genetic transformation of plants can be used, see, e.g., McCormac (1997) Mol. Biotechnol. 8:199-213; Hamilton (1997) Gene 200:107-116.
  • Binary clones were transferred to the Agrobacterium tumefacient strain LBA4404 (obtained from ATCC, http://www.atcc.org/).
  • most publicly available Agrobacterium strains can be used. Single colonies were selected and grown in LB medium to stationary phase in the presence of appropriate antibiotic selection (antibiotic selection for constructs based on pTFS-40 is tetracycline, at 1 microgram per milliliter), then subcultured 1 :10 in LB with antibiotic and grown overnight.
  • a further embodiment of the invention provides for expression of the constitutively active PDR polypeptides of the invention in transgenic plants using, e.g., constitutive or inducible, tissue-specific, developmentally specific, or environmentally (e.g., drought-, salt- or cold-) sensitive transcriptional control elements, such as promoters and enhancers.
  • the coding sequence for the PDR polypeptide can be expressed under the control of a constitutive or inducible promoter.
  • a constitutive promoter is the 35S promoter from Agrobacterium (see, e.g., Mengiste (1997) supra).
  • An exemplary inducible promoter is a viral sub-genomic promoter, e.g., from the tomato bushy stunt virus (see, e.g., Hillman (1989) supra; GenBank Accession Nos. M21958, M31019, U80935).
  • the gene expression cassette including the PDR coding sequence, selected inducible or constitutive promoter, and optionally, tagging, selection and marker genes (described in detail, above), are cloned into a binary vector between the left and right borders of a T-DNA, as described above.
  • the binary clones were transferred to a Agrobacterium sp. , which were infiltrated into plant cells, as described above.
  • the clones were also inserted into tomato plant genomic DNA by co-cultivation of the Agrobacterium and tomato tissue, as described by McCormick (1986) Plant Cell Rep. 5:81-84.
  • the binary clones are also introduced directly into the genomic DNA of the plant cell by electroporation, microinjection of plant cell protoplasts (Schnorf (1991) 57
  • Selection for tissue transgenic for insertion of the T-DNA containing the PDR coding sequence is effected by growth of the tissue on a selectable substance, such as Kanamycin, as discussed above.
  • Plantlets surviving the selection step are transferred to soil to mature into adult plants.
  • Standard techniques can be used to analyze the location and copy number of PDR transgenes and the presence and amounts of transcripts and polypeptides, e.g., by nucleic acid amplification or hybridization techniques, as described above. Rapid cell death induced by expression of the invention's polypeptide would prevent pathogen (e.g., viral) replication and dissemination in the infected plant, thus preventing death of the plant and minimizing the amount of pathogen-induced damage to the plant.
  • pathogen e.g., viral
  • constitutively active PDR polypeptide can be controlled on a transcriptional level, i.e., the amount of PDR polypeptide in a cell is regulated by the amount of message RNA transcribed, which in turn is regulated by an inducible promoter.

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Abstract

D'une manière générale, cette invention se rapporte à la résistance aux maladies chez les végétaux. Plus particulièrement, cette invention se rapporte à la découverte d'une nouvelle famille de gènes et polypeptides de résistance aux maladies mis au point par génie génétique, lesquels, lorsqu'il sont exprimés dans une cellule végétale, peuvent induire une réponse de résistance aux maladies.
PCT/US1999/001970 1998-01-30 1999-01-28 Genes et polypeptides de resistance aux phytopathies constitutivement actifs WO1999038989A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2799204A1 (fr) * 1999-10-01 2001-04-06 Agronomique Inst Nat Rech Nouvelle classe de proteines et leurs applications a la resistance de plantes a divers agents pathogenes
DE19958961A1 (de) * 1999-12-07 2001-06-28 Bioplant Biotechnologisches Fo Nukleinsäuren zur Herstellung von transgenen Pflanzen mit erhöhter Phytopathogen-Resistenz
WO2001029239A3 (fr) * 1999-10-15 2002-06-13 Plant Bioscience Ltd Genes de resistance modifies
WO2010022443A1 (fr) * 2008-08-25 2010-03-04 Commonwealth Scientific And Industrial Research Organisation Gènes de résistance

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MARTIN G B, ET AL.: "MAP-BASED CLONING OF A PROTEIN KINASE GENE CONFERRING DISEASE RESISTANCE IN TOMATO", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 262, 26 November 1993 (1993-11-26), US, pages 1432 - 1436, XP002924152, ISSN: 0036-8075, DOI: 10.1126/science.7902614 *
THILMONY R. L., ET AL.: "EXPRESSION OF THE TOMATO PTO GENE IN TOBACCO ENHANCES RESISTANCE TOPSEUDOMONAS SYRINGAE PV TABACI EXPRESSING AVRPTO.", THE PLANT CELL, AMERICAN SOCIETY OF PLANT BIOLOGISTS, US, vol. 07., 1 October 1995 (1995-10-01), US, pages 1529 - 1536., XP002053463, ISSN: 1040-4651, DOI: 10.1105/tpc.7.10.1529 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2799204A1 (fr) * 1999-10-01 2001-04-06 Agronomique Inst Nat Rech Nouvelle classe de proteines et leurs applications a la resistance de plantes a divers agents pathogenes
WO2001025458A1 (fr) * 1999-10-01 2001-04-12 Institut National De La Recherche Agronomique (Inra) Nouvelle classe de proteines et leurs applications a la resistance de plantes a divers agents pathogenes
WO2001029239A3 (fr) * 1999-10-15 2002-06-13 Plant Bioscience Ltd Genes de resistance modifies
DE19958961A1 (de) * 1999-12-07 2001-06-28 Bioplant Biotechnologisches Fo Nukleinsäuren zur Herstellung von transgenen Pflanzen mit erhöhter Phytopathogen-Resistenz
DE19958961B4 (de) * 1999-12-07 2004-09-16 Bioplant Biotechnologisches Forschungslabor Gmbh Nukleinsäuren zur Herstellung von transgenen Pflanzen mit erhöhter Phytopathogen-Resistenz
WO2010022443A1 (fr) * 2008-08-25 2010-03-04 Commonwealth Scientific And Industrial Research Organisation Gènes de résistance
AU2009287411B2 (en) * 2008-08-25 2013-11-07 Commonwealth Scientific And Industrial Research Organisation Resistance genes
US8581038B2 (en) 2008-08-25 2013-11-12 Grains Research And Development Corporation Resistance genes
US9115370B2 (en) 2008-08-25 2015-08-25 Grains Research And Development Corporation Resistance genes
EA031178B1 (ru) * 2008-08-25 2018-11-30 Коммонвелт Сайентифик Энд Индастриал Рисерч Организейшн Гены резистентности

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